Shunting Phenylacetic Acid Catabolism for Tropone Biosynthesis

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Shunting phenylacetic acid catabolism for tropone biosynthesis Yan Li, Mengyuan Wang, Qianjing Zhao, Xiaolin Shen, Jia Wang, Yajun Yan, Xinxiao Sun, and Qipeng Yuan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00013 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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ACS Synthetic Biology

A hybrid pathway was designed and de novo production of tropone was achieved in E. coli. 277x82mm (150 x 150 DPI)

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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

Shunting phenylacetic acid catabolism for tropone biosynthesis Yan Lia, Mengyuan Wanga, Qianjing Zhaoa, Xiaolin Shena, Jia Wanga, Yajun Yanb, Xinxiao Suna*, Qipeng Yuana* a

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, State Key Laboratory

of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China b

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

of Georgia, Athens, GA 30602, USA

*Corresponding authors: Xinxiao Sun [email protected] Telephone: +86-10-64431557 Address: 15# Beisanhuan East Road, Chaoyang District, Beijing 100029, China

Qipeng Yuan [email protected] Telephone: +86-10-64435710; fax: +86-10-64435710; Address: 15# Beisanhuan East Road, Chaoyang District, Beijing 100029, China


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Abstract Tropone is a seven-membered ring non-benzenoid aromatic compound. It is the core structure of tropolonoids, which have various biological activities. In this study, a hybrid tropone biosynthetic pathway was designed by connecting phenylacetic acid (PAA) degradation with its biosynthesis and reconstituted in Escherichia coli. To simplify pathway construction and optimization, the use of E. coli endogenous genes was maximized and only three exogenous genes were employed. The entire pathway was divided into four modules: the endogenous shikimate pathway module, the hybrid PAA biosynthetic module, the endogenous PAA catabolic module and the heterogenous tropone biosynthetic module. Efficiency of the PAA catabolic module was enhanced using PAA consumption rate as the indicator. Then, a single point mutation was introduced to inactivate the ALDH domain of PaaZ and the carbon flow was redirected towards tropone synthesis. Assembly of the full pathway led to de novo tropone production with the best titer of 65.2±1.4 mg/L in shake flask experiment. This study provides a potential alternative for sustainable production of tropone and its derivatives. Keywords: shikimate pathway, phenylacetic acid catabolism, tropone, tropolonoids, biosynthesis



Introduction The tropolonoid is a family of natural products with the seven-membered aromatic tropone ring as the core structure1. Tropolonoids have been isolated from bacteria, fungi, and plants and exhibit various bioactivities such as antimicrobial2, antitumor3, antiviral4 and anti-inflammatory5 activities. The representatives include tropolone, stipitatic acid, colchicine, hinokitiol, 3, 7-dihydroxytropolone (DHT) and tropodithietic acid (TDA). Owing to the broad biological activities, studies have long been conducted to elucidate their biosynthetic pathways. TDA, isolated from the marine roseobacters, is one of the first tropolonoids whose biosynthetic pathway and action mechanism have been characterized6-8. The carbon skeleton of TDA is derived from phenylacetic acid (PAA) catabolism (Fig.1). In this process, the coenzyme A (CoA) ligase PaaK activates PAA to produce phenylacetyl-CoA (PAA-CoA), which is subsequently converted into an epoxide 1 by the multicomponent oxygenase PaaABCDE. The isomerase PaaG converts epoxide 1 into a seven-membered cyclic ether 2. This ether ring is further opened by PaaZ, a bifunctional enzyme that contains two catalytic domains. The C-terminal enoyl-CoA hydratase (ECH) domain catalyzes the formation of the reactive aldehyde 3 while the N-terminal aldehyde dehydrogenase (ALDH) domain converts 3 into the corresponding carboxylic acid, which is further degraded into two units of acetyl-CoA and one succinyl-CoA via β-oxidation. If not oxidized immediately, 3 would be condensed intramolecularly to a stable cyclic derivative 4, which serves as the precursor for TDA synthesis. Deactivating ALDH domain of E. coli PaaZ by introducing a single mutation E256Q led to the conversion of 2 into 49. In P. inhibens, the gene cluster tdaA-F is responsible for TDA biosynthesis7. Compound 4 is oxidized by the acyl-CoA dehydrogenase TdaE to 5, which is further converted into 6


