嗜水气单胞菌WQ中PHBHHx的合成及其分子基础研究

聚羟基脂肪酸酯(Polyhydroxyalkanoate，PHA)是一系列生物合成的高分子材料，其单体可由多种3-羟基脂肪酸(3-hydroxyalkanoate，3HA)构成^1]。PHA物理和机械性能的变化很大，从高脆性到弹性体，这跟它们的单体成分有很大关系^2]。短链和中长链单体共聚的PHA比短链单体或中长链单体聚合得到的PHA有着更好的性能^3]。在1994年，豚鼠气单胞菌(Aeromonas caviae)FA440被发现能以偶数碳原子数脂肪酸或植物油作为碳源在体内积累PHBHHx^4]其PHA生物合成基因被成功克隆^5]。根据亚基数目和底物特异性，PHA合成的关键酶，即PHA合酶或PhaC，被分成了3种类型。A．caviae的PHA合酶属于第1类PHA合酶^6]。PHA合酶的一些类型含有一些保守的基因序列，该特征可被用于克隆，特别是第Ⅱ类PHA合酶^2,8]。嗜水气单胞菌(Aeromonas hydrophila)WQ和A．hydrophila 4AK4是能够合成PHBHHx的另外两种菌株，其中A．hydrophila 4AK4已被用作大规模生产PHBHHx。就目前来说，不管生长条件怎么改变，其合成的PHBHHx中3羟基己酸单体(3-hydroxyhexanoate，3HHx)的含量始终在12％～17％之间变化^9]。而A．hydrophila WQ合成的PHBHHx中则含有6％～14％ 3HHx。本论文研究了A．hydrophila WQ的PHA生物合成及其分子基础。

Synthesis of Copolyesters Consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonas hydrophila WQ and its Molecular Basis

Aeromonas hydrophila WQ isolated from lake water was found to be able to synthesize polyhydroxyalkanoates (PHA) copolymer consisting of 3-hydroxybutyrate (HB) and 3-hydroxyhexanoate (HHx) (PHBHHx). Lauric acid was found to be the most suitable carbon source for cell growth and PHBHHx accumulation. The bacteria accumulated 49% PHBHHx containing 6% HHx in terms of cell dry weight when grown on lauric acid for 72 h. 42% PHBHHx consisting of 14% HHx was obtained with 5 g/L glucose and 10 g/L lauric acid as co-substrate. Higher glucose concentration greatly reduced the cell concentration and PHA content. The PHA biosynthesis genes from A. hydrophila WQ was successfully cloned using a two-step PCR cloning strategy based on PHA biosynthesis genes organization of Aeromonas caviae. A. hydrophila WQ and A.caviae shared high identities in the PHA gene loci, namely, ORF1, phaC and phaJ had 100%, 97% and 97.5% identities respectively. PHA synthases of A. caviae and A. hydrophila were proposed to contain type IV PHA synthases which are different compared with type I PHA synthases on the substrate specificity and location arrangement of PHA metabolic genes.