Skip to main content
Log in

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Anthony C (1982) The biochemistry of methylotrophs. Academic, London

    Google Scholar 

  • ARPA-E (2013) REMOTE | ARPA-E. http://arpa-e.energy.gov/?q=arpa-e-programs/remote. Accessed 29 Mar 2015

  • Bar-Even A, Noor E, Flamholz A, Milo R (2013) Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes. Biochim Biophys Acta 1827:1039–47. doi:10.1016/j.bbabio.2012.10.013

    Article  PubMed  CAS  Google Scholar 

  • Behera S, Singh R, Arora R, Sharma NK, Shukla M, Kumar S (2015) Scope of algae as third generation biofuels. Front Bioeng Biotechnol 2:1–13. doi:10.3389/fbioe.2014.00090

    Article  Google Scholar 

  • Bélanger L, Figueira MM, Bourque D, Morel L, Béland M, Laramée L, Groleau D, Míguez CB (2004) Production of heterologous protein by Methylobacterium extorquens in high cell density fermentation. FEMS Microbiol Lett 231:197–204. doi:10.1016/S0378-1097(03)00956-X

    Article  PubMed  CAS  Google Scholar 

  • Bentley FK, Zurbriggen A, Melis A (2014) Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene. Mol Plant 7:71–86. doi:10.1093/mp/sst134

    Article  PubMed  CAS  Google Scholar 

  • Bohlmann J, Keeling CI (2008) Terpenoid biomaterials. Plant J 54:656–669. doi:10.1111/j.1365-313X.2008.03449.x

    Article  PubMed  CAS  Google Scholar 

  • Borzyskowski LS von, Remus-Emsermann M, Weishaupt R, Vorholt JA, Erb TJ (2014) A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in Methylobacterium extorquens AM1 and other Alphaproteobacteria.

  • Bourque D, Ouellette B, Andre G, Groleau D (1992) Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens. Appl Microbiol Biotechnol 37:7–12. doi:10.1007/BF00174194

    Article  CAS  Google Scholar 

  • Bourque D, Pomerleau Y, Groleau D (1995) High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB. Appl Microbiol Biotechnol 44:367–376. doi:10.1007/s002530050569

    Article  CAS  Google Scholar 

  • Cabañó J, Buesa C, Hegardt FG, Marrero PF (1997) Catalytic properties of recombinant 3-hydroxy-3-methylglutaryl coenzyme a synthase-1 from Blattella germanica. Insect Biochem Mol Biol 27:499–505

    Article  PubMed  Google Scholar 

  • Chistoserdova L, Chen S-W, Lapidus A, Lidstrom ME, Chen S (2003) Minireview: Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J Bacteriol 185:2980–2987. doi:10.1128/JB.185.10.2980

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Choi J, Kim JH, Daniel M, Lebeault JM (1989) Optimization of growth medium and poly-β-hydroxybutyric acid production from methanol in Methylobacterium organophilum. Korean J Appl Microbiol Bioeng 17:392–396

    CAS  Google Scholar 

  • Choi YJ, Morel L, Bourque D, Mullick A, Massie B, Míguez CB (2006) Bestowing inducibility on the cloned methanol dehydrogenase promoter (PmxaF) of Methylobacterium extorquens by applying regulatory elements of Pseudomonas putida F1. Appl Environ Microbiol 72:7723–7729. doi:10.1128/AEM.02002-06

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Chubiz LM, Purswani J, Carroll SM, Marx CJ (2013) A novel pair of inducible expression vectors for use in Methylobacterium extorquens. BMC Res Notes 6:183. doi:10.1186/1756-0500-6-183

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Delaney NF, Kaczmarek ME, Ward LM, Swanson PK, Lee MC, Marx CJ (2013) Development of an optimized medium, strain and high-throughput culturing methods for Methylobacterium extorquens. PLoS One 8:e62957

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Espah Borujeni A, Channarasappa AS, Salis HM (2014) Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res 42:2646–2659. doi:10.1093/nar/gkt1139

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Fiehn O, Kopka J, Trethewey RN, Willmitzer L (2000) Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Anal Chem 72:3573–3580. doi:10.1021/ac991142i

    Article  PubMed  CAS  Google Scholar 

  • Fitz GKA, Lidstrom ME (2003) Overexpression of a heterologous protein, haloalkane dehalogenase, in a poly-β-hydroxybutyrate-deficient strain of the facultative methylotroph Methylobacterium extorquens AM1. Biotechnol Bioeng 81:263–268. doi:10.1002/bit.10470

    Article  CAS  Google Scholar 

  • Hrtado-Guerrrero R, Pena-Diaz J, Montalvetti A, Ruiz-Perez LM, Gonzalez-Pacanowska D (2002) Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase. FEBS Lett 510:141–144

    Article  Google Scholar 

  • Hu B, Lidstrom ME (2014) Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production. Biotechnol Biofuels 7:156. doi:10.1186/s13068-014-0156-0

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kaczmarczyk A, Vorholt JA, Francez-Charlot A (2013) Cumate-inducible gene expression system for sphingomonads and other Alphaproteobacteria. Appl Environ Microbiol 79:6795–6802. doi:10.1128/AEM.02296-13

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kiefer P, Portais JC, Vorholt JA (2008) Quantitative metabolome analysis using liquid chromatography-high-resolution mass spectrometry. Anal Biochem 382:94–100. doi:10.1016/j.ab.2008.07.010

    Article  PubMed  CAS  Google Scholar 

  • Li Y, Pfeifer BA (2014) Heterologous production of plant-derived isoprenoid products in microbes and the application of metabolic engineering and synthetic biology. Curr Opin Plant Biol 19:8–13. doi:10.1016/j.pbi.2014.02.005

    Article  PubMed  CAS  Google Scholar 

  • Li H, Opgenorth PH, Wernick DG, Rogers S, Wu T-Y, Higashide W, Malati P, Huo Y-X, Cho KM, Liao JC (2012) Integrated electromicrobial conversion of CO2 to higher alcohols. Science 335(80):1596

    Article  PubMed  CAS  Google Scholar 

  • Ma SM, Garcia DE, Redding-Johanson AM, Friedland GD, Chan R, Batth TS, Haliburton JR, Chivian D, Keasling JD, Petzold CJ, Soon Lee T, Chhabra SR (2011) Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases. Metab Eng 13:588–597. doi:10.1016/j.ymben.2011.07.001

    Article  PubMed  CAS  Google Scholar 

  • 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. doi:10.1038/nbt833

    Article  PubMed  CAS  Google Scholar 

  • Marx CJ (2008) Development of a broad-host-range sacB-based vector for unmarked allelic exchange. BMC Res Notes 1:1. doi:10.1186/1756-0500-1-1

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Marx CJ, Lidstrom ME (2001) Development of improved versatile broad-host-range vectors for use in methylotrophs and other gram-negative bacteria. Microbiology 147:2065–2075

    Article  PubMed  CAS  Google Scholar 

  • Marx CJ, Lidstrom ME (2002) Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Biotechniques 33:1062–7

    PubMed  CAS  Google Scholar 

  • Marx CJ, Lidstrom ME (2004) Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch. Microbiology 150:9–19. doi:10.1099/mic.0.26587-0

    Article  PubMed  CAS  Google Scholar 

  • Metzger LC, Francez-Charlot A, Vorholt JA (2013) Single-domain response regulator involved in the general stress response of Methylobacterium extorquens. Microbiology (UK) 159:1067–1076. doi:10.1099/mic.0.066068-0

    Article  CAS  Google Scholar 

  • National Bureau of Statistics C (2014) Statistical Communiqué of the People’s Republic of China on the 2013 National Economic and Social Development. http://www.stats.gov.cn/english/PressRelease/201402/t20140224_515103.html. Accessed 29 Mar 2015

