Abstract

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or P3HB4HB was first synthesized by Ralstonia eutropha grown in the presence of expensive precursors 4-hydroxybutyrate (4HB) or 1,4-butanodiol (BDO). Ratios of 3HB and 4HB in P3HB4HB could only be adjusted by controlling feeding of 4HB or BDO, leading to significant changing material properties from highly crystalline nature to very soft elastomers. It is particularly important to construct pathways for P3HB4HB production from low cost glucose and find tools to adjust ratios of 3HB to 4HB, in order to get various materials with targeted properties. The CRISPRi tool is comprised of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In this study, CRISPRi, a novel genetic tool recently developed for editing genomes of mammalian cells, was utilized for the first time to manipulate the monomer ratios of P3HB4HB, which is synthesized by E. coli grown in glucose as the only carbon substrate by down regulating single or multiple genes in the Tricarboxylic acid cycle (TCA cycle). A series of sgRNAs were designed to target five key genes in P3HB4HB synthesis pathway, namely, sdhA, sdhB, sucC, sucD, and sad, respectively. Simultaneous targetings via the sgRNAs were also examined. RT-PCR results indicated that the expression