S-Adenosyl-L-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although L-methionine is generally used, the low-cost DLmethionine is more suitable as the substrate for industrial production of SAM. However, Dmethionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of DL-methionine, intracellular conversion of D-methionine to L-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding D-amino acid-N-acetyltransferase, D-methionine was accumulated in vivo and no N-acetyl-D-methionine production was observed. Further, codon-optimized D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and L-phenylalanine dehydrogenase gene (L-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert D-methionine to L-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L Dmethionine. Using the recently developed CRISPR tools, the DAAO and L-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L DL-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L DL-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost DL-methionine.
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