Abstract
Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems.
Similar content being viewed by others
Abbreviations
- DCW:
-
Dry cell weight
- DMAPP:
-
Dimethylallyl-diphosphate
- GPPS:
-
Geranyl-diphosphate synthase
- IPP:
-
Isopentenyl-diphosphate
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- MEP:
-
2-C-methyl-erythritol-4-phosphate
- MVA:
-
Mevalonic acid
- OD:
-
Optical density
- β-PHL:
-
β-Phellandrene
- PHLS:
-
β-Phellandrene synthase
- TIR:
-
Translation initiation region
References
Alonso-Gutierrez J, Chan R, Batth TS, Adams PD, Keasling JD, Petzold CJ, Lee TS (2013) Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab Eng 19:33–41
Banerjee A, Wu Y, Banerjee R, Li Y, Yan H, Sharkey TD (2013) Feedback inhibition of deoxy-D-xylulose 5-phosphate synthase regulates the methyl erythritol 4-phosphate pathway. J Biol Chem 288:16926–16936
Bentley FK, García-Cerdán JG, Chen H, Melis A (2013) Paradigm of monoterpene (β-phellandrene) hydrocarbons production via photosynthesis in cyanobacteria. Bioenerg Res 6:917–929
Bohlmann J, Phillips M, Ramachandiran V, Katoh S, Croteau R (1999) cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis). Arch Biochem Biophys 368:232–243
Bouhss A, Trunkfield AE, Bugg TD, Mengin-Lecreulx D (2008) The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS Microbiol Rev 32(2):208–233
Carter OA, Peters RJ, Croteau R (2003) Monoterpene biosynthesis pathway construction in Escherichia coli. Phytochemistry 64:425–433
Demissie ZA, Sarker LS, Mahmoud SS (2011) Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia. Planta 233:685–696
Dumelin CE, Chen Y, Leconte AM, Chen YG, Liu DR (2012) Discovery and biological characterization of geranylated RNA in bacteria. Nat Chem Biol 8:913–919
Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, Hadi MZ, Mukhopadhyay A (2011) Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 7:487
Englund E, Pattanaik B, Ubhayasekera SJ, Stensjö K, Bergquist J, Lindberg P (2014) Production of squalene in Synechocystis sp. PCC 6803. PLoS ONE 9(3):e90270. doi:10.1371/journal.pone.0090270
Farmer WR, Liao JC (2001) Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnol Prog 17:57–61
Fischer MJC, Meyer S, Claudel P, Bergdoll M, Karst F (2011) Metabolic engineering of monoterpene synthesis in yeast. Biotechnol Bioeng 108:1883–1892
Formighieri C, Melis A (2014) Regulation of β-phellandrene synthase gene expression, recombinant protein accumulation, and monoterpene hydrocarbons production in Synechocystis transformants. Planta. doi:10.1007/s00425-014-2080-8
Kim SW, Keasling JD (2001) Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol Bioeng 72:408–415
Lindberg P, Park S, Melis A (2010) Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. Metab Eng 12:70–79
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
Martin DM, Chiang A, Lund ST, Bohlmann J (2012) Biosynthesis of wine aroma: transcript profiles of hydroxymethylbutenyl diphosphate reductase, geranyl diphosphate synthase, and linalool/nerolidol synthase parallel monoterpenol glycoside accumulation in Gewürztraminer grapes. Planta 236:919–929
Matthews PD, Wurtzel ET (2000) Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase. Appl Microbiol Biotechnol 53:396–400
Melis A (2012) Photosynthesis-to-fuels: from sunlight to hydrogen, isoprene, and botryococcene production. Energy Environ Sci 5(2):5531–5539
Melis A (2013) Carbon partitioning in photosynthesis. Curr Opin Chem Biol 17:453–456
Sarria S, Wong B, Martin HG, Keasling JD, Peralta-Yahya P (2014) Microbial synthesis of pinene. ACS Synth Biol. doi:10.1021/sb4001382
Schilmiller AL, Schauvinhold I, Larson M, Xu R, Charbonneau AL, Schmidt A, Wilkerson C, Last RL, Pichersky E (2009) Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proc Nat Acad Sci USA 106:10865–10870
Schmidt A, Gershenzon J (2008) Cloning and characterization of two different types of geranyl diphosphate synthases from Norway spruce (Picea abies). Phytochemistry 69:49–57
Vadali RV, Fu Y, Bennett GN, San KY (2005) Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli. Biotechnol Prog 21:1558–1561
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 beta-carotene in E. coli. J Biotechnol 140:218–226
Zerbe P, Bohlmann J (2014) Bioproducts, biofuels, and perfumes: conifer terpene synthases and their potential for metabolic engineering. In: Jetter R (ed) Phytochemicals–biosynthesis, function and application. Springer, Berlin, pp 85–107
Zhang H, Liu Q, Cao Y, Feng X, Zheng Y, Zou H, Liu H, Yang J, Xian M (2014) Microbial production of sabinene — a new terpene-based precursor of advanced biofuel. Microb Cell Fact 13:20
Zurbriggen A, Kirst H, Melis A (2012) Isoprene production via the mevalonic acid pathway in Escherichia coli (bacteria). Bioenerg Res 5:814–828
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Erko Stackebrandt.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Formighieri, C., Melis, A. Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures. Arch Microbiol 196, 853–861 (2014). https://doi.org/10.1007/s00203-014-1024-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00203-014-1024-9