Construction of a Corynebacterium Glutamicum Platform Strain for the Production of High-Value Plant Secondary Metabolites

The soil-bacterium Corynebacterium glutamicum is an important microorganism in industrial biotechnology for the production of bulk chemicals, especially amino acids (van Ooyen et al., 2012, Vogt et al., 2014). However, until now, only a few C. glutamicum strains for the production of aromatic compounds are available, which can be mostly attributed to the presence of a complex (and partly unknown) catabolic network for a broad range of aromatic substances. We were able to uncover the complete catabolic route for phenylpropanoids, which allows growth of C. glutamicum using these compounds as sole carbon and energy source. Phenylpropanoids are channeled into the central carbon metabolism by the coupled action of an identified peripheral, CoA-dependent, β-oxidative degradation pathway and the well-known β-ketoadipate pathway (Kallscheuer et al., 2016). By deleting four gene clusters comprising altogether 21 genes being essential for degradation of phenylpropanoids and other aromatic compounds, we obtained a C. glutamicum platform strain that was subsequently applied for the production of (2S)-flavanones and stilbenes (Marienhagen and Bott, 2013). Heterologous expression of codon-optimized genes encoding a 4-coumarate: CoA-ligase (4CL), a chalcone synthase and a chalcone isomerase led to the production of 30 - 35 mg/L of corresponding (2S)-flavanones from supplemented phenylpropanoids in the constructed strain. The strain also produced stilbenes when genes coding for a 4CL and a stilbene synthase (STS) were heterologously expressed. Several cultivation parameters influencing growth and gene expression were optimized with respect to improved product titers. Under optimal conditions, stilbene concentrations ranging from 60 to 160 mg/L could be obtained from supplemented phenylpropanoids. A strain converting the phenylpropanoid p-coumaric acid into the stilbene resveratrol was further modified with a focus on the increased supply of the precursor metabolite l-tyrosine. By introduction of an additional gene coding for a tyrosine ammonia lyase combining aromatic amino acid metabolism with phenylpropanoid synthesis, the strain was able to produce 60 mg/L directly from glucose in defined medium simultaneous to biomass formation. Beyond production of polyphenols, the strain will help to overcome current drawbacks of C. glutamicum resulting from degradation of aromatic compounds, offering a huge potential for production of other high-value aromatic compounds with this industrially relevant organism.

Kallscheuer, N., Vogt, M., Kappelmann, J., Krumbach, K., Noack, S., Bott, M., & Marienhagen, J. (2016). Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum. Applied microbiology and biotechnology, 100(4), 1871-1881

Marienhagen, J., & Bott, M. (2013). Metabolic engineering of microorganisms for the synthesis of plant natural products. Journal of biotechnology, 163(2), 166-178.

van Ooyen, J., Noack, S., Bott, M., Reth, A., & Eggeling, L. (2012). Improved L‐lysine production with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity. Biotechnology and bioengineering, 109(8), 2070-2081.

Vogt, M., Haas, S., Klaffl, S., Polen, T., Eggeling, L., van Ooyen, J., & Bott, M. (2014). Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction. Metabolic engineering, 22, 40-52.