Fermentative production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by recombinant Aeromonas hydrophila 4AK4 (pTG01) / 生物工程学报

ABSTRACT

Copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (PHBHHx), a new type of biodegradable material, are receiving considerable attentions recently. The material properties are strongly related to the 3HHx fraction of PHBHHx. As the 3HHx fraction increase, crystallinity and melting point of PHBHHx decrease, flexibility and tractility increase. PHBHHx of different 3HHx fraction can meet different demands of commercial application and research. Aeromonas are the best studied PHBHHx-producing strains. Recent studies have been focused on optimizations of fermentative culture media and culture conditions for low-cost and efficient fermentative production. Aliphatic substrates such as long-chain fatty acid and soybean oil were used in the PHBHHx fermentation as the sole carbon source and energy source. Two-stage fermentation method was also developed for more efficient PHBHHx production. While studies on Aeromonas hydrophila revealed that the monomer composition of PHBHHx could not easily be regulated by fermentative process engineering methods such as changing substrates and fermentative conditions because precursors involved in the PHBHHx synthesis were all from the beta-oxidation pathway. In this study, phbA gene encoding beta-ketothiolase and phbB gene encoding acetoacetyl-CoA reductase were introduced into a PHBHHx-producing strain Aeromonas hydrophila 4AK4 so as to provide a new 3HB precursors synthesis way. phbA gene encodes beta-ketothiolase which can catalyze two acetyl-CoA to form acetoacetyl-CoA; phbB gene encodes acetoacetyl-CoA reductase catalyzing acetoacetly-CoA into 3HB-CoA which is the precursor of 3HB. The introduced novel 3-hydroxybutyrate precursor synthesis pathway allowed the recombinant strain to use unrelated carbon source such as gluconate to provide 3HB precursors for PHBHHx synthesis. Shake-flask experiments were carried out to produce PHBHHx of controllable monomer composition and fermentations in 5 L fermentor were also proceeded for confirmation of these result in large-scale culture. In flask culture, it was possible to reduce the 3HHx mol fraction in PHBHHx from 15 % in the wild type to 3% - 12% in the recombinant by simply changing the ratio of gluconate to lauric acid in the culture media. When lauric acid was used as the sole carbon source, 51.5 g/L Cell Dry Weight (CDW) containing 62 % PHBHHx with 9.7 % 3HHx mol fraction was obtained in 56 hours of fermentation in a 5 liter fermentor. When co-substrates of sodium gluconate and lauric acid (11) were used as carbon sources, 32.8 g/L CDW containing 52 % PHBHHx with 6.7% 3HHx mol fraction was obtained in 48 hours of fermentation. These results showed the possibility for fermentative production of PHBHHx with controllable monomer composition.