Establishment of Novel Biosynthetic Pathways for the Production of

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Establishment of novel biosynthetic pathways for the production of salicyl alcohol and gentisyl alcohol in engineered Escherichia coli Xiaolin Shen, Jia Wang, Bradley Gall, Eric M. Ferreira, Qipeng Yuan, and Yajun Yan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00051 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 24, 2018

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ACS Synthetic Biology

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Establishment of novel biosynthetic pathways for the production of salicyl alcohol and

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gentisyl alcohol in engineered Escherichia coli

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Xiaolin Shena,b,1, Jia Wang a,b,1, Bradley K. Gallc, Eric M. Ferreirac,

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Qipeng Yuana,b,*, Yajun Yand,*

6 7

a

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University of Chemical Technology, Beijing 100029, China

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing

9 10

b

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Technology, Beijing 100029, China

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

12 c

Department of Chemistry, The University of Georgia, Athens, GA 30602, USA

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d

School of Chemical, Materials and Biomedical Engineering, College of Engineering, The

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University of Georgia, Athens, GA 30602, USA

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17 18

1

XS and JW contributed equally to this work

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* Corresponding authors:

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Qipeng Yuan

22

15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China

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E-mail: [email protected] ; telephone: +86-10-64437610

1 ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Yajun Yan

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146 Riverbend Research Lab South, The University of Georgia, Athens, GA 30602, USA

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E-mail: [email protected] ; telephone: +1-706-542-8293


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Abstract

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Salicyl alcohol and gentisyl alcohol are two important phenolic alcohols that possess significant

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biological activities and pharmaceutical properties. Here, we report establishment of novel

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biosynthetic pathways for microbial production of salicyl alcohol and gentisyl alcohol from

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renewable feedstocks. We first examined the promiscuity of the carboxylic acid reductase CAR

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towards salicylic acid and 2,5-DHBA, which enabled efficient synthesis of salicyl alcohol and

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gentisyl alcohol. Then, we employed a novel salicylic acid 5-hydroxylase to achieve 2,5-DHBA

35

production from salicylic acid. After that, the de novo biosynthetic pathways were assembled and

36

optimized by programming the carbon flux into the shikimate pathway. The final titers of salicyl

37

alcohol and gentisyl alcohol reached to 594.4 mg/L and 30.1 mg/L, respectively. To our

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knowledge, this work achieved microbial production of salicyl alcohol and gentisyl alcohol for

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the first time. Our present study also demonstrated application of enzyme promiscuity to

40

establish non-natural biosynthetic pathways for the production of high-value compounds.

41 42

Keywords: Salicyl alcohol, Gentisyl alcohol, Shikimate pathway, Metabolic engineering



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Phenolic compounds are a large group of phytochemicals that are found in many vegetables and

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fruits1. Those compounds possess significant pharmaceutical importance owing to their

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antioxidant, anti-inflammatory, anti-cancer and antibacterial activities2, 3. Metabolic engineering

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of microorganisms for the synthesis of phenolic compounds has gained great attention in recent

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years4-6. Current research has mainly focused on synthesis of phenolic acids, such as salicylic

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acid7, gallic acid8, caffeic acid9, and p-coumaric acid10. In comparison, less effort has been made

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to achieve bio-production of phenolic alcohols due to the lack of natural biosynthetic pathways

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or the absence of the responsible pathway enzymes4. As a class of value-added phenolic

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compounds, phenolic alcohols have extensive applications in solvent, flavor, fragrance,

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cosmetics and pharmaceutical industries11,

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microbial cell factories for achieving their biosynthesis. In this study, we report establishment of

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a novel microbial platform for the production of two important phenolic alcohols, salicyl alcohol

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and gentisyl alcohol in Escherichia coli.

12

. Thus, it is of great significance to develop

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Salicyl alcohol, also named saligenin, or 2-hydroxybenzyl alcohol, is a drug or prodrug that has

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been widely used in pharmaceutical industry. It has been used for a long time as a local

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anesthetic agent13. It also possesses antiseptic, antibacterial and anti-pyretic activities14, 15. In

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addition, salicyl alcohol can be used as a precursor for the synthesis of salicin. Compared with

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the well-known anti-inflammatory drug aspirin, salicin exhibits similar pharmacological

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activities but less side effect to the human health16. Gentisyl alcohol, also named 2,5-dihydroxy

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benzyl alcohol, is another important biologically active compound that has great health benefits.

