Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast

Abstract

Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-l-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l−1) and ferulic acid (3.8 ± 0.3 g l−1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Biosynthesis of CaA and FA requires numerous cofactors.
Fig. 2: Construction of CaA/FA biosynthetic pathways in yeast.
Fig. 3: Engineering NADPH regeneration for CaA production by pulling nonoxidizing steps of the PPP during glucose-limited conditions.
Fig. 4: Manipulation of FADH2 biosynthesis and relocation for enhancing CaA production.
Fig. 5: Engineering SAM biosynthesis and recycling for FA production.

Data availability

The main data supporting the findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper. Extra data are available from the corresponding author upon request.

References

  1. Paddon, C. J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).

    CAS  PubMed  Article  Google Scholar 

  2. Luo, X. Z. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123–126 (2019).

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Chen, R. B., Yang, S., Zhang, L. & Zhou, Y. J. J. Advanced strategies for production of natural products in yeast. iScience 23, 100879 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Chen, X. L., Li, S. B. & Liu, L. M. Engineering redox balance through cofactor systems. Trends Biotechnol. 32, 337–343 (2014).

    CAS  PubMed  Article  Google Scholar 

  6. Thomas, D. & Surdin-Kerjan, Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol. Biol. Rev. 61, 503–532 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Abbas, C. A. & Sibirny, A. A. Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers. Microbiol Mol. Biol. R. 75, 321 (2011).

    CAS  Article  Google Scholar 

  8. Sporty, J. L. et al. Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae. J. Sep. Sci. 31, 3202–3211 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Pallotta, M. L. et al. Saccharomyces cerevisiae mitochondria can synthesise FMN and FAD from externally added riboflavin and export them to the extramitochondrial phase. FEBS Lett. 428, 245–249 (1998).

    CAS  PubMed  Article  Google Scholar 

  10. Silva, H. & Lopes, N. M. F. Cardiovascular effects of caffeic acid and its derivatives: a comprehensive review. Front Physiol. 11, 595516 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  11. Vanholme, R. et al. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341, 1103–1106 (2013).

    CAS  PubMed  Article  Google Scholar 

  12. Liu, L. Q. et al. Engineering the biosynthesis of caffeic acid in Saccharomyces cerevisiae with heterologous enzyme combinations. Eng.-Prc. 5, 287–295 (2019).

    CAS  Google Scholar 

  13. Phillips, I. R. & Shephard, E. A. Flavin-containing monooxygenases: mutations, disease and drug response. Trends Pharmacol. Sci. 29, 294–301 (2008).

    CAS  PubMed  Article  Google Scholar 

  14. Louie, T. M., Xie, X. S. & Xun, L. Y. Coordinated production and utilization of FADH(2) by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase. Biochem. 42, 7509–7517 (2003).

    CAS  Article  Google Scholar 

  15. Yang, J. Z. et al. Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis. Metab. Eng. 59, 44–52 (2020).

    CAS  PubMed  Article  Google Scholar 

  16. Rodriguez, A., Kildegaard, K. R., Li, M. J., Borodina, I. & Nielsen, J. 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 (2015).

    CAS  PubMed  Article  Google Scholar 

  17. Wang, J. P. et al. Complete proteomic-based enzyme reaction and inhibition kinetics reveal how monolignol biosynthetic enzyme families affect metabolic flux and lignin in Populus trichocarpa. Plant Cell 26, 894–914 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  19. Liu, Q. et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals. Nat. Commun. 10, 4976 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Yu, T. et al. Reprogramming yeast metabolism from alcoholic fermentation to lipogenesis. Cell 174, 1549–1558 (2018).

    CAS  PubMed  Article  Google Scholar 

  21. Frick, O. & Wittmann, C. Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis. Micro. Cell Fact. 4, 30 (2005).

    Article  CAS  Google Scholar 

  22. Campos-Bermudez, V. A., Bologna, F. P., Andreo, C. S. & Drincovich, M. F. Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation. FEBS J. 277, 1957–1966 (2010).

    CAS  PubMed  Article  Google Scholar 

  23. Bergman, A., Siewers, V., Nielsen, J. & Chen, Y. Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae. Amb. Express 6, 115 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. Mironov, V. N. et al. Functional organization of the riboflavin biosynthesis operon from Bacillus subtilis SHgw. Mol. Gen. Genet. 242, 201–208 (1994).

