valencene and n-dodecane

Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L(-1) (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.

Topics: Alkanes; Culture Media; DNA, Fungal; Metabolic Engineering; Metabolic Networks and Pathways; Molecular Sequence Data; Schizophyllum; Sequence Analysis, DNA; Sesquiterpenes

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.

Topics: Alkanes; Amino Acid Sequence; Carotenoids; Citrus; Corynebacterium glutamicum; Escherichia coli; Escherichia coli Proteins; Geranyltranstransferase; Metabolic Engineering; Polyisoprenyl Phosphates; Sequence Alignment; Sesquiterpenes