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Research article

Biotechnology and Bioengineering (BB)  |  Synthetic Biology and Metabolic Engineering

Reconstitution of the Mevalonate Pathway for Improvement of Isoprenoid Production and Industrial Applicability in Escherichia coli

Min-Kyoung Kang1†*, Minh Phuong Nguyen1,2†, Sang-Hwal Yoon1, Keerthi B. Jayasundera1, Jong-Wook Son1,2, Chonglong Wang3, Moonhyuk Kwon1,2,4*, and Seon-Won Kim1,2,5*

1Anti-aging Bio Cell factory Regional Leading Research Center, Gyeongsang National University, Jinju 52828, Republic of Korea
2Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Republic of Korea
3School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215123, People's Republic of China
4Research Institute of Molecular Alchemy (RIMA), Gyeongsang National University, Jinju 52828, Republic of Korea
5Plant Molecular Biology & Biotechnology Research Center, Gyeongsang National University, Jinju 52828, Republic of Korea

Correspondence to:
Moonhyuk Kwon,       mkwon@gnu.ac.kr
Seon-Won Kim,         swkim@gnu.ac.kr

†The authors who have contributed equally to this work.
Received: August 29, 2024; Revised: September 30, 2024; Accepted: October 1, 2024

J. Microbiol. Biotechnol. 2024; 34(11): 2338-2346

Published November 28, 2024 https://doi.org/10.4014/jmb.2408.08053

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Natural products, especially isoprenoids have many industrial applications, including medicine, fragrances, food additives, personal care and cosmetics, colorants, and even advanced biofuels. Recent advancements in metabolic engineering with synthetic biology and systems biology have drawn increased interest in microbial-based isoprenoid production. In order to engineer microorganisms to produce a large amount of value-added isoprenoids, great efforts have been made by employing various strategies from synthetic biology and systems biology. We also have engineered E. coli to produce various isoprenoids by targeting and engineering the isoprenoid biosynthetic pathways, methylerythritol phosphate (MEP), and mevalonate (MVA) pathways. Here, we introduced new combinations of the MVA pathway in E. coli with genes from biosafety level 1 (BSL 1) organisms. The reconstituted MVA pathway constructs (pSCS) are not only preferred to the living modified organism (LMO) regulation, but they also improved carotenoid production. In addition, the pSCS constructs resulted in enhanced lycopene production and cell-specific productivity compared to the previous MVA pathway combination (pSNA) in fed-batch fermentation. The pSCS constructs would not only bring an increase in isoprenoid production in E. coli, but they could be an efficient system to be applied for the industrial production of isoprenoids with industry-preferred genetic combinations.

