中长链聚羟基脂肪酸酯（mcl PHA）在嗜水气单胞菌I型PHA合酶缺失突变株中的合成

拥有Ⅰ型聚羟基脂肪酸酯（PHA）合酶基因的嗜水气单胞菌CGMCC 0911株可利用月桂酸而不能利用葡萄糖作为碳源积累PHBHHx。将氯霉素抗性基因（Cm）插入到该基因中,获得带有I型PHA合酶断裂基因（phaC::Cm）的自杀质粒pFH10。自杀质粒pFH10通过接合作用转入嗜水气单胞菌CGMCC 0911株中并发生体内同源重组,Cm被整合到基因组上,获得Ⅰ型PHA合酶缺失突变株。DNA序列测定证明了这一结果。GC分析表明,突变株不再产生PHBHHx,但却可利用月桂酸或葡萄糖积累中长链PHA,明显表明野生型嗜水气单胞菌基因组中存在另一个编码Ⅱ型PHA合酶的基因,且只有Ⅰ型PHA合酶被钝化后,这个功能被隐藏的Ⅱ型PHA合酶才可在细胞中发挥作用。     

中长链聚羟基脂肪酸酯(mcl PHA)在嗜水气单胞菌Ⅰ型PHA合酶缺失突变株中的合成

拥有Ⅰ型聚羟基脂肪酸酯(PHA)舍酶基因的嗜水气单胞菌CGMCC 0911株可利用月桂酸而不能利用葡萄糖作为碳源积累PHBHHx。将氯霉素抗性基因(cm)插入到该基因中,获得带有Ⅰ型PHA合酶断裂基因(phaC：：Cm)的自杀质粒pFH10。自杀质粒DFH10通过接合作用转入嗜水气单胞菌CGMCC 0911株中并发生体内同源重组,Cm被整合到基因组上,获得Ⅰ型PHA合酶缺失突变株。DNA序列测定证明了这一结果。GC分析表明,突变株不再产生PHBHHx,但却可利用月桂酸或葡萄糖积累中长链PHA,明显表明野生型嗜水气单胞菌基因组中存在另一个编码Ⅱ型PHA合酶的基因,且只有Ⅰ型PHA合酶被钝化后,这个功能被隐藏的Ⅱ型PHA合酶才可在细胞中发挥作用。     

嗜水气单胞菌合成含3-羟基戊酸单体的聚羟基脂肪酸共聚酯的研究

分别利用葡萄糖或葡萄糖酸钠与十一碳酸、月桂酸与十一碳酸为混合碳源进行嗜水气单孢菌 (Aeromonashydrophila)菌株 4AK4的摇瓶培养 ,实现了含有 3 羟基戊酸 (3HV)单体的聚羟基脂肪酸酯的微生物合成。当使用葡萄糖或葡萄糖酸钠与十一碳酸为混合碳源时 ,野生型A .hydrophila 4AK4及含有 3 羟基丁酸辅酶A合成基因phaA和phaB的重组A .hydrophila 4AK4 (pTG01)能够合成-3-羟基丁酸(3HB)与-3HV的共聚物 ,且葡萄糖或葡萄糖酸钠与十一碳酸比例为 1∶1时最利于细胞生长和PHA的积累。当使用月桂酸和十一碳酸为混合碳源时 ,A .hydrophila4AK4能够合成-3HB、3HV与 β-羟基己酸 (3HHx)的共聚物 ,且随着混合碳源中十一碳酸的含量增加 ,A .hydrophila4AK4合成的PHA中-3HV的比例增加 ,而-3HB和-3HHx的比例降低.     

盐单胞菌利用短链脂肪酸合成聚羟基脂肪酸酯

盐单胞菌(Halomonas)能够利用多种底物为碳源生长,由于其能在高盐条件下进行不灭菌的开放发酵,已被开发用作下一代生物技术的底盘细胞.包括乙酸、丙酸和丁酸在内的短链挥发性脂肪酸能够以生物质为原料制备,有望成为用于微生物发酵的新型碳源.利用10-50g/L浓度的丁酸为碳源对Halomonas sp.TD01和TD08...     