  • Nieves LM, Panyon LA, Wang X (2015) Engineering sugar utilization and microbial tolerance toward lignocellulose conversion. Front Bioeng Biotechnol 3:1–10. doi:10.3389/fbioe.2015.00017

    Article  Google Scholar 

  • Ochsner AM, Sonntag F, Buchhaupt M, Schrader J, Vorholt JA (2014) Methylobacterium extorquens: methylotrophy and biotechnological applications. Appl Microbiol Biotechnol. doi:10.1007/s00253-014-6240-3

    PubMed  Google Scholar 

  • OECD (2014) OECD-FAO agricultural outlook 2014. OECD, Paris

    Google Scholar 

  • Ohto C, Muramatsu M, Obata S, Sakuradani E, Shimizu S (2009) Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols. Appl Microbiol Biotechnol 82:837–845. doi:10.1007/s00253-008-1807-5

    Article  PubMed  CAS  Google Scholar 

  • Olah GA, Goeppert A, Prakash GKS (2009) Beyond oil and gas: the methanol economy: second edition. John Wiley, ISBN: 9783527644636

  • Orita I, Nishikawa K, Nakamura S, Fukui T (2014) Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions. Appl Microbiol Biotechnol 98:3715–25. doi:10.1007/s00253-013-5490-9

    Article  PubMed  CAS  Google Scholar 

  • Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Jiang H, Fleck M, Bajad S, Dang G, Dengrove D, Diola D, Dorin G, Ellens KW, Fickes S, Galazzo J, Gaucher SP, 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 LF, Xu L, Yu M, Zhang Y, Zhao L, Lievense J, Covello PS, Keasling JD, Reiling KK, Renninger NS, Newman JD (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496:528–532. doi:10.1038/nature12051

    Article  PubMed  CAS  Google Scholar 

  • Peel D, Quayle JR (1961) Microbial growth on C1 compounds. Biochem J 81:465–469

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Peña-Díaz J, Montalvetti A, Camacho A, Gallego C, Ruiz-Perez LM, Gonzalez-Pacanowska D (1997) A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi. Biochem J 324(Pt 2):619–626

    Article  PubMed Central  PubMed  Google Scholar 

  • Peyraud R, Kiefer P, Christen P, Massou S, Portais J-C, Vorholt JA (2009) Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics. Proc Natl Acad Sci U S A 106:4846–4851. doi:10.1073/pnas.0810932106

    Article  PubMed Central  PubMed  Google Scholar 

  • Peyraud R, Schneider K, Kiefer P, Massou S, Vorholt JA, Portais J-C (2011) Genome-scale reconstruction and system level investigation of the metabolic network of Methylobacterium extorquens AM1. BMC Syst Biol 5:189. doi:10.1186/1752-0509-5-189

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950. doi:10.1038/nbt.1568

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Schrader J, Schilling M, Holtmann D, Sell D, Filho MV, Marx A, Vorholt JA (2009) Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria. Trends Biotechnol 27:107–115. doi:10.1016/j.tibtech.2008.10.009

    Article  PubMed  CAS  Google Scholar 

  • Shi W (2014) Production of Fermentation Industry of China in 2013. http://unn.people.com.cn/n/2014/0428/c82452-24951188.html. Accessed 17 Feb 2015

  • Sirirote P, Yamane T, Shimizu S (1986) Production of l-serine from methanol and glycine by resting cells of a methylotroph under automatically controlled conditions. J Ferment Technol 64:389–396. doi:10.1016/0385-6380(86)90025-7

    Article  CAS  Google Scholar 

  • Sonntag F, Buchhaupt M, Schrader J (2014) Thioesterases for ethylmalonyl-CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1. Appl Microbiol Biotechnol 98:4533–4544. doi:10.1007/s00253-013-5456-y

    Article  PubMed  CAS  Google Scholar 

  • Tabata K, Hashimoto SI (2004) Production of mevalonate by a metabolically-engineered Escherichia coli. Biotechnol Lett 26:1487–1491. doi:10.1023/B:BILE.0000044449.08268.7d

