Impact of various ß-ketothiolase genes on PHBHHx production in Cupriavidus necator H16 derivatives.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active ß-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important ß-ketothiolases for C6 metabolism in ß-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six ß-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.

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.