Abstract
l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose.IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.
Highlights
L-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals
The L-homoserine-converting pathway was further strengthened by overexpression of thrA to “push” the carbon flux to L-homoserine production
The disruption of L-lysine biosynthesis increased the production of HS2 to 2.01 g/liter with the same specific production of 0.33 g/g cell dry weight (CDW) when L-lysine (0.025 g/liter, optimized amount) was added
Summary
L-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. Due to the lack of optimization upstream of glycolysis, as well as branched pathways [11], L-homoserine was produced with a low yield by mutant and metabolically engineered strains, including Corynebacterium glutamicum [12] and Escherichia coli [13, 14]. Some strategies have been applied to optimize the biosynthetic pathways, which consist of large numbers of genes, or to reprogram gene expression to manipulate complex phenotypes Prominent examples of such methods include the multidimensional heuristic process (MHP) [20], multiplex navigation of global regulatory networks (MINR) [21], and multibranched and multilevel regulated biosynthetic pathways (MBMRPs) [22], which rapidly engineer organisms with desired capabilities without the trial and error of iterative experimentation. “omics” profiling technologies, such as metabolomics, have facilitated an overview of cell metabolism, allowing a more in-depth insight into intracellular mechanisms in modified-organism analysis [23]
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