| Polyhydroxyalkanoates (PHAs) are a kind of biological polyester, which are produced by some bacteria under the condition of rich carbon source and nitrogen source. It has good biodegradable, biocompatible, water-repellent. The different functional groups are brought by the unique nature, and were used as drug delivery materials and tissue engineering materials. PHA are a good potential for development of biological materials. However, PHA biosynthesis can not satisfy the demand for new biomaterials. The wide application of PHA was seriously restricted by it’s single component, low production and high costs.At present, the microbial fermentation is the main method of PHA production, How to reduce the production cost of PHA and how to change the PHA component in order to change the physical and chemical properties of PHA, it has become a hot topic. The composition and content of PHA were affected by the composition of the substrate. The cost of the substrate can account for 28-50% of the total cost. Crude glycerol is the mainly by-product in the production process of biodiesel. PHA synthesis from glycerol can not only solve the problems of environmental pollution which are brought in the production process of biodiesel with excessive accumulation of waste water, exhaust and others, but also reduce the cost of production of the PHA.PHA synthase is a key enzyme in the process of PHA synthesis, which determines the type of PHA and the yield of PHA. It is feasible to change the PHA monomer composition and improve the yield of PHA by modifying the PHA synthase. Genetic engineering is the most widely used technology in enzyme modification. Through the integration of two or more PHA enzymes to construct chimeric enzyme, and to explore the molecular mechanism of PHA synthase transformation has become the focus of many scholars. The change of monomer composition and improving the catalytic activity are hoped when we remoulded the PHA synthase in the level of molecular. Through the integration of two or more PHA enzymes to construct chimeric enzyme, and exploring the molecular mechanism of PHA synthase transformation have become a hot topic for many scholars.In this paper, two aspects of works have been done, on one hand, Pseudomonas oleovorans was used for the the study on the fermentation of glycerol and levulinic acid mixed carbon sources. Not only can make full use of the glycerol which is produced in the process of biodiesel production, but also can solve a series of environmental problems. On the other hand, P. oleovorans and R. eutropha were used as the starting bacteria to construct the type of I PHA chimeric enzymes. Pseudomonas corrugata and Pseudomonas putida were used as the starting bacteria to construct the type of II PHA chimeric enzymes. The results were as follows.(1) The optimization of fermentation medium of P. oleovorans. A series of MS medium containing different concentration gradient of glycerol and acetyl propionate were prepared for this experiment. The yield and composition of PHA were detected by shake flask fermentation and GC detection. Results showed that the P. oleovorans could produce copolymer PHB-PHV when cultivated in medium of glycerol and levulinic acid as mixed carbon source, and its composition and concentration of copolymer have a certain relationship to the mixed carbon source.(2) Construction of a novel I chimeric enzyme. R. eutropha has a high level of produce PHA, but can only to produce PHB. P.oleovorans could take a better use of glycerol to produce PHA, but its production capacity is poor. Secondary structure of PhaCRe and PhaCAh were predicted using Predtor, GOR4 and SOPMA softwares so as to find the proper junction sites. With this junction sites, a chimeric PHA synthase genes PhaCPoRe was obtained.After that, recombinant plasmid pBBR1MCS2-PhaCPoRe was constructed by ligating. Then recombinant plasmid was transformed into R. eutropha PHB-4 with conjunction. Recombinant was cultured on mineral salt (MS) medium supplemented with different carbon sources in shaking flask. Dry cell was collecting, weighing cells and detecting PHA content with GC. Results showed that the recombinant bacterium R. eutropha PHB-4-pBBR1MCS2-phaCPoRe could grow and produced PHA monomer when used suitable carbon source. The I type of chimeric enzyme produced 3HB only when fructose, glycerol and gluconate as the sole carbon source. While it produced a new monomer 3-HO except 3-HB when octanoate or lauric acid as sole carbon source. The experimental results showed that the type of I chimeric enzyme was active, and could make use of the suitable carbon source to produce PHA.(3) Construction of a novel II chimeric enzyme. To construct a functional chimeric enzyme, PHA synthase gene from the P. corrugata (PhaCpc) and P. putida (PhaCpp) were chosed as parental enzymes. The amino acid sequence secondary structure of PhaCpc and PhaCPp were predicted successfully by the online database Predtor, GOR4 and SOPMA in order to find the proper junction sites, With this junction sites, a chimeric PHA synthase genes PhaCPcPp was obtained. After that, recombinant pBBRlMCS2-PhaCPcPp pwas constructed by ligating. Then recombinant recombinant strain P. putida GPp 104-pBBRlMCS2-PhaCPcPp was cultured on mineral salt medium supplemented with different carbon sources in shaking flask. Dry cell was collecting, weighing cells and detecting PHA content with GC. Results showed the recombinant bacterium P. putida GPp 104-pBBR1MCS2-PhaCPcPp could grow and produced PHA monomer when used suitable carbon source. When added fructose as carbon source of the MS culture medium, could grow and produced 3-HDD, and when the oleic acid used as the carbon source, the recombinant strain could grow and produced of 3-HD. While it produced a different proportion of 3-HD and 3-HDD when using sodium oleate as the carbon source. A conclusion was drawed that the type of II PHA chimeric enzyme had the function of producing PHA.In summary, in this study, a series of MS medium containing different concentration gradient of glycerol and acetyl propionate were prepared for this experiment. The results showed that the P. oleovorans could produce copolymer PHB-PHV when glycerol and levulinic acid were used as mixed carbon source, and its composition and concentration of copolymer have a certain relationship to the mixed carbon source. Thus, it can not only solve environmental problems caused by excessive accumulation of byproducts of biodiesel, but also reduce the cost of production of the PHA. At the same time, two chiemic enzymes phaCPoRe and PhaCPcPp were constructed which could produced novel PHA when appropriate carbon sources were added. The study will laid a theoretical foundantion for the construction of chiemic enzyme by gene engineering techniques and will provide a method to improve PHA monomer composition. It will largely expand the applications of PHA in future. |
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