title: Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol.
abstract: 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 18 mg/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 1.65 g/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.
authors.affiliation: DECHEMA Research Institute, Theodor-Heuss-Allee 25, Frankfurt am Main 60486, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Sonntag
authors.affiliation: DECHEMA Research Institute, Theodor-Heuss-Allee 25, Frankfurt am Main 60486, Germany.
authors.forename: Cora
authors.initials: C
authors.lastname: Kroner
authors.affiliation: DECHEMA Research Institute, Theodor-Heuss-Allee 25, Frankfurt am Main 60486, Germany.
authors.forename: Patrice
authors.initials: P
authors.lastname: Lubuta
authors.affiliation: INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, F-31326 Castanet-Tolosan, France; CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, F-31326 Castanet-Tolosan, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Peyraud
authors.affiliation: DECHEMA Research Institute, Theodor-Heuss-Allee 25, Frankfurt am Main 60486, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Horst
authors.affiliation: DECHEMA Research Institute, Theodor-Heuss-Allee 25, Frankfurt am Main 60486, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Buchhaupt
authors.affiliation: DECHEMA Research Institute, Theodor-Heuss-Allee 25, Frankfurt am Main 60486, Germany. Electronic address: schrader@dechema.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Schrader
mesh_terms: D019149:Bioreactors; D002338:Carotenoids / Q000096:biosynthesis; D003198:Computer Simulation; D003470:Culture Media; D005285:Fermentation; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D000432:Methanol / Q000378:metabolism*; D020601:Methylobacterium extorquens / Q000235:genetics* / Q000378:metabolism*; D008798:Mevalonic Acid / Q000378:metabolism; D000081222:Monocyclic Sesquiterpenes; D010957:Plasmids; D012717:Sesquiterpenes / Q000378:metabolism*
chemical_list: D003470:Culture Media; D000081222:Monocyclic Sesquiterpenes; D012717:Sesquiterpenes; D002338:Carotenoids; C042686:humulene; D008798:Mevalonic Acid; D000432:Methanol
keywords: Alpha-humulene; Diapocarotenoids; Methanol; Methylobacterium extorquens; Mevalonate pathway; Terpenoids