    Article  PubMed  CAS  Google Scholar 

  • Ueda S, Matsumoto S, Takagi A, Yamane T (1992) Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from methanol and n-amyl alcohol by the methylotrophic bacteria Paracoccus denitrificans and Methylobacterium extorquens. Appl Environ Microbiol 58:3574–3579

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vuilleumier S, Chistoserdova L, Lee MC, Bringel F, Lajus A, Yang Z, Gourion B, Barbe V, Chang J, Cruveiller S, Dossat C, Gillett W, Gruffaz C, Haugen E, Hourcade E, Levy R, Mangenot S, Muller E, Nadalig T, Pagni M, Penny C, Peyraud R, Robinson DG, Roche D, Rouy Z, Saenempechek C, Salvignol G, Vallenet D, Zaining W, Marx CJ, Vorholt JA, Olson MV, Kaul R, Weissenbach J, Médigue C, Lidstrom ME (2009) Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources. PLoS One. doi:10.1371/journal.pone.0005584

    PubMed Central  PubMed  Google Scholar 

  • Wang C, Yoon SH, Shah AA, Chung YR, Kim JY, Choi ES, Keasling JD, Kim SW (2010) Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway. Biotechnol Bioeng 107:421–429. doi:10.1002/bit.22831

    Article  PubMed  CAS  Google Scholar 

  • Wilding EI, Brown JR, Bryant A, Chalker AF, Holmes D, Ingraham K, So CY, Iordanescu S, Rosenberg M, Gwynn MN (2000) Identification, essentiality and evolution of the mevalonate pathway for isopentenyl diphosphate biosynthesis in {Gram}-positive cocci. J Bacteriol 182:4319–4327. doi:10.1128/JB.182.15.4319-4327.2000.Updated

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Xiaofeng GMEL (2008) Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. Biotechnol Bioeng 99:929–940. doi:10.1002/bit

    Article  CAS  Google Scholar 

  • Yang J, Xian M, Su S, Zhao G, Nie Q, Jiang X, Zheng Y, Liu W (2012) Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli. PLoS One 7:1–7. doi:10.1371/journal.pone.0033509

    Article  Google Scholar 

  • Yang S, Matsen JB, Konopka M, Green-saxena A, Clubb J, Sadilek M, Orphan VJ, Beck D, Kalyuzhnaya MG (2013) Global molecular analyses of methane metabolism in methanotrophic alphaproteobacterium, Methylosinus trichosporium OB3b. Part II. Metabolomics and 13C-labeling study. Front Microbiol 4:1–13. doi:10.3389/fmicb.2013.00070

    Google Scholar 

  • Yoon SH, Lee SH, Das A, Ryu HK, Jang HJ, Kim JY, Oh DK, Keasling JD, Kim SW (2009) Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. J Biotechnol 140:218–226. doi:10.1016/j.jbiotec.2009.01.008

    Article  PubMed  CAS  Google Scholar 

  • Zahiri HS, Yoon SH, Keasling JD, Lee SH, Won Kim S, Yoon SC, Shin YC (2006) Coenzyme Q10 production in recombinant Escherichia coli strains engineered with a heterologous decaprenyl diphosphate synthase gene and foreign mevalonate pathway. Metab Eng 8:406–416. doi:10.1016/j.ymben.2006.05.002

    Article  PubMed  CAS  Google Scholar 

  • Zhang F, Rodriguez S, Keasling JD (2011) Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 22:775–783. doi:10.1016/j.copbio.2011.04.024

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by National Natural Science Foundation of China (NSFC 21376137) and Tsinghua University Initiative Scientific Research Program (2013Z02-1).

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Chong Zhang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 157 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhu, WL., Cui, JY., Cui, LY. et al. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway. Appl Microbiol Biotechnol 100, 2171–2182 (2016). https://doi.org/10.1007/s00253-015-7078-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-7078-z

Keywords

Navigation