L-Homoserine is an important non-proteinogenic amino acid widely used in the synthesis of various high-value chiral chemicals, including glufosinate-P, 2,4-dihydroxybutyric acid, 1,4-butanediol, and L-methionine. In recent years, microbial synthesis of L-homoserine has advanced rapidly; however, most production strains rely on plasmid-based expression systems or auxotrophic genetic backgrounds, which face critical bottlenecks in industrial applications, including poor genetic stability and limited scalability.

Recently, the research team led by YU Bo at the Institute of Microbiology, Chinese Academy of Sciences, published a research article titled "Boosting L-homoserine production by non-auxotrophic E. coli strain via iterative rational design and tolerance engineering" in Metabolic Engineering. In this study, the team established a new-generation plasmid-free, non-auxotrophic E. coli strain for high-level L-homoserine production through a combined strategy of iterative rational design and tolerance engineering. Using a low-cost minimal salt medium for fermentation, the strain achieved the highest titer and productivit y reported to date.

Building on the previously constructed L-homoserine-producing strain HS15 (Metabolic Engineering, 2021), the team first eliminated multiple amino acid auxotrophies through dynamic regulation of competing essential amino acid biosynthetic pathways. They then employed a rationally designed 5′ untranslated region (5′UTR) library to fine-tune expression of the key pathway gene thrA*, achieving chromosomal single-copy expression levels that surpassed those of plasmid-based systems, and yielding the plasmid-free, non-auxotrophic strain GHS03. Next, the team combined genome-wide mutagenesis with adaptive laboratory evolution to rapidly increase tolerance to L-homoserine beyond 100 g/L. After fermentation process optimization, strain NS18 produced 144.5 g/L of L-homoserine in 48 hours using low-cost minimal salt fermentation medium, with a yield of 0.48 g/g. Finally, the team employed comparative genomics, transcriptomics, and metabolite profiling to elucidate the molecular mechanisms underlying the evolved strain's high productivity and tolerance.

The results revealed that inactivation of key genes such as kdgK and mobB played a critical role in enhancing tolerance and production. In the evolved strain NS18, key genes in the tricarboxylic acid cycle and pentose phosphate pathway were significantly upregulated, and intracellular NADPH levels were markedly elevated, providing ample reducing power for efficient L-homoserine biosynthesis. Furthermore, the upregulation of branched-chain amino acid biosynthetic pathways suggested that the strain acquired improved physiological adaptation under high-concentration product stress.

This study effectively overcame the product toxicity challenge in L-homoserine biosynthesis and proposed a systematic cell factory optimization strategy integrating rational design with non-rational evolution, laying the technical foundation for the industrial production of L-homoserine and its downstream derivatives.

This research was supported by grants including the Beijing Municipal Science and Technology Program.

High-yield and tolerance mechanisms of strain NS18 (image by Prof. YU Bo’s group)

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