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by the dehydratase TdaC. In the following steps, TdaB and TdaF catalyze the dithiet moiety formation. Finally, TdaD catalyzes thioester hydrolysis to generate TDA. By thioester hydrolysis and decarboxylation, intermediate 6 can be converted into tropone, which was detected in headspace extracts of Phaeobacter inhibens10. Recently, the biosynthetic pathway of DHT in Streptomyces species was also proposed and tropone was shown to be a biosynthetic intermediate11. DHT biosynthesis involves the similar truncated PAA catabolic pathway till compound 3. However, the following steps involve a different combination of enzymes to produce tropone, indicating the diversity of tropolonoid biosynthesis. Considering their versatile functions, tropolonoids production using the native strains has been explored. For example, Silicibacter sp. TM1040 produced 15 mg/L of TDA in 120 h12. Streptomyces cyaneofuscatus Soc7 was able to produce 380 mg/L of DHT11. However, the limitations such as slow growth and lack of genetic manipulation tools hinder further improvement of tropolonoid production in the native producers. Therefore, reconstitution of the biosynthetic pathway in a genetic tractable host represents a promising alternative for efficient and sustainable production of tropolonoids. In recent years, a variety of valuable compounds such as artimisinic acid13, taxol precursors14, opioids15 and 4-hydroxycoumarin16 have been successfully synthesized using metabolic engineered microorganisms. Considering the central role of tropone in tropolonoid biosynthesis and its relatively simple structure, in this study we constructed a hybrid biosynthetic pathway and achieved its production from simple carbon sources (Fig.1). From PAA, the pathway involves 6 enzymatic steps (10 genes). Plasmid-based expression of all the pathway genes may cause heavy metabolic burden. Fortunately, E. coli contains native PAA catabolon in the choromosome17. To release this catabolon from native metabolic regulation, its



promoter was replaced with strong inducible ones. Assembly of the full pathway, which contains only three heterogenous genes, led to de novo production of tropone. This study represents as a key step toward biosynthesis of tropolonoids in E. coli. Results and Discussion Design a hybrid tropone biosynthetic pathway In some roseobacters, tropone is a metabolite derived from PAA catabolism10. To achieve de novo tropone biosynthesis, it is necessary to establish a PAA biosynthetic pathway and connect it with PAA catabolism. So far, PAA has not been reported to be a native metabolite of E. coli although it contains phenylacetaldehyde dehydrohenase FeaB. Phenylacetaldehyde can be derived from phenylpyruvate via the keto-acid pathway. This pathway has been used for biosynthesis of higher alcohols, tyrosol, and hydroxytyrosol via the sequential catalysis of keto-acid decarboxylase (KDC) and alcohol dehydrogenase18-20. Co-expression of KDC and FeaB would lead to PAA production. Accordingly, a tropone biosynthetic pathway was designed and divided into four modules (Fig.1). The shikimate pathway module provides phenylpyruvate for the PAA biosynthetic module. And the PAA catabolic and tropone biosynthetic modules act consecutively to convert PAA into tropone. Among the four modules, the PAA catabolic module is considered as the centerpiece. Therefore, our first effort was focused on optimization of this module. Enhancing E. coli native PAA catabolism by promoter replacement Tropone native producers such as P. inhibens contain PAA catabolic pathway and the upstream part till PaaZ is involved in tropone biosynthesis. After searching the genome sequence, we noticed that these genes are scattered in the genome of P. inhibens DSM 17395 (Fig.2). It would be time-consuming to achieve coordinated heterologous expression of these genes in E. coli. Furthermore, plasmid-based


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expression may lead to heavy cell burden. Fortunately, E. coli also contains PAA catabolic genes and these genes form a well-organized cluster. The gene cluster is located in the chromosome and consists of 12 structural genes (paaABCDEFGHIJK and paaZ). The genes paaABCDEFGHIJK are transcribed as one operon. Gene paaZ is adjacent to the operon but is transcribed as a monocistron in the opposite direction (Fig.2). As a result of long-term evolution, gene expression in an operon is expected to be coordinated. This would greatly facilitate the holistic regulation and the pathway efficiency would be improved by replacing the native promoter with stronger ones while pathway balance is still maintained. Therefore, we first evaluated PAA degradation efficiency of two E. coli strains. One is the commonly used BW25113 (BW) and the other is a phenylalanine overproducer ATCC31884 (ATCC). Both strains were able to grow on PAA and the latter showed better capability of PAA consumption (Fig. 3). In 18 h, BW consumed 833.8 ± 41.6 mg/L of PAA while ATCC consumed nearly 2 g/L of PAA. As the objective of this study is to achieve de novo tropone production, we further tested their growth on mixed carbon sources containing glucose, glycerol and PAA. As expected, the existence of glucose and glycerol significantly hindered PAA consumption probably due to the carbon catabolite repression effect (Fig. 4). Within 48 h, only 140.6±15.1 mg/L and 187.4±7.0 mg/L of PAA were consumed by BW and ATCC, respectively. To solve this problem, the transcriptional repressor gene paaX was knocked out. However, the effort led to negative effect on PAA consumption (Fig. 3). We then tried to replace the promoter of the paa operon with strong inducible promoters PlacO1, Ptac and PT7. It should be mentioned here that this manipulation simultaneously disrupted the adjacent gene paaZ. Therefore, to supplement paaZ, a low-copy plasmid pSA-paaZ was constructed and introduced into



the promoter-replaced strains. In addition, for the function of PT7, a copy of T7 RNA polymerase gene under the control of PlacO1 was integrated into the chromosome. As expected, the resultant strains showed improved PAA consumption. When grown on PAA, all six strains completely consumed 2 g/L of PAA in 18 h (Fig. 3). And the improved capability of PAA consumption also led to higher cell densities than the wide type strains. Among the six strains, those with Ptac and PT7 (BWZtac, BWZT7, ATCCZtac and ATCCZT7) performed better than those with PlacO1 (BWZlac and ATCCZlac). Therefore, the former four strains were used for further studies. When grown on the mixed carbon sources, BWZT7 and ATCCZT7 performed better than strains BWZtac and ATCCZtac (Fig. 4). Within 48 h, 649.3±72.1 mg/L and 1033.6 ± 48.6 mg/L of PAA were consumed by BWZT7 and ATCCZT7, respectively. PaaK catalyzes the committed step in PAA catabolism and gene paaK is located at the far end away from the promoter. To test whether expressing paaK could further improve the consumption efficiency, plasmid pSA-paaKZ was constructed. Strains containing this plasmid (BWKZtac, BWKZT7, ATCCKZtac and ATCCKZT7) showed decreased PAA consumption (Fig. 4). Therefore, strain BWZT7 and ATCCZT7 were used in the following studies. Tropone production from PAA To achieve tropone production from PAA, two exogenous enzymes TdaE and TdaC are required (Fig. 1). The encoding genes from P. inhibens DSM 17395 were codon-optimized, synthesized and cloned into plasmid pZE12-luc, generating plasmid pZE-tdaEC. Strains BWZT7 and ATCCZT7 were transformed with pZE-tdaEC, generating strain BW1 and ATCC1, respectively. The results of feeding experiments showed that both strains were able to consume PAA but produce no detectable tropone, which indicated that with the intact PAA catabolic pathway PAA was mainly