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It has been identified as a potent antioxidant with radical-scavenging activity17. It also has been

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reported that gentisyl alcohol could induce new blood vessel formation18 and inhibit etoposide-


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induced apoptosis19. Besides, gentisyl alcohol also exhibits antibacterial activity against multi

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drug resistant Staphylococcus aureus20 and antifungal activity against Colletotrichum

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gloeosporioides21. Although many studies have reported the outstanding bioactivities of salicyl

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alcohol and gentisyl alcohol, to the best of our knowledge, biosynthesis of those two compounds

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in microorganisms from renewable feedstocks has not yet been achieved so far. In this work, we

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constructed novel artificial pathways for the production of salicyl alcohol and gentisyl alcohol in

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E. coli by extending its endogenous shikimate pathway (Fig. 1).

73 74

To assemble the salicyl alcohol and gentisyl alcohol biosynthetic pathways, their appropriate

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precursors need to be identified first. Based on the structural similarity, we selected salicylic acid

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and 2,5-dihydroxy benzoic acid (2,5-DHBA) as the direct precursors, bio-reduction of those two

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aromatic carboxylic acids into their corresponding alcohols might be a promising mechanism for

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the synthesis of salicyl alcohol and gentisyl alcohol. However, the enzymes responsible for

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catalyzing such biological reactions have not yet been identified so far. To overcome this issue,

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we searched for the similar catalytic reactions, and found that a carboxylic acid reductase (CAR)

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from Mycobacterium marinum is capable of converting a series of aromatic and short-chain

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carboxylic acids into their corresponding aldehydes22. Those aldehydes could be automatically

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reduced to the respective alcohols by the E. coli endogenous alcohol dehydrogenases. Given the

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broad-substrate specificity of the CAR, we speculated that it might also possess the capability of

85

reducing salicylic acid and 2,5-DHBA.

86 87

To verify our hypothesis, we performed feeding experiments to test the catalytic activities of

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CAR toward salicylic acid and 2,5-DHBA. The high-copy number plasmid pZE12-luc carrying



89

genes car from M. marinum and sfp (encoding CAR maturation factor phosphopantetheinyl

90

transferase) from Bacillus subtilis was constructed and transferred into E. coli BW25113 (F’),

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yielding strain XJ01. In this strain, E. coli native alcohol dehydrogenases were supposed to

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convert the produced aldehydes into alcohols. Given the toxicity of the salicylic acid and 2,5-

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DHBA7, 23, they were fed into the medium at 300 mg/L every three hours, respectively. When fed

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with salicylic acid as the substrate, the titer of salicyl alcohol rapidly increased to 882.1 mg/L at

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the end of 24 h (Fig. 2A), indicating the high catalytic efficiency of CAR towards salicylic acid.

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When using 2,5-DHBA as the substrate, the production rate was much slower, only 363.1 mg/L

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gentisyl alcohol was produced by the end of 60 h (Fig. 2B). Those results suggested that CAR is

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able to accept both salicylic acid and 2,5-DHBA as the substrates but exhibits higher activity

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towards salicylic acid. To further improve the production efficiency, we over-expressed a

100

heterologous alcohol dehydrogenase (ADH6) from Saccharomyces cerevisiae. The plasmids

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pZE-car-sfp and pCS-adh6 were co-transferred into E. coli BW25113 (F’), yielding strain XJ02.

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As shown in Fig. 2A, XJ02 generated 849.2 mg/L salicyl alcohol, which was comparable with

103

the strain XJ01. Remarkably, the titer of gentisyl alcohol in JX02 was enhanced to 669.7 mg/L,

104

representing an 84.4 % improvement in titer compared with XJ01 (Fig. 2B). Thus, we concluded

105

that the E. coli native alcohol dehydrogenases were not sufficient for reduction of gentisyl

106

aldehyde to gentisyl alcohol.