    CAS  PubMed  Article  Google Scholar 

  25. Zakal’skii, A. E. et al. Cloning of the RIB1 gene coding for the enzyme of the first stage of flavinogenesis in the yeast Pichia guilliermondi, GTP cyclohydrolase, in Escherichia coli cells. Genetika 26, 614–620 (1990).

    PubMed  Google Scholar 

  26. Mack, M., van Loon, A. P. & Hohmann, H. P. Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC. J. Bacteriol. 180, 950–955 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Reihl, P. & Stolz, J. The monocarboxylate transporter homolog Mch5p catalyzes riboflavin (vitamin B2) uptake in Saccharomyces cerevisiae. J. Biol. Chem. 280, 39809–39817 (2005).

    CAS  PubMed  Article  Google Scholar 

  28. Fischer, M. & Bacher, A. Biosynthesis of flavocoenzymes. Nat. Prod. Rep. 22, 324–350 (2005).

    CAS  PubMed  Article  Google Scholar 

  29. Vogl, C. et al. Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum. J. Bacteriol. 189, 7367–7375 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Chen, H., Wang, Z., Cai, H. & Zhou, C. Progress in the microbial production of S-adenosyl-l-methionine. World J. Microbiol. Biotechnol. 32, 153 (2016).

    CAS  PubMed  Article  Google Scholar 

  31. Thomas, D. & Surdin-Kerjan, Y. SAM1, the structural gene for one of the S-adenosylmethionine synthetases in Saccharomyces cerevisiae. J. Biol. Chem. 262, 16704–16709 (1987).

    CAS  PubMed  Article  Google Scholar 

  32. Suliman, H. S., Sawyer, G. M., Appling, D. R. & Robertus, J. D. Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae. Arch. Biochem. Biophys. 441, 56–63 (2005).

    CAS  PubMed  Article  Google Scholar 

  33. Murphy, J. T. & Spence, K. D. Transport of S-adenosylmethionine in Saccharomyces cerevisiae. J. Bacteriol. 109, 499–504 (1972).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Gaynor, P. M. & Carman, G. M. Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties. Biochim. Biophys. Acta 1045, 156–163 (1990).

    CAS  PubMed  Article  Google Scholar 

  35. Hoffman, D. R., Haning, J. A. & Cornatzer, W. E. Microsomal phosphatidylethanolamine methyltransferase: inhibition by S-adenosylhomocysteine. Lipids 16, 561–567 (1981).

    CAS  PubMed  Article  Google Scholar 

  36. Tehlivets, O., Hasslacher, M. & Kohlwein, S. D. S-adenosyl-l-homocysteine hydrolase in yeast: key enzyme of methylation metabolism and coordinated regulation with phospholipid synthesis. FEBS Lett. 577, 501–506 (2004).

    CAS  PubMed  Article  Google Scholar 

  37. Hoffman, D. R., Marion, D. W., Cornatzer, W. E. & Duerre, J. A. S-Adenosylmethionine and S-adenosylhomocystein metabolism in isolated rat liver. Effects of L-methionine, L-homocystein, and adenosine. J. Biol. Chem. 255, 10822–10827 (1980).

    CAS  PubMed  Article  Google Scholar 

  38. Lecoq, K., Belloc, I., Desgranges, C. & Daignan-Fornier, B. Role of adenosine kinase in Saccharomyces cerevisiae: identification of the ADO I gene and study of the mutant phenotypes. Yeast 18, 335–342 (2001).

    CAS  PubMed  Article  Google Scholar 

  39. Gerber, A., Grosjean, H., Melcher, T. & Keller, W. Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2. EMBO J. 17, 4780–4789 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Xavier, K. B. & Bassler, B. L. LuxS quorum sensing: more than just a numbers game. Curr. Opin. Microbiol. 6, 191–197 (2003).

    CAS  PubMed  Article  Google Scholar 

  41. Kunjapur, A. M., Hyun, J. C. & Prather, K. L. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway. Micro. Cell Fact. 15, 61 (2016).

    Article  CAS  Google Scholar 

  42. Perl, M., Kearney, E. B. & Singer, T. P. Transport of riboflavin into yeast cells. J. Biol. Chem. 251, 3221–3228 (1976).

    CAS  PubMed  Article  Google Scholar 

  43. Wang, M., Chen, B., Fang, Y. & Tan, T. Cofactor engineering for more efficient production of chemicals and biofuels. Biotechnol. Adv. 35, 1032–1039 (2017).