Keywords

Isoprenoids, carotenoids, MVA pathway, Microbial cell factory, synthetic biology

Graphical Abstract


References

  1. Zu Y, Prather KL, Stephanopoulos G. 2020. Metabolic engineering strategies to overcome precursor limitations in isoprenoid biosynthesis. Curr. Opin. Biotechnol. 66: 171-178.
    Pubmed
  2. Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G. 2008. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol. Pharm. 5: 167-190.
    Pubmed
  3. Sacchettini JC, Poulter CD. 1997. Creating isoprenoid diversity. Science 277: 1788-1789.
    Pubmed
  4. Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS. 2011. Identification and microbial production of a terpene-based advanced biofuel. Nat. Commun. 2: 483.
    Pubmed PMC
  5. George KW, Alonso-Gutierrez J, Keasling JD, Lee TS. 2015. Isoprenoid drugs, biofuels, and chemicals--artemisinin, farnesene, and beyond. Adv. Biochem. Eng. Biotechnol. 148: 355-389.
    Pubmed
  6. Wang C, Zada B, Wei G, Kim S-W. 2017. Metabolic engineering and synthetic biology approaches driving isoprenoid production in Escherichia coli. Bioresour. Technol. 241: 430-438.
    Pubmed
  7. Nielsen J, Keasling JD. 2016. Engineering cellular metabolism. Cell 164: 1185-1197.
    Pubmed
  8. Wang C, Liwei M, Park JB, Jeong SH, Wei G, Wang Y, et al. 2018. Microbial platform for terpenoid production: Escherichia coli and Yeast. Front. Microbiol. 9: 2460.
    Pubmed PMC
  9. Kang MK, Yoon SH, Kwon M, Kim SW. 2024. Microbial cell factories for bio-based isoprenoid production to replace fossil resources. Curr. Opin. Syst. Biol. 37:100502.
  10. Immethun CM, Hoynes-O'Connor AG, Balassy A, Moon TS. 2013. Microbial production of isoprenoids enabled by synthetic biology. Front. Microbiol. 4: 75.
  11. Tippmann S, Chen Y, Siewers V, Nielsen J. 2013. From flavors and pharmaceuticals to advanced biofuels: production of isoprenoids in Saccharomyces cerevisiae. Biotechnol. J. 8: 1435-1444.
    Pubmed
  12. Wang C, Pfleger BF, Kim SW. 2017. Reassessing Escherichia coli as a cell factory for biofuel production. Curr. Opin. Biotechnol. 45: 92103.
    Pubmed
  13. Yoon SH, Lee SH, Das A, Ryu HK, Jang HJ, Kim JY, et al. 2009. Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. J. Biotechnol. 140: 218-226.
    Pubmed
  14. Yang L, Wang C, Zhou J, Kim S-W. 2016. Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production. Microb. Cell Fact. 15: 14.
    Pubmed PMC
  15. Wang Y, Zhou S, Liu Q, Jeong SH, Zhu L, Yu X, et al. 2021. Metabolic engineering of Escherichia coli for production of α-santalene, a precursor of sandalwood oil. J. Agric. Food Chem. 69: 13135-13142.
    Pubmed
  16. Han GH, Kim SK, Yoon PK, Kang Y, Kim BS, Fu Y, et al. 2016. Fermentative production and direct extraction of (−)-α-bisabolol in metabolically engineered Escherichia coli. Microb. Cell Fact. 15: 185.
    Pubmed PMC
  17. Kim SR, Kim W, Yeom S-J, Choi K-Y, Shin Y, Shin J, et al. 2023. Improvement of the approval system for the development and experimentation of living modified organisms in synthetic biology. Public Health Rep. 16: 1141-1161.
    Pubmed
  18. Quinlan MM, Smith J, Layton R, Keese P, Agbagala ML, Palacpac MB, et al. 2016. Experiences in engaging the public on biotechnology advances and regulation. Front. Bioeng. Biotechnol. 4: 3.
    Pubmed PMC
  19. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6: 343-345.
    Pubmed
  20. Eiben CB, de Rond T, Bloszies C, Gin J, Chiniquy J, Baidoo EEK, et al. 2019. Mevalonate pathway promiscuity enables noncanonical terpene production. ACS Synth. Biol. 8: 2238-2247.
    Pubmed
  21. Yoon SH, Lee YM, Kim JE, Lee SH, Lee JH, Kim JY, et al. 2006. Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate. Biotechnol. Bioeng. 94: 1025-1032.
    Pubmed
  22. Yoon SH, Park HM, Kim JE, Lee SH, Choi MS, Kim JY, et al. 2007. Increased β-Carotene production in recombinant Escherichia coli harboring an engineered isoprenoid precursor pathway with mevalonate addition. Biotechnol. Prog. 23: 599-605.
    Pubmed
  23. Shin J, South EJ, Dunlop MJ. 2022. Transcriptional tuning of mevalonate pathway enzymes to identify the impact on limonene production in Escherichia coli. ACS Omega 7: 18331-18338.
    Pubmed PMC
  24. Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21: 796-802.
    Pubmed
  25. Yang J, Zhao G, Sun Y, Zheng Y, Jiang X, Liu W, et al. 2012. Bio-isoprene production using exogenous MVA pathway and isoprene synthase in Escherichia coli. Bioresour. Technol. 104: 642-647.
    Pubmed
  26. Liu H, Cheng T, Zou H, Zhang H, Xu X, Sun C, et al. 2017. High titer mevalonate fermentation and its feeding as a building block for isoprenoids (isoprene and sabinene) production in engineered Escherichia coli. Process Biochem. 62: 1-9.
  27. Cheng T, Wang L, Sun C, Xie C. 2022. Optimizing the downstream MVA pathway using a combination optimization strategy to increase lycope ne yield in Escherichia coli. Microb. Cell Fact. 21: 205.
    Pubmed PMC
  28. Sun C, Dong X, Zhang R, Xie C. 2021. Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol. BMC Biotechnol. 21: 3.
    Pubmed PMC
  29. Yu Q, Schaub P, Ghisla S, Al-Babili S, Krieger-Liszkay A, Beyer P. 2010. The lycopene cyclase CrtY from Pantoea ananatis (formerly Erwinia uredovora) catalyzes an FADred-dependent non-redox reaction. J. Biol. Chem. 285: 12109-12120.
    Pubmed PMC
  30. Zhang C, Chen X, Zou R, Zhou K, Stephanopoulos G. 2013. Combining genotype improvement and statistical media optimization for isoprenoid production in E. coli. PLoS One 8: e75164.
    Pubmed PMC
  31. Alper H, Miyaoku K, Stephanopoulos G. 2006. Characterization of lycopene-overproducing E. coli strains in high cell density fermentations. Appl. Microbiol. Biotechnol. 72: 968-974.
    Pubmed

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Article

Research article

J. Microbiol. Biotechnol. 2024; 34(11): 2338-2346

Published online November 28, 2024 https://doi.org/10.4014/jmb.2408.08053

Copyright © The Korean Society for Microbiology and Biotechnology.

Reconstitution of the Mevalonate Pathway for Improvement of Isoprenoid Production and Industrial Applicability in Escherichia coli

Min-Kyoung Kang1†*, Minh Phuong Nguyen1,2†, Sang-Hwal Yoon1, Keerthi B. Jayasundera1, Jong-Wook Son1,2, Chonglong Wang3, Moonhyuk Kwon1,2,4*, and Seon-Won Kim1,2,5*

1Anti-aging Bio Cell factory Regional Leading Research Center, Gyeongsang National University, Jinju 52828, Republic of Korea
2Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Republic of Korea
3School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215123, People's Republic of China
4Research Institute of Molecular Alchemy (RIMA), Gyeongsang National University, Jinju 52828, Republic of Korea
5Plant Molecular Biology & Biotechnology Research Center, Gyeongsang National University, Jinju 52828, Republic of Korea

Correspondence to:Moonhyuk Kwon,       mkwon@gnu.ac.kr
Seon-Won Kim,         swkim@gnu.ac.kr

†The authors who have contributed equally to this work.

Received: August 29, 2024; Revised: September 30, 2024; Accepted: October 1, 2024

Abstract

Natural products, especially isoprenoids have many industrial applications, including medicine, fragrances, food additives, personal care and cosmetics, colorants, and even advanced biofuels. Recent advancements in metabolic engineering with synthetic biology and systems biology have drawn increased interest in microbial-based isoprenoid production. In order to engineer microorganisms to produce a large amount of value-added isoprenoids, great efforts have been made by employing various strategies from synthetic biology and systems biology. We also have engineered E. coli to produce various isoprenoids by targeting and engineering the isoprenoid biosynthetic pathways, methylerythritol phosphate (MEP), and mevalonate (MVA) pathways. Here, we introduced new combinations of the MVA pathway in E. coli with genes from biosafety level 1 (BSL 1) organisms. The reconstituted MVA pathway constructs (pSCS) are not only preferred to the living modified organism (LMO) regulation, but they also improved carotenoid production. In addition, the pSCS constructs resulted in enhanced lycopene production and cell-specific productivity compared to the previous MVA pathway combination (pSNA) in fed-batch fermentation. The pSCS constructs would not only bring an increase in isoprenoid production in E. coli, but they could be an efficient system to be applied for the industrial production of isoprenoids with industry-preferred genetic combinations.

Keywords: Isoprenoids, carotenoids, MVA pathway, Microbial cell factory, synthetic biology

