Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil
Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil
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DOI：
10.1139/v08-047
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发表时间：
2008-06-01
期刊：
影响因子：
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通讯作者：
Fukui, Toshiaki
中科院分区：
文献类型：
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作者：
Mifune, Jun;Nakamura, Satoshi;Fukui, Toshiaki
先前的研究已经证明，异源表达能够接受C（4）-C（7）的（R）-3-羟基酰基-CoA作为底物的来自嗜水气单胞菌的PHA合酶（PhaC（Ac））可以赋予钩虫贪铜菌PHB（-）4的PHA阴性突变体从植物油合成聚（（R）-3-羟基丁酸酯-co-（R）-3-羟基己酸酯）[P（3 HB-co-3 HHx）]的能力。经确定，在PHB（-）4中的pha操纵子内的突变点是PHA合酶基因（phaC（Cn））内的单个无义突变，表明在该菌株中观察到的低得多的β-酮硫解酶和NADPH依赖性乙酰乙酰辅酶A还原酶活性将是突变的极性效应。为了进一步有效地合成P（3 HB-co-3 HHx）β，C.通过靶向染色体phaC（Cn）的同源重组来工程化necator野生菌株H16，并将PHA生产率与先前的在多拷贝质粒（PHB（-）4/pJRDEE 32 d13）上携带phaC（Ac）的PHB（-）4衍生菌株进行比较。将染色体上的phaC（Cn）替换为phaC（Ac）的菌株H16 C（Ac）能以较高的产量从大豆油中产生P（3 HB-co-3 HHx），但积累的聚合物中的3 HHx组分减少。同时，H16 Δ C/pJRDEE 32 d13，其失去了原始合酶基因的区域并表达外染色体phaC（Ac），生长并积累具有与PHB（-）4衍生菌株相似性质的PHA。酶活性测定结果表明，以大豆油为唯一碳源时，β-酮硫解酶活性降低可能与生长能力下降和3 HHx组分增加有关。
Previous studies have demonstrated that heterologous expression of PHA synthase from Aeromonas caviae (PhaC(Ac)), capable of accepting (R)-3-hydroxyacyl-CoA of C(4)-C(7) as substrates, could confer the ability to PHA-negative mutant of Cupriavidus necator PHB(-)4 to synthesize poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from vegetable oils. The mutation point within pha operon in PHB(-)4 was determined to be a single nonsense mutation within the PHA synthase gene (phaC(Cn)), suggesting the much lower beta-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase activities observed in this strain would be a polar effect of the mutation. For further efficient biosynthesis of P(3HB-co-3HHx) copolyester, C. necator wild strain H16 was engineered by homologous recombination targeting the chromosomal phaC(Cn), and the PHA productivity was compared with previous PHB(-)4-derived strain harboring phaC(Ac) on a multi-copy plasmid (PHB(-)4/pJRDEE32d13). A strain H16C(Ac), in which phaC(Cn) was substituted for phaC(Ac) on the chromosome, could produce P(3HB-co-3HHx) from soybean oil with high productivity, but the 3HHx fraction in the accumulated polymer was decreased. Meanwhile, H16 Delta C/pJRDEE32d13, that lost region for the original synthase gene and expresses exochromosomal phaC(Ac), grew and accumulated PHA with similar properties to the PHB(-)4-derived strain. The results of enzyme assay suggested that low beta-ketothiolase activity might be relevant for decrease of growth ability accompanied by increase of 3HHx composition when soybean oil was fed as a sole carbon source.