Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol

• Published in: Metabolic engineering Vol. 32; pp. 82 - 94
• Main Authors: Sonntag, Frank, Kroner, Cora, Lubuta, Patrice, Peyraud, Rémi, Horst, Angelika, Buchhaupt, Markus, Schrader, Jens
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.11.2015; Elsevier
• Subjects: Alpha-humulene; binding sites; Bioreactors; Biotechnology; carbon; carotenoids; Carotenoids - biosynthesis; Computer Simulation; Culture Media; Diapocarotenoids; farnesene; Fermentation; Life Sciences; metabolic engineering; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; Methanol; Methanol - metabolism; Methylobacterium extorquens; Methylobacterium extorquens - genetics; Methylobacterium extorquens - metabolism; Mevalonate pathway; Mevalonic Acid - metabolism; microorganisms; mutants; Myxococcus xanthus; Plasmids; prices; raw materials; ribosomes; Saccharomyces cerevisiae; Sesquiterpenes - metabolism; sugars; Terpenoids; Zingiber zerumbet; Terpenoids; Alpha-humulene; Methylobacterium extorquens; Mevalonate pathway; Diapocarotenoids; Methanol; HETEROLOGOUS MEVALONATE PATHWAY; ESCHERICHIA-COLI; BIOSYNTHETIC-PATHWAY; METHANOTROPHIC BACTERIA; CODON ADAPTATION INDEX; CORDIA-VERBENACEA; SACCHAROMYCES-CEREVISIAE; ETHYLMALONYL-COA PATHWAY; GENE-EXPRESSION; MICROBIAL-PRODUCTION
• ISSN: 1096-7176; 1096-7184; 1096-7184
• DOI: 10.1016/j.ymben.2015.09.004

Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol. •First de novo terpenoid production in an engineered methylotrophic bacterium. •Introduction of the mevalonate pathway from Myxococcus xanthus.•Flux balancing by ribosome binding site optimizations.•Up to 75mg/L α-humulene in shake flask cultivations.•Up to 1.65g/L α-humulene in methanol limited fed-batch fermentation.