107 108

Since we have successfully achieved efficient synthesis of salicyl alcohol and gentisyl alcohol by

109

recruiting salicylic acid and 2,5-DHBA as the direct precursors, our next focus is to construct

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salicylic acid and 2,5-DHBA biosynthetic pathways. We previously reported that the

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isochorismate synthase EntC from E. coli and the isochorismate pyruvate lyase PchB from


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Pseudomonas fluorescens have high efficiency for generation of salicylic acid from chorismate,

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a key intermediate in the shikimate pathway24. Wild type E. coli strain carrying those two genes

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produced 158.5 mg/L of salicylic acid24. However, few studies have reported the biosynthesis of

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2,5-DHBA. Previously, we identified an anthranilate 5-hydroxylase (SalABCD) from Ralstonia

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eutropha H16 that could hydroxylate anthranilate at the C5 position to form 5-

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hydroxyanthranilate25. Additionally, we confirmed that combinatorial expression of anthraniloyl-

118

CoA synthetase (PqsA) from P. aeruginosa and salicyloyl-CoA 5-hydroxylase (SdgC) from

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Streptomyces sp. strain WA46 also lead to 5-hydroxyanthranilate production from anthranilate25.

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Given the similarity in catalytic mechanisms and structures of the substrates anthranilate and

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salicylic acid, we speculated that 2,5-DHBA could be generated from salicylic acid by

122

employment of SalABCD or the combination of PqsA and SdgC. To examine our hypothesis,

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plasmids pZE-salABCD and pZE-pqsA-sdgC were individually transferred into E. coli

124

BW25113 (F’), yielding strain XJ03 and XJ04, respectively. As shown in Fig. 2C, at the end of

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24 h, strain XJ03 produced 622.4 mg/L 2,5-DHBA by feeding salicylic acid into the cultures, the

126

titer slightly declined to 601.6 mg/L at 48 h, probably caused by the autoxidation of 2,5-DHBA.

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Although the titer was much lower, strain XJ04 also synthesized 11.6 mg/L 2,5-DHBA at 24 h

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(Fig. 2C). Those results indicated that SalABCD was the best salicylate 5-hydroxylase for the

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generation of 2,5-DHBA from salicylic acid.

130 131

Next, we attempted to produce gentisyl alcohol from salicylic acid by coupling SalABCD with

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Car-Sfp. Plasmid pZE-car-sfp-salABCD was transferred into E. coli BW25113 (F’), generating

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strain XJ05. Salicylic acid was fed into the cultures as the substrate. At the end of 60 h, XJ05

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only synthesized 89.7 mg/L gentisyl alcohol (Fig. 2D), while salicyl alcohol as the dominant by-



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product was produced at 587.2 mg/L (Fig. S1A), indicating that salicylic acid was directly

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reduced to salicyl alcohol without being hydroxylated. This was because the CAR demonstrated

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higher substrate affinity and enzyme activity towards salicylic acid than 2,5-DHBA. To further

138

enhance the conversion efficiency, pCS-adh6 was introduced into strain XJ05 to generate strain

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XJ06. As shown in Fig. 2D, the titer of gentisyl alcohol was slightly improved to 109.8 mg/L at

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60 h, while the production of by-product salicyl alcohol was also enhanced to 692.7 mg/L (Fig.

141

S1A). We also observed that strains XJ05 and XJ06 showed similar production profiles of the

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intermediate 2,5-DHBA and accumulated the highest titers at 12 h. Strain XJ06 consumed all the

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2,5-DHBA at the end of 60h, while strain XJ05 left 113.1 mg/L 2,5-DHBA unconsumed in the

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cultures (Fig. S1B).

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After achieving the synthesis of salicyl alcohol and gentisyl alcohol from salicylic acid, we

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began to assemble the entire pathways for de novo production of those two chemicals. The E.

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coli native metabolism was extended to synthesize salicylic acid by employment of the plasmid

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pCS-EP carrying entC and pchB. Plasmids pCS-EP and pZE-car-sfp were co-transferred into E.

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coli BW25113 (F’), generating strain XJ07, for de novo production of salicyl alcohol. As shown

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in Fig. 3, the highest amount of salicyl alcohol reached to 271.0 mg/L at the end of 72 h,

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suggesting that our established artificial pathway towards salicyl alcohol was functional in E.

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coli. Then, we introduced Adh6 into this pathway by placing the gene adh6 into plasmid pCS-EP.

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The E. coli BW25113 (F’) was transformed with the generated plasmid pCS-EPA and pZE-car-

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sfp, yielded strain XJ08. The results showed that XJ08 produced 272.9 mg/L salicyl alcohol,

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indicating that over-expression of adh6 has a negligible effect on salicyl alcohol titer (Fig. 3),

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this was consistent with the feeding experiments. However, this titer is less than that obtained in

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our feeding experiments, indicating the supply of the salicylic acid is insufficient in our pathway.


