Development of recombinant escherichia coli strain and the process for the production of poly(3-hydroxyalkanoate)

Abstract

Polyhydroxyalkanoates (PHAs) including poly(3-hydroxybutyrate) [P(3HB)], the most well known member of PHAs, can be produced from renewable source and are biodegradable with similar material properties and processibility to conventional plastic material. From the studies on the design and economic evaluation of the processes for the production of PHAs, it was found that the PHA productivity, PHA content and recovery method are the most important factors determining the overall production cost. Recombinant Escherichia coli harboring the heterologous PHA biosynthesis genes has several advantages over other wild-type PHA producers including Ralstonia eutropha and Alcaligenes latus. But, the highest P(3HB) productivity obtained by the fed-batch culture of recombinant E. coli was lower than that achieved by of A. latus. It was reasoned that the efficient synthesis of P(3HB) in A. latus is due to the greater catalytic power of the PHA biosynthetic enzymes in this bacterium. Therefore, economical production of P(3HB) by recombinant E. coli was thought to be possible if P(3HB) productivity can be increased by cloning the A. latus PHA biosynthesis genes. A 6.4-kb A. latus genomic DNA (AL64) coding for the PHA biosynthetic enzymes were cloned in E. coli by screening the transformants that appeared as opaque colonies in LB medium containing glucose. The nucleotide sequence analysis of the cloned AL64 fragment indicated that phaCAl, phaAAl and phaBAl genes, coding PHA synthase, ketothiolase and reductase, respectively, were clustered in a single operon (phaCAB) as in R. eutropha. Several stable recombinant plasmids containing the A. latus PHA biosynthesis genes were constructed. P(3HB) production rate was higher for recombinant E. coli strains harboring the A. latus PHA biosynthesis genes compared with recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes. Fed-batch cultures of XL1-Blue(pJC3) and XL1-Blue(pJC4), recombinant E. coli strains harboring ...