Potential natural food preservatives and their sustainable production

Mar 7, 2019 - Food Chem. , Just Accepted Manuscript ... using the generally recognized as safe (GRAS) strain, Saccharomyces cerevisiae, as a cell fact...
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Potential natural food preservatives and their sustainable production in yeast: terpenoids and polyphenols Xiaomei Lyu, Jaslyn Lee, and Wei Ning Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07141 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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Journal of Agricultural and Food Chemistry

Potential natural food preservatives and their sustainable production in yeast: terpenoids and polyphenols Xiaomei Lyu, Jaslyn Lee, Wei Ning Chen* School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore

* Corresponding author: Wei Ning Chen Address: School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore Tel: (+65)6316 2870 Email: [email protected]

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Abstract

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Terpenoids and polyphenols are high-valued plant secondary metabolites. Their high antimicrobial

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activities demonstrate their huge potential as natural preservatives in the food industry. With the

4

rapid development of metabolic engineering, it has become possible to realize large-scale production

5

of non-native terpenoids and polyphenols by using the generally recognized as safe (GRAS) strain,

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Saccharomyces cerevisiae, as a cell factory. This review will summarize the major terpenoid and

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polyphenol compounds with high antimicrobial properties, describe their native metabolic pathways

8

as well as antimicrobial mechanisms, and highlight current progress on their heterologous

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biosynthesis in S. cerevisiae. Current challenges and perspectives for the sustainable production of

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terpenoid and polyphenol as natural food preservatives via S. cerevisiae will also be discussed

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herein.

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Keywords: terpenoids; polyphenols; food preservatives; sustainable production; antimicrobial

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mechanism

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Journal of Agricultural and Food Chemistry

1. Introduction

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Food safety is an important concern for the food industry. Despite the tremendous advances in

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production technologies, the cases of food poisoning worldwide continue to grow. It has been

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estimated that 600 million people suffered from foodborne illnesses, with approximately 230,000

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deaths caused from foodborne diarrhoeal disease agents in 20101. Microorganisms are the main

20

cause of food poisoning, with Salmonella and Campylobacter species causing the majority of food

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poisoning cases. The toxigenic organism Staphylococcus aureus and Clostridium botulinum, are the

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cause of far fewer cases2. In addition to foodborne illnesses, spoilage of foods by microbes also leads

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to enormous losses. One-quarter of the world’s food supply was estimated to be lost, due to the

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growth of spoilage and pathogenic microorganisms3. To combat this, various synthetic

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antimicrobials, e.g., nitrates, benzoates, sorbic acid, sulphites, potassium lactate, and citric acid, have

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been in use as food preservatives, for decades. However, their usage presents toxic risk on human

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and animal health as reported in many studies 4, 5. For example, nitrate can get converted into nitrous

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acid when consumed and is suspected of causing stomach cancer, and benzoates may cause allergies

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or skin rashes5. Hence there is growing consumer demands and an urgent need for a natural food

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preservative, which does not compromise on food safety, convenience, or sensory characteristics.

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Essential oils (EO) are concentrated hydrophobic liquids extracted from diverse plants. In

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addition to their original usage as food additives due to their fragrant properties, their demonstrated

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antimicrobial activities against a wide range of microorganisms makes them good candidates as

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natural food preservatives6-14. Composition analysis showed that the volatile portion of EO mainly

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consists of monoterpenes and sesquiterpene hydrocarbons, alcohols, aliphatic aldehydes, and esters15.

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In addition to terpenoids, polyphenols extracted from the plant also have high antimicrobial activity 3

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and thus display high potential for applications as food preservatives16-18. To date, many terpenoid

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and polyphenol compounds (e.g. linalool, thymol, eugenol, carvone, cinnamaldehyde, vanillin,

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carvacrol, citral, and limonene) have been accepted by the European Commission for use as

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flavoring in food products and are recognized as safe by the United States Food and Drug

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Administration (FDA)19. Currently, the supply of natural terpenoids and polyphenols is largely

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dependent on plant extraction, which is subject to the limitations of climate and long growth cycles.

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Based on the advancement of mature high-density fermentation and metabolic engineering, microbial

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biosynthesis could provide an environmentally sustainable and cost-effective alternative for mass

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production of such high-value chemicals in a short period of time20, 21. Its collaboration with plant

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extraction methods showed good prospects to meet the growing needs of the food market.

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Considering that both terpenoids and polyphenols have long biosynthetic pathways and are used in

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the field of foods and medicine, the desired cell factory should have advantageous features inclusive

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of high security and ease of genetic manipulation. Saccharomyces cerevisiae (S. cerevisiae) is an

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attractive cell factory, due to its safety, genetic tractability, and mature high-density fermentation

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technology. Its eukaryotic nature allows expression of multiple complex proteins and production of

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various bio-based products22. Furthermore, the development of excellent pathway assembly tools23

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and advanced dynamic metabolic regulation systems24 makes it possible to complement the

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construction and metabolic optimization of the heterologous biosynthetic pathways in yeast. Up to

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now, there have been many studies on sustainable production of terpenoids and polyphenols by

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metabolic engineering of S. cerevisiae and exploring their antimicrobial activities for use in the food

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area. As no review has been published in this context, the aim of this paper was to summarize major

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terpenoid and polyphenol compounds, which could potentially be used as natural food preservatives, 4

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their antimicrobial mechanisms, as well as metabolic pathways, and metabolic engineering strategies

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for the biosynthesis of them in engineered S. cerevisiae. In addition, current challenges and

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perspectives are also discussed herein.

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2. Terpenoids

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Terpenoids comprise a vast family of the most abundant natural products which are widely

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distributed in nature. They exist in almost every organism, having important physiological,

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metabolic, communication and defense functions. Naturally occurring isoprenoids have important

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application value to humans: the primary components of oil metabolites in plants can be used as

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flavoring agents and aromatics; secondary metabolites have important medicinal value including

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anti-inflammatory, antibacterial, anti-hypertensive, immune regulation, anti-oxidation, anti-cancer,

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and other functions.

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2.1 Biosynthetic pathway of terpenoids

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Terpenoids are modified forms of terpenes ((C5H8)n). This basic terpene form (C5H8)n, can have

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oxygen molecules added to it, or have its methyl group removed, to form terpenoid derivatives

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including alcohols, esters, aldehydes, ketones, ethers, phenols, and epoxides. Also, they can be

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classified into hemi-(C5), mono- (C10), sesqui- (C15), di- (C20), sester- (C25), tri- (C30), tetra-

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(C40) and polyterpenes (C5)n depending on the number of isoprene units25. The biosynthetic

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pathway of terpenoids can be divided into four basic steps (Figure 1)26: (1) synthesis of monomer

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isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) from the initial acetyl-CoA

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molecule; (2) polymerization of DMAPP and IPP under the action of isopentenyl transferase to form

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the linear prenyl diphosphate precursor - geranyl pyrophosphate (GPP, C10, monoterpenoid),

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farnesyl pyrophosphate (FPP, C15, sesquiterpenoid), and geranylgeranyl diphosphate (GGPP, C20, 5

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biguanide); (3) cyclization or rearrangement of linear isopentenyl pyrophosphate precursors under

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catalysis of terpene synthases to form an initial terpene carbon skeleton; (4) tailoring and

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modification of the carbon skeleton to biologically active chemicals.

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Monomer isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are the

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universal precursors for the biosynthesis of various downstream terpenoids (hemiterpenoid,

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monoterpenoid,sesquiterpenoid, diterpenoid, etc.). There are two distinct pathways for biosynthesis

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of IPP and DMAPP27-30: the mevalonate pathway (MVA pathway) and the methylerythritol

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phosphate pathway (MEP pathway) (Figure 1). The MVA pathway is ubiquitous in the cytoplasm of

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all organisms including: some bacteria, most fungi, and plants, which initiates from acetyl-CoA and

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ends up with the production of DMAPP31, 32. As the primary precursor, two molecules of acetyl-CoA

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are condensed to acetoacetyl-CoA in a reversible reaction via acetoacetyl-CoA thiolase (ERG10).

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Under

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3-hydroxy3-methylglutaryl-CoA (HMG-CoA) and subsequently generated in mevalonate (MVA)

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within two reduction steps which consumes NADPH. The latter reaction is catalyzed through

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3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme of the MVA pathway

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33.

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phosphor-MVA kinase (PMK). Lastly, IPP is generated from MVA 5-diphosphate under an

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ATP-dependent decarboxylation reaction via diphospho-MVA decarboxylase (MVD1). This can be

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interconverted to DMAPP by isomerization via IPP isomerase (IDI). In 1996, Eisenvaich et al. found

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that the taxane carbon skeleton of taxol was not of mevalonoid origin, in an experiment using(13)C

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stable isotope labelling34. This triggered the discovery of the MEP pathway which is present in most

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bacteria, green algae, and plants27, 28. The MEP pathway, also referred to as the 1-deoxy-D-xylulose

the

catalysis

of

HMG

synthase

(HMGS),

Acetoacetyl-CoA

is

converted

to

MVA is then phosphorylated twice to MVA 5-diphosphate through MVA kinase (MK) and

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5-phosphate (DXP) pathway, starts from the condensation of glyceraldehyde-3-phosphate (G3P) and

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pyruvateto generate DXP via 1-deoxy-D-xylulose 5-phosphate synthase (DXS). Under catalysis of

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DXP reductoisomerase (DXR), DXP is intramolecularly rearranged and reduced to form MEP. Then,

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MEP is further modified to yield hydroxy-2-methyl-2-butenyl-4-diphosphate (HMBPP) within four

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steps catalyzed by 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ISPD), 4-(cytidine 5’ -

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diphospho)-2-C-methyl-D-erythritol kinase (ISPE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate

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synthase (ISPF) and 4-hydroxy-3-methylbut-2-enyldiphosphate (HMBPP) synthase (ISPG). HMBPP

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is subsequently converted into IPP and DMAPP by HMBPP reductase (ISPH). Finally, the

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conversion and balance of IPP to DMAPP35 is controlled by IPP isomerase (IPPI), in a reversible

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reaction. In the MEP pathway, DXS36-38, DXR37,

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rate-limiting enzymes. Compared with the MVA pathway, the MEP pathway is energetically

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balanced and theoretically more efficient than the MVA pathway in converting sugars (30.2% vs.

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25.2% mass yield on glucose) or glycerol to terpenoids40, 41.

