Microbial production of polyhydroxyalkanoates

Abstract

Recent interest in biodegradable plastics has prompted growing concern over the recalcitrance of pollutants created by many of the current producers of synthetic polymers. Many countries are introducing more and more stringent environmental regulations on plastic usage. From 1986-1998, about 15% of total domestic, commercial and industrial waste in Hong Kong was petroleum-derived plastics. Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates that are biodegradable, biocompatible, microbial thermoplastics and are regarded as potentially useful in replacing conventional petroleum-derived thermoplastics. Two PHA producing strains were isolated from activated sludge. Both strains produced PHB most effectively when hydrolyzed malt waste was used as a medium. The PHB can reach up to 37% of cell dry weight. Only homopolymer PHB has accumulated in the cells as other carbon substrates were used and the isolated bacteria were characterized by DNA sequence alignment and PHB inclusion body-associated proteins. The two most abundant proteins have a molecular mass of approximately 20 kDa and 41 kDa, respectively, and are found in all three strains. The biopolymers accumulated were analyzed by GC, 1H NMR, DSC and FT-IR. Bacillus strains are widely used as industrial production organisms. Bacillus subtilis is a Gram-positive non-PHA producer that is nevertheless easy to grow at very high cell density when utilizing inexpensive carbon and nitrogen sources. It is the best-studied member of the genus Bacillus and well characterized in both genetics and physiology. It is generally recognized as nonpathogenic and does not produce endotoxins. Bacillus megaterium, the first PHA-producing strain to be discovered, was identified in the 1920s. In our laboratory, the B. megaterium PHA biosynthetic genes were isolated and cloned into a ?-105 prophage-based B. subtilis expression system that was shown to be good for protein overproduction. We demonstrated for the first time that the PHA biosynthetic genes from B. megaterium were functionally expressed in this B. subtilis system. Because the expression in the recombinant B. subtilis without heat shock was controlled by the native promoters of the pha genes PQRBC, the PHA yield was not very high and the experiments also suggested that the pha PQ genes were essential for the PHA production. Heat shock process in B. subtilis 1A304 (?105 MU331) could trigger its strong phage promoter. However, the recombinant B. subtilis showed a PHA accumulation after fermentation with the yield lower than the same strain without heat shock. This finding suggests that the heat shock process might impose adverse effects on the expression of the pha genes. The use of recombinant B. subtilis together with inexpensive carbon sources, such as soy and malt wastes, as culture media in our laboratory would make PHA production economical and allow easier commercialization of PHA. The pha genes were inserted into the vector pYCL18 and finally transformed into B. subtilis 168. The pha genes were not integrated into the chromosomal DNA and resulted in a multi-copy of the genes and a higher PHA yield which was expected since the copy number of the pha genes is directly proportional to the yield. The expression showed that there was no PHA in the recombinant strains. The Pseudomonas pseudoalcaligenes HBQ06 PHA synthase 1 (phaCl) gene was cloned into the vector pKS- and pUC19 with pha AB genes of Ralstonia. eutrophus and expressed in E. coli LS1298. The target of the subcloning was to produce short chain length and medium chain length PHA copolymers. However, there was also no PHA accumulation. There might be some reasons for the absence of PHA accumulation in both cases, such as plasmid stability and mutation on PCR. The sub-cloning and expression of the phaCAB Gene in E. coli XL1-Blue and HMS174 were successful. The expression showed that the majority of biopolymer accumulated in cells was PHB and its yield was much higher in E. coli HMS174/PHA than that in E. coli XL1-Blue/PHA. In an experiment using fed-batch fermentation in a computer-controlled 3L fermenter, the cell dry weight could reach 10.27 g/L at 59 h and the P(HB-HV) content, as analyzed by GC, could reach 45% (g/g) of the cell dry weight.