High Efficient Fermentative Production Of A New Bio-material Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a novel completely biodegradable material belonging to the large family of polyhydroxyalkanoates (PHA). For its excellent material properties and biocompatibility which are strongly related to the 3-hydroxyhexanoate (3HHx) content in the polymer, PHBHHx has great potential in various applications. In this paper, Aeromonas hydrophila 4AK4 was chosen as the host strain to study the PHBHHx synthesis and production. It was found that addition of 4 g L-1 butanol to the culture media could decrease 3HHx fraction from 14.4 mol% to 5.8 mol%. In order to freely control the monomer composition of PHBHHx synthesized by Aeromonas hydrophila 4AK4, phbA gene encoding β-ketothiolase and phbB gene encoding acetoacetyl-CoA reductase were introduced into Aeromonas hydrophila 4AK4. In flask culture and fermentor culture, it was possible to reduce the 3HHx fraction in PHBHHx from about 15 mol% in the wild type to 3-12 mol% in the recombinant strain by simply changing the ratio of gluconate to dodecanoate in the culture media. These results showed the possibility for fermentative production of PHBHHx with controllable monomer composition. Further study on the optimization of the fermentation culture media indicated that phosphorus limitation could increase PHBHHx content.Based on the above result, vgb gene encoding vitreoscilla hemoglobin protein and phbA phbB genes were introduced into A. hydrophila 4AK4, the new recombinant strain A. hydrophila 4AK4 (pVGAB) could efficiently synthesize PHBHHx with controllable monomer composition. In flask culture using co-substrates of 1:1 dodecanoate to sodium gluconate as carbon sources, it could produce 20.5 g L-1 dry cell weight (DCW) containing 49.6 wt% PHBHHx. The PHBHHx productivity was 85.7% and 148.5% more than the recombinant A. hydrophila 4AK4 only harboring phbA, phbB genes and the wild type, respectively. Fermentation culture in 6L fermentor also indicated that the expression of vgb gene in 4AK4 resulted in much more rapid cell growth and PHBHHx production. When 1:1 co-substrates were used as carbon sources, 38.4 g L-1 DCW containing 52 wt% PHBHHx with 7.4 mol% 3HHx fraction was obtained in 42 hours of fermentation culture in 6L fermentor; when only dodecanoate was used, 54.0 g L-1 DCW containing 52.7 wt% PHBHHx with 12.4 mol% 3HHx fraction was obtained in 36 hours. Further study on plasmid stability during high cell density culture revealed vgb expression in A. hydrophila 4AK4 could stabilize plasmid, which would have remarkable significance for large-scale culture.