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In order to boost the availability of the salicylic acid, we employed our previously constructed

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plasmid pCS-APTA containing aroL (encoding shikimate kinase II), ppsA (encoding

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phosphoenolpyruvate synthetase), tktA (encoding transketolase) and aroGfbr (encoding the

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feedback inhibition resistant mutant of 2-dehydro-3-deoxyphosphoheptonate aldolase) to redirect

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the carbon flux into the shikimate pathway24. Strain XJ09 carrying plasmids pCS-APTA-EPA

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and pZE-car-sfp was constructed and used for shake flask fermentation. We observed that the

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resulting strain generated 594.4 mg/L salicyl alcohol at 72 h (Fig. 3), indicating a 117.8 %

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improvement in titer compared with strain XJ08. In addition, we observed that the growth profile

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of all three strains was very similar. They all reached the highest OD600 value of around 8~9 after

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24 h cultivation. Those results suggested that over-expression of pCS-APTA is effective to

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enhance the production of salicyl alcohol.

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Next, we tried to extend the salicyl alcohol biosynthetic pathway to realize the de novo

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production of gentisyl alcohol. The salicylic acid 5-hydroxylase SalABCD was incorporated into

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the salicyl alcohol producers XJ07, XJ08 and XJ09 to create the gentisyl alcohol producers XJ10,

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XJ11 and XJ12, respectively. As shown in Fig. 4, XJ10 carrying the entire pathway synthesized

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7.2 mg/L gentisyl alcohol. Even the gene adh6 was over-expressed, the titer in JX11 only

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reached to 11.5 mg/L. The intermediate 2,5-DHBA was completely consumed after 72 h (Fig.

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S2A). We also observed that both XJ10 and XJ11 generated negligible titer of salicyl alcohol

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throughout the fermentation (Fig. S2B), indicating the insufficiency of the precursor supply.

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When the operon APTA was introduced into the pathway, strain XJ12 produced 30.1 mg/L

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gentisyl alcohol at 72 h (Fig 4). In addition, 264.2 mg/L salicyl alcohol was also generated as the

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by-product (Fig. S2B), and the accumulation of the intermediate 2,5-DHBA reached to 374.6



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mg/L (Fig. S1A). All three strains showed similar growth pattern that reached highest cell

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density (OD600 = 8~9) at the end of 72 h. Those results suggested that the catalytic efficiency of

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the CAR towards 2,5-DHBA is the limiting step in the gentisyl alcohol biosynthetic pathway.

184 185

In this study, we constructed and demonstrated novel biosynthetic pathways for the de novo

186

production of salicyl alcohol and gentisyl alcohol from renewable feedstocks for the first time.

187

Validating the promiscuity of the carboxylic acid reductase CAR towards the precursors salicylic

188

acid and 2,5-DHBA enabled efficient synthesis of their corresponding products salicyl alcohol

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and gentisyl alcohol. Then, a novel salicylic acid 5-hydroxylase was employed to convert

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salicylic acid to 2,5-DHBA. After that, the whole artificial pathways were assembled and

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optimized by strengthening the shikimate pathway. The final titers of salicyl alcohol and gentisyl

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alcohol reached to 594.4 mg/L and 30.1 mg/L, respectively. However, production of the gentisyl

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alcohol was accompanied with the generation of salicyl alcohol due to the promiscuity of the

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CAR, resulting in a relatively low titer of gentisyl alcohol. To overcome this issue, it is necessary

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to enhance the substrate specificity of CAR towards 2,5-DHBA. Recently, rational protein

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engineering has been proven to be an effective approach to improve the performance of the

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target

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compartmentalization of metabolic pathways28 also serve as promising strategies to increase the

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accessibility of 2,5-DHBA to CAR. Overall, our work presented here provides an example for

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applying enzyme promiscuity to establish non-natural biosynthetic pathways for the production

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of high-value compounds.

enzyme26.

