Maehara A, Ikai K, Ueda S, Yamane T
Laboratory of Molecular Biotechnology, Division of Molecular Cell Mechanisms, Department of Biological Mechanisms and Functions, Graduate School of Bio- & Agro-Sciences, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan.
Biotechnol Bioeng. 1998 Oct 5;60(1):61-9.
Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA). (g total biomass)-1] and the highest rates of PHA accumulation [(g PHA). (g residual biomass)-1. h-1] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass. h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.
已鉴定出反硝化副球菌聚羟基脂肪酸酯（PHA）合成基因的假定启动子。在反硝化副球菌重组体中研究了PHA合成的基因剂量效应，这些重组体中每种PHA合成酶的表达水平均有所提高。在使用乙醇或正戊醇作为碳源的摇瓶培养中，编码phaC的PHA合酶的自克隆重组体在这些重组体中显示出最高的含量[（g PHA）·（g总生物量）-1]和最高的PHA积累速率[（g PHA）·（g残留生物量）-1·h-1]。自克隆重组体的PHA含量和PHA积累速率（g PHA/g残留生物量·h-1）分别比野生菌株高2倍和2.7倍。这一结果有力地表明，在通过β-氧化从正戊醇经3-酮戊酰辅酶A以及从乙醇经乙酰辅酶A进行体内PHA合成的过程中，PHA合酶步骤受到限制。在5-L生物反应器中进行的补料分批培养研究中，保持酒精浓度恒定（0.02%），结果表明，尽管在生长相关的PHA合成阶段细胞生长速率受到抑制，但PHA合酶的基因剂量提高了PHA生物合成能力。从PHA合酶基因剂量增加的菌株中分离出的聚合物的分子量低于野生菌株聚合物的分子量，这表明体内PHA合酶的量会影响聚合物的分子量。
