Engineered Corynebacterium glutamicum as an endotoxin-free platform strain for lactate-based polyester production.

The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D: -lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by ß-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.

Improved polyhydroxybutyrate (PHB) production in transgenic tobacco by enhancing translation efficiency of bacterial PHB biosynthetic genes.

Polyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves.