Genetic Analysis of Comamonas acidovorans Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaC(Ca)) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (phaA(Ca)). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaA(Ca). The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB(−)4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.

以真养产碱菌（Alcaligenes eutrophus）的合酶基因为异源杂交探针，克隆得到食酸丛毛单胞菌DS-17（Comamonas acidovorans DS-17）的聚羟基脂肪酸酯（polyhydroxyalkanoate, PHA）合酶基因phaC(Ca)。对4.0千碱基对的SmaI-HindIII（SH40）亚片段进行全序列测定，结果显示其包含一段长1893碱基对的PHA合酶编码区，下游紧邻一段长1182碱基对的β-酮硫解酶基因phaA(Ca)。上述两个基因的翻译产物分别与真养产碱菌对应基因产物的一级结构存在51.1%和74.2%的显著同源性。食酸丛毛单胞菌的PHA生物合成基因排布模式与真养产碱菌高度相似，仅存在一处差异：phaA(Ca)下游未发现编码乙酰乙酰辅酶A还原酶的第三基因phaB。该克隆片段可互补真养产碱菌的PHA缺陷突变株PHB(-)4，当以果糖作为唯一碳源时，可使宿主细胞积累高达干细胞重73%的聚3-羟基丁酸酯。该异源表达体系还可实现4-羟基丁酸（4HB）与3-羟基戊酸单体的整合。食酸丛毛单胞菌的PHA合酶似乎并未对4-羟基丁酰辅酶A底物表现出特异性偏好。据此可以推断，在食酸丛毛单胞菌中，介导4HB单体整合并高效合成高4HB含量PHA的关键因素是代谢途径，而非该菌株PHA合酶的底物特异性。