Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle

Abstract

Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO 2 -fixing system. Herein, EcN was equipped with a simultaneous CO 2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO 2 -assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the pf kAB and zwf genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO 2 fixation. The CO 2 -fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO 2 release by 77%. CO 2 fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.

Utilizing Ribose 1,5-Bisphosphate Isomerase and RuBisCO-Equipped Escherichia coli Nissle for Low-Carbon Footprint GABA Production

“…Taken together, these results aimed to integrate the CO 2 assimilation pathway in EcN for greener GABA production. The artificial CO 2 -fixing system was reconstructed via R15P by synergizing R15Pi from Thermococcus kodakarensis and RuBisCO from Synechococcus elongatus PCC6301, which has successfully enhanced carbon dioxide assimilation for higher 5-aminolevulinic acid production recently . Intriguingly, EcN was identified to possess a unique R15P-producing gene compared with other E. coli lineages.…”