    CAS  PubMed  Article  Google Scholar 

  44. Minard, K. I. & McAlister-Henn, L. Sources of NADPH in yeast vary with carbon source. J. Biol. Chem. 280, 39890–39896 (2005).

    CAS  PubMed  Article  Google Scholar 

  45. Li, S., Li, Y. & Smolke, C. D. Strategies for microbial synthesis of high-value phytochemicals. Nat. Chem. 10, 395–404 (2018).

    CAS  PubMed  Article  Google Scholar 

  46. Cao, X., Yang, S., Cao, C. & Zhou, Y. J. Harnessing sub-organelle metabolism for biosynthesis of isoprenoids in yeast. Synth. Syst. Biotechnol. 5, 179–186 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  47. Hammer, S. K. & Avalos, J. L. Harnessing yeast organelles for metabolic engineering. Nat. Chem. Biol. 13, 823–832 (2017).

    CAS  PubMed  Article  Google Scholar 

  48. Huang, Y. et al. Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation. J. Appl. Microbiol. 112, 683–694 (2012).

    CAS  PubMed  Article  Google Scholar 

  49. Heo, K. T., Kang, S. Y. & Hong, Y. S. De novo biosynthesis of pterostilbene in an Escherichia coli strain using a new resveratrol O-methyltransferase from Arabidopsis. Micro. Cell Fact. 16, 30 (2017).

    Article  CAS  Google Scholar 

  50. Kuras, L., Cherest, H., Surdin-Kerjan, Y. & Thomas, D. A heteromeric complex containing the centromere binding factor 1 and two basic leucine zipper factors, Met4 and Met28, mediates the transcription activation of yeast sulfur metabolism. EMBO J. 15, 2519–2529 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Kuras, L., Barbey, R. & Thomas, D. Assembly of a bZIP-bHLH transcription activation complex: formation of the yeast Cbf1-Met4-Met28 complex is regulated through Met28 stimulation of Cbf1 DNA binding. EMBO J. 16, 2441–2451 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Li, Y. et al. De novo biosynthesis of caffeic acid from glucose by engineered Saccharomyces cerevisiae. ACS Synth. Biol. 9, 756–765 (2020).

    CAS  PubMed  Article  Google Scholar 

  53. Zhou, P. et al. Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid. Appl. Microbiol. Biotechnol. 105, 5809–5819 (2021).

    CAS  PubMed  Article  Google Scholar 

  54. Lin, Y. & Yan, Y. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex. Micro. Cell Fact. 11, 42 (2012).

    Article  CAS  Google Scholar 

  55. Huang, Q., Lin, Y. & Yan, Y. Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain. Biotechnol. Bioeng. 110, 3188–3196 (2013).

    CAS  PubMed  Article  Google Scholar 

  56. Yang, S., Cao, X., Yu, W., Li, S. & Zhou, Y. J. Efficient targeted mutation of genomic essential genes in yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 104, 3037–3047 (2020).

    CAS  PubMed  Article  Google Scholar 

  57. Zhou, Y. J. et al. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J. Am. Chem. Soc. 134, 3234–3241 (2012).

    CAS  PubMed  Article  Google Scholar 

  58. Mikkelsen, M. D. et al. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab. Eng. 14, 104–111 (2012).

    CAS  PubMed  Article  Google Scholar 

  59. Mans, R. et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res. 15, fov004 (2015).

  60. Verduyn, C., Postma, E., Scheffers, W. A. & Van Dijken, J. P. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501–517 (1992).

    CAS  PubMed  Article  Google Scholar 

  61. Sinha, T., Makia, M., Du, J., Naash, M. I. & Al-Ubaidi, M. R. Flavin homeostasis in the mouse retina during aging and degeneration. J. Nutr. Biochem. 62, 123–133 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Lu, H. et al. A consensus S. cerevisiae metabolic model Yeast8 and its ecosystem for comprehensively probing cellular metabolism. Nat. Commun. 10, 3586 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Heirendt, L. et al. Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v.3.0. Nat. Protoc. 14, 639–702 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Key Research and Development Program of China (grant no. 2018YFA0900300 to Y.J.Z.), National Natural Science Foundation of China (grant nos. 31970316 to L.Z., 21922812 to Y.J.Z. and 32000231 to R.C.), LiaoNing Revitalization Talents Program (grant no. XLYC1807191 to Y.J.Z.) and Shanghai Sail Program (grant no. 19YF1459300 to R.C.). We appreciate Y. Zhao (East China University of Science and Technology) for helpful discussion about FAD quantification. We also thank J. Yang and C. Song for raw data summary and AiMi Academic Services (www.aimieditor.com) for English language editing.

