丛毛单胞菌CNB-1合成PHA及相关基因的研究

Biosynthesis of polyhydroxyalkanoates and its metabolic pathway in Comamonas sp. strain CNB-1

摘要:

分析了丛毛单胞菌(Comamonas sp.)CNB-1菌株在不同条件下合成聚羟基烷酸(polyhydroxyalkanoic acids,PHAs)的组分和含量,同时克隆了与PHA合成相关的基因。结果表明,该菌可以多种短链有机酸及醇类为碳源合成PHA多聚物或共聚物,以戊酸和1,4-丁二醇为底物时,可达菌体干重的57%;同时发现小分子醇类的存在能显著促进PHA的合成,推测与醇类氧化过程中提供了更多的还原力有关。为了克隆相关基因,利用已知phaC的保守区简并引物筛选基因组文库,将得到的阳性克隆质粒测序,发现phaC、phaA、phaB组成一个基因簇phaC-A-B。将phaC、phaA、phaB连接到pET载体在E.coli中共表达,重组E.coli菌株能合成PHA;将这3个基因单独连接到pET载体,在E.coli中表达后检测到相应酶活,分别约为原始菌株的4.1、71和2882倍。

Abstract:

Comamonas sp. strain CNB-1 degrades chloronitrbenzene and nitrobenzene for carbon and nitrogen sources. In this study，accumulation of polyhydroxyalkanoic acids (PHAs) within strain CNB-1 cells was investigated under various conditions. Results indicated that strain CNB-1 was able to synthesize PHA from various short-chain fatty acid and alcohols，and 57 w% of the dry cell weight (DCW) PHA was obtained when valerate and 1，4-butanediol were co-fed. Supplements of short-chain alcohols stimulated the accumulation of PHAs，and this stimulatory effect was attributed to the more amount of reductant generated from alcohol dehydrogenation. The genes encoding for PHA polymerase (phaC)，for acetoacetyl-CoA thiolase (phaA)，and acetoacetyl-CoA reductase (phaB) were cloned in Escherichia coli，and the recombinant E. coli synthesized PHA and showed enzymatic activities of PHA polymerase，acetoacetyl-CoA thiolase，and acetoacetyl-CoA reductase. The three genes occurred as a cluster of pha(C-A-B). To optimize their expression，the three genes were cloned to the pET vector and expressed respectively. Mass of expressed protein was detected and the enzyme activities increased greatly in contrast to wild CNB-1 strain，which is about 4.1，71，and 2882 folds of activities of CNB-1.