title: Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum.
abstract: The sesquiterpene (+)-valencene is an aroma compound of citrus fruits and is used to flavor foods and drinks. Biosynthesis of (+)-valencene starts from farnesyl pyrophosphate, an intermediate of carotenoid biosynthesis. Corynebacterium glutamicum, the workhorse of the million-ton scale amino acid industry, is naturally pigmented as it synthesizes the rare fifty carbon atoms (C50) containing carotenoid decaprenoxanthin. Since the carotenoid pathway of this Gram-positive bacterium has previously been engineered for efficient production of several C50 and C40 carotenoids, its potential to produce a sesquiterpene was assessed. Growth of C. glutamicum was negatively affected by (+)-valencene, but overlaying n-dodecane as organic phase for extraction of (+)-valencene was shown to be biocompatible. Heterologous expression of the (+)-valencene synthase gene from the sweet orange Citrus sinensis was not sufficient to enable (+)-valencene production, likely because provision of farnesyl pyrophosphate (FPP) by endogenous prenyltransferases was too low. However, upon deletion of two endogenous prenyltransferase genes and heterologous expression of either FPP synthase gene ispA from Escherichia coli or ERG20 from Saccharomyces cerevisiae (+)-valence production by C. sinensis valencene synthase was observed. Employing the valencene synthase from Nootka cypress improved (+)-valencene titers 10 fold to 2.41±0.26mgl(-1) (+)-valencene, which is equivalent to 0.25±0.03mgg(-1) cell dry weight (CDW). This is the first report on sesquiterpene overproduction by recombinant C. glutamicum.
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld 33615, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Frohwitter
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld 33615, Germany.
authors.forename: Sabine A E
authors.initials: SA
authors.lastname: Heider
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld 33615, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Peters-Wendisch
authors.affiliation: Plant Research International, Wageningen, The Netherlands.
authors.forename: Jules
authors.initials: J
authors.lastname: Beekwilder
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld 33615, Germany. Electronic address: volker.wendisch@uni-bielefeld.de.
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
mesh_terms: D000473:Alkanes / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D002338:Carotenoids / Q000096:biosynthesis* / Q000235:genetics; D002957:Citrus / Q000737:chemistry; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D051228:Geranyltranstransferase; D060847:Metabolic Engineering; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis / Q000378:metabolism; D016415:Sequence Alignment; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000473:Alkanes; D029968:Escherichia coli Proteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C007548:n-dodecane; D002338:Carotenoids; C004808:farnesyl pyrophosphate; C506706:valencene; D051228:Geranyltranstransferase; C573354:IspA protein, E coli
keywords: (+)-Valencene; Corynebacterium glutamicum; Metabolic engineering; Sesquiterpene; Terpenoid