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used for cell growth (Fig. 5). To shunt PAA for tropone production, the ALDH domain of PaaZ was mutated by introducing the site mutation E256Q. E. coli strains BW2 and ATCC2 containing pZE-tdaEC and pSA-paaZE256Q were able to produce 18.0±0.4 mg/L and 17.2±0.6 mg/L of tropone, respectively. To check if PaaK and PaaG are rate-limiting, plasmid pSA-paaKZE256QG was constructed. Strain BW3 and ATCC3 containing this plasmid produced decreased amount of tropone (16.5 ± 2.0 mg/L and 13.7±0.4 mg/L, respectively) (Fig. 5). Biosynthesis of PAA After achieving tropone production from PAA, we continued to achieve PAA biosynthesis. E. coli natively contains phenylpyruvate biosynthetic pathway and PAA degradation pathway. To fill the gap between phenylpyruvate and PAA, the well-characterized keto-acid pathway was used. In this pathway, phenylpyruvate was converted to phenylacetaldehyde by the phenylpyruvate decarboxylase Aro10 from S. cerevisia. Phenylacetaldehyde was further converted into PAA by FeaB. Strains BW and ATCC were transformed with pCS-Aro10-feaB, generating strains BW4 and ATCC4, respectively. Shake flask experiments were performed to test their capacity for PAA production. In 48 h strain BW4 produced 249.3 ±5.6 mg/L of PAA while strain ATCC4 produced 1477.1 ± 21.0 mg/L of PAA. To enhance the carbon flux through the shikimate pathway, four key genes (pheA, ppsA, tktA and aroGfbr) was co-expressed on plasmid pCS-Aro10-feaB-PPTA. In 48 h strain BW5 and ATCC5 containing this plasmid produced 472.1±20.4 mg/L and 1528.7±28.1 mg/L of PAA, respectively (Fig. 6). These results demonstrated that ATCC is a better host than BW for PAA production. Assembly of the full pathway for de novo production of tropone To achieve de novo production of tropone, E. coli strain BW2 and BW3 were



transformed with plasmid pCS-Aro10-feaB, generating strain BW6 and BW7, respectively. In 48 h strain BW6 produced 39.8±0.9 mg/L of tropone with 134.8±6.2 mg/L PAA accumulated. Strain BW7 containing pSA-paaKZE256QG instead of pSA-paaZE256Q produced 65.2±1.4 mg/L of tropone with 149.5±17.9 mg/L of PAA accumulated (Fig. 7). As significant amount of PAA was already accumulated, we reasoned that it is not necessary to further enhance the upstream shikimate pathway. Although ATCC is a better host for PAA production, when it was used for de novo tropone production the corresponding strains produced no detectable product for yet unknown reasons. In this study, we achieved heterologous biosynthesis of tropone in E. coli, which is an important step toward heterogenous tropolonoid biosynthesis. Tropone biosynthesis is via the degradation of PAA. In nature, aromatic compounds are degraded via several common intermediates such as catechol and protocatechuic acid21. Artificial pathways have been constructed for bioproduction of muconic acid by connecting the degrading pathways with the corresponding biosynthetic pathways22-25. This work provided another successful example of this strategy. Materials and methods Media and strains Luria-Bertani (LB) medium was used for plasmid propagation and seed culture. The modified M9 medium was used for feeding experiments and de novo production of tropone and PAA. LB medium contains 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl. The modified M9 medium contains 10 g/L glycerol, 2.5 g/L glucose, 6 g/L Na2HPO4, 0.5 g/L NaCl, 3 g/L KH2PO4, 1 g/L NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 2 g/L yeast extract and 2 g/L MOPS. When needed, ampicillin, kanamycin and chloramphenicol were supplemented to the final concentrations of 100 mg/L, 50 mg/L


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and 34 mg/L, respectively. E. coli DH5α was used for plasmid propagation and extraction. The derivative strains of E. coli BW25113 and ATCC31884 were used for feeding experiments and de novo production. Plasmid construction Plasmids pZE12-luc (high copy), pCS27 (medium copy), and pSA74 (low copy), are used for plasmid construction and pathway assembly26 . Plasmid pZE-tdaEC was constructed by inserting tdaE and tdaC into pZE12-luc using EcoRI, KpnI and XbaI. Plasmid pSA-paaZ was constructed by inserting paaZ into KpnI/BamHI sites of pSA74. Gene paaZE256Q was obtained by site directed mutagenesis. Plasmid pSA-paaZE256Q was constructed as pSA-paaZ. Plasmid pSA-paaKZ was constructed by inserting paaK and paaZ into pSA74 using KpnI, SalI and BamHI. Plasmid pSA-paaKZG was constructed by inserting paaK, paaZ and paaG into KpnI/BamHI sites of pSA74. Plasmid pSA-paaKZE256QG was constructed as plasmid pSA-paaKZG. Plasmid pCS-Aro10-feaB was constructed by inserting Aro10 and feaB into pCS27 using KpnI, SphI and BamHI. Plasmid pCS-PPTA was constructed in our previous study and was used as the template to amplify the expressing cassette PlacO1-PPTA27. Plasmid pCS-Aro10-feaB-PPTA was constructed by inserting the expression cassette PlacO1-PPTA into SpeI/SacI sites of pSA-Aro10-feaB. Plasmid pZE-tdaEC-paaZE256Q was constructed by inserting the expressing cassette PlacO1-paaZE256Q into SpeI/SacI sites of pZE-tdaEC. The details of the strains and plasmids used in this study are summarized in Table 1. Feeding experiments Feeding experiments were carried out to examine PAA consumption capability of different strains and tropone production from PAA. For PAA consumption experiments, E. coli strains were cultivated in M9 medium containing 2 g/L PAA or



