O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum

Abstract

Background O-Acetyl-L-homoserine (OAH) is an important potential platform chemical. However, low levels of production of OAH are greatly limiting its industrial application. Furthermore, as a common and safe amino acid-producing strain, Corynebacterium glutamicum has not yet achieved efficient production of OAH. Results First, exogenous L-homoserine acetyltransferase was introduced into an L-homoserine-producing strain, resulting in the accumulation of 0.98 g/L of OAH. Second, by comparing different acetyl-CoA biosynthesis pathways and adding several feedstocks (acetate, citrate, and pantothenate), the OAH titer increased 2.3-fold to 3.2 g/L. Then, the OAH titer further increased by 62.5% when the expression of L-homoserine dehydrogenase and L-homoserine acetyltransferase was strengthened via strong promoters. Finally, the engineered strain produced 17.4 g/L of OAH in 96 h with acetate as the supplementary feedstock in a 5-L bioreactor. Conclusions This is the first report on the efficient production of OAH with C. glutamicum as the chassis, which would provide a good foundation for industrial production of OAH.

Identification of key genes through the constructed CRISPR-dcas9 to facilitate the efficient production of O-acetylhomoserine in Corynebacterium glutamicum

“…The key genes of competition and degradation pathways including mcbR , thrB , metB , and metY were knocked out to obtain a L -homoserine-producing strain. The exogenous l -homoserine acetyltransferase variant MetX r from Leptospira meyeri was introduced to obtain an engineered strain Cg17 ( Figure 1 ), which accumulated 0.98 g/L of OAH after 48 h. To drain pyruvate flow to the L -aspartate biosynthesis pathway, reduce the use of pyruvate for the acetyl-CoA biosynthesis and promote the OAH accumulation, the expression of bifunctional L -aspartokinase and L -homoserine dehydrogenase (ThrA S345F ) from E. coli and MetX r were enhanced through the plasmid pEC-XK99E to generate strain Cg17-3 ( Li et al, 2022 ). With the addition of 5 g/L acetate at 24 and 36 h, the resulting strain Cg17-3 produced 5.2 g/L of OAH at 48 h. Although the C. glutamicum achieved efficient OAH biosynthesis, few of the limiting factors of OAH biosynthesis were identified.…”

“…The strains culture conditions were described as our previous study ( Li et al, 2022 ). For the shake flask culture, the engineered C. glutamicum strains were precultured in seed medium, cultured in fermentation medium with 4% inoculum.…”

“…To drain pyruvate flow to the L-aspartate biosynthesis pathway, reduce the use of pyruvate for the acetyl-CoA biosynthesis and promote the OAH accumulation, the expression of bifunctional L-aspartokinase and L-homoserine dehydrogenase (ThrA S345F ) from E. coli and MetX r were enhanced through the plasmid pEC-XK99E to generate strain Cg17-3 (Li et al, 2022). With the addition of 5 g/L acetate at 24 and 36 h, the resulting strain Cg17-3 produced 5.2 g/L of OAH at 48 h. Although the C. glutamicum achieved efficient OAH biosynthesis, few of the limiting factors of OAH biosynthesis were identified.…”

“…Phosphoenolpyruvate and pyruvate produced by glycolysis are precursors of l -aspartic acid biosynthesis ( Piao et al, 2019 ) and there are many pathways to consume l -aspartic acid and acetyl-CoA ( Yin et al, 2012 ; Milke et al, 2019 ; Sagong et al, 2022 ). Currently, regulating the expression of genes related to the L -aspartate metabolic pathway and strengthening the biosynthesis of acetyl-CoA are the main strategies to improve the production of OAH in Corynebacterium glutamicum and Escherichia coli ( Wei et al, 2019 ; Li et al, 2022 ). However, these cannot further improve the titer and yield of OAH, especially in C. glutamicum .…”

“…However, in addition to regulating the L -aspartate metabolic pathway, the lack of research on other pathways such as glucose transport, glycolysis and TCA cycle has hampered the strain improvement in the accumulation of OAH. Previous studies found that identifying the limiting factors of OAH biosynthesis by overexpression and knockout of a single gene in C. glutamicum was a very time- and labor-consuming method ( Li et al, 2022 ), and thereby the application of a more efficient way to quickly identify the limiting step of OAH biosynthesis would be very significant.…”