In

addition,

construction

of

synthetic


protein

scaffolds27

and

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Methods

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Media, Strains and Plasmids

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Luria-Bertani (LB) medium containing 10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract was

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used for plasmid propagation and strain inoculation. The modified M9 medium containing 2.5

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g/L glucose, 20 g/L glycerol, 5 g/L yeast extract, 6 g/L Na2HPO4, 0.5 g/L NaCl, 3 g/L KH2PO4,

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1 g/L NH4Cl, 246.5 mg/L MgSO4·7H2O and 14.7 mg/L CaCl2·2H2O was used for feeding

208

experiments and total biosynthesis of salicyl alcohol and gentisyl alcohol. If necessary, the

209

antibiotics ampicillin and kanamycin were supplemented into the medium at the final

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concentration of 100 and 50 µg/mL, respectively. E. coli strain XL1-Blue was used for DNA

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manipulation experiments and E. coli BW25113 (F’) was used as the host for salicyl alcohol and

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gentisyl alcohol production. Plasmids pZE12-luc (high-copy number) and pCS27 (medium-copy

213

number) were used as the backbone for pathway construction. All of used strains and plasmids in

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this study were listed in Table 1.

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Chemical synthesis of gentisyl alcohol

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The gentisyl alcohol standard is not commercially available. It was chemically synthesized using

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2,5-DHBA as the precursor. To a solution of 2,5-dihydroxybenzoic acid (49.5 mg, 0.321 mmol)

219

in THF (1.6 mL, 0.2 M) in a flame-dried flask under argon at 0 °C was added BH3•THF (2.10

220

mL, 1.0 M solution in THF, 2.10 mmol) dropwise. The reaction mixture was allowed to stir at

221

0 °C and monitored by TLC. After completion (2 h), the reaction mixture was quenched with sat.

222

aq. NH4Cl/H2O (1:1, 10 mL), and the mixture was extracted with EtOAc (3 x 10 mL). The

223

organic layers were combined, dried over Na2SO4, and concentrated in vacuo. The crude product



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was purified by flash chromatography (2:1 EtOAc/hexanes eluent), affording 2,5-

225

dihydroxybenzyl alcohol (26.9 mg, 60% yield) as a white solid.

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DNA manipulations

228

Plasmids pZE-salABCD, pZE-pqsA-sdgC25, pZE-EP7 and pCS-APTA24 were constructed in our

229

previous studies. The genes car from Mycobacterium marinum and sfp from Bacillus subtilis

230

were cloned into the backbone of pZE12-luc using Acc65I, NdeI and XbaI to create plasmid

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pZE-car-sfp. The gene adh6 from Saccharomyces cerevisiae was inserted into the backbone of

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pCS27 using Acc65I and BamHI to create plasmid pCS-adh6. The expression cassette PLlacO1-

233

SalABCD was amplified by PCR from pZE-salABCD and inserted into plasmid pZE-car-sfp

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between SacI and SpeI to create plasmid pZE-car-sfp-salABCD. The gene fragment entC-pchB

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was amplified by PCR from pZE-EP and inserted into plasmid pCS27 between Acc65I and

236

BamHI to create plasmid pCS-EP. The gene fragments entC-pchB and adh6 were inserted into

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the backbone of pCS27 using Acc65I, BamHI and HindIII to create plasmid pCS-EPA. The

238

expression cassette PLlacO1-APTA was amplified by PCR from pCS-APTA and inserted into

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plasmid pCS-EPA between SacI and SpeI to create plasmid pCS-EPA-APTA.

240 241

Feeding experiments

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The corresponding plasmids were transferred into E. coli strain BW25113 (F’). The fresh

243

colonies were inoculated into 3 ml LB liquid medium and grown at 37ºC. 200 µL of overnight

244

cultures were transferred into 20 mL M9 liquid medium with 20 g/L glucose. When OD600

245

reached 0.6, the cultures were supplemented with 0.5 mM IPTG and transferred to a 30ºC shaker.


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The cultures were supplemented with the corresponding substrates at 300 mg/L per 3 h. Samples

247

were taken every few hours and the product concentrations were analyzed by HPLC.

248 249

De novo biosynthesis of salicyl alcohol and gentisyl alcohol

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Production colonies were grown in 3 ml LB medium (37ºC, 270 rpm) for overnight. 200 µL of

251

the pre-inoculum along with appropriate antibiotics were added into flasks containing the

252

modified M9 medium and grown for 3 hours (37ºC, 270 rpm). Then 0.5 mM IPTG was added to

253

the cultures and the cultures were transferred to a 30ºC shaker (270 rpm) till 72 hours. 1 mL

254

samples were collected every 12 hours. OD600 values were measured and the products and

255

intermediates concentrations were analyzed by HPLC.

