Corynebacterium glutamicum as a platform strain for the production of a broad variety of terpenoids

Corynebacterium glutamicum is a natural carotenoid producing bacterium used in the million-ton-scale amino acid biotechnology that has been engineered for isoprenoid production1. The native membrane-bound carotenoid decaprenoxanthin is a rare C50 carotenoid. Volatile terpenoids such as valencene2 and patchoulol3 could be produced upon deletion of the first step of the specific carotenoid pathway and heterologous expression of the FPP synthase gene ispA from E. coli and terpene synthases from plant origin. However, these strains produced a yet unidentified carotenoid and only when all carotenoid biosynthetic genes were deleted, a colorless strain resulted. Expressing a codon optimized ADS from Artemisia annua in the white strain, amorphadiene, the volatile precursor for artemisinin was produced. For production of volatile terpenoids a dodecane overlay was used, a condition in which C. glutamicum benefits from its robust myco-membrane. Recently, we showed production of membrane-bound carotenoids with different length and/or cyclization status: bicyclic C50 sarcinaxanthin4, bicyclic C40 astaxanthin5, the linear lycopene6 and the linear C50 bisanhydrobacterioruberin7. This indicated that the C. glutamicum myco-membrane accepts these linear and bicyclic carotenoids. Please click Additional Files below to see the full abstract

Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum

Frohwitter J, Heider S, Peters-Wendisch P, Beekwilder J, Wendisch VF. Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum. Journal of Biotechnology. 2014;191:205-213

Patchoulol Production with Metabolically Engineered Corynebacterium glutamicum

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1

Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia

Milijasevic-Marcic S, Gartemann K-H, Frohwitter J, et al. Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia. European Journal Of Plant Pathology. 2012;134(4):697-711.Sixty-eight Clavibacter michiganensis subsp. michiganensis (Cmm) strains from recent outbreaks of bacterial wilt and canker in Serbia were collected from several tomato growing regions during a three-year period. The pathogen was identified based on bacteriological characteristics and pathogenicity tests and the identity of strains was confirmed by DAS ELISA and PCR amplification using primers CMM5/6 and PSA4/R. The strains showed homogeneity in biochemical and physiological properties. However, pathogenicity tests revealed differences in virulence that are presumably due to a loss of the pat-1 gene. Further strain characterization using DNA-based methods revealed a high diversity of the Serbian Cmm strains. Based on multi-locus sequence typing (MLST) analyses of five genes, Cmm strains were divided into seven groups. The pulsed-field gel electrophoresis (PFGE) pattern of a selection of strains supported the groupings based on trees of the kdpA/sdhA sequences. On the other hand, groupings made according to PFGE and MLST were not correlated to plasmid content in all cases. This study suggested that high genetic variability of the Serbian Cmm strains was detected both in MLST and PFGE analyses, and could have resulted either from new Cmm strains being introduced by seeds from different origins or as a consequence of an intraspecific hybridization process. In addition, this study proposed MLST as an efficient tool in epidemiological studies, population biology investigations and tracking the routes of transmission of pathogens. Four of the five house-keeping genes (kdpA, sdhA, ligA and gyrB) selected to characterize Cmm strains proved to be suitable for the MLST analysis. This is the first study carried out on the characterization of Cmm using MLST