Author information

Authors and Affiliations

Authors

Contributions

Y.J.Z. and L.Z., conceived the original research. R.C designed and performed experiments and analyzed the data. R.C., J.G., X.Z. and W.Y. performed the fed-batch fermentations. R.C. and X.C performed the metabolic analysis. Y.C. performed the MFA. R.C., L.Z. and Y.J.Z. wrote the manuscript.

Corresponding authors

Correspondence to Lei Zhang or Yongjin J. Zhou.

Ethics declarations

Competing interests

Authors have filed two patents (PCT/CN2021/138511 and PCT/CN2021/138510) for protection of the cofactor engineering strategies of the work described herein.

Peer review

Peer review information

Nature Chemical Biology thanks Rodrigo Ledesma-Amaro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Biosynthesis of phenolic acids and their derivatives.

Structures of pCA- (green), CaA- (orange) and FA-derived (pink) natural products, and their pharmaceutical properties.

Extended Data Fig. 2 Engineering strategies for improving the production of CaA and FA.

The engineered gene targets for pathway optimization were marked as golden and the cofactor engineering was marked as blue. See Fig. 2 and Fig. 3 legends regarding abbreviations of other metabolites.

Extended Data Fig. 3 Engineering the biosynthesis of precursor pCA in yeast.

a. Overview of the engineered metabolic pathway for pCA biosynthesis. Optimization of the shikimic acid and aromatic amino acid (AAA) pathways together with the introduction of bacteria- and plant-derived phenolic acid pathways were used for pCA production. ARO10 encoding phenylpyruvate decarboxylase, PDC5 encoding pyruvate decarboxylase, ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, ARO7 encoding chorismate mutase, EcAROL encoding E. coli shikimate kinase II, FjTAL encoding Flavobacterium johnsoniae tyrosine ammonia lyase, SbPAL1 encoding Isatis indigotica phenylalanine ammonia lyase 1, AtCPR1 encoding Arabidopsis thaliana cytochrome P450 reductase, PtrC4H1/2 encoding Populus trichocarpa cinnamic acid hydroxylase 1/2, PtrC3H encoding P. trichocarpa coumarate-3-hydroxylase, CYP complex consisting of PtrC4H1 + PtrC4H2 + PtrC3H. PEP, phosphoenolpyruvate; DHQ, 3-dehydroquinate; DHS, 3-dehydro-shikimate; SHIK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvyl-shikimate-3-phosphate; CHA, chorismic acid; PPA, prephenate; PPY, phenylpyruvate; HPP, para-hydroxy-phenylpyruvate; Phe, phenylalanine; Tyr, tyrosine; CA, cinnamic acid; pCA, coumaric acid; CaA, caffeic acid; FA, ferulic acid. b. Production of pCA in yeast. c. Introduction of the CYP complex further improved pCA biosynthesis. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. Statistical analysis was performed by using Student’s t test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).

Source data

Extended Data Fig. 4 Optimization of HPAB/C and the shikimic acid pathway for CaA production.

a. Overview of the yeast metabolic pathway for CaA biosynthesis. ARO1 encoding shikimate dehydrogenase, ARO2 encoding chorismate synthase, ARO3 encoding DAHP synthase, MtPDH1 encoding Medicago truncatula prephenate dehydrogenase, PHA2 encoding S. cerevisiae prephenate dehydratase 2. See Supplementary Fig. 5 legend regarding other abbreviations. The fusion EcHPAB-EcHPAC (a) and PaHPAB-SeHPAC (b) result in a negative effect on CaA production. PaHPAB, encoding Pseudomonas aeruginosa HpaB; SeHPAC, encoding Salmonella enterica HpaC; EcHPAB: encoding E. coli HpaB; EcHPAC: encoding E. coli HpaC. d. Further optimization of the shikimic acid pathway increased CaA titers with the CYP system. Cells were grown in defined minimal medium containing 20 g/L glucose and cultures were extracted after 96 h of growth for phenolic acid detection. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. Statistical analysis was performed by using Student’s t test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).

Source data

Extended Data Fig. 5 Effect of combining MCH5 overexpression with FADH2 strategies on CaA production.

a. Schematic illustration of the combinations of FADH2 strategies. See Fig. 4 legend regarding abbreviations. (+) indicates expression under strong constitutive promoters. Strain RB19 is the reference strain for regulating FADH2. b. Relative titers of engineered strains normalized to the titers of reference strain RB19. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation.

