Decoupling sucrose utilization from oxygen-responsive regulation for high-efficiency L-lactic acid production in Escherichia coli

The shift toward sustainable biomanufacturing necessitates microbial platforms that efficiently convert low-cost, non-food feedstocks into high-value chemicals. Sucrose, a widely available and economical carbon source, remains underutilized in industrial Escherichia coli fermentation due to its low metabolic efficiency. This study investigates the production of L-lactic acid monomer in E. coli using sucrose, a cost-effective carbon source. Initially, we found that the recombinant strain 090S with the cscR gene knocked out exhibited an enhanced aerobic growth rate; however, during anaerobic fermentation for acid production, synthesis of the lactic acid monomer ceased after 3–4 h, indicating an impediment in sucrose metabolism under anaerobic conditions. Furthermore, we analyzed its transcriptional characteristics under aerobic-anaerobic phases through dynamic transcriptomic profiling and found significant differences. Specifically, for the csc operon, all three genes (cscB, cscK, and cscA) saw a significant decrease in expression when transferred into anaerobic conditions, retaining less than 10% of their aerobic expression levels. Here, we address this critical challenge by engineering optimized anaerobically active promoters to decouple sucrose utilization from native transcriptional constraints. Ultimately, the recombinant strain 091S, in which overexpresses the cscA and cscB genes by using the gapA promoter, produced 129.7 g/L of L-lactic acid in a 5-L bioreactor within 30 h of fermentation, with an average volumetric productivity of 4.32 g/(L·h), marking a 3.04-fold increase over the control. Additionally, an industrial fermentation process was simulated in a 30-L bioreactor under scaled-up conditions, resulting in a higher L-lactic acid yield of 145.7 g/L and a productivity of 4.96 g/(L·h), which was similar to that of glucose as a carbon source. This study elucidates the impact of oxygen content changes on gene transcription levels during the fermentation of E. coli using sucrose as a carbon source, offering a scalable and economically viable strategy for the efficient production of bio-products from sucrose or sucrose-rich feedstocks by E. coli.