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。     

活性污泥法生产聚羟基烷酸（PHA）

介绍了厌氧-好氧活性污泥法生产生物降解塑料PHA的生化机制及增加活性污泥中PHA含量的新方法.     

门多萨假单胞菌NK-01合成中长链聚羟基脂肪酸酯的发酵条件优化

为了提高PHA_MCL在门多萨假单胞菌NK-01中的积累,采用单因素实验和正交实验确立了发酵生产PHA_MCL的最佳条件,即以PHA产量为指标的最佳发酵条件为15 g/L葡萄糖浓度、C/N=50、发酵时间48 h,该条件下获得产量0.8 g/L以上的PHA;以PHA占菌体干重百分含量为指标的最佳发酵条件为10 g/L葡萄糖浓度、C/N=60、发酵时间48 h,该条件下获得占菌体干重50%以上的PHA。该研究将为门多萨假单胞菌NK-01用于PHA_MCL的规模化生产提供理论依据。     

短须嗜热单孢菌聚羟基脂肪酸酯解聚酶的表达、热稳定性改造及在PHB降解中的应用

聚羟基脂肪酸酯解聚酶(polyhydroxyalkanoate depolymerase,PHAD)可用于聚羟基脂肪酸酯(polyhydroxyalkanoate,PHA)的降解回收,为开发热稳定性好的PHAD,本研究在大肠杆菌(Escherichiacoli)BL21(DE3)中成功表达了来自短须嗜热单孢菌(Thermomonospora umbrina)的PHA解聚酶(TumPHAD),并通过二硫键理性设计获得了热稳定性提升的突变体A190C/V240C,其最适温度为60℃,比野生型提高20℃,50℃半衰期为7h,是野生型酶的21倍。将突变体A190C/V240C用于典型PHA之一的聚羟基丁酸酯(polyhydroxybutyrate,PHB)降解,在50℃条件下,PHB的2 h和12 h降解率较野生型分别提高了2.1倍和3.8倍。本研究获得的TumPHAD突变体A190C/V240C具有耐高温、热稳定性好和PHB降解能力强的特点,对PHB的降解回收具有重要意义。     

嗜水气单胞菌非核糖体肽合成酶基因功能研究

非核糖体肽是微生物体内一类具有天然生物活性的次生代谢物,由非核糖体肽合成酶催化生成。而AHA2474和AHA2476是嗜水气单胞菌ATCC7966中两个编码非核糖体肽合成酶的基因。利用同源重组技术分别构建了AHA2474、AHA2476基因缺失株,并对其生理特性进行测定。结果表明,与野生株相比,缺失株的溶血性和胞外蛋白酶活性均显著增强,而产铁能力明显减弱;在缺铁条件下,缺失株的生长能力较弱,补充铁离子后又能恢复生长。同时在过氧化氢应激下ΔAHA2474菌株具有更大的耐受性。以上研究结果提示AHA2474和AHA2476基因可能通过影响铁离子动态平衡过程来调控该菌的生理特性,同时也表明非核糖体肽在该菌致病性方面起作用,为探究该菌的致病机制及防治策略提供理论依据。     

Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp

Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.     

Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC_Ac, was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC_Ac gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) {by LS5218 [fadR601 atoC(Con)] strain}. The expression vector for the phaC_Ac gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).     

Polyhydroxyalkanoate (PHA) Synthesis by Class IV PHA Synthases Employing Ralstonia eutropha PHB−4 as Host Strain

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC_YB4) or B. megaterium NBRC15308^T (PhaRC_Bm) was expressed in Ralstonia eutropha PHB^?4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC_YB4 produced significant amounts of PHA and had broader substrate specificity than PhaRC_Bm.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.