Sustainable Polyhydroxybutyrate Production via Metabolic Flux Redirection Using Clustered Regularly Interspaced Short Palindromic Repeats Interference in Escherichia coli and Carbon Capture with Microalgae

Abstract

The pursuit of biobased alternatives to petrochemical plastics has been driven by the oil crisis and climate change challenges, with polyhydroxybutyrate (PHB) emerging as a promising candidate due to its biodegradability and material properties. This study leverages metabolic engineering strategies in Escherichia coli WT7L to enhance PHB production. By employing the phaCAB operon from Caldimonas manganoxidans under a T7 promoter and implementing clustered regularly interspaced short palindromic repeats interference (CRISPRi)-mediated gene downregulation, we systematically optimized metabolic pathways. Target genes (ldhA, poxB, and pta) were selected for downregulation based on their roles in competing pathways that divert acetyl-CoA from PHB synthesis, leading to a 1.3-fold increase in PHB production, reaching 1.94 g/L. However, further inhibition of adhE, arcA, and gltA disrupted metabolic balance, reducing the PHB titer to 0.7 g/L. Integrative enhancements using GroELS on the chromosome boosted biomass and PHB titer by 3.06-fold in the optimal CRISPRi design. Further expression of phosphoenolpyruvate carboxylase (ppc) for CO₂ assimilation elevated the PHB yield to 3.1 g/L. Finally, fed-batch fermentation significantly increased biomass to 17.06 g/L and PHB titer to 5.6 g/L after 72 h. Notably, released CO₂ was utilized to cultivate Chlorella sorokiniana, reaching 2.26 g/L for the first time. Our results demonstrate a novel closed-loop bioprocess with the potential for net-zero emissions in sustainable biopolymer production.