The accD3 gene for mycolic acid biosynthesis as a target for improving fatty acid production by fatty acid-producing Corynebacterium glutamicum strains

Abstract

We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers.A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3 A433T ), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3 A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3 A433T , increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3 A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3 A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.

A Futile Metabolic Cycle of Fatty Acyl Coenzyme A (Acyl-CoA) Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production

“…These data suggest that fadD5 and fadD15 play significant roles in getting free fatty acids back to their CoA derivatives. Although the pCfadD32 carrier showed a 15% decrease in production, we believed that this was probably due to the redirection of carbon into mycolic acid synthesis, considering the predicted role of FadD32, namely, activation of free fatty acids to form acyl-AMP, a precursor for mycolic acid synthesis ( 14 , 30 ).…”

“…To date, however, there have been no reports on their functions, except for fadD32 , which is located in a chromosomal cluster with accD3 and pks (Fig. S1A) and has been assumed to be involved in the synthesis of mycolic acids by activating free fatty acids to form acyl-AMP ( 14 , 30 ). On the other hand, C. glutamicum possesses two annotated tes genes, tesB (Cgl1664, NCgl1600) and tesA (Cgl2451, NCgl2365), whose functions remain to be clarified (Fig.…”

“…The TesFad method differs in production mechanism from the general method, namely, deregulation of fatty acid synthesis, and is expected to be a new strategy for fatty acid production in C. glutamicum . To substantiate the method, we applied it to our best fatty acid producer, PCCA-3 ( 14 ). This producer was developed by assembling four positive mutations ( fasR20 , fasA63 up , fasA2623 , and accD3 A433T ) in the wild-type genome, and it has the capability of producing approximately 600 mg/liter of fatty acids, which consist mainly of oleic acid (449 mg/liter), palmitic acid (133 mg/liter), and stearic acid (40 mg/liter), in flask cultivation with 1% glucose ( Fig.…”

“…Our group and others have developed the amino acid-producing microorganism Corynebacterium glutamicum as another platform for the production of fatty acids and their related compounds (9)(10)(11)(12)(13)(14). With respect to fatty acid metabolism, this organism has at least three inherent properties that are distinct from those of E. coli (Fig.…”

Evolutionary engineering of Corynebacterium glutamicum

“…While Jiang et al did not perform whole genome sequencing, Takeno et al reported three mutations in the genes responsible for fatty acid synthesis. Takeno et al repeated the selection step on cerulenin plates and identified one additional mutation. Re‐engineering of this mutation in the previously evolved strain resulted in a 1.2‐fold higher oleic acid titer.…”