Solaiman Daniel K Y, Ashby Richard D, Licciardello Grazia, Catara Vittoria
US Department of Agriculture, Eastern Regional Research Center, Agricultural Research Service, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA.
J Ind Microbiol Biotechnol. 2008 Feb;35(2):111-20. doi: 10.1007/s10295-007-0272-y. Epub 2007 Nov 7.
The complete sequence of the pha locus responsible for the biosynthesis of poly(hydroxyalkanoates) (PHAs) in Pseudomonas corrugata 388 was determined. As with the other known pseudomonad pha gene loci, the one in P. corrugata 388 also consists of phaC1 (1,680 bps; PHA synthase 1), phaZ (858 bp; PHA depolymerase) and phaC2 (1,683 bp; PHA synthase 2) genes. A BLAST search showed that the nucleotide sequences of these genes and the amino-acid sequences of their respective gene products are homologous to those of P. corrugata CFBP5454 and P. mediterranea CFBP5447. A putative intrinsic transcription terminator consisting of a dyad symmetry (24 bp; Delta G = -41.8 kcals) that precedes a stretch of dA residues was located in the phaC1-phaZ intergenic region. P. corrugata mutant-clones XI 32-1 and XI 32-4 were constructed in which this intergenic region was replaced with a selectable kanamycin-resistance gene. These mutant clones when grown on oleic acid for 48 h showed 4.7-to 7.0-fold increases of phaC1 and phaC2 relative expression in comparison to the initial inoculants, whereas the parental strain showed only 1.2- to 1.4-fold increases. Furthermore, in comparison to parental P. corrugata with only a few large PHA inclusion bodies, the mutants grown on oleic acid produce numerous smaller PHA granules that line the periphery of the cells. With glucose as a substrate, XI 32-1 and XI 32-4 clones produce mcl-PHA with a high content (26-31 mol%) of the mono-unsaturated 3-hydroxydodecenoate as a repeat-unit monomer. Our results show for the first time the effects of the phaC1-phaZ intergenic region on the substrate-dependent temporal expression of phaC1 and phaC2 genes, the repeat-unit composition of mcl-PHA, and the morphology of the PHA granules.
测定了皱纹假单胞菌388中负责聚羟基脂肪酸酯（PHA）生物合成的pha基因座的完整序列。与其他已知的假单胞菌pha基因座一样，皱纹假单胞菌388中的pha基因座也由phaC1（1680 bp；PHA合酶1）、phaZ（858 bp；PHA解聚酶）和phaC2（1683 bp；PHA合酶2）基因组成。BLAST搜索表明，这些基因的核苷酸序列及其各自基因产物的氨基酸序列与皱纹假单胞菌CFBP5454和地中海假单胞菌CFBP5447的同源。在phaC1-phaZ基因间区域发现了一个假定的固有转录终止子，它由一段dA残基之前的二元对称序列（24 bp；ΔG = -41.8 kcals）组成。构建了皱纹假单胞菌突变克隆XI 32-1和XI 32-4，其中该基因间区域被一个可选择的卡那霉素抗性基因取代。这些突变克隆在油酸上生长48小时后，phaC1和phaC2的相对表达量相较于初始接种物增加了4.7至7.0倍，而亲本菌株仅增加了1.2至1.4倍。此外，与仅含有少数大的PHA包涵体的亲本皱纹假单胞菌相比，在油酸上生长的突变体产生了许多较小的PHA颗粒，这些颗粒排列在细胞周边。以葡萄糖为底物时，XI 32-1和XI 32-4克隆产生的中链PHA中，单不饱和3-羟基十二碳烯酸作为重复单元单体的含量很高（26-31 mol%）。我们的结果首次展示了phaC1-phaZ基因间区域对phaC1和phaC2基因的底物依赖性瞬时表达、中链PHA的重复单元组成以及PHA颗粒形态的影响。
