Hang Xiaoming, Zhang Guang, Wang Guanlin, Zhao Xianghui, Chen Guo-Qiang
Department of Bioengineering, Dalian University of Technology, 116024, PR China.
FEMS Microbiol Lett. 2002 Apr 23;210(1):49-54. doi: 10.1111/j.1574-6968.2002.tb11158.x.
The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741. The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned. The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified. Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively. The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes. Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E. coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate. At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E. coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate. Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case. This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD.
先前为假单胞菌建立的用于II型聚羟基脂肪酸酯（PHA）生物合成基因的PCR克隆策略成功应用于石竹伯克霍尔德菌菌株AS 1.2741。包含PHA合酶基因phaC1、phaC2和PHA解聚酶基因phaZ的整个pha基因座被克隆。确定了phaC1（Bc）、phaC2（Bc）和phaZ（Bc）的完整开放阅读框。phaC1（Bc）、phaZ（Bc）和phaC2（Bc）的序列分析表明，与已知假单胞菌菌株的相应pha基因座相比，同一性分别超过77.7%、73.7%和68.5%。phaC1（Bc）或phaC2（Bc）在大肠杆菌菌株KM32B（fadB缺失突变体）中的功能表达显示了估计的PHA合酶基因产生PHA的能力。从在辛酸上生长的携带phaC1（Bc）的重组大肠杆菌KM32B（pHXM11）细胞中检测到超过1%由3-羟基己酸（3HHx）、3-羟基辛酸（3HO）和3-羟基癸酸（3HD）组成的PHA。同时，从在癸酸上生长的携带phaC2（BC）的重组大肠杆菌KM32B（pHXM21）细胞中产生了超过3%由3HO和3HD组成的PHA。结果表明，先前开发的PCR克隆策略可应用于非假单胞菌菌株，如本例中的伯克霍尔德菌。该结果也为伯克霍尔德菌菌株不仅拥有聚羟基丁酸合酶基因，还拥有由3HHx、3HO和3HD组成的中链长度聚羟基脂肪酸酯合酶的推测提供了证据。
