Targeted silencing of exopolysaccharide biosynthesis genes via CRISPR/dCas9 to enhance polyhydroxybutyrate production from biodiesel-derived crude glycerol in Burkholderia sp.

The newly isolated Burkholderia sp. SCN-KJ, which is capable of simultaneously producing intracellular polyhydroxybutyrate (PHB) and exopolysaccharide (EPS) from biodiesel-derived crude glycerol, presented a significant challenge in this study because of interference from EPS synthesis. The bceA gene, which encodes a bifunctional protein responsible for mannose-6-phosphate isomerization and the mannose-1-phosphate guanylyltransferase reaction, was identified as a key regulator of EPS production. To address this issue, CRISPR interference (CRISPRi) was employed to target the bceA gene, leading to the complete suppression of EPS synthesis and a significant improvement in PHB yield. Under optimized culture conditions, the ΔbceA mutant strain exhibited complete inhibition of EPS production, resulting in a 1.75-fold increase in the PHB concentration and PHB of a higher molecular weight than that of the wild-type Burkholderia sp. SCN-KJ. These results highlight the effectiveness of dCas9-mediated silencing of the bceA gene in overcoming the challenges posed by EPS interference and underscore its potential for increasing PHB production in Burkholderia sp. SCN-KJ.