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2.2 Antimicrobial property of terpenoids

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and IPPI38 have been identified as the

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Terpenoids are one of the classes of compounds which are responsible for the antimicrobial

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properties in EO. In the study by Dorman, H. J. D. et al., fourteen major compounds in EO from

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eight plants were assessed for their antibacterial activity against 25 different genera of bacteria,

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whereby most of the terpenoids in the forms of monoterpenoids and sesquiterpenes possessed strong

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antimicrobial activity42. The typical terpenoids which possess antimicrobial properties are

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summarized in Table 1 and discussed as follows.

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Monoterpenoids are active against a broad range of microorganisms, with monoterpenoid phenol

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- carvacrol and thymol being the most active components43. GC-MS analysis demonstrated their high 7

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content in plant EO, for example, EO from Moroccan Labiatae contained 58.1% of carvacrol in

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Origanum compactum and 95.5% of thymol in Thymus pectinatus44. On the other hand, many in

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vitro assays of carvacrol45-47 and thymol45,

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towards diverse microbes (e.g. S. cerevisiae, Candida strains, Enterococcus faecalis, E. amylovora,

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E. carotovora, E. coli, L. monocytogenes, S. aureus, Staphylococcus epidermidis, S. typhimurium, P.

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fluorescens, Vibrio vulnificus, and Yersinia enterocolitica) (Table 1). For instance, the antibacterial

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activity of major components in EO, from leaves of Lippia multiflora, Mentha x piperita and

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Ocimum basilicum, have been evaluated against L. monocytogenes, E. aerogenes, E. coli, P.

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aeruginosa, amongst which thymol and carvacrol demonstrated the strongest antibacterial activity

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with an MIC (minimum inhibitory concentration) of 300 and 800 μg/mL, respectively48. Another in

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vitro assay exhibited their antifungal activity against Botrytis cinerea with 100% of inhibition at 100

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ppm

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antimicrobial activity mainly contain linalool50, 51, citral46, carvone52, 53, linalyl acetate54, menthol55,

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56,

49.

47

revealed their outstanding antimicrobial properties

Apart from carvacrol and thymol, the other monoterpenoid compounds with strong

geraniol57-59, and terpineol 47 (see Table 1).

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Sesquiterpenoids are another major constituent of antimicrobial terpenoids, such as the

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compound artemisinin60, 61, farnesene62-64, patchoulol65 and farnesol66-68 as summarized in Table 1.

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Besides, eudesmanes and cuparanes, isolated from the organic extract of the red alga Laurencia

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obtusa Lamouroux have been reported to have antimicrobial activities against a wide range of

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microbes, including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas

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aeruginosa, Enterococcus faecalis and Staphylococcus aureus (especially Gram-positive bacteria)69.

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In addition to monoterpenoids and sesquiterpenoids, rare triterpenoids showed microbial

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inhibition activity. Triterpenoid canophyllol isolated from Elaeodendron buchananii Loescan 8

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reported to inhibit the growth of Staphylococcus aureus and Scaphirhynchus albus with MIC value

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of 62.5 μg/ml. It also had promising antibacterial activity against Neisseria meningitidis with an MIC

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value of 31.25 μg/ml. Friedelin, exhibited antibacterial activities against S. aureus and S. albus, as

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well antifungal activity against Trichophyton schoenleinii; wheras umbelliferone showed antifungal

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activity with an MIC value of 62.5 μg/ml against both Crytococcus neoformans and C. albicans70.

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3. Polyphenols

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Polyphenols are natural secondary metabolites derived from the shikimate/phenylpropanoid

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and/or the polyketide pathway, featuring in more than one phenolic unit and deprived of

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nitrogen-based functions in plants. They carry out many important functions in plants, e.g., the

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formation of pigments, resisting environmental stresses and acting as chemical messengers.

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3.1 Biosynthetic pathway of polyphenols

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As shown in Figure 2, depending on the number of phenolic rings and chemical structure

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differences, polyphenols are usually classified into i) phenolic acids; ii) flavonoids; iii) lignans; iv)

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stilbenoids; v) coumarins71. The shikimate and phenylpropanoid pathways are the most common and

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essential routes for polyphenol biosynthesis in plants. Specifically, the shikimate pathway is present

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in bacteria, fungi, and plants. It consists of seven metabolic steps, beginning with the condensation of

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phosphoenolpyruvate with erythrose-4-phosphate and ending with the production of chorismate. The

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end product chorismate is the precursor for postchorismate pathways leading to tryptophan and

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phenylalanine/tyrosine biosynthesis. The phenylpropanoid pathway begins with the conversion of

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phenylalanine into cinnamic acid via phenylalanine ammonia lyase (PAL). Cinnamic acid is then

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activated to p-coumaric acid by a membrane-bound P450 monooxygenase, cinnamate 4-hydroxylase

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(C4H). Alternatively, p-coumaric acid can also be indirectly generated from tyrosine through 9

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tyrosine ammonia lyase (TAL), circumventing the use of P450 enzymes. Once p-coumaric acid is

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generated, the next step is catalyzed by p-coumaric acid: CoA ligase (4CL) which will mediate the

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formation of the corresponding CoA ester, 4-coumaroyl-CoA from p-coumaric acid. The

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intermediate p-coumaric acid will lead to the production of lignans, coumarins, and hyroxycinnamic

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acids (e.g. caffeic acid, ferulic acid, and sinapic acid). And the end product of the phenylpropanoid

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pathway, 4-coumaroyl CoA, will direct the carbon flow to branches of flavonoids and stilbenes.

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3.2 Antimicrobial property of polyphenols

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The antimicrobial properties of polyphenols have been well clarified from both plant extraction

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assay and in vitro analysis with pure specific compound. Typical polyphenols harboring

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antimicrobial property are summarized in Table 1.

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From plant extracts, it was found that extracts from red fruits, plum skins, Italian red grape skins,

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and different parts of elderberry, had a high concentration of polyphenols (100-1005 μg/g of phenolic

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acids, 50-380μg/g of anthocyanins, 30-530 μg/g of flavonoid catechin), which showed inhibitory

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effect towards almost all the pathogenic strains tested, including Bacillus cereus DSM

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345, Lactobacillus paracasei IMC 502®, Lactobacillus plantarum IMC 509, Lactobacillus

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rhamnosus IMC 501®,

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SY-SYNBIO®,

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(Balsaminaceae) revealed the presence of nineteen antimicrobial constituents against 10

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microorganisms tested, comprising phenolic acids (1.4-4.7 mg CAE/g DW) and flavonoids (3.2 to

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6.3 mg QE/g DW) 74

E.

Listeria monocytogenes 306,

coli ATCC

13706,

Candida

Staphylococcus aureus ATCC 25923, albicans ATCC

1026172,

73.

Impatiens

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In addition, from in vitro analysis, phenolic acids and flavonoids have proved their good

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antimicrobial performance. According to numerous of reports, the phenolic acids (including gallic 10

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acid, caffeic acid, ferulic acid, chlorogenic acid, and quinic acid) and flavonoid derivatives (including

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quercetin, apigenin, genistein, naringenin, silymarin, and silibinin) exerted robust antibacterial effect

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towards Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae,

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Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis,

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etc75-80(see details in Table 1). In food test, phenolic acids - caffeic acid and p-coumaric acid were

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found can completely inhibit chicken soup contaminant bacterium Staphylococcus aureus in food

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assay, (above 0.1 mg/mL)81, and 18 prenylated flavonoids (having a prenyl chain on the flavonoid

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backbone) isolated from medicinal plants showed high inhibiting activity against Candida albicans,

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Saccaromyces cerevisiae, Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis, and

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S. aureus82. In our previous study83, metabolites of engineered yeast producing naringenin (a kind of

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flavonoid) exhibited strong antimicrobial activity. Pure naringenin had weak antimicrobial effect

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whereas prenylnaringenin can inhibit Staphylococcus aureus. According to the study of Zemek, J. et

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al., a total of 25 aromatic compounds including guaiacyl- and syringyl-like structures

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(low-molecular-weight part of lignin), gallic acid and its derivatives, cinnamic acid and its

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derivatives, veratrie acid, were analyzed and been demonstrated their effective antibiotic activity

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against bacteria, yeast-like organisms and protozoa84.

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Besides phenolic acids and flavonoids, some lignans and stilbenoids also displayed good

208

antimicrobial

properties.

For

instance,

six

lignans

including

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(7′R,8′S)-4,4'-Dimethoxy-strebluslignanol,

210

3,3'-Methylene-bis(4-hydroxybenzaldehyde,

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isomagnolol inhibited S. cerevisiae (ATCC 9763), Bacillus subtilis (ATCC 6633), Pseudomonas

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aeruginosa (ATCC 9027), E. coli (ATCC 11775), and Staphylococcus aureus (ATCC 25923), with

3'-Hydroxy-isostrebluslignaldehyde, 4-Methoxy-isomagnaldehyde),

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MIC values ranging from 0.0150 to 0.0940 Μm85. Stibenoid blestriarene B and blestriarene C

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exhibited antibacterial activity against two Gram positive strains, Staphylococcus aureus, and S.

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epidermidis, with the MIC values of 6.25–25 μg/mL86.

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4. Antimicrobial mechanisms of terpenoids and polyphenols, and their synergetic effects

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4.1 Antimicrobial mechanism

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The mechanisms of action of terpenoids and polyphenols against microorganisms have not been

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entirely understood. Nevertheless, there are some fundamental mechanisms which are widely

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accepted (Figure 3). They can be summarized as: 1) destabilization of the cytoplasmic membrane

221

and mitochondria due to the lipophilic nature of hydrocarbon skeleton and, consequently, the leakage

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of ions; 2) collapse of the proton motive force, depletion of ATP pool and loss of macromolecules

223

caused by the presence of hydroxyl group and delocalized electrons; 3) inhibiting energy

224

metabolism, DNA synthesis, and enzyme activity.

225

For terpenoids and polyphenols, the lipophilic nature of the hydrocarbon skeleton plays an

226

important role in their antimicrobial activities due to the damage to biological membranes. They can

227

partition the lipophilic lipids of the cytoplasmic membrane and mitochondria, and cause an

228

expansion of the membrane. This results in a destabilization of the membrane and, consequently, the

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leakage of ions87,

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polyphenols than Gram-negative bacteria89,

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terpenoids and polyphenols. The cell wall of Gram-positive bacteria is composed of peptidoglycan

232

(90-95%) and proteins as well as teichoic acid linked to it. Since major components are hydrophobic,

233

they can prone to pass through the cell wall. For Gram-negative bacteria, the monolayer of

234

peptidoglycan is surrounded by an outer complex membrane, namely the lipopolysaccharide

88.

In general, Gram-positive bacteria are more sensitive to terpenoids and 90,

which are pertained to the lipophilic character of

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envelope. The outer cell boundary is charged, having a hydrophilic nature, hence it slows down the

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passage of phytochemicals91, 92.

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In addition to the lipophilic nature of hydrocarbon skeleton, specific functional groups are

238

additionally effective42. Under the umbrella of effective terpenoids and polyphenol chemicals as

239

described above, phenolic compounds are the one with greatest antimicrobial activities, followed in

240

order by aldehydes, ketones, alcohols, ethers, and hydrocarbons93. The effects of each chemical

241

group are summarized as follows:

242

1) Phenol structure (like phenolic terpenoids and polyphenols). The most typical representatives

243

are carvacrol, thymol, and eugenol (Figure 3). Their hydrophobic cyclic hydrocarbon

244

structure, benzene ring, lead to destabilization of the membrane94. The other more important

245

reason for the antimicrobial activity of phenolic compounds proposed by many researches is

246

that they act as a trans-membrane carrier for monovalent cations, carrying H+ into the

247

cytoplasm while transporting K+ back out95. Thereby this reduces the pH gradient across the

248

cytoplasmic membrane, due to both the presence of hydroxyl group and delocalized

249

electrons (benzene ring). The resulting collapse of the proton motive force, depletion of ATP

250

pool and loss of macromolecules87, leads to an impairment of cellular essential activity and

251

finally to cell death. The combination of a hydroxyl group and delocalized electrons is

252

important for this antimicrobial mechanism, as shown in the case of comparison between

253

menthol and carvacrol96. Since the structure of menthol lacks delocalized electrons (double

254

bonds), its hydroxyl group has no ability to release its proton (Figure 3). Thus this results in

255

it having a lower antimicrobial activity than carvacrol. Furthermore, the relative position of

256

the OH group on the benzene ring also influences the antimicrobial activity of phenolic 13

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compounds97.

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2) Aldehyde group, especially unsaturated aldehyde (-C=C-CHO) (like citral, linalool).

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Aldehyde group might help to interfere with membrane-integrated or associated enzyme

260

proteins, stopping their production or activity98. Many studies on assays of antifungal

261

activity of components of EO found that biochemicals with –CHO group in conjugation with

262

a carbon to carbon double bond (C=C) have higher antifungal effect. This suggested that an

263

increase in electronegativity was responsible for the antifungal effects of these compounds99,

264

100.

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(moderate activity to broad microorganism) and citronellal (only active to very few

266

strains)42.

Its effects could be seen from the differences in antibacterial ability between citral

267

3) Ketone group, especially unsaturated ketone (-CO-C=C-) (like piperitone and carvone). The

268

presence of a ketone group in the framework of terpenoids increases the antimicrobial

269

properties101. This results in a decrease in MIC values and to a wider spectrum of action,

270

including Gram-positive and Gram-negative bacteria, as well as fungi. Besides, a

271

conjugation to carbon double bond usually benefits its antimicrobial effects, as in the case

272

that antifungal activity of carvone is much higher than camphor, a saturated ketone100. The

273

mechanism of the ketone group on inhibition of microorganism is unclear yet.

274

4) Acetate moiety and alcohol moiety. The presence of an acetate moiety or alcohol moiety in

275

the chemical structure can increase its antimicrobial activity. For example, in the case of

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geraniol, a replacement of –OH group with –COOH lead to an increase in the activity of

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geranyl acetate against tested microorganisms42, while the alcohol terpenoids do exhibit

278

higher antimicrobial activity than alkyl terpenoids, by acting as protein denaturing agents or 14

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dehydrating agents102. In addition, chemicals with allylic alcohol moiety (-C=C-C-OH), like

280

geraniol, nerol, and linalool, showed weak to medium effects against key enzymatic activity

281

to inhibit microorganism, which is better than non-terminal one103.

282

Furthermore, the structural feature of terpenoids and polyphenols may act by inhibiting or

283

disturbing energy metabolism and DNA synthesis, as well as influencing enzyme expression/activity

284

to a lesser extent97. For instance, coumarins can reduce the cell respiration, lower the ergosterol

285

content of cells and increase the trans-membrane leakage of amino acids104. Flavonoids were proved

286

to inhibit DNA gyrase and β-hydroxyacyl-acyl carrier protein dehydratase105, thus the synthesis of

287

DNA and RNA is inhibited. A rapid collapse in membrane potential and a substantial decrease in

288

total DNA content were detected in the cells treated with resveratrol-trans-dihydrodimer, which

289

showed the impact of polyphenols on DNA synthesis106. As mentioned earlier, the MEP pathway

290

exists in many pathogenic bacteria, but it is not included in humans. Therefore, the key enzyme in

291

the MEP pathway, DXR, becomes a promising target for screening of novel antibiotics. In the study

292

by Gao et al., carvacrol and eugenol were reported to display weak to medium inhibition against

293

DXR; Thymol, geraniol, linalool, and nerol exhibited weak DXR inhibitory activity103. It was

294

suspected to be due to the presence of a delocalized electrons system in these above compounds,

295

while the reason why terpenoids inhibit DXR is unclear. Another excellent example of enzyme

296

activity inhibition is FtsZ, a key protein involved in bacterial cell division. Coumarins have been

297

recognized as a promising candidate for its inhibition107. In addition, sesquiterpene germacrene D

298

will interact with the FtsZ binding pocket so as to disturb cell division108.

299

4.2 Synergetic effects of combination of terpenoids and/or polyphenols

300

It was noticeable that a combination of different terpenoids and/or polyphenols played an 15

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important role in increasing the antimicrobial activity of EO109, 110. In Thymus vulgaris essential oil

302

assays, most of these prominent synergistic interactions are usually between the stronger and weaker

303

antimicrobial constituents111, an excellent example of which, is the combination between carvacrol

304

(monoterpenoid) and ρ-cymene (its monoterpene precursor). Carvacrol has antimicrobial activity

305

against broad bacteria whilst ρ-cymene has no inhibition on microbial growth112. It is interesting that

306

when both are used together, antimicrobial properties were improved, as compared to pure carvacrol

307

alone. As described in several studies, it was suspected that ρ-cymene acted as a substitutional

308

impurity in the bacterial membrane, which affected the membrane potential of intact cells and thus

309

facilitated the activity of carvacrol 55.

310

The synergetic effects of the combination were also been demonstrated by in vitro assays of

311

specific terpenoids or polyphenols. For example, in the study by Pei et al., a combination of

312

cinnamaldehyde/eugenol, thymol/eugenol, carvacrol/eugenol, and thymol/carvacrol revealed

313

synergistic effects depending on the corresponding microbes113. This synergistic effect of combining

314

thymol/eugenol was also shown in the study by Gallucci, M. N. et al114. For thymol/eugenol and

315

carvacrol/eugenol, it was suspected that their synergistic effects were caused by combination of the

316

effects from terpenoid thymol or carvacrol and from polyphenol eugenol. Thymol, or carvacrol are

317

hydrophobic in nature, and prone to disturb the outer membrane of Gram-negative bacteria, thus

318

increasing the permeability of the cytoplasmic membrane to ATP45,

319

combined easier with proteins due to the hydroxyl group116.

320

5. Metabolic engineering of S. cerevisiae for production of terpenoids and polyphenols

115.

In addition, eugenol then

321

Today, metabolic engineering for the production of high-value biochemicals, focus on the use of

322

E. coli and S. cerevisiae. The use of S. cerevisiae is more attractive for food applications due to its 16

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safety. Regardless of the products and organisms, the common strategies used for metabolic

324

engineering are mainly: 1) Pathway construction: discovery and introduction of novel heterologous

325

enzymes and pathways in yeast, or engineering endogenous pathways for biosynthesis of target

326

products; 2) Pathway evaluation: applying reverse metabolic engineering or -omics technology to

327

identify limiting steps involved in target pathways; 3) Protein engineering: improving expression

328

level of target key enzymes by modifying regulatory elements, as well as improving enzyme

329

activity/specificity by directed evolution or rational design techniques; 4) Pathway engineering:

330

elimination of limiting steps involved in target pathways (including genes overexpression, cofactor

331

engineering and transporter engineering), down-regulation of competing pathways to reduce loss of

332

carbon flow, balancing metabolism and cell growth via dynamic engineering, and comprehensive

333

regulation by modular/compartmentalization engineering.

334

Herein we mainly introduce efforts on the biosynthesis of monoterpenoids & sesquiterpenes and

335

phenolic acids & flavonoids in S. cerevisiae as summarized in Table 2, since they are the major

336

constitutes of antimicrobial terpenoids and polyphenols. The main strategies in biosynthesis of

337

terpenoids and polyphenols are depicted in Figure 4. Detailed research progress as well as regulation

338

information are discussed below.

339

5.1 Biosynthesis of antimicrobial terpenoids in S. cerevisiae

340

5.1.1 Biosynthesis of antimicrobial monoterpenoids

341

Monoterpenes are a class of terpenes consisting of two isoprene units. The precursor of

342

monoterpenoids, geranyl diphosphate (GPP) is synthesized from two common C5 intermediates, IPP

343

and DAMPP, by geranyl diphosphate synthase (GPPS) (Figure 1). It is subsequently converted into

344

monoterpenoids by various monoterpene synthases (MTS) (Figure 1). In plants, monoterpenoids are 17

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synthesized in the plastid via the MEP pathway, while sesquiterpenes are produced in the cytosol via

346

the MVA pathway via farnesyl diphosphate (FPP) as the precursor. However, in S. cerevisiae, the

347

MVA pathway is used for synthesis of terpenoids to maintain cell growth. The production of

348

terpenoid ergosterol can reach 5% of the dry weight of yeast, demonstrating its high inherent

349

capacity for biosynthesis of specific heterologous compounds. Currently, the most popular

350

antimicrobial monoterpenoids which have been engineered in S. cerevisiae are mainly geraniol and

351

linalool (Table 2), as described below.

352

Enhancement of precursor supply is one of the most common strategies to improve production

353

of biochemicals. For the production of terpenoids, overexpression of the key enzymes in the MVA

354

pathway, or introduction of a whole heterologous MEP pathway is the most usual strategy for

355

improving IPP and DMAPP supply. In the case of monoterpenoids biosynthesis, tHMG1 (the key

356

rate-limiting enzyme in the MVA pathway of yeast) and IDI1 (IPP isomerase) have been

357

over-expressed in S. cerevisiae. This resulted in an increased production of geraniol, from 0.73 mg/L

358

to 6.77 mg/L as reported117.

359

In addition to improving precursor supply, much effort was focused on identification of highly

360

active enzymes for the downstream extended biosynthetic pathways by gene screening and enzyme

361

modification. Terpene synthase from various sources have been cloned and functionally

362

characterized for the synthesis of different monoterpenoids118, 119, amongst which geraniol synthase

363

(GES) of Ocimum basilicum is the most popular option. It was reported that overexpressing ObGES

364

in yeast enabled a stronger and more specific excretion of geraniol out into the growth medium120.

365

Under microvinification conditions, 750 μg/L of geraniol was detected and it could be further

366

metabolized up to production of 1,558 μg/L additional monoterpenes and esters (230-fold 18

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improvement as compared with the control). This included citronellol, linalool, nerol,

368

citronellylacetate and geranyl acetate, by alcohol acetyltransferase (ATF1) and NADPH

369

oxidoreductase (OYE2)121. In addition to enzyme screening, enzyme modification provided an

370

additional powerful approach to further improve the catalytic efficiency of MTS. Through protein

371

structure analysis and site-directed mutation, Y436 and D501 were revealed to be highly conserved

372

amino acid residues located in the active pocket of CrGES (GES from Catharanthus roseus). The

373

transit peptide from the N-terminus of CrGES was found to be an obstacle for protein expression and

374

activity. By eliminating this transit peptide, a truncated CrGES (at S43) resulted in improved

375

production of geraniol122. Moreover, to improve catalytic rate of GES, a high-throughput screen

376

method was developed based on a coupled enzyme-based fluorogenic assay, in which geraniol was

377

converted to geranial by GeDH with a reduction of NAD+ to NADH. Subsequently it was utilized by

378

diaphorase to reduce oxidized resazurin, into fluorescent resorufin. Saturation mutagenesis was

379

performed based on this design, which resulted in a mutation of F418Q, with improved production of

380

geraniol123.

381

Furthermore, heterologous production of industrially useful monoterpenoids in yeast also suffers

382

from competition in the native downstream branches. In S. cerevisiae, Erg20p is a bifunctional

383

enzyme, both GPPS and farnesyl pyrophosphate synthase (FPPS) activities, which converts DMAPP

384

and IPP into GPP (Figure 1). It subsequently adds another IPP to produce FPP124 (Figure 1), thus

385

limiting the availability of GPP for monoterpene production. ERG20 plays an essential function in

386

the synthesis of sterols, ubiquinone and protein prenylation, thus it could not be deleted. Other than

387

deletion, deregulation strategies including genetic mutation, promoter replacement, dynamic control

388

as well as protein degradation, were successfully applied to balance monoterpenoids metabolism and 19

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cell growth. Specifically, a set of ERG20 mutants have been constructed to decrease the FPPS

390

function and to also screen for GPPS preferred mutants. The mutant variants F96W-N127 W and

391

K197G were identified to have greater GPPS activity, and subsequently significant increased

392

monoterpene titers125,

393

HXT1 promoter, together with the optimization of glucose and ethanol ratio were shown to increase

394

the production of geraniol by 3.8-fold127. Expression of Erg20p under the repressible MET3

395

promoter, and in the presence of methionine, resulted in a more than two fold improvement in

396

linalool production128. It is interesting to note that recombining sterol-responsive transcriptional

397

regulation and N-degron-mediated protein degradation, successfully reduced Erg20p to a minimal

398

level which was required, to maintain sterol flux for normal cell growth as well as to orient carbon

399

into monoterpene production. Under this regulation, linalool titer increased by 27-fold/17-fold with

400

either constitutive promoter constructs or diauxie-inducible promoter constructs. A final titer of 76

401

mg/L of limonene in batch cultivation was obtained. In addition to ERG20, other key competing

402

steps existed. For example, MAF1 is a negative regulator of MOD5 which encodes for tRNA

403

isopentenyl transferase. This tRNA molecule helps to redirect the carbon flux from DMAPP to tRNA

404

biosynthesis. Overexpression of MAF1 reportedly resulted in an obvious increase in geraniol

405

production117, 127. A recent genetic analysis of geraniol metabolism during wine fermentation by S.

406

cerevisiae demonstrated that geraniol can be converted into citronellol by NADPH oxidoreductase

407

(OYE2). It can also be acetylated under the catalysis of alcohol acetyltransferase (ATF1)129. Deletion

408

of OYE2 and ATF1 were found to result in a 1.7-fold or 1.6-fold improvement of geraniol

409

production in batch fermentation respectively127. All these studies demonstrated the benefits of

410

down-regulating the competitive downstream flux, towards the accumulation of target compounds.

126.

In recent studies, the dynamic control of Erg20p expression under the

20

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Journal of Agricultural and Food Chemistry

5.1.2 Biosynthesis of antimicrobial sesquiterpenoids

412

Sesquiterpenoids are the most diverse class of terpenoids with more than 300 identified carbon

413

skeletons and more than 7000 characterized compounds. They are derived from precursor farnesyl

414

diphosphate (FPP) and catalyzed by a series of sesquiterpene synthases (sesui-TPSs) (Figure 1). At

415

present, many sesquiterpenes have been successfully produced via metabolic engineering in S.

416

cerevisiae such as artemisinin130, farnesene131, a-santalene132, epi-cedrol133, germacrene134,

417

trichodermol135, cubebol136, 137, amorpha-4,11-Diene138, 139, patchoulol, and farnesol140.

418

For accumulation of high-yield sesquiterpenes, an efficient FPP supply is the key. For this, the

419

most commonly used strategies were to overexpress key enzymes in the MVA pathway (tHMGR)137,

420

down-regulate ERG9 (squalene synthase) (Figure 1) to reduce its competing flux137, 141, as well as

421

codon optimization of key enzymes142. Promoter replacement was the first option to downregulate

422

ERG9 in recent studies with regards to sesquiterpenes synthesis. The replacement of the native

423

promoter of ERG9, with a repressible methionine (MET3) promoter, enhanced cubeboltiters107 as

424

well as amorpha-4,11-dieneyield109. However, due to the high cost of using methionine as a

425

repressor, this application was limited on an industrial-scale. Other than the MET3 promoter, the use

426

of glucose respondent promoters, such as HXT1, provided a more feasible solution for ERG9

427

down-regulation. This has been successfully applied in production of sesquiterpenes, such as

428

a-santalene132.

429

Enzyme fusion is used as an additive method to increase pathway efficiency by reducing

430

substrate loss. As was in the case of sesquiterpene amorphadiene synthesis (a key precursor molecule

431

of artemisinin), FPPS was coupled with amorphadiene synthase (ADS) in yeast to overcome the loss

432

of FPP in to competing pathways. This improved artemisinin yield130. In addition, enzyme scaffolds 21

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have arisen as an alternative strategy, to create custom enzyme complexes. Hudson et al. described a

434

design which used affibody scaffolds for the colocalization of farnesyl diphosphate synthase and

435

farnesene synthase. This reduced side product formation from FPP and redirected flux towards the

436

final product, farnesene. To do this, two pairs of anti-idiotypicaffibodies were fused to enzymes and

437

the corresponding affibody scaffolds were expressed separately131. As a result, the yield of farnesene

438

was improved by 135% in fed-batch cultivations. In addition, gene integration on the genome is

439

more stable and energy-saving as compared with plasmids. In the study by Ching et al.,

440

combinatorial genome integration of the MVA pathway was performed in yeast to improve the

441

production of amorpha-4,11-diene. By using carotenoid as a color indicator, a library of yeast with

442

various intensities of carotenoids were constructed and used to boost the titer of amorpha-4,11-diene,

443

based on the shared upstream pathway. As a result, one mutant strain with a 13-fold improvement of

444

amorpha-4,11-diene was obtained. This was approximately the equivalent to 64 mg/L of

445

caryophyllene138.

446

Furthermore, with increasing genome sequence data available, genome-scale models with more

447

simple assumptions to predict cellular metabolic behavior have arisen. This was carried out by the

448

use of constraint-based models, such as flux balance analysis (FBA)143-145. In addition to extensive

449

genetic engineering, this method provided a more rational approach to identify potential candidate

450

steps which limited the accumulation of target compounds. In the study by Jens N. et al., silico

451

driven metabolic engineering was performed to identify new target genes, for the enhanced

452

biosynthesis of sesquiterpenes in S. cerevisiae. This was done using OptGene as the modeling

453

framework, and by minimization of the metabolic adjustments (MOMA) as the objective function136.

454

NADPH-dependent glutamate dehydrogenase encoded by GDH1 were identified to be the best target 22

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gene for the knockout, to enhance the available NADPH supply in the cytosol, for other NADPH

456

requiring enzymes which are involved in the terpenoid biosynthetic pathway. The deletion of GDH1

457

resulted in an 85% increase in the final cubebol titer.

458

5.2 Biosynthesis of antimicrobial polyphenols in S. cerevisiae

459

5.2.1

Biosynthesis of phenolic acids

460

Phenolic acids typically have a phenolic ring and at least one organic carboxylic acid function.

461

Depending on the carbon units of the lateral chain attached to the phenolic ring, they can be

462

classified into C6-C3, C6-C2 or C6-C1 compounds. The most important C6-C3 compounds are

463

derived from the cinnamic acid skeleton while C6-C1 compounds typically have a hydroxybenzoic

464

structure146,

465

hydroxylation and methoxylation of the aromatic ring. In nature, the most abundant benzoic acid

466

derivatives include p-hydroxybenzoic, vanillic, syringic and gallic acids, while common cinnamic

467

acid derivatives are p-coumaric, caffeic, ferulic and sinapic acids148, 149.

147

(Figure 2, green boxes). The derivatives differ in degree and position, of the

468

There are many pathways involved in the biosynthesis of hydroxybenzoic acids. More than one

469

pathway may exists in a single organism146. For example, some of the simple hydroxybenzoic acids,

470

like 4-hydroxybenzoic acid (pHBA), can be produced directly from chorismate or from the

471

degradation of hydroxycinnamic acids150. Currently, there are several studies which use E. coli to

472

produce various hydroxybenzoic acids, such as p-hydroxybenzoic, gallic acids, and salicylic acids,

473

whereas the production in yeast is mainly limited to pHBA. The theoretical mass yield of pHBA in S.