M9 medium containing 2.5 g/L glucose, 10 g/L glycerol and 2 g/L PAA. For tropone production from PAA, the latter was used. Overnight cultures were inoculated into 50 mL of M9 medium containing appropriate antibiotics. The cultures were induced with 0.1 mM IPTG at 0 h and were left to grow at 37 °C. Samples were taken at several time points. Cell growth was monitored by measuring the optical density at 600 nm (OD600) and the products were analyzed by HPLC. De novo production of PAA and tropone Overnight cultures of the producing strains were inoculated at 1% into the M9 medium containing appropriated antibiotics and cultivated at 37 °C and 200 rpm. IPTG was added at 0 h to a final concentration of 0.1 mM. Samples were taken every 12 h. OD600 values were measured and the concentrations of the products and intermediates were analyzed by high performance liquid chromotography (HPLC). HPLC analysis The standards of PAA and tropone were purchased from Aladdin Chemical Industry. Both the standards and samples were analyzed and quantified by HPLC (HITACHI) equipped with a reverse phase Diamonsil C18 column and UV-VIS detector. Solvent A was methanol, and solvent B was water with 0.1 % formic acid. The column temperature was set to 30 °C. The following was used at a flow rate of 1 ml/min: 25 % solvent A for 28 min. Quantification of PAA and tropone was based on the peak areas at absorbance of specific wavelengths (220 nm for PAA and 231 nm for tropone). Tropone was extracted with ethyl acetate and analyzed by ESI-MS. The molecular weight was accordance with the calculated value (Figure S1). Author information Corresponding authors Email: [email protected]. Phone: +86-10-64431557


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Email:[email protected]. Phone: +86-10-64437610 Author Contributions XS and QY conceived the study and wrote the manuscript. YL, MW, QZ, XS and JW performed the experiments. XS, QY and YY directed the research and revised the manuscript. Notes The authors declare no competing financial interest.

Acknowledgements The authors would like to acknowledge financial support of the National Natural Science Foundation of China (21606012, 21636001 and 21776008) and the Fundamental Research Funds for the Central Universities (buctrc201613).

Supporting Information. Primers used in this study; ESI-MS results of tropone. References 1.

Bentley, R. (2008) A fresh look at natural tropolonoids, Nat. Prod. Rep. 25, 118-138.

2.

Nakano, K., Chigira, T., Miyafusa, T., Nagatoishi, S., Caaveiro, J. M., and Tsumoto, K. (2015) Discovery and characterization of natural tropolones as inhibitors of the antibacterial target CapF from Staphylococcus aureus, Sci. Rep. 5, 15337.

3.

Li, J., Falcone, E. R., Holstein, S. A., Anderson, A. C., Wright, D. L., and Wiemer, A. J. (2016) Novel alpha-substituted tropolones promote potent and selective caspase-dependent leukemia cell apoptosis, Pharmacol. Res. 113, 438-448.

4.

Lu, G., Lomonosova, E., Cheng, X., Moran, E. A., Meyers, M. J., Le Grice, S. F., Thomas, C. J., Jiang, J. K., Meck, C., Hirsch, D. R., D'Erasmo, M. P., Suyabatmaz, D. M., Murelli, R. P., and Tavis, J. E. (2015) Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity, Antimicrob. Agents Chemother. 59, 1070-1079.

