Sustainable Production of <sc>l</sc>-Homoserine Solely from CO2-Derived Acetate and Formate by Engineered E. coli Strain

l-Homoserine is proven to be a promising precursor for the synthesis of l-phosphinothricin, which is a broad-spectrum herbicide with low toxicity. To enhance the sustainability of the process, the utilization of nonfood carbon sources for l-homoserine production is highly sought-after. Acetate, which can be captured from C1 gases, has the potential to become the most abundant feedstock for biomanufacturing. In this study, we systematically engineered the Escherichia coli strain to generate l-homoserine from acetate. The production of l-homoserine from acetate was initially achieved by deleting branch pathways, activating the glyoxylate shunt, and enhancing the synthetic pathway. The strain's tolerance and acetate assimilation capabilities were strengthened through adaptive laboratory evolution. Reducing the flux toward gluconeogenesis further facilitated the production of l-homoserine. Finally, the engineered strain produced 15.96 g/L l-homoserine solely from CO2-derived acetate and formate in a 5 L fermentor via a fed-batch process. This study provides insights into the development of a low-carbon bioeconomy for the herbicide industry.