474

cerevisiae was evaluated to be 0.58 g/gglucose from metabolic network analysis. This revealed the

475

huge potential of S. cerevisiae as a microbial cell factory for the production of pHBA. Besides, the

476

minimal inhibitory concentration of pHBA in S. cerevisiae was detected to be 38.3 g/L, which is 23

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much lower than that observed in other microbes. Since no native genes and routes for pHBA

478

biosynthesis have been found in yeast, the ubiC enzyme from E. coli was used as the target gene for

479

over-expression in the construction of pHBA biosynthesis pathway (Figure 2). By combining this

480

with the knockout of aro7 gene (encoding for chorismate mutase) to reduce chorismate flux into

481

tyrosine and phenylalanine (Figure 2), 650 M pHBA were accumulated from 83.3 mM of glucose150.

482

However, most productive metabolic engineering strategies caused a severe metabolic burden on cell

483

growth. To address this problem, a dynamic regulation strategy which used a synthetic quorum

484

sensing circuit was performed in S. cerevisiae. In this study, the circuit autonomously triggered gene

485

expression at high population density, and were linked with an RNA interference module to enable

486

target gene silencing. Specifically, TKL1p (Transketolase), ARO4p (DAHP synthase), and UbiCp

487

were expressed under QS regulation to dynamically control production. In addition, ARO7p and

488

TRP3p (indole-3-glycerol-phosphate synthase) were selected for conditional knockdown using QS

489

circuit in combination with RNAi. As a result, 1.1mM pHBA were obtained151. More recently, a

490

strain which combined multiple regulation strategies including the deletions of TRP3 and ARO7, and

491

expression of the feedback inhibition resistant ARO4K229L as well as E. coli shikimate kinase (aroL),

492

were constructed for pHBA production. It was further optimized by a fed-batch bioreactor process,

493

which resulted in a final titer of 2.9 g/L (carbon-yield was up to 3.1 mgpHBA/gglucose)152.

494

Currently, the biosynthetic pathway for hydroxycinnamic acids is clearly established (Figure 2).

495

p-coumaric is an intermediate involved in the phenylpropanoid pathway. It is generated via the

496

introduction of TAL, and grown on tyrosine as the substrate, or by conversion of PAL&C4H from

497

phenyalanine. It can also be converted into caffeic acid via the introduction of a second hydroxyl

498

group, which is catalyzed by monophenol mono-oxygenases, a well-known group of plant 24

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499

enzymes153. The methylation of caffeic acids leads to the formation of ferulic acid and is

500

subsequently converted to rare 5-hydroxyferulic acid. This can yield sinapic acid as a result of

501

O-methylation. At present, there are several studies about the production of p-coumaric acid in yeast

502

whereas the other hydroxycinnamic acids are rarely reported. Biosynthesis of p-coumaric acid in S.

503

cerevisiae was firstly reported by Douglas et al. via introduction of PAL from Populuskitakamiensis,

504

C4H and CPR from Populustrichocarpa X Populusdeltiodes. He demonstrated that PAL and C4H

505

were sufficient to drive the carbon flux into p-coumaric acid without the need for multi-enzyme

506

complexes154. Apart from using phenylalanine as the precursor, biosynthesis of p-coumaric acid in S.

507

cerevisiae has been achieved by growing on tyrosine as the substrate, and by over-expression of

508

TAL155. Today, the highest titer of p-coumaric acid can reached up to 1.93 g/L, as reported by Jens

509

Nielsen et al. In their study, three strategies were applied to improve production of p-coumaric acid.

510

This including the reduction of by-product formation by knocking out phenylpyruvate decarboxylase

511

ARO10 and pyruvate decarboxylase PDC5 (Figure 2), overexpressing different versions of

512

feedback-resistant DAHP synthase (ARO4) and chorismate mutase (ARO7) (Figure 2), and

513

introduction of homologous genes for biosynthesis of aromatic acids from Escherichia coli. In

514

addition, shikimate kinase was identified as another important flux-controlling step156. When ARO4

515

and ARO7 were deregulated, the ARO2 and TYR1 genes which coded for chorismate synthase and

516

prephenate dehydrogenase, were further identified as a new and important rate-limiting step for

517

further optimization

518

the regulation mechanisms in genetic engineering. In order to study how the production of

519

p-coumaric acid influenced the cellular metabolism of S. cerevisiae, the metabolic response in two

520

background strains, S288c or CEN.PK which over-produced p-coumaric acid was compared by using

157.

Besides the above macro-control strategies, systems biology sheds light on

25

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omics analysis and transcriptome analysis. It revealed the superiority of CEN.PK in production of

522

p-coumaric acid as compared to S288c. It also demonstrated the importance of transporters in the

523

engineering of cell factories for the production of small molecules158. This information was useful

524

for further optimization.

525

5.2.2

Biosynthesis of flavonoids

526

Flavonoids are a large family of phytochemicals, encompassing more than 9000 substituted

527

moieties. The core flavonoid structure consists of two benzene rings interconnected by a heterocyclic

528

ring, in the formula of C6–C3–C6 (Figure 2). Depending on their structure, the differences in

529

position and modification in the heterocyclic ring, flavonoids are divided into six subclasses:

530

isoflavones, flavanones, flavones, flavonols, catechins, and anthocyanins. Amongst thousands of

531

flavonoid compounds, naringenin159-161, kaempferol162-164, genistein162, fisetin160, resveratrol165,

532

pinocembrin166, and anthocyanin167 were the most popular products studied using S. cerevisiae due to

533

their superior properties. The production of these compounds is summarized in Table 2.

534

Microbial production of flavonoids for industrial application is hampered by many obstacles: 1)

535

the poor expression and activities of heterologous enzymes; 2) a heavy reliance on the addition of

536

expensive phenylpropanoic precursors; 3) the low availability of malonyl-CoA as well as aromatic

537

amino acids tyrosine and phenylalanine. Based on previous studies, current progress in solving these

538

problems are summarized and discussed as follows.

539

The formation of the key flavanoid intermediate p-coumaric acid has two distinct paths. It is

540

either from phenylalanine through deamination to cinnamic acid (CA) by PAL and subsequent

541

hydroxylase activity by C4H, or through deamination of tyrosine by TAL. PAL is a key enzyme in

542

the path from phenylalanine. Hence various PAL genes were discovered, characterized and screened 26

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543

for biosynthesis of flavonoids168-172. Among the PAL gene from plant Centaureadepressa, Brassica

544

oleracea var. capitate, Lactuca sativa, Musa sp, Petroselinum crispum, the PAL gene from

545

Centaureadepressa showed a higher activity than the others. The subsequent step, catalyzed by C4H,

546

was found to be a limited step in the engineered microorganisms for production of flavonoids. For

547

bacteria, there is no complementary reductase (CPR), which is required for P450 cytochrome

548

monooxygenase to activity. Besides, the absence of endoplasmic reticulum causes the translational

549

incompatibility of the membrane signal modules of P450 enzymes with the bacterium173. In yeast,

550

the function of 4CL is also limited to CPR supply even though it has native CPR enzyme170. Taking

551

into consideration that the route from phenylalanine required the activity of a P450 enzyme, the route

552

from tyrosine via TAL might be more preferable. In the study by Nielsen et al., 22 sequences were

553

identified in silico using synteny information, which aimed for sequence divergence. Enzymes from

554

Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of

555

p-coumaric acid, a 5-fold improvement over that of strains expressing tyrosine ammonia-lyases174.

556

The early work on microbial production of flavonoids relied on external addition of flavonoid

557

intermediates, p-Coumaric acid, cinnamic acid, tyrosine or phenylalanine into the culture. For

558

example, 28 mg/L of naringenin was produced in engineered S. cerevisiae via the introduction of a

559

four-step flavone biosynthetic pathway composed by C4H, 4CL, CHS (encoding chalcone synthase)

560

and CHI (encoding chalcone isomerase). This was supplemented with p-Coumaric acid. However, a

561

heavy reliance on the addition of expensive phenylpropanoic precursors highly limited its large-scale

562

production. To solve this problem, a whole pathway which begins from tyrosine/phenylalanine was

563

introduced into target host cells. This consisted of phenylalanine/tyrosine ammonia lyase

564

(PAL/TAL), 4CL, CHS, and CHI. By combining with other metabolic engineering strategies, this led 27

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565

to 108 mg/L of de novo production of naringenin from glucose160. In the study by Borodina et al., six

566

different flavonoids (naringenin, liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin)

567

were produced in engineered S. cerevisiae grown on glucose. The production reached 26.5 mg/L of

568

kaempferol and 20.4 mg/L of quercetin, which exceeded previously reported titers in yeast. It

569

showed for the first time de novo biosynthesis of resokaempferol and fisetin in yeast164.

570

Phenylalanine and tyrosine are key intermediates in flavonoid biosynthesis. In yeast, the key

571

enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase in the shikimate pathway,

572

encoding by ARO3 and ARO4 gene, are feedback inhibited by phenylalanine and tyrosine175,

573

(Figure 2). Another key enzyme, ARO7, in the aromatic branch is also inhibited by tyrosine177

574

(Figure 2). These feedback inhibition mechanisms severely impacted the biosynthesis of aromatic

575

acid as well as flavonoids. Mutant ARO4K229L and ARO7G141S proved to be good options to abolish

576

the feedback inhibition effects from aromatic amino acids178, 179. The introduction of these mutant

577

genes resulted in a significant increase of intracellular phenylalanine and tyrosine concentration180,

578

and have been widely applied to improve the production of many flavonoids160,

579

Luttik et al. showed that the introduction of mutated ARO4 and ARO7 resulted in a 200-fold yield

580

improvement of aromatic compounds180. Besides, accumulation of aromatic amino acids was also

581

subjected to the competing biosynthesis of the byproduct phenylethanol. As described in the study of

582

Jean-Marc et al., restraining the phenylethanol branch resulted in a 3-fold increase of flavonoid

583

naringenin, by knocking-out the most active phenylpyruvate decarboxylase PDC5 and ARO10180. In

584

addition to aromatic acids, malonyl-CoA is another essential precursor of flavonoids (Figure 2).

585

Improvement of flavonoids in E. coli has been achieved by improving available malonyl-CoA

586

supply. This was carried out by modification of Acetyl-CoA carboxylase (ACC1), introducing 28

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161.

176

For example,

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587

heterologous pathways and down-regulating competing pathways181-183. However, in our previous

588

study, operations such as up-regulation of acetyl CoA biosynthesis and down-regulation of fatty acid

589

biosynthesis by the addition of the inhibitor cerulenin, did not result in flavonoid accumulation in S.

590

cerevisiae161.

591

6. Challenges and future perspectives

592

Terpenoids and polyphenols are secondary high-value metabolites in plants. In recent years, they

593

have attracted extensive interests as potential natural food preservatives. This is due to their

594

outstanding antimicrobial properties towards a diverse range of microorganisms, especially

595

pathogenic bacterium. The commercial terpenoids and polyphenols are traditionally extracted from

596

plants, which have long growth cycles and are influenced by environmental conditions. In the past

597

years, production of many terpenoids and polyphenols has been achieved by using engineered S.

598

cerevisiae as a cell factory. Many strategies were applied to improve their production. This included,

599

the traditional pull (expression of key enzymes)-push (improving precursor supply)-block (knockout

600

of key genes involved in competing pathway) strategy, discovery and introduction of novel

601

heterologous pathways, protein modification, as well as dynamic regulation as described in this

602

review. However, the production capacity is still far from industrial application due to: 1) the low

603

yield caused by insufficient efficient regulations, limited knowledge on metabolism and unclear

604

biosynthesis routes for some complex compounds; 2) a low toxicity resistance of yeast to terpenoids

605

and polyphenols; 3) the high production costs from feedstock supply.

606

With advances in system biology184, 185, protein engineering186-188, emerging and upgrading gene

607

editing tools e.g. CRISPR/cas9 189, 190, knowledge and tools can be combined to maximize the carbon

608

flux into target products. For instances, “omics” technique could help uncover the mechanisms 29

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609

behind the biosynthesis of terpenoids and polyphenols, and identify the potential limiting steps. This

610

would propel the technological advances in the biosynthesis of terpenoids and polyphenols. Besides,

611

as in the case of production of flavonoid anthocyanins using genetically engineered microbes167,

612

isolation and identification of the genes involved in downstream extended biosynthetic pathways is

613

another significant challenge for metabolic engineering of microbes26. With the advanced

614

development of genome and expressed sequence tag sequencing technique, the use of genomics in

615

combination with traditional methods have revealed many extended pathways for essential

616

flavonoids. In future, more rapid gene identification is expected.

617

Since many terpenoids and polyphenols are toxic to the yeast itself, the solution on how to

618

eliminate their inhibition on cell growth is critical for large-scale production. The two-phase system

619

offers an attractive bioprocessing option for alleviating this toxicity as described in the biosynthesis

620

of Jet Fuel Mixtures in S. cerevisiae191. Besides, dynamic engineering provides another feasible

621

strategy for balancing the metabolism of toxic compounds and cell growth, such as using modified

622

GAL system24, dynamic RNA repression151, and temperature control using Tm-sensitive

623

promoters/protein swich192,

624

industrial production. More excellent designs are still in urgent need.

193,

etc. Realistically dynamic control is of significance for their

625

Currently, most studies use glucose and YP (yeast extract-peptone) as the carbon and nitrogen

626

source for fermentation of yeast, which contributes to the high cost for industrial production of

627

terpenoids and polyphenols. Recently, studies about reusing bio-waste for production of other

628

high-value products have been reported. For example, based on the synergistic effect of

629

Rhizopusoligosporus and Phaffia rhodozyma, carotenoids could be produced from spent grain194, 195.

630

To reduce the cost of carbon source, it is important to consider the use of cheap feedstocks, such as 30

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631

biomass hydrolysates containing glucose, xylose, and arabinose. It is feasible to create a yeast strain

632

which could ferment xylose by integrating either xylose reductase and xylitol dehydrogenase or

633

xylose isomerase. Although it is possible that its uptake of xylose is relatively low, there is a

634

possibility for further optimization.

635

In addition to productivity issues, assessing the safety of using terpenoids and polyphenols as

636

food preservatives is of vital importance, especially for potential cytotoxic activity and Acceptable

637

Daily Intake (ADI). In contrast to beneficial effects, some terpenoids and polyphenol compounds

638

have been found to be pro-oxidant or mutagenic and to produce toxicity196. Comprehensive testing of

639

various terpenoids and polyphenol compounds is necessary to establish their safety for use in foods.

640

In addition to cytotoxicity assays, animal tests and transcriptomic/ proteomic analysis will no doubt

641

further advance this field. Furthermore, it is worth noting that too high concentrations of certain

642

natural compounds may also be lethal to humans197. Regulatory limitations on their daily intake

643

should be evaluated prior to use in foods.

644

Recent progress has clearly demonstrated the potential of S. cerevisiae for high-yield production

645

of terpenoids and polyphenols. Although some challenges exit, we believe that more advances in

646

metabolic engineering and processing engineering would overcome the current problems and enable

647

higher yield of terpenoids and polyphenols by S. cerevisiae, for commercial use, in the future.

31

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Associated content

Author information Corresponding Author * Tel: (+65)6316 2870. Email: [email protected]

Funding This work was supported by Nanyang Technological University, Singapore (iFood Research grant).

Notes The authors declare no competing financial interest

32

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195. Cooray, S. T.; Lee, J. J. L.; Chen, W. N. Evaluation of brewers' spent grain as a novel media for yeast growth. Amb Express. 2017, 7. 196. Galati, G.; Sabzevari, O.; Wilson, J. X.; O'Brien, P. J. Prooxidant activity and cellular effects of the phenoxyl radicals of dietary flavonoids and other polyphenolics. Toxicology. 2002, 177, 91-104. 197. Li, N.; Liu, J.-H.; Zhang, J.; Yu, B.-Y. Comparative evaluation of cytotoxicity and antioxidative activity of 20 flavonoids. J Agr Food Chem. 2008, 56, 3876-3883. 198. Carson, C. F.; Riley, T. V. Antimicrobial activity of the major components of the essential oil of melaleuca-alternifolia. J Appl Bacteriol. 1995, 78, 264-269. 199. Zemek, J.; Valent, M.; Podova, M.; Kosikova, B.; Joniak, D. Antimicrobial properties of aromatic-compounds of plant-origin. Folia Microbiol. 1987, 32, 421-+. 200. Sorrentino, E.; Succi, M.; Tipaldi, L.; Pannella, G.; Maiuro, L.; Sturchio, M.; Coppola, R.; Tremonte, P. Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool to improve the shelf life of fresh black truffles. Int J Food Microbiol. 2018, 266, 183-189. 201. Maddox, C. E.; Laur, L. M.; Tian, L. Antibacterial activity of phenolic compounds against the phytopathogen Xylella fastidiosa. Curr Microbiol. 2010, 60, 53-58. 202. Kepa, M.; Miklasinska-Majdanik, M.; Wojtyczka, R. D.; Idzik, D.; Korzeniowski, K.; Smolen-Dzirba, J.; Wasik, T. J. Antimicrobial potential of caffeic acid against Staphylococcus aureus Clinical Strains. BioMed Res Int. 2018. 203. Andrade, M.; Benfeito, S.; Soares, P.; Silva, D. M. E.; Loureiro, J.; Borges, A.; Borges, F.; Simoes, M. Fine-tuning of the hydrophobicity of caffeic acid: studies on the antimicrobial activity against Staphylococcus aureus and Escherichia coli. Rsc Adv. 2015, 5, 53915-53925. 204. Alves, M. J.; Ferreira, I. C. F. R.; Froufe, H. J. C.; Abreu, R. M. V.; Martins, A.; Pintado, M. Antimicrobial activity of phenolic compounds identified in wild mushrooms, SAR analysis and docking studies. J Appl Microbiol. 2013, 115, 346-357. 205. Redko, F.; Clavin, M. L.; Weber, D.; Ranea, F.; Anke, T.; Martino, V. Antimicrobial isoflavonoids from Erythrina crista galli infected with Phomopsis sp. Zeitschrift Fur Naturforschung C-a Journal of Biosciences. 2007, 62, 164-168. 206. Tiza Ng, T. M., Raymond Daniels; , J. K.; Fielding, B. C. Additive antibacterial activity of naringenin and antibiotic combinations against multidrug resistant Staphylococcus aureus. Afr J Microbiol Res. 2015, 9, 1513-1518. 207. Nakayama, M.; Shimatani, K.; Ozawa, T.; Shigemune, N.; Tsugukuni, T.; Tomiyama, D.; Kurahachi, M.; Nonaka, A.; Miyamoto, T. A study of the antibacterial mechanism of catechins: Isolation and identification of Escherichia coli cell surface proteins that interact with epigallocatechin gallate. Food Control. 2013, 33, 433-439. 208. Hosseinzadeh, H.; Fazly Bazzaz, B. S.; Sarabandi, S.; Khameneh, B., Effect of catechins, green tea extract and methylxanthines in combination with gentamicin against Staphylococcus aureus and Pseudomonas aeruginosa: Combination therapy against resistant bacteria. J Pharmacopuncture. 2016, 19, 312-318. 209. Mabe, K.; Yamada, M.; Oguni, I.; Takahashi, T. In vitro and in vivo activities of tea catechins against Helicobacter pylori. Antimicrob Agents Ch. 1999, 43, 1788-1791. 210. Bai, L.; Takagi, S.; Ando, T.; Yoneyama, H.; Ito, K.; Mizugai, H.; Isogai, E. Antimicrobial activity of tea catechin against canine oral bacteria and the functional mechanisms. J Vet Med Sci. 2016, 78, 1439-1445. 211. Rakelly de Oliveira, D.; Relison Tintino, S.; Morais Braga, M. F. B.; Boligon, A. A.; Linde Athayde, M.; Douglas Melo Coutinho, H.; de Menezes, I. R. A.; Fachinetto, R. In vitro antimicrobial and modulatory activity of the natural products silymarin and silibinin. BioMed Res Int. 2015, 1-7. 212. Peng, B. Y.; Nielsen, L. K.; Kampranis, S. C.; Vickers, C. E. Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae. Metab Eng. 2018, 47, 83-93. 213. Carrau, F. M.; Medina, K.; Boido, E.; Farina, L.; Gaggero, C.; Dellacassa, E.; Versini, G.; Henschke, P. A. De novo 43

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synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. Fems Microbiol Lett. 2005, 243, 107-115. 214. Deng, Y.; Sun, M. X.; Xu, S.; Zhou, J. W. Enhanced (S)-linalool production by fusion expression of farnesyl diphosphate synthase and linalool synthase in Saccharomyces cerevisiae. J Appl Microbiol. 2016, 121, 187-195. 215. Liu, Q.; Majdi, M.; Cankar, K.; Goedbloed, M.; Charnikhova, T.; Verstappen, F. W. A.; de Vos, R. C. H.; Beekwilder, J.; van der Krol, S.; Bouwmeester, H. J. Reconstitution of the costunolide biosynthetic pathway in yeast and nicotiana benthamiana. PloS one. 2011, 6.

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Figure captions Figure 1. Schematic presentation of biosynthetic pathway of terpenoids. Intermediates: DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP2ME, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol; CDP-MEP,

2-phospho-4-(cytidine

5′-di-phospho)-2-C-methyl-D-erythritol;

ME-cPP,

2-C-methyl-D-erythritol

2,4-cyclodiphosphate; HMBPP, 4-hydroxy-3-methylbut-2-enyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl diphosphate. Enzymes: DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR,

1-Deoxy-D-xylulose

5-phosphate

cytidylyltransferase;

ISPE,

4-cyclodiphosphate

synthase;

reductoisomerase;

ISPD,

4-diphosphocytidyl-2C-methyl-D-erythritol

4-diphosphocytidyl-2C-methyl-D-erythritol kinase; ISPF, 2C-methyl-D-erythritol-2, ISPG,

1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate

1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate 3-hydroxy-3-methylglutaryl- CoA

reductase.

ERG10,

acetoacetyl-CoA

synthase; thiolase;

ISPH, HMGS,

(HMG-CoA) synthase; HMGR, HMG-CoA reductase; MK, Mevalonate kinase;

PMK, Phosphomevalonate kinase; MVD1, Mevalonate pyrophosphate decarboxylase; IPP1/IDI, IPP isomerase; ERG20, Farnesyl pyrophosphate synthase; MTS, monoterpene synthase; TPS, sesquiterpene synthases; GGPPS, geranylgeranyl diphosphate synthase; ERG9, squalene synthase. Key enzymes are marked in red.

Figure 2. Schematic presentation of biosynthetic pathway of polyphenols. Green areas present phenolic acids, pink areas present lignans, yellow areas present coumarins, purple areas present stilbenes and blue areas present flavonoids. Intermediates:

PEP,

phosphoenolpyruvate;

E4P,

erythrose4-phosphate;

DAHP,

3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; Enzymes: ARO4/ARO3, DAHP synthase; ARO1, pentafunctional arom protein; ARO2, bifunctional chorismate synthase and flavin reductase; ARO7, chorismate mutase; TRP2, anthranilate synthase; TRP3, indole-3-glycerol-phosphate synthase; ARO10, phenylpyruvate decarboxylase; PDC5, minor isoform of pyruvate decarboxylase; TAL, tyrosine ammonia lyase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, p-coumaric acid: CoA ligase; CHS, chalcone synthase. Key enzymes are marked in red.

Figure 3. Schematic presentation of antimicrobial mechanisms of terpenoids and polyphenols.

Figure 4. Schematic presentation of recent metabolic engineering strategies used for biosynthesis of terpenoids and polyphenols in S. cerevisiae. 45

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Table 1 Overview of essential antimicrobial terpenoids and polyphenols and their identified target organisms Compound

Chemical classification

Target organisms (MIC)

Reference

Monoterpenoids

Enterococcus faecalis (225 μg/mL), E. carotovora (1600 μg/mL), E. amylovora

Terpenoids Carvacrol

19, 45-47, 115

(800 μg/mL), E. coli (225–2500 μg/mL), S. cerevisiae (79.8–112.5 μg/mL), Staphylococcus epidermidis (450 μg/mL), S. aureus (450–1250 μg/mL), Salmonella typhimurium (150–250 μg/mL), P. fluorescens (1.84 μg/mL), Candida strains (75–100 μg/mL), Yersinia enterocolitica (225 μg/mL), L. monocytogenes (450–1500 μg/mL), Vibrio vulnificus (250 μg/mL) Thymol

Monoterpenoids

E. coli (225-450 μg/mL), Salmonella typhimurium (56.25μg/mL )

45, 47

Y. enterocolitica (225μg/mL), S. aureus (225μg/mL), S. epidermidis (225μg/mL), E. faecalis (225μg/mL), L. monocytogenes (450 μg/mL), B. cereus (450μg/mL), C. albicans (112.5μg/mL), S. cerevisiae (112.5μg/mL) Linalool

Monoterpenoids

Staphylococcus aureus (1%, v/v),

Carnobacterium divergens (2%, v/v),

50, 51, 198

Listeria innocua (1%, v/v), Serratia liquefaciens (1%, v/v), Salmonella typhimurium (0.7, v/v), E. coli (0.6%, v/v) Porphylomonas gingivalis (100-800 μg/mL) Prevotella nigrescens (800μg/mL) Fusobacterium nucleatum (100-200μg/mL) Aggregatibacter actinomycetemcomitans (100μg/mL) Citral

Monoterpenoids

E. coli (500μg/mL), S. typhimurium (500μg/mL),

46

L. monocytogenes (500 μg/mL), V. vulnificus (100 μg/mL) Carvone

Monoterpenoids

Candida krusei (625-1250 μg/mL), Candida albicans (312-625μg/mL),

52, 53

Staphylococcus aureus (190-520μg/mL), Bacillus subtilis (30-160 μg/mL), Pasturella multocida (200-280 μg/mL), Escherichia coli (100-330 μg/mL), Aspergillus niger (30 μg/mL), Mucor mucedo (70-90 μg/mL), Fusarium solani (110 μg/mL), Botryodiplodia theobromae (90-100 μg/mL), Rhizopus solani (100-150 μg/mL) Linalyl acetate

Monoterpenoids

S. aureus ATCC 6538P (1250 μg/mL)

54

E. coli ATCC 15221 (5000 μg/mL) Menthol

Monoterpenoids

Bacillus cereus, Bacillus subtilis, Enterobacter cloacae, Escherichia coli,

55, 56

Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, S. epidermidis, S. typhimurium, Staphylococcus aureus Geraniol

Monoterpenoids

Mycobacterium smegmatis, Staphylococcus epidermidis,

57-59

Streptococcus mutans, Candida albicans, Trichophytron rubrum, Microsporum gypseum, Aspergillus niger, Sporothrix schenckii, Aspergillus flavus, Escherichia coli (2500μg/mL), Salmonella enterica (5000 μg/mL), Staphylococcus aureus (5000 μg/mL), Listeria monocytogenes (2500μg/mL), Enterobacter aerogenes (64μg/mL) Terpineol

Monoterpenoids

E. coli (450μg/mL), S. typhimurium (225μg/mL) C. albica (225μg/mL), S. cerevisiae (225μg/mL)

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Sesquiterpenoids

Aggregatibacter actinomycetemcomitans,

60, 62

Fusobacterium nucleatum subsp. animalis, Fusobacterium nucleatum subsp. polymorphum, Prevotella intermedia. Bacillus subtilis (90μg/mL), Staphylococcus aureus (90μg/mL), Salmonella sp. (90μg/mL) Farnesene

Sesquiterpenoids

S. aureus, S. Setubal, P. aeruginosa, B. subtilis

64

Patchoulol

Sesquiterpenoids

E. coli (1 mg/mL), Pseudomonas aeruginosa (3.5 mg/mL)

65

Bacillus proteus (3.5 mg/mL), Shigella dysenteriae (3 mg/mL) Typhoid bacillus (6.5 mg/mL), Staphylococcus aureus (2 mg/mL) Farnesol

Sesquiterpenoids

Paracoccidioides brasiliensis (25 mM)

66-68

P. aeruginosa Staphylococcus aureus (150 μM) Eudesmanes

Sesquiterpenoids

Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas

69

aeruginosa, Enterococcus faecalis and Staphylococcus aureus Cuparanes

Sesquiterpenoids

Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas

69

aeruginosa, Enterococcus faecalis and Staphylococcus aureus Canophyllol

Triterpenoids

Staphylococcus aureus and Scaphirhynchus albus (62.5 μg/ml)

70

Neisseria meningitides (31.25 μg/ml) Friedelin

Triterpenoids

S. aureus, S. albus, Trichophyton schoenleinii

70

Umbelliferone

Triterpenoids

Crytococcus neoformans and C. albicans (62.5 μg/ml)

70

Phenolic acids

Herpes simplex virus (0.05μg/mL)

Polyphenols Gallic acid

75-77, 199-201

Parainfluenza (type-3) (0.05μg/mL) S. aureus (1500 -1750 μg/mL), T. vaginalis (200-500 μg/mL) P. aeruginosa (500-2500 μg/mL), E. coli (1500 μg/mL), L. monocytogenes (2000 μg/mL) Pseudomonas putida DSMZ 291T (2.5 mg/mL), P. fluorescens DSMZ 50090T (5 mg/mL), P. fragi DSMZ 3456T (2.5 mg/mL), Pseudomonas spp. P30-4 (2.5 mg/mL), B. cereus (2.5 mg/mL), S. epidermidis (630μg/mL), M. albican (5 mg/mL), S. typhimurium (2.5 mg/mL), S. flexneri (2.5 mg/mL), Xylella fastidiosa (200-400 μM) Caffeic acid

Phenolic acids

herpes simplex virus (0.4μg/mL),

75, 81, 201-203

S. aureus (256 μg/mL to 1024 μg/mL) E. coli (39 mM), Xylella fastidiosa (200 μM) Ferulic acid

Phenolic acids

E. coli (100 μg/mL), P. aeruginosa (100 μg/mL) S. aureus (1100 μg/mL),

76, 201

L. monocytogenes (1250 μg/mL)

Xylella fastidiosa (800-2000 μM) Chlorogenic acid

Phenolic acids

herpes simplex virus (0.4μg/mL), parainfluenza (type-3) (0.4μg/mL), S. aureus (5 mg/mL), B. cereus (2.5 mg/mL), S. epidermidis (0.63 mg/mL), M. albican (5 mg/mL), E. coli (5 mg/mL), S. typhimurium (2.5 mg/mL), S. flexneri (2.5 mg/mL), P. aeruginosa (2.5mg/mL), Stenotrophomonas maltophilia (8-16 μg/ mL)

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Quinic acid

Phenolic acids

herpes simplex virus (0.05μg/mL), parainfluenza (type-3) (0.4μg/mL),

ρ-coumaric acids/

Phenolic acids

S. aureus (1 mg/mL), Xylella fastidiosa (200-400 μM)

derivatives

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75, 77 79, 81, 201, 204

Escherichia coli (0.22 μM/mL), Staphylococcus aureus (0.21μM/mL), Bacillus subtilis (0.28μM/mL), Aspergillus niger ( 0.23μM/mL), Candida albicans (0.27μM/mL), pMICam (0.16μM/mL)

Quercetin

Flavonoid derivatives

herpes simplex virus (0.1μg/mL)

, 72, 201

75

S. aureus (2.5 mg/mL), B. cereus (2.5 mg/mL), S. epidermidis (1.25 mg/mL), M. albican (2.5 mg/mL), E. coli (2.5 mg/mL), S. typhimurium (2.5 mg/mL), S. flexneri (2.5 mg/mL), P. aeruginosa (2.5 mg/mL), Apigenin

Flavonoid derivatives

Xylella fastidiosa (200-400 μM)

herpes simplex virus (0.4μg/mL)

75, 80

Pseudomonas aeruginosa, Salmonella typhimurium, Proteus mirabilis, Klebsiella pneumoniae, Enterobacter aerogenes Genistein

Flavonoid derivatives

herpes simplex virus (0.4μg/mL)

75, 205

parainfluenza (type-3) (0.2 μg/mL) Bacillus brevis (17.5μg/mL) Naringenin

Flavonoid derivatives

herpes simplex virus (0.4μg/mL)

75, 201, 206

Staphylococcus aureus Xylella fastidiosa (200-400 μM) Catechin

Flavonoid derivatives

Xylella fastidiosa (200-400 μM)

201, 207-210

E. coli, Staphylococcus aureus (62.5-250 μg/ mL), Pseudomonas aeruginosa (62.5-250 μg/ mL) Helicobacter pylori, Streptococcus mutans Silymarin

Flavonoid derivatives

herpes simplex virus (0.8μg/mL)

75, 211

E. coli, P. aeruginosa, S. aureus C. albicans, C. tropicalis, C. krusei Silibinin

Flavonoid derivatives

herpes simplex virus (0.1 μg/mL)

75, 211

E. coli, P. aeruginosa, S. aureus C. albicans, C. tropicalis, C. krusei Prenylated -flavonoids Flavonoid derivatives

Candida albicans, Saccaromyces cerevisiae,

82, 83

Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis, S. aureus (7′R,8′S)-4,4'-Dimetho Lignans

85

xy-strebluslignanol, 3'-Hydroxy-isostreblus lignaldehyde,

S. cerevisiae (ATCC 9763), Bacillus subtilis(ATCC 6633), Pseudomonas

3,3'-Methylene-bis(4-h

aeruginosa (ATCC 9027), Escherichia coli (ATCC 11775), and

ydroxybenzaldehyde,

Staphylococcus aureus (ATCC 25923)

4-Methoxy-isomagnal dehyde), magnolol and isomagnolol Blestriarene B

Stibenoids

Staphylococcus aureus, and S. epidermidis (6.25–25 μg/mL)

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Blestriarene C

Stibenoids

Staphylococcus aureus, and S. epidermidis (6.25–25 μg/mL)

Eugenol

Other polyphenols

E. coli (1000μg/mL), S. typhimurium (>1000μg/mL), L. monocytogenes(500 μg/mL), V. vulnificus (500μg/mL) S. aureus (750 μg/mL), T. vaginalis (15-90 μg/mL)

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Table 2 Production of certain terpenoids and polyphenols potencial as food preservitives though metabolic engineering in S. cerevisiae. Product

Substrate

Strategy

Titer

Reference

66.8 mg/L

123

750 μg/L

121

1.69 g/L

127

293 mg/L

119

1.68 g/L

122

36 mg/L

117

76 mg/L

212

Monoterpenoids Over-expression of SeACS (L641 P),tHMG1, IDI1 and ERG20 (K197 G); Geraniol

Glucose

deletion of OYE2 and

ATF1;

Introduce mutant tCrGES (F418Q) by developing an enzyme-coupled assay enable rapid protein engineering for geranoil production

Geraniol

Geraniol

Glucose

Expression of the Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in a Saccharomyces cerevisiae wine strain

Glucose

Dynamic control of ERG20 expression and OYE2 deletion in LEU2

+ethanol

prototrophic strain with pure ethanol feeding in fed batch fermentation Gene screening of geraniol synthases, GPP synthase, and farnesyl diphosphate synthase gene variants.

Geraniol

Glucose

Over-expression of IDI1, tHMG1, and UPC2-1. Construction of an Erg20p(F96WN127W)-tVoGES fusion protein fed-batch cultivation GESs screening, protein structure analysis and site-directed mutation in

Geraniol

Glucose

Geraniol

Glucose

CrGES, co-expression of the reverse fusion of Erg20ww/t3CrGES, Over-expression of tHMGR, IDI1, and Erg20WW Fed-batch fermentation under carbon restriction strategy Introduction of geraniol synthase, over-expression of IDI1 and MAF1 N-degron-dependent protein degradation strategy to down-regulate Erg20p,

Linalool

Glucose

Introduction of terpene biosynthetic pathway under either constitutive or diauxie-inducible promoters

Linalool /Limonene

Gene discovery (LaLIMS, LaLINS, LaBERS), using a homology-based PCR

/Terpinole

strategy

118

/Camphene /Pinene Biosynthesis of monoterpenes by S. cerevisiae in the absence of grape Linalool /Citronellol

Glucose

derived precursors

/fructose

Higher concentration of assimilable nitrogen

213

Microaerobic compared with anaerobic conditions Linalool

Glucose

Fusion expression of farnesyl diphosphate synthase and linalool synthase

240 μg/L

214

95μg/L

128

16 mg/L

131

Expression of linalool synthase gene from Lavandula angustifolia Linalool

Glucose

Downregulation of ERG9 gene with the repressible MET3 promoter Overexpression of tHMG1

Sesquiterpenoids Use of affibodies for enzyme tagging and scaffolding on farnesyl

Farnesene

Glucose

Cubebol

Galactose

Overexpression of the catalytic domain of HMG1

/Farnesol

/ethanol

Down-regulation of ERG9 with the regulatable MET3 promoter

diphosphate synthase and farnesene synthase

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9.9 mg/L ocubebol; 18.4 farnesol

mg/L

of

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16.9 Cubebol /Patchoulol/Farnesol

Glucose

mg/L

Two-phase fermentation using dodecane as the secondary phase

(patchoulol); 20.2

Down-regulation of ERG9 with the regulatable MET3 promoter

mg/L

140

(farnesol);

1.5 mg/L (cubebol) Cubebol

Galactose

In silico driven metabolic engineering Down-regulation of ERG9 with the regulatable MET3 promoter;

Amorphadiene

Over-expression of amorphadiene synthase (ADS).

8.4 mg/L

136

~120 mg/L/OD600

139

25 mg/L

130

64 mg/L

138

92 mg/l

132

6,535 μg/L

135

190.7 mg/L

134

370 µg/L

133

28µg/mL

215

2.9 g/L

152

650 μM

150

1.1 mM

151

21.3 mg/L

157

Down-regulation of ERG9 with the regulatable MET3 promoter; Amorphadiene

Glucose

Enzyme Fusion with farnesyldiphosphate synthase (FPPS) and amorphadiene synthase (ADS).

Amorpha-4,11-Diene

Glucose

Combinatorial engineering of Mevalonate Pathway by exploiting carotenoid

/galactose

biosynthesis as screening module

a-santalene

Glucose

Trichodermol

Glucose

Dynamic control of ERG9 expression and over-expression of tHMGR, Deletion of DPP1 Co-expression FgTRI5 and tHMGR. Screening of germacrene A synthases

Germacrene A

Glucose

Over-expression of tHMGR fusion of FPP synthase with GAS,

Epi-cedrol

Glucose

Over-expression of epi-cedrol synthase, Over-expression of tHMGR in a upc2-1 mating type a background Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and

Costunolide

chicory COS (CiCOS)

Phenolic acids Para-hydroxybenzoic acid (pHBA)

Glucose

PHBA

Glucose

PHBA

Glucose

p-coumaric acid

Glucose

Deletion of TRP3 and ARO7, expression of ARO4K229L as well as aroL, fed-batch bioreactor process Over-expression of Ubic and knock-out of Aro7 Dynamic metabolic pathway control via quorum-sensing linked RNA interference Downregulation of ARO4 and ARO7, Over-expression of ARO2 or TYR1 Comparison of the metabolic response to over-production of p-coumaric

p-coumaric acid

Glocose

acid in two yeast strains; systematically

158

overexpressed

or

deleted

genes

with

significant

transcriptional changes p-coumaricacid

Glucose

p-coumaricacid

Glucose

Naringenin

Over-expression of TAL, ARO4K229L, ARO7G141S, knock-out of PDC5 and

1.93 g/L

156

De novo synthesis of six different flavonoids

69 mg/L

164

Glucose

Introduction of PAL, 4CL, and CHS UNDER GAL10 promoter

7 mg/L

166

Pinocembrin

Glucose

Introduction of PAL, 4CL, and CHS UNDER GAL10 promoter

0.8 mg/L

166

Naringenin

Glucose

De novo pathway design

108 mg/L

160

p-coumaricaci

Construction of a gene cluster that contains four plant-derived genes of the early flavonoid biosynthetic pathway

28mg/L

170

d Glucose

Intra-modular engineering among the core flavonoid biosynthetic pathway,

90 mg/L

161

ARO10

Flavonoids

Naringenin Naringenin

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malonyl-coA biosynthetic pathway, and tyrosine biosynthetic pathway Naringenin

p-coumaricaci d

Introduction of PAL, 4CL, C4H, CHS, CHI, CPR

15.6 mg/L

162

Genistein

Naringenin

Introduction of PAL, C4H, CPR, CHS, CHI, 4CL, IFS

7.7 mg/L

162

Kaempferol

Naringenin,

Introduction of PAL, C4H, CPR, CHS, CHI, 4CL, FLS

4.6 mg/L

162

66.29 mg/L

163

Introduction of FLS from Populu deltoides, introduction of de novo Kaempferol

Glucose

Biosynthetic pathway, overexpression of acetyl-coA biosynthesis pathway, supplement of p-coumaric acid, fed-batch process

Kaempferol

Glucose

De novo synthesis of six different flavonoids

26 mg/L

164

Quercetin

Glucose

De novo synthesis of six different flavonoids

20 mg/L

164

Resokaempferol

Glucose

De novo synthesis of six different flavonoids

0.03 mg/L

164

Fisetin

Glucose

De novo synthesis of six different flavonoids

1.65 mg/L

164

Pinocembrin

Glucose

Introduction of PAL, 4CL, and CHS UNDER GAL10 promoter

0.8 mg/L

166

Anthocyanin

Glucose

1.5-2 mg/L

167

De novo biosynthesis of ACNs Gene screening

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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