Polyhydroxyalkanoate production from sucrose by Cupriavidus necator strains harboring csc genes from Escherichia coli W.

Cupriavidus necator H16 is the most promising bacterium for industrial production of polyhydroxyalkanoates (PHAs) because of their remarkable ability to accumulate them in the cells. With genetic modifications, this bacterium can produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which has better physical properties, as well as poly(3-hydroxybutyrate) (PHB) using plant oils and sugars as a carbon source. Considering production cost, sucrose is a very attractive raw material because it is inexpensive; however, this bacterium cannot assimilate sucrose. Here, we used the sucrose utilization (csc) genes of Escherichia coli W to generate C. necator strains that can assimilate sucrose. Especially, glucose-utilizing recombinant C. necator strains harboring the sucrose hydrolase gene (cscA) and sucrose permease gene (cscB) of E. coli W grew well on sucrose as a sole carbon source and accumulated PHB. In addition, strains introduced with a crotonyl-CoA reductase gene (ccr), ethylmalonyl-CoA decarboxylase gene (emd), and some other genetic modifications besides the csc genes and the glucose-utilizing mutations produced PHBHHx with a 3-hydroxyhexanoate (3HHx) content of maximum approximately 27 mol% from sucrose. Furthermore, when one of the PHBHHx-producing strains was cultured with sucrose solution in a fed-batch fermentation, PHBHHx with a 3HHx content of approximately 4 mol% was produced and reached 113 g/L for 65 h, which is approximately 1.5-fold higher than that produced using glucose solution.

A study on the relation between poly(3-hydroxybutyrate) depolymerases or oligomer hydrolases and molecular weight of polyhydroxyalkanoates accumulating in Cupriavidus necator H16.

Cupriavidus necator H16 has nine genes of poly(3-hydroxybutyrate) (PHB) depolymerases or oligomer hydrolases (intracellular PHB mobilization enzymes). In this study, we evaluated the relation between these genes and the accumulation, consumption, and molecular weight of polyhydroxyalkanoates (PHAs) accumulating in strain H16 and in a recombinant C. necator strain, KNK-005, which harbors an NSDG mutant of the PHA synthase gene (phaCAc) from Aeromonas caviae. PhaZ6 had a significant influence on the molecular weight of PHA when palm kernel oil was used as a carbon source. The 005dZ6 strain (ΔphaZ6 mutant of KNK-005) could produce ultra-high-molecular-weight poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with weight-average molecular weight (Mw) >3.0×10(6) (approximately double that of KNK-005). Under PHA consumption conditions, deletion of phaZ1 and phaZ2 had a significant and slight attenuating effect, respectively, on the reduction in PHA content of KNK-005 cells. Regardless of the PHA consumption, its Mw did not decrease. Thus, 005dZ126 (the ΔphaZ1ΔphaZ2ΔphaZ6 triple mutant of KNK-005) is a promising strain capable of producing PHBHHx of ultra-high-molecular-weight and barely degrades PHBHHx enzymatically intracellularly. This is the first report examining the relation between intracellular PHB mobilization enzymes and molecular weight of PHAs accumulating in C. necator H16 and the derivatives.

Regulation of 3-hydroxyhexanoate composition in PHBH synthesized by recombinant Cupriavidus necator H16 from plant oil by using butyrate as a co-substrate.

A (R)-3-hydroxyhexanoate (3HH) composition-regulating technology for poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) production was developed using recombinant Cupriavidus necator H16 with butyrate as a co-substrate. A new (R)-3-hydroxyhexanoyl-CoA ((R)-3HH-CoA) synthesis pathway was designed and enhanced by replacing the PHA synthase gene (phaC1) of C. necator by the phaCAcNSDG (encoding the N149S and D171G mutant of PHA synthase from Aeromonas caviae) and deactivation of the phaA gene (encoding (ß-ketothiolase) from C. necator H16 chromosome). The effect of butyrate as co-substrate was assessed in high-cell-density fed-batch cultures of several C. necator mutants, and the 3HH fraction was successfully increased by adding butyrate to the culture. Moreover, overexpression of BktB (encoding the second ß-ketothiolase with broad substrate specificity) enhanced the (R)-3HH-CoA synthesis pathway in the phaA deactivated mutant of C. necator by promoting the condensation of acetyl-CoA and butyryl-CoA into 3-ketohexanoyl-CoA. Consequently, PHBH containing 4.2-13.0 mol% 3HH was produced from butyrate and palm kernel oil by the genetically modified C. necator H16 strains.