5.

Ben-Chetrit, E., Bergmann, S., and Sood, R. (2006) Mechanism of the anti-inflammatory effect of colchicine in rheumatic diseases: a possible new outlook through microarray analysis, Rheumatology 45, 274-282.

6.

Berger, M., Brock, N. L., Liesegang, H., Dogs, M., Preuth, I., Simon, M., Dickschat, J. S., and Brinkhoff, T. (2012) Genetic analysis of the upper phenylacetate catabolic pathway in the



production of tropodithietic acid by Phaeobacter gallaeciensis, Appl. Environ. Microbiol. 78, 3539-3551. 7.

Brock, N. L., Nikolay, A., and Dickschat, J. S. (2014) Biosynthesis of the antibiotic tropodithietic acid by the marine bacterium Phaeobacter inhibens, Chem. Commun. 50, 5487-5489.

8.

Wilson, M. Z., Wang, R., Gitai, Z., and Seyedsayamdost, M. R. (2016) Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink, Proc. Natl. Acad. Sci. U. S. A. 113, 1630-1635.

9.

Teufel, R., Gantert, C., Voss, M., Eisenreich, W., Haehnel, W., and Fuchs, G. (2011) Studies on the mechanism of ring hydrolysis in phenylacetate degradation: a metabolic branching point, J. Biol. Chem. 286, 11021-11034.

10.

Thiel, V., Brinkhoff, T., Dickschat, J. S., Wickel, S., Grunenberg, J., Wagner-Döbler, I., Simon, M., and Schulz, S. (2010) Identification and biosynthesis of tropone derivatives and sulfur volatiles produced by bacteria of the marine Roseobacter clade, Org. Biomol. Chem. 8, 234-246.

11.

Chen, X., Xu, M., Lu, J., Xu, J., Wang, Y., Lin, S., Deng, Z., and Tao, M. (2018) Biosynthesis of tropolones in Streptomyces spp: Interweaving biosynthesis and degradation of phenylacetic acid and hydroxylations on tropone ring, Appl. Environ. Microbiol. 84:e00349-18

12.

Geng, H., and Belas, R. (2010) Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer, J. Bacteriol. 192, 4377-4387.

13.

Paddon, C. J., Westfall, P. J., Pitera, D. J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M. D., Tai, A., Main, A., Eng, D., Polichuk, D. R., Teoh, K. H., Reed, D. W., Treynor, T., Lenihan, J., Fleck, M., Bajad, S., Dang, G., Dengrove, D., Diola, D., Dorin, G., Ellens, K. W., Fickes, S., Galazzo, J., Gaucher, S. P., Geistlinger, T., Henry, R., Hepp, M., Horning, T., Iqbal, T., Jiang, H., Kizer, L., Lieu, B., Melis, D., Moss, N., Regentin, R., Secrest, S., Tsuruta, H., Vazquez, R., Westblade, L. F., Xu, L., Yu, M., Zhang, Y., Zhao, L., Lievense, J., Covello, P. S., Keasling, J. D., Reiling, K. K., Renninger, N. S., and Newman, J. D. (2013) High-level semi-synthetic production of the potent antimalarial artemisinin, Nature 496, 528-532.

14.

Ajikumar, P. K., Xiao, W.-H., Tyo, K. E. J., Wang, Y., Simeon, F., Leonard, E., Mucha, O., Phon, T. H., Pfeifer, B., and Stephanopoulos, G. (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli, Science 330, 70-74.

15.

Galanie, S., Thodey, K., Trenchard, I. J., Interrante, M. F., and Smolke, C. D. (2015) Complete biosynthesis of opioids in yeast, Science 349, 1095-1100.

16.

Lin, Y., Shen, X., Yuan, Q., and Yan, Y. (2013) Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin, Nat. Commun. 4, 2603.

17.

Teufel, R., Mascaraque, V., Ismail, W., Voss, M., Perera, J., Eisenreich, W., Haehnel, W., and Fuchs, G. (2010) Bacterial phenylalanine and phenylacetate catabolic pathway revealed, Proc. Natl. Acad. Sci. U. S. A. 107, 14390-14395.