256 257

Analytical procedures

258

Genesys 10S UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA) was used to

259

measure the optical density at 600 nm. The samples were centrifuged at 13,000 rpm for 20 min,

260

the supernatant was analyzed by HPLC (Dionex Ultimate 3000) equipped with a reverse phase

261

ZORBAX SB-C18 column and an Ultimate 3000 Photodiode Array Detector. Solvent A was

262

water with 0.1% trifluoroacetic acid, and solvent B was methanol. The column temperature was

263

set to 28 °C. The following gradient was used at a flow rate of 1 mL/min: 5–50% solvent B for

264

15 min, 50–5% solvent B for 1 min, and 5% solvent B for an additional 4 min. Quantification of

265

the products and intermediates were based on the peak areas at UV absorbance at 220 nm.



266

AUTHOR INFORMATION

267

Corresponding Authors

268

*E-mails: [email protected] (Q. Yuan), [email protected] (Y. Yan).

269 270

Author Contributions

271

1

272

manuscript. XS and JW performed the experiments. BKG and EMF chemically synthesized

273

gentisyl alcohol standard. QY and YY directed the research. XS, JW, QY and YY revised the

274

manuscript.

XS and JW contributed equally to this work. XS and JW conceived the study and wrote the

275 276

Notes

277

The authors declare no competing financial interest.

278 279

ACKNOWLEDGMENTS

280

This work was supported by National Natural Science Foundation of China (21636001,

281

21776008), the Program of Introducing Talents of Discipline to Universities (“111” project,

282

B13005), the Program for Changjiang Scholars and Innovative Research Team in Universities in

283

China (No. IRT13045). We would also like to thank the College of Engineering, The University

284

of Georgia, Athens.


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285 286


Table 1. List of strains and plasmids used in this study. Strain

Genotype

Reference

BW25113 (F’)

rrnBT14 ∆lacZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78 F‫׳‬

Yale CGSC

[traD36 proAB lacIqZ∆M15 Tn10(Tetr)] XL1-Blue

recA1 endA1gyrA96thi-1hsdR17supE44relA1lac

Stratagene

[F’ proAB lacIqZDM15Tn10 (TetR)] XJ01

BW25113 (F’) harboring pZE-car-sfp

This study

XJ02

BW25113 (F’) harboring pZE-car-sfp and pCS-adh6

This study

XJ03

BW25113 (F’) harboring pZE-salABCD

This study

XJ04

BW25113 (F’) harboring pZE-sdgC-pqsA

This study

XJ05

BW25113 (F’) harboring pZE-car-sfp-salABCD

This study

XJ06

BW25113 (F’) harboring pZE-car-sfp-salABCD and pCS-adh6

This study

XJ07

BW25113 (F’) harboring pCS-EP and pZE-car-sfp

This study

XJ08

BW25113 (F’) harboring pCS-EPA and pZE-car-sfp

This study

XJ09

BW25113 (F’) harboring pCS-EPA-APTA and pZE-car-sfp

This study

XJ10

BW25113 (F’) harboring pCS-EP and pZE-car-sfp-salABCD

This study

XJ11

BW25113 (F’) harboring pCS-EPA and pZE-car-sfp-salABCD

This study

XJ12

BW25113 (F’) harboring pCS-EPA-APTA and pZE-car-sfp-

This study

salABCD Plasmid

Description

Reference

pZE12- luc

pLlacO-1; luc; ColE1 ori; AmpR

5

pCS27

pLlacO-1; p15A ori; KanR

5

pZE-salABCD

pZE12-luc carrying salAB, salCD from Ralstonia eutropha

25



pZE-pqsA-sdgC

pZE12-luc carrying pqsA from Pseudomonas aeruginosa and sdgC

Page 16 of 26

25

from Streptomyces sp. Strain WA46 and pZE-EP

pZE12-luc carrying entC from E. coli and pchB from P.

