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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] 1
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Abstract
2
Terpenoids and polyphenols are high-valued plant secondary metabolites. Their high antimicrobial
3
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,
6
Saccharomyces cerevisiae, as a cell factory. This review will summarize the major terpenoid and
7
polyphenol compounds with high antimicrobial properties, describe their native metabolic pathways
8
as well as antimicrobial mechanisms, and highlight current progress on their heterologous
9
biosynthesis in S. cerevisiae. Current challenges and perspectives for the sustainable production of
10
terpenoid and polyphenol as natural food preservatives via S. cerevisiae will also be discussed
11
herein.
12
Keywords: terpenoids; polyphenols; food preservatives; sustainable production; antimicrobial
13
mechanism
14
2
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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
17
production technologies, the cases of food poisoning worldwide continue to grow. It has been
18
estimated that 600 million people suffered from foodborne illnesses, with approximately 230,000
19
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
21
poisoning cases. The toxigenic organism Staphylococcus aureus and Clostridium botulinum, are the
22
cause of far fewer cases2. In addition to foodborne illnesses, spoilage of foods by microbes also leads
23
to enormous losses. One-quarter of the world’s food supply was estimated to be lost, due to the
24
growth of spoilage and pathogenic microorganisms3. To combat this, various synthetic
25
antimicrobials, e.g., nitrates, benzoates, sorbic acid, sulphites, potassium lactate, and citric acid, have
26
been in use as food preservatives, for decades. However, their usage presents toxic risk on human
27
and animal health as reported in many studies 4, 5. For example, nitrate can get converted into nitrous
28
acid when consumed and is suspected of causing stomach cancer, and benzoates may cause allergies
29
or skin rashes5. Hence there is growing consumer demands and an urgent need for a natural food
30
preservative, which does not compromise on food safety, convenience, or sensory characteristics.
31
Essential oils (EO) are concentrated hydrophobic liquids extracted from diverse plants. In
32
addition to their original usage as food additives due to their fragrant properties, their demonstrated
33
antimicrobial activities against a wide range of microorganisms makes them good candidates as
34
natural food preservatives6-14. Composition analysis showed that the volatile portion of EO mainly
35
consists of monoterpenes and sesquiterpene hydrocarbons, alcohols, aliphatic aldehydes, and esters15.
36
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,
39
carvacrol, citral, and limonene) have been accepted by the European Commission for use as
40
flavoring in food products and are recognized as safe by the United States Food and Drug
41
Administration (FDA)19. Currently, the supply of natural terpenoids and polyphenols is largely
42
dependent on plant extraction, which is subject to the limitations of climate and long growth cycles.
43
Based on the advancement of mature high-density fermentation and metabolic engineering, microbial
44
biosynthesis could provide an environmentally sustainable and cost-effective alternative for mass
45
production of such high-value chemicals in a short period of time20, 21. Its collaboration with plant
46
extraction methods showed good prospects to meet the growing needs of the food market.
47
Considering that both terpenoids and polyphenols have long biosynthetic pathways and are used in
48
the field of foods and medicine, the desired cell factory should have advantageous features inclusive
49
of high security and ease of genetic manipulation. Saccharomyces cerevisiae (S. cerevisiae) is an
50
attractive cell factory, due to its safety, genetic tractability, and mature high-density fermentation
51
technology. Its eukaryotic nature allows expression of multiple complex proteins and production of
52
various bio-based products22. Furthermore, the development of excellent pathway assembly tools23
53
and advanced dynamic metabolic regulation systems24 makes it possible to complement the
54
construction and metabolic optimization of the heterologous biosynthetic pathways in yeast. Up to
55
now, there have been many studies on sustainable production of terpenoids and polyphenols by
56
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
58
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
61
perspectives are also discussed herein.
62
2. Terpenoids
63
Terpenoids comprise a vast family of the most abundant natural products which are widely
64
distributed in nature. They exist in almost every organism, having important physiological,
65
metabolic, communication and defense functions. Naturally occurring isoprenoids have important
66
application value to humans: the primary components of oil metabolites in plants can be used as
67
flavoring agents and aromatics; secondary metabolites have important medicinal value including
68
anti-inflammatory, antibacterial, anti-hypertensive, immune regulation, anti-oxidation, anti-cancer,
69
and other functions.
