Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

Abstract

The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

Fermentative production of L‐pipecolic acid from glucose and alternative carbon sources

“…In this pathway, L‐lysine is oxidatively deaminated by L‐lysine 6‐dehydrogenase to yield α‐aminoadipic semialdehyde. After spontaneous cyclization to 1‐piperideine 6‐carboxylic acid (P6C), pyrroline 5‐carboxylate reductase, an enzyme of L‐proline biosynthesis encoded by proC , reduces P6C to L‐PA . We have previously shown that this pathway can be used for fermentative production of L‐PA by recombinant C. glutamicum .…”

Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate l-pipecolic acid in Escherichia coli

“…l -PA can be biosynthesized using crude enzymes or whole-cell biocatalysts [14, 15, 19], and several of the different catalytic steps involved have been investigated, including some that are unstable and accompanied by high manufacturing costs that limit their applications. However, few researchers have focused on the direct fermentation of l -PA, even though fermentation potentially offers an alternative, efficient, low cost method [22]. In the present study, we expanded the E. coli metabolic pathway by introducing a heterologous lysine cyclodeaminase that can biosynthesize l -PA directly from glucose.…”

“…Wendisch et al, previously attempted to develop the l -PA producers by engineering the industrial lysine producer C. glutamicum to fermentatively synthesized l -PA from glucose via the P6C pathway through heterologous expression of lysine dehydrogenase ( lysDH ) and pyrroline 5-carboxylate reductase ( proC ) [22]. Although they fully optimized the glucose uptake system and achieved an acceptable level of by-product elimination, the l -PA titer, yield and productivity were low (below 1.9 g/L or 15 mM, 0.1 g/g and 0.05 g/L h, respectively).…”

“…The catalytic efficiency of LCD has been described as poor by various researches [22]. For example, Wendisch et al noted that in comparison with the P6C pathway, the relatively low turnover number of LCD may limit l -PA biosynthesis.…”

“…Compared with traditional catalytic processes, fermentation can simplify the production procedures, potentially lowering cost and improving efficiency [21]. Very recently, Wendisch et al described a fermentation method for the production of l -PA from glucose via the P6C pathway that include two enzymes and a spontaneous chemical reaction in Corynebacterium glutamicum , achieving a l -PA titer of 14 mM (1.73 g/L) [22]. Given the complexity if the l -PA biosynthesis pathway (P6C), it may be possible to improve this method considerably.…”