An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain

Abstract

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMI Cg ::I Sm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDH Cg ) by NADP-GapDH Ca from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ signi cantly between these strains (14.1 ± 1.8 g/L v.s. 16.2 ± 1.1 g/L). Enhancing the carbon ux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icd Cg were introduced into C. glutamicum with NADP-GapDH Ca and pEC-ABNCE increased L-leucine yield (32.8 ± 1.7 g/L) and improved cell growth (OD 562 = 41.3 ± 4.2) because the resultant strain C. glutamicum XL-1 ΔMI Cg ::I Sm G Cg ::G Ca P zwf -D1 P icd -D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the rst report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.

Construction of a plasmid-free l-leucine overproducing Escherichia coli strain through reprogramming of the metabolic flux

“…However, despite the accumulation of l -leucine achieved with these above-mentioned strategies, the low titer and/or yield still limit translation of the process to the industrial scale. In addition, NADPH is also required in the l -leucine biosynthesis pathway and cofactor must be balanced to ensure efficient synthesis of l -leucine [ 12 ]. Wang et al achieved an l -leucine production of 23.31 g/L by converting the cofactor requirements of l -leucine biosynthesis and glutamate dehydrogenase from NADPH to NADH [ 13 ].…”