70
2.1 Biosynthetic pathway of terpenoids
71
Terpenoids are modified forms of terpenes ((C5H8)n). This basic terpene form (C5H8)n, can have
72
oxygen molecules added to it, or have its methyl group removed, to form terpenoid derivatives
73
including alcohols, esters, aldehydes, ketones, ethers, phenols, and epoxides. Also, they can be
74
classified into hemi-(C5), mono- (C10), sesqui- (C15), di- (C20), sester- (C25), tri- (C30), tetra-
75
(C40) and polyterpenes (C5)n depending on the number of isoprene units25. The biosynthetic
76
pathway of terpenoids can be divided into four basic steps (Figure 1)26: (1) synthesis of monomer
77
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) from the initial acetyl-CoA
78
molecule; (2) polymerization of DMAPP and IPP under the action of isopentenyl transferase to form
79
the linear prenyl diphosphate precursor - geranyl pyrophosphate (GPP, C10, monoterpenoid),
80
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
82
catalysis of terpene synthases to form an initial terpene carbon skeleton; (4) tailoring and
83
modification of the carbon skeleton to biologically active chemicals.
84
Monomer isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are the
85
universal precursors for the biosynthesis of various downstream terpenoids (hemiterpenoid,
86
monoterpenoid,sesquiterpenoid, diterpenoid, etc.). There are two distinct pathways for biosynthesis
87
of IPP and DMAPP27-30: the mevalonate pathway (MVA pathway) and the methylerythritol
88
phosphate pathway (MEP pathway) (Figure 1). The MVA pathway is ubiquitous in the cytoplasm of
89
all organisms including: some bacteria, most fungi, and plants, which initiates from acetyl-CoA and
90
ends up with the production of DMAPP31, 32. As the primary precursor, two molecules of acetyl-CoA
91
are condensed to acetoacetyl-CoA in a reversible reaction via acetoacetyl-CoA thiolase (ERG10).
92
Under
93
3-hydroxy3-methylglutaryl-CoA (HMG-CoA) and subsequently generated in mevalonate (MVA)
94
within two reduction steps which consumes NADPH. The latter reaction is catalyzed through
95
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme of the MVA pathway
96
33.
97
phosphor-MVA kinase (PMK). Lastly, IPP is generated from MVA 5-diphosphate under an
98
ATP-dependent decarboxylation reaction via diphospho-MVA decarboxylase (MVD1). This can be
99
interconverted to DMAPP by isomerization via IPP isomerase (IDI). In 1996, Eisenvaich et al. found
100
that the taxane carbon skeleton of taxol was not of mevalonoid origin, in an experiment using(13)C
101
stable isotope labelling34. This triggered the discovery of the MEP pathway which is present in most
102
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
6
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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
105
DXP reductoisomerase (DXR), DXP is intramolecularly rearranged and reduced to form MEP. Then,
106
MEP is further modified to yield hydroxy-2-methyl-2-butenyl-4-diphosphate (HMBPP) within four
107
steps catalyzed by 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ISPD), 4-(cytidine 5’ -
108
diphospho)-2-C-methyl-D-erythritol kinase (ISPE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate
109
synthase (ISPF) and 4-hydroxy-3-methylbut-2-enyldiphosphate (HMBPP) synthase (ISPG). HMBPP
110
is subsequently converted into IPP and DMAPP by HMBPP reductase (ISPH). Finally, the
111
conversion and balance of IPP to DMAPP35 is controlled by IPP isomerase (IPPI), in a reversible
112
reaction. In the MEP pathway, DXS36-38, DXR37,
113
rate-limiting enzymes. Compared with the MVA pathway, the MEP pathway is energetically
114
balanced and theoretically more efficient than the MVA pathway in converting sugars (30.2% vs.
115
25.2% mass yield on glucose) or glycerol to terpenoids40, 41.