18.

Atsumi, S., Hanai, T., and Liao, J. C. (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels, Nature 451, 86-89.

19.

Bai, Y., Bi, H., Zhuang, Y., Liu, C., Cai, T., Liu, X., Zhang, X., Liu, T., and Ma, Y. (2014) Production of salidroside in metabolically engineered Escherichia coli, Sci. Rep. 4, 6640.

20.

Li, X., Chen, Z., Wu, Y., Yan, Y., Sun, X., and Yuan, Q. (2018) Establishing an artificial pathway for efficient biosynthesis of hydroxytyrosol, ACS Synth. Biol. 7, 647-654.


Page 14 of 25

Page 15 of 25 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


21.

Díaz, E., Jiménez, J. I., and Nogales, J. (2013) Aerobic degradation of aromatic compounds, Curr. Opin. Biotechnol. 24, 431-442.

22.

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.

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.

Sun, X., Lin, Y., Huang, Q., Yuan, Q., and Yan, Y. (2013) A novel muconic acid biosynthesis approach by shunting tryptophan biosynthesis via anthranilate, Appl. Environ. Microbiol. 79, 4024-4030.

25.

Sun, X., Lin, Y., Yuan, Q., and Yan, Y. (2014) Biological production of muconic acid via a prokaryotic 2,3-dihydroxybenzoic acid decarboxylase, ChemSusChem 7, 2478-2481.

26.

Chen, Z., Sun, X., Li, Y., Yan, Y., and Yuan, Q. (2017) Metabolic engineering of Escherichia coli for microbial synthesis of monolignols, Metab. Eng. 39, 102-109.

27.

Sun, J., Lin, Y., Shen, X., Jain, R., Sun, X., Yuan, Q., and Yan, Y. (2016) Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase, Metab. Eng. 35, 75-82.



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Table 1 Strains and plamids used in this study. Strains

Description

Source

DH5α

F-ψ80dZlac△ (ZlacYA-argF) U169 endA1 recA1 hsdR17 (rk-, mk+)

