Solaiman D K, Ashby R D, Foglia T A
US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA.
Appl Microbiol Biotechnol. 2001 Sep;56(5-6):664-9. doi: 10.1007/s002530100692.
Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.
恶臭假单胞菌和食油假单胞菌因其合成中链长度（mcl）聚羟基脂肪酸酯（PHA）而受到广泛研究。这些细菌无法代谢三酰甘油（TAGs）。我们构建了能够利用TAGs作为生长和mcl-PHA合成底物的重组恶臭假单胞菌和重组食油假单胞菌。携带假单胞菌脂肪酶基因的重组质粒pCN51lip-1用于对这些生物体进行电转化。如罗丹明B荧光平板试验所示，转化体表现出TAG水解活性。转基因生物体在含TAG的培养基中生长至细胞干重为2 - 4 g/l。重组恶臭假单胞菌以猪油或椰子油（Co）为底物，粗产率为0.9 - 1.6 g/l生产mcl-PHA。虽然食油假单胞菌转化体不产生mcl-PHA，但野生型和重组菌株的混合培养发酵方法可从Co生产聚合物，粗产率为0.5 g/l。气相色谱/质谱组成分析表明，β-羟基辛酸（31 - 45 mol%）和β-羟基癸酸（28 - 35 mol%）是基于TAG的PHA的主要重复单元。通过凝胶渗透色谱法测定的PHA的数均分子量和重均分子量分别为82 - 170×10³ g/mol和464 - 693×10³ g/mol。这种重组方法可以大大增加可用于从油脂底物生产PHA的生物体数量。