7

fluorescens Migula pCS-APTA

pCS27 carrying aroL, ppsA, tktA, aroGfbr from E. coli

24

pZE-car-sfp

pZE12-luc carrying car from Mycobacterium marinum and sfp

This study

from Bacillus subtilis pCS-adh6

pCS27 carrying adh6 from Saccharomyces cerevisiae

This study

pZE-car-sfp-

pZE12-luc carrying car, sfp and PLlacO1-salABCD, two operons

This study

pCS-EP

pCS27 carrying entC and pchB

This study

pCS-EPA

pCS27 carrying entC, pchB and adh6

This study

pCS-EPA-

pCS27 carrying entC, pchB, adh6 and PLlacO1-APTA, two

This study

APTA

operons

salABCD

287


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288


Figure Legends

289 290

Fig. 1. Engineering artificial biosynthetic pathways for the production of salicyl alcohol and

291

gentisyl alcohol. PPP pathway: pentose phosphate pathway; E4P: erythrose-4-phosphate; PEP:

292

phosphoenolpyruvate; DAHP: 3-deoxy-D-heptulosonate-7-phosphate. The blue arrow indicated

293

heterologous pathways, solid lines indicate a single step, dotted lines indicate multiple steps. The

294

E. coli endogenous genes which were overexpressed in this study depicted in yellow. Gene ppsA

295

encodes

296

aroG/aroH/aroF encode 2-dehydro-3-deoxyphosphoheptonate aldolase; gene aroL encodes

297

shikimate kinase I; gene entC encodes isochorismate synthase; gene pchB encodes isochorismate

298

pyruvate lyase; gene car encodes carboxylic acid reductase; gene sfp encodes CAR maturation

299

factor phosphopantetheinyl transferase; gene adh6 encodes alcohol dehydrogenase; gene

300

salABCD encodes anthranilate 5-hydroxylase.

phosphoenolpyruvate

synthetase;

gene

tktA

encodes

transketolase;

genes

301 302

Fig. 2. Results of shake flask studies for feeding experiments. (A) Salicyl alcohol produced from

303

salicylic acid; (B) Gentisyl alcohol produced from 2,5-DHBA; (C) 2,5-DHBA produced from

304

salicylic acid; (D) Gentisyl alcohol produced from salicylic acid. The data were generated from

305

three independent experiments (n=3; s.d. represented by ±).

306 307

Fig. 3. De novo biosynthesis of salicyl alcohol. The data were generated from three independent

308

experiments (n=3; s.d. represented by ±).

309



310

Fig. 4. De novo biosynthesis of gentisyl alcohol. The data were generated from three

311

independent experiments (n=3; s.d. represented by ±).

312


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313

Supporting Information.

314

The production profiles of salicyl alcohol and 2,5-DHBA.

315

Figure S1

316

Figure S2

317



318

Reference

319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362

[1] Rice-Evans, C., Miller, N., and Paganga, G. (1997) Antioxidant properties of phenolic compounds, Trends Plant Sci. 2, 152-159. [2] Cicerale, S., Lucas, L., and Keast, R. (2012) Antimicrobial, antioxidant and antiinflammatory phenolic activities in extra virgin olive oil, Curr. Opin. Biotechnol. 23, 129-135. [3] Huang, W.-Y., Cai, Y.-Z., and Zhang, Y. (2009) Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention, Nutr. Cancer 62, 1-20. [4] Wang, J., Shen, X., Rey, J., Yuan, Q., and Yan, Y. (2017) Recent advances in microbial production of aromatic natural products and their derivatives, Appl. Microbiol. Biotechnol. , 1-15. [5] Wang, J., Shen, X., Yuan, Q., and Yan, Y. (2018) Microbial synthesis of pyrogallol using genetically engineered Escherichia coli, Metab. Eng. 45, 134-141. [6] Shen, X., Wang, J., Wang, J., Chen, Z., Yuan, Q., and Yan, Y. (2017) High-level De novo Biosynthesis of Arbutin in Engineered Escherichia coli, Metab. Eng. [7] Lin, Y., Sun, X., Yuan, Q., and Yan, Y. (2014) Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli, Metab. Eng. 23, 62-69. [8] Chen, Z., Shen, X., Wang, J., Wang, J., Yuan, Q., and Yan, Y. (2017) Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway, Biotechnol. Bioeng. [9] Lin, Y., and Yan, Y. (2012) Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex, Microb. Cell Fact. 11, 42. [10] Rodriguez, A., Kildegaard, K. R., Li, M., Borodina, I., and Nielsen, J. (2015) Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis, Metab. Eng. 31, 181-188. [11] Pugh, S., McKenna, R., Halloum, I., and Nielsen, D. R. (2015) Engineering Escherichia coli for renewable benzyl alcohol production, Metab. Eng. Commun. 2, 39-45. [12] Chen, Z., Shen, X., Wang, J., Wang, J., Zhang, R., Rey, J. F., Yuan, Q., and Yan, Y. (2017) Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl Alcohol in Escherichia coli, ACS Synth. Biol. 6, 1784-1792. [13] Harischfelder, A., and Wynne, H. (1920) Saligenin as a local anesthetic for the female urethra, JAMA 75, 1770. [14] Harischfelder, A., Lundholm, A., and Norrgard, H. (1920) The local anaesthetic actions of saligenin and other phenyl carbinols, J. Pharmacol. Exp. Ther. 15, 227-261. [15] Charles, O., and Tony, E. (1956) The American drug index, J. B Lippincott Co, Philadelphia. [16] Kumar, S., Singh, S. K., Calabrese, C., Maris, A., Melandri, S., and Das, A. (2014) Structure of saligenin: Microwave, UV and IR spectroscopy studies in a supersonic jet combined with quantum chemistry calculations, Phys. Chem. Chem. Phys 16, 1716317171. [17] Alfaro, C., Urios, A., González, M. C., Moya, P., and Blanco, M. (2003) Screening for metabolites from Penicillium novae-zeelandiae displaying radical-scavenging activity and oxidative mutagenicity: isolation of gentisyl alcohol, Mutat. Res. Genet. Toxicol. Environ. Mutagen. 539, 187-194. 20 ACS Paragon Plus Environment