TransGen

BW25113

supE44λ-thi-1 gyrA96hsdR514 relA1 phoA rrnBT14 ΔZlacWJ16 ΔaraBADAH33 ΔrhaBADLD78

CGSC

ATCC31884

aroH367, tyrR366, tna-2, lacY5,

aroF394fbr,

malT384,

pheA101fbr,

aroG397fbr

ATCC

BWΔX

pheO352, BW25113 ΔpaaX

This study

ATCCΔX

ATCC31884ΔpaaX

This study

BWZlac

BW25113 PlacO1::PpaaA-K, pSA-paaZ

This study

BWZtac

BW25113 Ptac::PpaaA-K, pSA-paaZ

This study

BWT7

BW25113 PlacO1-T7RNAPol, PT7::PpaaA-K

This study

BWZT7

BW25113 PlacO1-T7RNAPol, PT7::PpaaA-K, pSA-paaZ

This study

BWKZtac

BW25113 Ptac::PpaaA-K, pSA-paaKZ,

This study

BWKZT7

BW25113 PlacO1-T7RNAPol, PT7::PpaaA-K, pSA-paaKZ

This study

ATCCZlac

ATCC31884 PlacO1::PpaaA-K, pSA-paaZ

This study

ATCCZtac

ATCC31884 Ptac::PpaaA-K, pSA-paaZ,

This study

ATCCZT7

ATCC31884 PlacO1-T7RNAPol, PT7::PpaaA-K, pSA-paaZ

This study

ATCCKZtac

ATCC31884 Ptac::PpaaA-K, pSA-paaKZ

This study

ATCCKZT7

ATCC31884 PT7::PpaaA-K, PlacO1-T7RNAPol, pSA-paaKZ

This study

BW1

BWT7, pSA-PaaZ and pZE-TdaEC

This study

BW2

BWT7, pSA-PaaZE256Q and pZE-TdaEC

This study

pSA-PaaKZE256QG

BW3

BWT7,

and pZE-TdaEC

ATCC1

ATCCT7, pSA-PaaZ and pZE-TdaEC

This study

ATCC2

ATCCT7, pSA-PaaZE256Q and pZE-TdaEC

This study

ATCC3

ATCCT7, pSA-PaaKZE256QG and pZE-TdaEC

This study

BW4

BW25113, pCS-Aro10-feaB

This study

BW5

BW25113, pCS-Aro10-feaB-PPTA

This study

ATCC4

ATCC31884, pCS-Aro10-feaB

This study

ATCC5

ATCC31884, pCS-Aro10-feaB-PPTA

This study

BWT7, pCS-Aro10-feaB,

pSA-PaaZE256Q

BW7

BWT7, pCS-Aro10-feaB,

pSA-PaaKZE256QG

Plasmids

Description

pZE12-luc

PLlacO1, colE ori, luc, Ampr

BW6

This study

and pZE-TdaEC and pZE-TdaEC

This study This study Source

Kanr

Ref. 26

pCS27

PLlacO1, P15A ori,

Ref. 26

pSA74

PLlacO1, pSC101 ori, Cmr

Ref. 26

pSA-paaZ

pSA74,

paaZ from E. coli

This study

pSA-paaKZ

pSA74,

paaK and paaZ from E. coli

This study

pSA-paaZE256Q

pSA74,

paaZE256Q

This study

pSA-paaKZE256QG

pSA74,

paaK, paaZE256Q and paaG from E. coli

pZE-tdaEC

pZE12-luc, tdaE and tdaC from P. inhibens DSM17395

This study

pZE-tdaEC-PaaZE256Q

pZE12-luc, tdaE and tdaC from P. inhibens DSM17395

This study

and

paaZE256Q

and

from E. coli

paaG from E. coli


This study

Page 17 of 25 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


pCS-Aro10-feaB

pCS27, Aro10 from S. cerevisiae and feaB from E. coli

This study

pCS-Aro10-feaB-PPTA

pCS27, Aro10 from S. cerevisiae, feaB from E. coli,

This study

pheA, ppsA, tktA and aroG from E. coli, two operons pCS-Aro10-feaB-paaZE

pCS27, Aro10 from S. cerevisiae, feaB from E. coli,

256Q

and paaZE256Q from E. coli, , two operons


This study


Figure Legends Fig. 1 The artificial pathway for tropone biosynthesis. Blue-color indicates E. coli native genes; red-color indicates foreign genes.

Fig. 2 Location of genes involved in PAA catabolism in P. inhibens DSM17395 and E. coli MG1655. Fig. 3 PAA consumption by different strains. The strains were cultivated in M9 medium with 2 g/L of PAA as the sole carbon source. A, E. coli BW25113 derivative strains; B, E. coli ATCC31884 derivative strains. Genetic information of the strains was listed in Table 1. Data points are reported as mean ± s.d. from three independent experiments. Fig. 4 PAA consumption by different strains. The strains were cultivated in M9 medium with 2 g/L PAA, 2.5 g/L glucose and 10 g/L glycerol as the carbon sources. A, E. coli BW25113 derivative strains; B, E. coli ATCC31884 derivative strains. Genetic information of the strains was listed in Table 1. Data points are reported as mean ± s.d. from three independent experiments. Fig. 5 Tropone production from PAA. The strains were cultivated in M9 medium with 2 g/L PAA, 2.5 g/L glucose and 10 g/L glycerol as the carbon sources. Genetic information of the strains was listed in Table 1. Data points are reported as mean ± s.d. from three independent experiments. Fig. 6 De novo production of PAA. Genetic information of the strains was listed in Table 1. Data points are reported as mean ± s.d. from three independent experiments.

Fig. 7 De novo production of tropone. Genetic information of the strains was listed in Table 1. Data points are reported as mean ± s.d. from three independent experiments.


Page 18 of 25

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Fig. 1 203x235mm (300 x 300 DPI)



Fig. 2


Page 20 of 25

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Fig. 3



Fig. 4


Page 22 of 25

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Fig. 5



Fig. 6


Page 24 of 25

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Fig. 7


Shunting Phenylacetic Acid Catabolism for Tropone Biosynthesis

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