Enhancement of L-arginine production by increasing ammonium uptake in an AmtR-deficient Corynebacterium crenatum mutant.

L-Arginine is an important amino acid with extensive application in the food and pharmaceutical industries. The efficiency of nitrogen uptake and assimilation by organisms is extremely important for L-arginine production. In this study, a strain engineering strategy focusing on upregulate intracellular nitrogen metabolism in Corynebacterium crenatum for L-arginine production was conducted. Firstly, the nitrogen metabolism global transcriptional regulator AmtR was deleted, which has demonstrated the beneficial effect on L-arginine production. Subsequently, this strain was engineered by overexpressing the ammonium transporter AmtB to increase the uptake of NH4+ and L-arginine production. To overcome the drawbacks of using a plasmid to express amtB, Ptac, a strong promoter with amtB gene fragment, was integrated into the amtR region on the chromosome in the Corynebacterium crenatum/ΔamtR. The final strain results in L-arginine production at a titer of 60.9 g/L, which was 35.14% higher than that produced by C. crenatum SYPA5-5.

Improved L-ornithine production in Corynebacterium crenatum by introducing an artificial linear transacetylation pathway.

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.