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[18] Kim, H.-J., Kim, J.-H., Lee, C.-H., and Kwon, H.-J. (2006) Gentisyl alcohol, an antioxidant from microbial metabolite, induces angiogenesis in vitro, J. Microbiol. Biotechnol. 16, 475-479. [19] Kim, J., Kim, D., Kim, M., Kwon, H., Oh, T., and Lee, C. (2005) Gentisyl alcohol inhibits apoptosis by suppressing caspase activity induced by etoposide, J. Microbiol. Biotechnol. 15, 532-536. [20] Abbott Jr, L. D., Smith, J. D., and Reid, J. D. (1948) Antibacterial Activity of Gentisyl Alcohol and Homogentisic Acid, Proc. Soc. Exp. Biol. Med. 69, 201-202. [21] Gupta, S., Kaul, S., Singh, B., Vishwakarma, R. A., and Dhar, M. K. (2016) Production of Gentisyl Alcohol from Phoma herbarum Endophytic in Curcuma longa L. and Its Antagonistic Activity Towards Leaf Spot Pathogen Colletotrichum gloeosporioides, Appl. Biochem. Biotechnol. 180, 1093-1109. [22] Venkitasubramanian, P., Daniels, L., and Rosazza, J. (2006) Biocatalytic Reduction of Carboxylic Acids: Mechanism and Applications Biocatalysis in Pharmaceutical and Biotechnology Industries, ed Patel R (CRC, Boca Raton, FL). [23] Noda, S., Shirai, T., Mori, Y., Oyama, S., and Kondo, A. (2017) Engineering a synthetic pathway for maleate in Escherichia coli, Nat. Commun. 8, 1153. [24] Lin, Y., Shen, X., Yuan, Q., and Yan, Y. (2013) Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin, Nat. Commun. 4, 2603. [25] Sun, X., Lin, Y., Yuan, Q., and Yan, Y. (2014) Precursor-directed biosynthesis of 5hydroxytryptophan using metabolically engineered E. coli, ACS Synth. Biol. 4, 554-558. [26] Wang, J., Jain, R., Shen, X., Sun, X., Cheng, M., Liao, J. C., Yuan, Q., and Yan, Y. (2017) Rational engineering of diol dehydratase enables 1, 4-butanediol biosynthesis from xylose, Metab. Eng. 40, 148-156. [27] Dueber, J. E., Wu, G. C., Malmirchegini, G. R., Moon, T. S., Petzold, C. J., Ullal, A. V., Prather, K. L., and Keasling, J. D. (2009) Synthetic protein scaffolds provide modular control over metabolic flux, Nat. Biotechnol. 27, 753-759. [28] Avalos, J. L., Fink, G. R., and Stephanopoulos, G. (2013) Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols, Nat. Biotechnol. 31, 335-341.

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Establishment of Novel Biosynthetic Pathways for the Production of

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