References

  1. Zu Y, Prather KL, Stephanopoulos G. 2020. Metabolic engineering strategies to overcome precursor limitations in isoprenoid biosynthesis. Curr. Opin. Biotechnol. 66: 171-178.
    Pubmed
  2. Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G. 2008. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol. Pharm. 5: 167-190.
    Pubmed
  3. Sacchettini JC, Poulter CD. 1997. Creating isoprenoid diversity. Science 277: 1788-1789.
    Pubmed
  4. Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS. 2011. Identification and microbial production of a terpene-based advanced biofuel. Nat. Commun. 2: 483.
    Pubmed KoreaMed
  5. George KW, Alonso-Gutierrez J, Keasling JD, Lee TS. 2015. Isoprenoid drugs, biofuels, and chemicals--artemisinin, farnesene, and beyond. Adv. Biochem. Eng. Biotechnol. 148: 355-389.
    Pubmed
  6. Wang C, Zada B, Wei G, Kim S-W. 2017. Metabolic engineering and synthetic biology approaches driving isoprenoid production in Escherichia coli. Bioresour. Technol. 241: 430-438.
    Pubmed
  7. Nielsen J, Keasling JD. 2016. Engineering cellular metabolism. Cell 164: 1185-1197.
    Pubmed
  8. Wang C, Liwei M, Park JB, Jeong SH, Wei G, Wang Y, et al. 2018. Microbial platform for terpenoid production: Escherichia coli and Yeast. Front. Microbiol. 9: 2460.
    Pubmed KoreaMed
  9. Kang MK, Yoon SH, Kwon M, Kim SW. 2024. Microbial cell factories for bio-based isoprenoid production to replace fossil resources. Curr. Opin. Syst. Biol. 37:100502.
  10. Immethun CM, Hoynes-O'Connor AG, Balassy A, Moon TS. 2013. Microbial production of isoprenoids enabled by synthetic biology. Front. Microbiol. 4: 75.
  11. Tippmann S, Chen Y, Siewers V, Nielsen J. 2013. From flavors and pharmaceuticals to advanced biofuels: production of isoprenoids in Saccharomyces cerevisiae. Biotechnol. J. 8: 1435-1444.
    Pubmed
  12. Wang C, Pfleger BF, Kim SW. 2017. Reassessing Escherichia coli as a cell factory for biofuel production. Curr. Opin. Biotechnol. 45: 92103.
    Pubmed
  13. Yoon SH, Lee SH, Das A, Ryu HK, Jang HJ, Kim JY, et al. 2009. Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. J. Biotechnol. 140: 218-226.
    Pubmed
  14. Yang L, Wang C, Zhou J, Kim S-W. 2016. Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production. Microb. Cell Fact. 15: 14.
    Pubmed KoreaMed
  15. Wang Y, Zhou S, Liu Q, Jeong SH, Zhu L, Yu X, et al. 2021. Metabolic engineering of Escherichia coli for production of α-santalene, a precursor of sandalwood oil. J. Agric. Food Chem. 69: 13135-13142.
    Pubmed
  16. Han GH, Kim SK, Yoon PK, Kang Y, Kim BS, Fu Y, et al. 2016. Fermentative production and direct extraction of (−)-α-bisabolol in metabolically engineered Escherichia coli. Microb. Cell Fact. 15: 185.
    Pubmed KoreaMed
  17. Kim SR, Kim W, Yeom S-J, Choi K-Y, Shin Y, Shin J, et al. 2023. Improvement of the approval system for the development and experimentation of living modified organisms in synthetic biology. Public Health Rep. 16: 1141-1161.
    Pubmed
  18. Quinlan MM, Smith J, Layton R, Keese P, Agbagala ML, Palacpac MB, et al. 2016. Experiences in engaging the public on biotechnology advances and regulation. Front. Bioeng. Biotechnol. 4: 3.
    Pubmed KoreaMed
  19. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6: 343-345.
    Pubmed
  20. Eiben CB, de Rond T, Bloszies C, Gin J, Chiniquy J, Baidoo EEK, et al. 2019. Mevalonate pathway promiscuity enables noncanonical terpene production. ACS Synth. Biol. 8: 2238-2247.
    Pubmed
  21. Yoon SH, Lee YM, Kim JE, Lee SH, Lee JH, Kim JY, et al. 2006. Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate. Biotechnol. Bioeng. 94: 1025-1032.
    Pubmed
  22. Yoon SH, Park HM, Kim JE, Lee SH, Choi MS, Kim JY, et al. 2007. Increased β-Carotene production in recombinant Escherichia coli harboring an engineered isoprenoid precursor pathway with mevalonate addition. Biotechnol. Prog. 23: 599-605.
    Pubmed
  23. Shin J, South EJ, Dunlop MJ. 2022. Transcriptional tuning of mevalonate pathway enzymes to identify the impact on limonene production in Escherichia coli. ACS Omega 7: 18331-18338.
    Pubmed KoreaMed
  24. Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21: 796-802.
    Pubmed
  25. Yang J, Zhao G, Sun Y, Zheng Y, Jiang X, Liu W, et al. 2012. Bio-isoprene production using exogenous MVA pathway and isoprene synthase in Escherichia coli. Bioresour. Technol. 104: 642-647.
    Pubmed
  26. Liu H, Cheng T, Zou H, Zhang H, Xu X, Sun C, et al. 2017. High titer mevalonate fermentation and its feeding as a building block for isoprenoids (isoprene and sabinene) production in engineered Escherichia coli. Process Biochem. 62: 1-9.
  27. Cheng T, Wang L, Sun C, Xie C. 2022. Optimizing the downstream MVA pathway using a combination optimization strategy to increase lycope ne yield in Escherichia coli. Microb. Cell Fact. 21: 205.
    Pubmed KoreaMed
  28. Sun C, Dong X, Zhang R, Xie C. 2021. Effectiveness of recombinant Escherichia coli on the production of (R)-(+)-perillyl alcohol. BMC Biotechnol. 21: 3.
    Pubmed KoreaMed
  29. Yu Q, Schaub P, Ghisla S, Al-Babili S, Krieger-Liszkay A, Beyer P. 2010. The lycopene cyclase CrtY from Pantoea ananatis (formerly Erwinia uredovora) catalyzes an FADred-dependent non-redox reaction. J. Biol. Chem. 285: 12109-12120.
    Pubmed KoreaMed
  30. Zhang C, Chen X, Zou R, Zhou K, Stephanopoulos G. 2013. Combining genotype improvement and statistical media optimization for isoprenoid production in E. coli. PLoS One 8: e75164.
    Pubmed KoreaMed
  31. Alper H, Miyaoku K, Stephanopoulos G. 2006. Characterization of lycopene-overproducing E. coli strains in high cell density fermentations. Appl. Microbiol. Biotechnol. 72: 968-974.
    Pubmed