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2.2 Antimicrobial property of terpenoids
39
and IPPI38 have been identified as the
117
Terpenoids are one of the classes of compounds which are responsible for the antimicrobial
118
properties in EO. In the study by Dorman, H. J. D. et al., fourteen major compounds in EO from
119
eight plants were assessed for their antibacterial activity against 25 different genera of bacteria,
120
whereby most of the terpenoids in the forms of monoterpenoids and sesquiterpenes possessed strong
121
antimicrobial activity42. The typical terpenoids which possess antimicrobial properties are
122
summarized in Table 1 and discussed as follows.
123
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
127
vitro assays of carvacrol45-47 and thymol45,
128
towards diverse microbes (e.g. S. cerevisiae, Candida strains, Enterococcus faecalis, E. amylovora,
129
E. carotovora, E. coli, L. monocytogenes, S. aureus, Staphylococcus epidermidis, S. typhimurium, P.
130
fluorescens, Vibrio vulnificus, and Yersinia enterocolitica) (Table 1). For instance, the antibacterial
131
activity of major components in EO, from leaves of Lippia multiflora, Mentha x piperita and
132
Ocimum basilicum, have been evaluated against L. monocytogenes, E. aerogenes, E. coli, P.
133
aeruginosa, amongst which thymol and carvacrol demonstrated the strongest antibacterial activity
134
with an MIC (minimum inhibitory concentration) of 300 and 800 μg/mL, respectively48. Another in
135
vitro assay exhibited their antifungal activity against Botrytis cinerea with 100% of inhibition at 100
136
ppm
137
antimicrobial activity mainly contain linalool50, 51, citral46, carvone52, 53, linalyl acetate54, menthol55,
138
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).
139
Sesquiterpenoids are another major constituent of antimicrobial terpenoids, such as the
140
compound artemisinin60, 61, farnesene62-64, patchoulol65 and farnesol66-68 as summarized in Table 1.
141
Besides, eudesmanes and cuparanes, isolated from the organic extract of the red alga Laurencia
142
obtusa Lamouroux have been reported to have antimicrobial activities against a wide range of
143
microbes, including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas
144
aeruginosa, Enterococcus faecalis and Staphylococcus aureus (especially Gram-positive bacteria)69.
145
In addition to monoterpenoids and sesquiterpenoids, rare triterpenoids showed microbial
146
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
149
value of 31.25 μg/ml. Friedelin, exhibited antibacterial activities against S. aureus and S. albus, as
150
well antifungal activity against Trichophyton schoenleinii; wheras umbelliferone showed antifungal
151
activity with an MIC value of 62.5 μg/ml against both Crytococcus neoformans and C. albicans70.
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3. Polyphenols
153
Polyphenols are natural secondary metabolites derived from the shikimate/phenylpropanoid
154
and/or the polyketide pathway, featuring in more than one phenolic unit and deprived of
155
nitrogen-based functions in plants. They carry out many important functions in plants, e.g., the
156
formation of pigments, resisting environmental stresses and acting as chemical messengers.
157
3.1 Biosynthetic pathway of polyphenols
158
As shown in Figure 2, depending on the number of phenolic rings and chemical structure
159
differences, polyphenols are usually classified into i) phenolic acids; ii) flavonoids; iii) lignans; iv)
160
stilbenoids; v) coumarins71. The shikimate and phenylpropanoid pathways are the most common and
161
essential routes for polyphenol biosynthesis in plants. Specifically, the shikimate pathway is present
162
in bacteria, fungi, and plants. It consists of seven metabolic steps, beginning with the condensation of
163
phosphoenolpyruvate with erythrose-4-phosphate and ending with the production of chorismate. The
164
end product chorismate is the precursor for postchorismate pathways leading to tryptophan and
165
phenylalanine/tyrosine biosynthesis. The phenylpropanoid pathway begins with the conversion of
166
phenylalanine into cinnamic acid via phenylalanine ammonia lyase (PAL). Cinnamic acid is then
167
activated to p-coumaric acid by a membrane-bound P450 monooxygenase, cinnamate 4-hydroxylase
168
(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
170
generated, the next step is catalyzed by p-coumaric acid: CoA ligase (4CL) which will mediate the
171
formation of the corresponding CoA ester, 4-coumaroyl-CoA from p-coumaric acid. The
172
intermediate p-coumaric acid will lead to the production of lignans, coumarins, and hyroxycinnamic
173
acids (e.g. caffeic acid, ferulic acid, and sinapic acid). And the end product of the phenylpropanoid
174
pathway, 4-coumaroyl CoA, will direct the carbon flow to branches of flavonoids and stilbenes.
175
3.2 Antimicrobial property of polyphenols
176
The antimicrobial properties of polyphenols have been well clarified from both plant extraction
177
assay and in vitro analysis with pure specific compound. Typical polyphenols harboring
178
antimicrobial property are summarized in Table 1.
179
From plant extracts, it was found that extracts from red fruits, plum skins, Italian red grape skins,
180
and different parts of elderberry, had a high concentration of polyphenols (100-1005 μg/g of phenolic
181
acids, 50-380μg/g of anthocyanins, 30-530 μg/g of flavonoid catechin), which showed inhibitory
182
effect towards almost all the pathogenic strains tested, including Bacillus cereus DSM
183
345, Lactobacillus paracasei IMC 502®, Lactobacillus plantarum IMC 509, Lactobacillus
184
rhamnosus IMC 501®,
185
SY-SYNBIO®,
186
(Balsaminaceae) revealed the presence of nineteen antimicrobial constituents against 10
187
microorganisms tested, comprising phenolic acids (1.4-4.7 mg CAE/g DW) and flavonoids (3.2 to
188
6.3 mg QE/g DW) 74
E.
Listeria monocytogenes 306,
coli ATCC
13706,
Candida
Staphylococcus aureus ATCC 25923, albicans ATCC
1026172,
73.
Impatiens
189
In addition, from in vitro analysis, phenolic acids and flavonoids have proved their good
190
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
192
quercetin, apigenin, genistein, naringenin, silymarin, and silibinin) exerted robust antibacterial effect
193
towards Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae,
194
Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis,
195
etc75-80(see details in Table 1). In food test, phenolic acids - caffeic acid and p-coumaric acid were
196
found can completely inhibit chicken soup contaminant bacterium Staphylococcus aureus in food
197
assay, (above 0.1 mg/mL)81, and 18 prenylated flavonoids (having a prenyl chain on the flavonoid
198
backbone) isolated from medicinal plants showed high inhibiting activity against Candida albicans,
199
Saccaromyces cerevisiae, Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis, and
200
S. aureus82. In our previous study83, metabolites of engineered yeast producing naringenin (a kind of
201
flavonoid) exhibited strong antimicrobial activity. Pure naringenin had weak antimicrobial effect
202
whereas prenylnaringenin can inhibit Staphylococcus aureus. According to the study of Zemek, J. et
203
al., a total of 25 aromatic compounds including guaiacyl- and syringyl-like structures
204
(low-molecular-weight part of lignin), gallic acid and its derivatives, cinnamic acid and its
205
derivatives, veratrie acid, were analyzed and been demonstrated their effective antibiotic activity
206
against bacteria, yeast-like organisms and protozoa84.
207
Besides phenolic acids and flavonoids, some lignans and stilbenoids also displayed good
208
antimicrobial
properties.
For
instance,
six
lignans
including
209
(7′R,8′S)-4,4'-Dimethoxy-strebluslignanol,
210
3,3'-Methylene-bis(4-hydroxybenzaldehyde,
211
isomagnolol inhibited S. cerevisiae (ATCC 9763), Bacillus subtilis (ATCC 6633), Pseudomonas
212
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
214
exhibited antibacterial activity against two Gram positive strains, Staphylococcus aureus, and S.
215
epidermidis, with the MIC values of 6.25–25 μg/mL86.
216
4. Antimicrobial mechanisms of terpenoids and polyphenols, and their synergetic effects
217
4.1 Antimicrobial mechanism
218
The mechanisms of action of terpenoids and polyphenols against microorganisms have not been
219
entirely understood. Nevertheless, there are some fundamental mechanisms which are widely
220
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
222
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
229
leakage of ions87,
230
polyphenols than Gram-negative bacteria89,
231
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
236
passage of phytochemicals91, 92.
237
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).
259
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.
265
(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
276
geraniol, a replacement of –OH group with –COOH lead to an increase in the activity of
277
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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433
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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455
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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477
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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521
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 4
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