Source data

Extended Data Fig. 6 Quantification of flavin derived cofactors in CaA producing strains.

a. Representative chromatographs of standards and samples. Total concentration of intracellular FMN(H2) (b), intracellular riboflavin (c) and extracellular riboflavin (d) was detected at different time points. μmol gDCW-1 refers to the number of moles of cofactor per gram of dry cell weight (DCW). All strains were grown at 30 °C in 20 mL of Delft-D medium containing 20 g/L glucose with an initial OD600 of 0.1. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. Statistical analysis was performed by using Student’s t test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).

Source data

Extended Data Fig. 7 Metabolic flux analysis (MFA) in the CaA producer RB209HU (right, with cofactor-engineering strategy) and RB103HU (left, without cofactor-engineering strategy).

Based on MFA, the fluxes to the different products were represented relative to the uptake of 100 mmol of glucose. Cofactor-related pathways is highlighted in blue. 1,3BPG, 1,3-biphosphoglycerate. See Fig. 2 and Fig. 3 legends regarding abbreviations of other metabolites.

Extended Data Fig. 8 Cofactor engineering and pathway optimization for FA production.

a. Schematic illustration of the SAM cycle. LiMETK1 encoding Leishmania infantum S-adenosylmethionine synthetases, EcMET6 encoding E. coli methionine synthase, cMTHFR encoding chemeric methylenetetrahydrofolate reductase. SAM, S-adenosylmethionine; Met, methionine; Hcys, homocysteine; SAH, S-adenosylhomocysteine; CH3-THF, 5-methyl tetrahydrogen folic acid; 5, 10-CH2-THF, 5, 10-methylene tetrahydrogen folic acid; ATP, adenosine triphosphate. b. Overexpression of the SAM pathway reduced phenolic acid titers. c. Enhancing CaA biosynthesis improved FA production. ARO1 encoding shikimate dehydrogenase, ARO2 encoding chorismate synthase, ARO3 encoding DAHP synthase, MtPDH1 encoding Medicago truncatula prephenate dehydrogenase, PHA2 encoding S. cerevisiae prephenate dehydratase 2, LmXFPK, encoding Leuconostoc mesenteroides phosphoketolase; CkPTA, encoding Clostridium kluyveri phosphotransacetylase. gpp1Δ indicates deletion of GPP1 (encoding GAP phosphatase). d. Overexpression of NtCOMT1 using multiple gene copies under of the control of PGAL or strong constitutive promoters improved FA production and the CaA conversion rate. The # indicates expression under strong constitutive promoters (PtHXT7 and PTDH3). All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. Statistical analysis was performed by using Student’s t test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).

Source data

Extended Data Fig. 9 Quantification of cellular SAM and SAH in FA producing strains.

a. Representative chromatographs of standards and samples. Corresponding mass spectra of SAM (b) and SAH (c). d. Cellular SAM/SAH ratios were detected at different time points. All strains were grown at 30 °C in 20 mL of Delft-D medium containing 20 g/L glucose. All data represent the mean of n = 3 independent biological samples and error bars show standard deviation. Statistical analysis was performed by using Student’s t test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).

Source data

Extended Data Fig. 10 Metabolic flux analysis (MFA) of the FA producer RB218HU (right, with cofactor-engineering strategy) and RB101HU (left, without cofactor-engineering strategy).

Based on MFA, the fluxes to the different products were represented relative to the uptake of 100 mmol of glucose. Cofactor-related pathways is highlighted in blue. See Fig. 2 and Fig. 3 legends regarding abbreviations of other metabolites.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Tables 1–5 and data legends.

Reporting Summary

Supplementary Table 1

Primers used in this study.

Supplementary Table 2

Overview of DNA constructs used in this study.

Supplementary Table 3

Codon-optimized genes used in this study.

Supplementary Table 4

Homology sequences for integration at selected chromosomal loci.

Supplementary Table 5

New reactions for metabolic flux analysis.

Supplementary Data 1

Code of culture simulation for metabolic flux analysis.

Supplementary Data 2

Code of yeast genome-scale metabolic model for metabolic flux analysis.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, R., Gao, J., Yu, W. et al. Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast. Nat Chem Biol 18, 520–529 (2022). https://doi.org/10.1038/s41589-022-01014-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-022-01014-6

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing