利用基因突变手段研究pseudomonas+putida的pha合成途径相关酶的功能及新型中长链pha材料的生物合成.pdf

汕头大学 博士学位论文 利用基因突变手段研究Pseudomonas putida的PHA合成途径相关 酶的功能及新型中长链PHA材料的生物合成 姓名：刘倩 申请学位级别：博士 专业：生物化学与分子生物学 指导教师：陈国强 2010-05 中 文 摘 要 III 摘摘 要要 聚羟基脂肪酸酯(PHA)是细菌胞内发现的一种多聚物，具有生物可降 解性、生物相容性等优良性能，有广泛的应用前景可是已经实现商业化 生产的几种 PHA 材料在性能上还不能完全满足实际需求，传统中长链 PHA 含 有多种单体，经过水解后各种单体难以分离，因此，各国研究者都致力于开 发性能更加优良的中长链 PHA 以及长链 PHA 均聚物 本文围绕利用基因工程手 段来改造 PHA 合成菌，通过提高其长链单体含量来改善其物化性能，并从而有 可能在应用上有所突破 本论文通过基因突变的手段来缺失恶臭假单胞菌 P.putida KTOY08 中的十 个与 PHA 合成以及菌体本身的β-氧化循环相关的基因，来分析基因的功能并 筛选出高产菌株一株，能够合成聚羟基癸酸酯及新型中长链聚羟基脂肪 酸酯(mcl-PHA)的突变株一株并对其材料性能进行研究。

通过对各种突变菌菌在以月桂酸为单一底物的培养基中生物合成 PHA 的气 相色谱分析， 我们发现基因 PP3280、PP4636 以及基因 PP2051、PP2047、PP2048 对于 PHA 合成的影响比较大其中基因 PP2047 编码 3-羟基脂酰辅酶 A 脱氢酶 (3-hydroxyactyl-CoA dehydrogenase, FadB)，PP2048 编码脂酰辅酶 A 脱氢酶 (Actyl-CoA dehydrogenase, FadE)，而基因 PP2051、PP3280 和 PP4636 编码的都 是乙酰辅酶 A 乙酰基转移酶(Acetyl-CoA acetyl-transferase, FadA) 的同功酶基 因PP3280和PP4636的缺失可以使突变菌株的PHA含量从缺失前的50wt%升高 到 90wt%另外三个基因 PP2051、PP2047 及 PP2048 的缺失则可以极大的削弱 突变菌株的β-氧化，使得与底物脂肪酸链长相同的 mcl-PHA 中单体含量从 15mol%增加到 80mol%左右， 而且可以合成聚羟基癸酸酯均聚物和只含有两个长 链单体的 mcl-PHA。

所有的突变菌中筛选出两种比较理想的菌株 KT12 和 KT20 KT12 是一株高产菌，在以月桂酸为单一底物的情况下，可以合成出占细胞干重(CDW) 90wt%以上的 PHA这在中长链 PHA(mcl-PHA)的生物合成中从来没有过的另 外一株突变菌 KT20 可以在以癸酸为单一底物的情况下，合成出羟基 癸酸酯，这也是首次在生物体内合成长链的 PHA 均聚物在以月桂酸和 十四酸为单一底物的情况下，其合成的共聚物 P(16mol% 3HD-co-84mol% 中 文 摘 要 IV 3HDD) 和 P(21mol% 3HDD-co-79mol% 3HTD)中与底物链长相同的单体的含 量非常高，而且共聚物中只有两个单体相对于传统 mcl-PHA 来说，单 体的均一性要高得多，从而对材料的热力学性能有了较大的改善 最后，分析了所得的PHD、P(16mol% 3HD-co-84mol% 3HDD) 和P(21 mol% 3HDD-co-79mol% 3HTD)的三种mcl-PHA材料的分子量分布，热学性 能，机械性能，热稳定性并与其他典型的mcl-PHA性能比较均聚物PHD 与长链PHA P(16mol% 3HD-co-84mol% 3HDD)和P(21mol% 3HDD-co-79mol% 3HTD)的玻璃化转变温度Tg比野生型的KT2442 所合成的典型PHA的玻璃化转变 温度高7到11℃。

均聚物PHD以及长链PHAP(16mol% 3HD-co-84mol% 3HDD) 和 P(21mol% 3HDD-co-79mol% 3HTD) 的Tm分别是 72℃、78℃和 72℃P(16 mol% 3HD-co-84 mol% 3HDD)的Tm是目前为止报道过的最高的mcl-PHA的Tm均聚物 PHD的拉伸强度、断裂拉伸强度和断裂伸长率分别是 12 MPa, 12 MPa, 313%，明 显高于典型的mcl-PHA甚至是本文所做的另外一种长链PHA材料P(16 mol% 3HD-co-84 mol% 3HDD)说明PHD及新型mcl-PHA具有更好的结晶性、机械 强度，更利于PHA的广泛应用 关键词：关键词：PHA；聚羟基脂肪酸酯；PHD；聚羟基癸酸酯；中长链聚羟基脂肪酸 酯；mcl-PHA；代谢工程；β-氧化；Pseudomonas putida；基因工程 Abstract V ABSTRACT Polyhydroxyalkanoates (PHA) are produced by various bacteria as intracellular carbon and energy storage materials under nutrient-limitation conditions in the presence of excess carbon source. They have attracted increasing attention from scientific and industrial communities due to their interesting properties including biodegradability, biocompatibility and piezoelectricity. However, the physical characters of PHA which had been developed as commercial materials could not fit their utilization completely， it was hard to separate typical medium-chain-length PHA hydrolyzed product because of its various of monomers component. Therefore, researchers all over the world are focusing on mcl-PHA with better physical characters or mcl-PHA homopolymers. This study mainly focused on using genetic engineering technology to construct mutant recombinants to biosynthesis better physical character material with high content of long-chain-length monomer. P.putida KTOY08 was used as the parent to construct mutant recombinants by knocking out ten genes which related to PHA synthesis or β-oxidation. The functions of the genes which knocked out in this study were analyzed. Lauric acid was used as the single substrate to culture the mutant strains. By analysis the PHA and its monomer content detected by Gas chromatography, five genes were found important to PHA synthesis and β-oxidation. Deletion of gene PP3280 and PP4636, which were encoding isozymes of Acetyl-CoA acetyl-transferase (FadA), lead a high-yield of PHA about 90wt% of CDW. The lack of other three genes PP2051 (encoding Acetyl-CoA acetyl-transferase, FadA), PP2047 (encoding 3-hydroxyactyl- CoA dehydrogenase, FadB) and PP2048 (encoding Actyl-CoA dehydrogenase, FadE) would weaken β-oxidation strongly. Meanwhile, one high-yielding strain KT12 and one long-chain-length homopolymer and novel medium-chain-length PHA production strain KT20 were obtained. When KT12 were cultured by single substrate lauric acid, the product of PHA would be 40wt% higher than the parent strain. The long-chain-length Abstract VI homopolymer polydecanoate (PHD) which firstly obtained in vivo could be biosynthesized by KT20 under the single substrate decanoate. In addition, the mutant KT20 could also produce P(16mol% 3HD-co-84mol% 3HDD) and P(21 mol% 3HDD-co-79mol% 3HTD) by using dodecanoate and tetradecanoate as single substrate respectively. At last, molecular weight, physical properties of PHD, P(16mol% 3HD-co-84 mol% 3HDD) and P(21mol% 3HDD-co-79mol% 3HTD) were characterized. P(16 mol% 3HD-co-84mol% 3HDD) has the highest melting temperature of 78oC among mcl-PHA been reported. The tensile strength, strength at break and elongation at break were 12 MPa, 12 MPa, 313%, which were obviously higher than typical mcl-PHA. Therefore, there would be a wider application of novel mcl-PHA: P(16 mol% 3HD-co-84m。