聚羟基脂肪酸酯在大肠杆菌中的合成、降解以及分子改造
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摘要
聚羟基脂肪酸酯(PHA)是微生物体内合成的一类生物聚酯,PHA不仅具有与传统化学合成高分子相似的材料性质,而且还具有一般传统塑料没有的性质,如生物可降解性、生物相容性、压电性、光学活性等特殊性质。因此,PHA的应用范围非常广,可用作工业、农业、医药、食品业中各种绿色包装材料的原材料。由于PHA可以通过很多可再生资源获得,因此具有巨大的经济价值。除了大批量生产PHA用于包装材料之外,PHA还是一种具有特殊应用的极好的医学材料,由于PHA能够与哺乳动物的组织相容,并且缓慢降解;另外,PHA具有一定的力学性能,可用作骨移植的支架。近年来,越来越多的学者注重于研究PHA在医药及组织工程等方面的应用。在全球环境面临白色污染威胁、人类健康面临严重器官和组织的障碍及丧失的今天,更应该大力发展PHA产业,开发更好性能的PHA以适应各种特殊应用。
对于PHA的研究已经有七十多年的历史了,从最初聚-3-羟基丁酸酯(PHB)天然颗粒的提取,到聚羟基丁酸羟基戊酸酯(PHBV)的工业化生产,再到PHA的新单体、新基因、新菌种,不同的发酵底物、合成途径以及代谢调控等等的新发现,加上蛋白质组学和代谢组学的相辅相成,人们对PHA的研究越来越广、也越来越深入,可开发的资源就越来越少。因此,怎样从一个新的视角入手研究PHA的新功能是本论文的目的之一。
在利用大肠杆菌基因工程菌高产PHB时,我们发现大肠杆菌在积累大量的PHB(达细胞干重的90%)后,不单没有影响到细胞内的渗透压,而且仍然可以生产琥珀酸等一些有机酸类,因此我们致力于发现和找到PHB对重组大肠杆菌的“贡献”以及它的机制。
PHA是细菌在生长条件不平衡时的产物(比如碳源过剩,氮源缺乏时),在环境不利时消耗掉以躲过恶劣环境的伤害,提高菌体对外界的适应能力。本论文首次在大肠杆菌中研究了PHB合成和降解对于宿主菌的有利作用。模仿PHB自然合成菌,成功地构建了一种压力诱导的启动系统来表达PHB合成和降解酶基因,无需进行人工诱导,只是在在压力情况下诱导PHB的合成和降解,就如同自然合成菌一样;顺利的表达了PHB合成及降解酶基因,得到了一株高产PHB以及3-羟基丁酸的工程菌。同时还验证了大肠杆菌利用3-羟基丁酸途径的存在,我们对大肠杆菌中3HB-CoA合成酶的活性进行了体外检测,发现在ATP以及CoA存在时,即能够将3-羟基丁酸转化为CoA形式而进入能量代谢循环,从而为细胞提供能量。
菌体不仅在碳源丰富时积累PHB,在氮源缺乏的环境中,这种不理想的条件非常有益于PHB的产生和积累。氮源的利用率是一个限制性因素,为了研究氮源限制也能够在重组菌E.coli DH5α(pSCP-CAB/pQWQ2)中诱导PHB产生,本实验以M9为基础培养基(含2%的葡萄糖),而变化NH_4Cl的浓度(0.2 g/L,1 g/L,2.5 g/L,5 g/L),在这四种培养条件下观察PHB的诱导情况。实验结果表明:低浓度氮源会诱导重组菌更快的更多的积累PHB。
同时,重组菌对外界压力的适应能力是通过饥饿实验和抗性试验检验的:积累PHB的工程菌E.coli DH5α(pSCP-CAB/pQWQ2)在经过32天的饥饿条件下,仍然可以存活和繁殖,这是普通大肠杆菌所不具有的,实验证明普通大肠杆菌在饥饿培养32天之后,存活率不到1%;同时探究了PHB合成和降解的这种循环机制对大肠杆菌抗性的影响,分别对大肠杆菌进行了热击、紫外照射、酸、高渗溶液的处理,发现产并能降解PHB的菌株E.coli DH5α(pSCP-CAB/pQWQ2)均具有较高的存活率,说明重组菌获得PHB生产能力后,菌体的各种抗性指标(如:耐饥饿存活率,抗氧化,抗热,抗辐射能力以及抗酸能力)均有较大提高,为工业上利用大肠杆菌生产各种物质,提供一个稳定抗逆菌株打下良好基础。
PHA家族是一个非常庞大的家族,目前已经发现PHA至少有150种不同的单体结构,并且还在不断地发掘出新的单体。在PHA家族中,单纯的PHB或PHA都由于其性能的缺陷影响了它的应用范围。因此开发具有高性能的PHA-co-PHB共聚物是国际上的研究热点。PHA聚合酶是生产共聚物的关键。
本研究以改变PHA聚合酶的底物特异性入手,通过对PHA聚合酶分子的改造,使其能够聚合本来不能利用的前体物,成为一种具备广泛底物特异性的酶,从而也就增加了PHA单体的种类。我们的目的是构建一个既有SCL-PHA合成能力又有mcl-PHA合成能力的PHA合酶,那么这个酶应该同时具有Ⅰ类合酶和Ⅱ类合酶的性质。本论文首次引入了递增截短法来构建一个具有广泛底物特异性的PHA合酶的杂合酶。它的优点是避免了在构建杂合酶时融合位点难于把握的问题,并且通过文库筛选可以得到更多有活性的广底物特异性的酶。
将Ⅰ类PHB合酶和Ⅱ类的mcl-PHA合酶同时克隆于一个载体上,转化大肠杆菌E.coli LS5218ΔfadA进行发酵生产,结果表明重组菌能够生产PHB/mcl-PHA的共混物,而且惊喜的发现PHB含量在85～95%。为进一步大规模生产优良性能的PHB/PHA共混物提供了良好的开端。然后,对PHA合酶进行的分子进化改造,我们通过递增截短文库法将合成mcl-PHA的合酶基因phaCl基因从3′端向5′端酶切,并将合成SCL-PHA的合酶基因phbC基因从5′端向3′端酶切,得到了不同程度酶切的杂合酶基因文库,通过对文库的进行尼罗红筛选和基因大小的筛选得到了25株能够合成PHB的重组茵,发现随着杂合酶phbC的5′端的逐渐切除,其PHB合酶的活性也逐步降低。根据最近的报道是将PhbC氨基端82个氨基酸切除后还能保持一定体内活性,85个氨基酸就完全失去活性,也就是是说其最小功能区既是切掉82个氨基酸之后的区域。我们通过构建缺失体库而获得的杂合酶首次发现了在83-100甚至120个氨基酸缺失后仍然有活性。这就说明,PHB合酶的递增缺失发现其氮端的缺失可以通过PHA合酶PhaCl的互补进行弥补。将失去活性的PHB合酶能够再次合成PHB;通过递增截短文库法构建的杂合酶,经筛选得到了3株克隆,检测这些酶发现,在体内既可以利用葡萄糖为碳源合成PHB,也可以利用脂肪酸(癸酸盐)为碳源合成P(HB-co-HA)共聚物,说明了PhaCl的氮端不仅仅是对杂合酶起到保护作用,而且可能含有底物的结合区域,因此能够结合中长链的羟脂酰CoA底物。这种人工改造的杂合酶性质稳定,易于控制表达,其延伸强度好等更加优越的性能,接近于低密度聚乙烯,在不久的将来必会得到广泛应用。
PHA合酶的性质研究也是近年来备受关注的,PHA生产的关键技术之一就是提高PHA合酶的聚合能力。而聚合能力的提高与了解PHA合酶的催化以及结构性质密切相关。近年来普遍认为,PHA聚合酶是以二聚体的形式催化3-羟基丁酰CoA合成高分子量聚酯PHA的,从酶的催化机制可以推测在体内酶的存在形式是单体与双体的平衡状态。并且认为聚合酶的二聚体是酶存在的活性形式;PHB颗粒的形成存在两个假说,近来越来越倾向于“出芽”模型,即PHB颗粒是从内膜上形成并脱落的,但是没有直接证据证明以上理论的正确性。本论文首次通过绿色荧光蛋白(GFP)片段重组装系统对PHB合酶的相互作用进行了检测,利用的是两个在大肠杆菌中相兼容的载体分别为pET11a-NGFP,及pMRBAD-CGFP,两个载体上分别有GFP的两部分片断,通过将待检测相互作用的两个基因分别与NGFP、CGFP的融合表达,再将两个载体同时转化大肠杆菌E.coliBL21(DE3),表达融合蛋白后,只有当蛋白发生相互作用时,GFP两部分相互靠近重组装,从而发出荧光。结果证明:只有在有底物3-羟基丁酰CoA存在的情况下,重组菌才会发出荧光,说明PHB合酶发生了相互作用,在体内形成二聚体然后催化PHB的形成的。在没有底物3-羟基丁酰CoA的情况下,PHB合酶并不或不主要以二聚体形式存在,GFP无法靠近形成重组装。因此观察不到GFP的绿色荧光。同时,若存在二聚体,通过对产荧光二聚体的实时示踪,可以观察在PHB颗粒形成早期,脂单层膜的来源。从而验证“出芽”模型的理论的正确性。该过程将会利用电镜技术帮助更好的验证。由于荧光显微镜不易观察到PHB颗粒的发生和形成,对这部分实验进行了进一步探索,利用荧光能量共振转移技术对其的研究正在进行中。
Polyhydroxyalkanoates(PHAs) are synthesized by micro-organism within the cell as a kind of bio-polyester.PHAs not only possess the properties as well as traditional petrochemical plastics but also are biodegradable,biocompatible,piezoelectric, optically active etc.these features makes them suitable for many applications in the packaging industry,medicine,pharmacy,agriculture,food industry,as raw materials for enantiomerically pure chemicals and in the production of paints.PHA can be obtained from renewable resources;hence,there has been a tremendous amount of commercial interest in these polymers.In contrast to bulk uses,PHAs have also been established as excellent materials in several niche applications,in particular for medical purposes,because PHAs are generally biocompatible with mammalian tissue and are resorbed at a slow rate;PHA can be used to develop tissue engineering scaffolds,which support cell growth and are degraded after implantation,leaving a viable tissue.Thus nowadays more and more efforts have been made for the applications in medical and tissue engineering.In conclusion,efforts in biopolymer research must be made to develop and enhance PHA production processes at low cost levels,and the development of PHA with better properties is necessary. PHA has been intensively studied for seventy years since the first PHA, poly(3-hydroxybutyrate)(PHB),was discovered in Bacillus megaterium by the French scientist Lemoigne in 1926.In the following years,research on purification of PHB granules from the cell and other forms of PHAs e.g.PHBV's production,and in succession investigations with other monomers,genes,microorganisms,metabolic pathways and the potential use of these biopolymers was realized.Recent research has focused on the use of alternative substrates,novel extraction methods,genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive.
When we engineered Escerichia coli to produce different kind of PHAs,we found that Escerichia coli could not only accumulate approximate 90%PHB of the cell dry weight without any harmful effect on cell growth but also produce succinic acid and other organic acid.Thus we were devoted to investigate the mechanism that PHB can benefit the host Escerichia coli for survival under stress conditions such as cold shock, pH and cell density.
A wide variety of microorganisms are able to accumulate polyhydroxyalkanoates (PHAs) as intracellular carbon/ energy storage compounds or reducing power for coping with changing,often oligotrophic environments.Various PHAs,as well as the best-known poly 3-β-hydroxybutyrate(PHB),were found to be accumulated and degraded as required under environmental conditions by most natural PHAs producing bacteria.When the environment is sufficient with carbon source or the C/ N ratio is quite high(＞20),the PHAs accumulation is much faster than degradation. While facing different stresses,such as low nutrient availability and detrimental physical,chemical,or biological factors,these bacteria begin to mobilize PHAs to conquer those unfavorable environments.This paper we reported for the first time that a stress-induced system enabled E.coli,a non-PHB producer,to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.In this study,the successful expression of PHB biosynthesis and PHB depolymerase genes in E.coli was confirmed by PHB production and 3-hydroxybutyrate secretion.To confirm the capability of 3HB utilization by E.coli,an in vitro experiment of 3-hydroxybutyral-CoA synthetase activity measurement was performed.The consumption of 3HB as carbon and energy source when CoA and ATP existed in the reaction mixture indicated that the complete PHB mobilization in engineered E.coli was realized and 3HB can serve as an energy material.
Starvation experiment demonstrated that the complete PHB mobilization system in E.coli served as an intracellular energy and carbon storage system,which increased the survival rate of the host when carbon resources were limited.Stress tolerance experiment indicated that E.coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.This engineered E.coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation.
So far,PHAs with more than 150 types of monomers have been synthesized, ranging from stiff plastics to flexible elastomers,which properties are mainly depending on the monomer composition and molecular weight.PHB is a highly crystalline material which is stiffer and more brittle than synthetic plastics, Medium-chain-length(mcl)-PHAs are elastic polyesters but hard to manipulate, therefore,their industrial applications are limited.The copolymers consisting of both scl and mcl-PHAs greatly improve the flexibility and toughness owing to its diverse monomers.To a considerable extent,the substrate specificity of the PHA synthases determines the composition of the accumulated PHA.
This section is aimed at obtain a broad substrate specificity hybrid PHA synthase which will posseses the substrate specificity of both parent PHA synthases and thus can incorporate both scl and mcl-hydroxyalkanyl-CoA precursors into copolymers. This paper introduced an incremental truncation hybrid enzyme library to create a broad substrate specificity hybrid enzyme.For hybrid or chimeric enzyme construction,it is difficult to predict exactly which fusion-points in domain swapping will produce an active hybrid enzyme.Thus,Incremental truncation was thought as a powerful strategy in the engineering of novel biocatalysts.In this study,we created a hybrid library with PHA synthase gene from Ralstonia eutropha as C-terminus and the gene from Pseudomonas aeruginosa as N-terminus using incremental truncation method.
First,a recombinant strain harboring two PHA synthase genes was constructed,the polymer accumulated in this strain is a blend of PHB(about 90%) and PHA(about 10%).Then the incremental truncation was used to create a phaCl_(pa)-phbC_(Re) hybrid protein library.As revealed by PHB production in recombinant E.coli,25 hybrids with different length were functional.The truncated mutants of PhbC_(Re) showed a gradually decreased in vivo enzyme activity trends along with the increased truncation degree of PhbC_(Re).High degree truncation of PhbC_(Re) at N-terminus can be complemented by N-terminus of typeⅡPHA synthase-PHA synthase from Pseudomonas aeruginosa.Importantly,three of the hybrids were found to have altered product specificity.They can produce P(3HB-co-3HA) with different monomer composition,which will broaden the variation of engineered PHA synthase. Accordingly,these results suggested that the N-terminal sequence of PHA synthase contributed to both enzyme activity and product specificity.The incremental truncation provides us a novel method to generate functional PHA synthase with desired properties and to study the reaction mechanism of the enzyme.
PHB synthase from Ralstonia eutropha purified from recombinant E.coli cells exists in aqueous solution in both monomeric(single subunit) and homodimeric(two subunits) forms in equilibrium.Several lines of evidence suggest that the homodimer is the active form of the synthase.The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site.The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis,but a second thiol group on the same protein molecule has not yet been identified.
In this paper,we confirmed the presence of synthase dimer in reaction by using complement GFP method based on reassembly of dissected fragments of green fluorescent protein fused to interacting proteins.The system used in this study consists of two plasmid vectors for indenendent expression of fusions with N-and C-terminal fragments of GFP,and allows for simple visual detection of protein-protein interactions.We demonstrate that a dimer synthase that has initiated the polymerization reaction(primed synthase) when the precursor is present,which means the dimer form of PHB synthase is significantly more stable against dissociation than the unprimed(unreacted) dimer synthase.Further study should be carried out to detect whether PHB granule formation begins at the inner site of the cytoplasmic membrane which is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.
对于PHA的研究已经有七十多年的历史了,从最初聚-3-羟基丁酸酯(PHB)天然颗粒的提取,到聚羟基丁酸羟基戊酸酯(PHBV)的工业化生产,再到PHA的新单体、新基因、新菌种,不同的发酵底物、合成途径以及代谢调控等等的新发现,加上蛋白质组学和代谢组学的相辅相成,人们对PHA的研究越来越广、也越来越深入,可开发的资源就越来越少。因此,怎样从一个新的视角入手研究PHA的新功能是本论文的目的之一。
在利用大肠杆菌基因工程菌高产PHB时,我们发现大肠杆菌在积累大量的PHB(达细胞干重的90%)后,不单没有影响到细胞内的渗透压,而且仍然可以生产琥珀酸等一些有机酸类,因此我们致力于发现和找到PHB对重组大肠杆菌的“贡献”以及它的机制。
PHA是细菌在生长条件不平衡时的产物(比如碳源过剩,氮源缺乏时),在环境不利时消耗掉以躲过恶劣环境的伤害,提高菌体对外界的适应能力。本论文首次在大肠杆菌中研究了PHB合成和降解对于宿主菌的有利作用。模仿PHB自然合成菌,成功地构建了一种压力诱导的启动系统来表达PHB合成和降解酶基因,无需进行人工诱导,只是在在压力情况下诱导PHB的合成和降解,就如同自然合成菌一样;顺利的表达了PHB合成及降解酶基因,得到了一株高产PHB以及3-羟基丁酸的工程菌。同时还验证了大肠杆菌利用3-羟基丁酸途径的存在,我们对大肠杆菌中3HB-CoA合成酶的活性进行了体外检测,发现在ATP以及CoA存在时,即能够将3-羟基丁酸转化为CoA形式而进入能量代谢循环,从而为细胞提供能量。
菌体不仅在碳源丰富时积累PHB,在氮源缺乏的环境中,这种不理想的条件非常有益于PHB的产生和积累。氮源的利用率是一个限制性因素,为了研究氮源限制也能够在重组菌E.coli DH5α(pSCP-CAB/pQWQ2)中诱导PHB产生,本实验以M9为基础培养基(含2%的葡萄糖),而变化NH_4Cl的浓度(0.2 g/L,1 g/L,2.5 g/L,5 g/L),在这四种培养条件下观察PHB的诱导情况。实验结果表明:低浓度氮源会诱导重组菌更快的更多的积累PHB。
同时,重组菌对外界压力的适应能力是通过饥饿实验和抗性试验检验的:积累PHB的工程菌E.coli DH5α(pSCP-CAB/pQWQ2)在经过32天的饥饿条件下,仍然可以存活和繁殖,这是普通大肠杆菌所不具有的,实验证明普通大肠杆菌在饥饿培养32天之后,存活率不到1%;同时探究了PHB合成和降解的这种循环机制对大肠杆菌抗性的影响,分别对大肠杆菌进行了热击、紫外照射、酸、高渗溶液的处理,发现产并能降解PHB的菌株E.coli DH5α(pSCP-CAB/pQWQ2)均具有较高的存活率,说明重组菌获得PHB生产能力后,菌体的各种抗性指标(如:耐饥饿存活率,抗氧化,抗热,抗辐射能力以及抗酸能力)均有较大提高,为工业上利用大肠杆菌生产各种物质,提供一个稳定抗逆菌株打下良好基础。
PHA家族是一个非常庞大的家族,目前已经发现PHA至少有150种不同的单体结构,并且还在不断地发掘出新的单体。在PHA家族中,单纯的PHB或PHA都由于其性能的缺陷影响了它的应用范围。因此开发具有高性能的PHA-co-PHB共聚物是国际上的研究热点。PHA聚合酶是生产共聚物的关键。
本研究以改变PHA聚合酶的底物特异性入手,通过对PHA聚合酶分子的改造,使其能够聚合本来不能利用的前体物,成为一种具备广泛底物特异性的酶,从而也就增加了PHA单体的种类。我们的目的是构建一个既有SCL-PHA合成能力又有mcl-PHA合成能力的PHA合酶,那么这个酶应该同时具有Ⅰ类合酶和Ⅱ类合酶的性质。本论文首次引入了递增截短法来构建一个具有广泛底物特异性的PHA合酶的杂合酶。它的优点是避免了在构建杂合酶时融合位点难于把握的问题,并且通过文库筛选可以得到更多有活性的广底物特异性的酶。
将Ⅰ类PHB合酶和Ⅱ类的mcl-PHA合酶同时克隆于一个载体上,转化大肠杆菌E.coli LS5218ΔfadA进行发酵生产,结果表明重组菌能够生产PHB/mcl-PHA的共混物,而且惊喜的发现PHB含量在85～95%。为进一步大规模生产优良性能的PHB/PHA共混物提供了良好的开端。然后,对PHA合酶进行的分子进化改造,我们通过递增截短文库法将合成mcl-PHA的合酶基因phaCl基因从3′端向5′端酶切,并将合成SCL-PHA的合酶基因phbC基因从5′端向3′端酶切,得到了不同程度酶切的杂合酶基因文库,通过对文库的进行尼罗红筛选和基因大小的筛选得到了25株能够合成PHB的重组茵,发现随着杂合酶phbC的5′端的逐渐切除,其PHB合酶的活性也逐步降低。根据最近的报道是将PhbC氨基端82个氨基酸切除后还能保持一定体内活性,85个氨基酸就完全失去活性,也就是是说其最小功能区既是切掉82个氨基酸之后的区域。我们通过构建缺失体库而获得的杂合酶首次发现了在83-100甚至120个氨基酸缺失后仍然有活性。这就说明,PHB合酶的递增缺失发现其氮端的缺失可以通过PHA合酶PhaCl的互补进行弥补。将失去活性的PHB合酶能够再次合成PHB;通过递增截短文库法构建的杂合酶,经筛选得到了3株克隆,检测这些酶发现,在体内既可以利用葡萄糖为碳源合成PHB,也可以利用脂肪酸(癸酸盐)为碳源合成P(HB-co-HA)共聚物,说明了PhaCl的氮端不仅仅是对杂合酶起到保护作用,而且可能含有底物的结合区域,因此能够结合中长链的羟脂酰CoA底物。这种人工改造的杂合酶性质稳定,易于控制表达,其延伸强度好等更加优越的性能,接近于低密度聚乙烯,在不久的将来必会得到广泛应用。
PHA合酶的性质研究也是近年来备受关注的,PHA生产的关键技术之一就是提高PHA合酶的聚合能力。而聚合能力的提高与了解PHA合酶的催化以及结构性质密切相关。近年来普遍认为,PHA聚合酶是以二聚体的形式催化3-羟基丁酰CoA合成高分子量聚酯PHA的,从酶的催化机制可以推测在体内酶的存在形式是单体与双体的平衡状态。并且认为聚合酶的二聚体是酶存在的活性形式;PHB颗粒的形成存在两个假说,近来越来越倾向于“出芽”模型,即PHB颗粒是从内膜上形成并脱落的,但是没有直接证据证明以上理论的正确性。本论文首次通过绿色荧光蛋白(GFP)片段重组装系统对PHB合酶的相互作用进行了检测,利用的是两个在大肠杆菌中相兼容的载体分别为pET11a-NGFP,及pMRBAD-CGFP,两个载体上分别有GFP的两部分片断,通过将待检测相互作用的两个基因分别与NGFP、CGFP的融合表达,再将两个载体同时转化大肠杆菌E.coliBL21(DE3),表达融合蛋白后,只有当蛋白发生相互作用时,GFP两部分相互靠近重组装,从而发出荧光。结果证明:只有在有底物3-羟基丁酰CoA存在的情况下,重组菌才会发出荧光,说明PHB合酶发生了相互作用,在体内形成二聚体然后催化PHB的形成的。在没有底物3-羟基丁酰CoA的情况下,PHB合酶并不或不主要以二聚体形式存在,GFP无法靠近形成重组装。因此观察不到GFP的绿色荧光。同时,若存在二聚体,通过对产荧光二聚体的实时示踪,可以观察在PHB颗粒形成早期,脂单层膜的来源。从而验证“出芽”模型的理论的正确性。该过程将会利用电镜技术帮助更好的验证。由于荧光显微镜不易观察到PHB颗粒的发生和形成,对这部分实验进行了进一步探索,利用荧光能量共振转移技术对其的研究正在进行中。
Polyhydroxyalkanoates(PHAs) are synthesized by micro-organism within the cell as a kind of bio-polyester.PHAs not only possess the properties as well as traditional petrochemical plastics but also are biodegradable,biocompatible,piezoelectric, optically active etc.these features makes them suitable for many applications in the packaging industry,medicine,pharmacy,agriculture,food industry,as raw materials for enantiomerically pure chemicals and in the production of paints.PHA can be obtained from renewable resources;hence,there has been a tremendous amount of commercial interest in these polymers.In contrast to bulk uses,PHAs have also been established as excellent materials in several niche applications,in particular for medical purposes,because PHAs are generally biocompatible with mammalian tissue and are resorbed at a slow rate;PHA can be used to develop tissue engineering scaffolds,which support cell growth and are degraded after implantation,leaving a viable tissue.Thus nowadays more and more efforts have been made for the applications in medical and tissue engineering.In conclusion,efforts in biopolymer research must be made to develop and enhance PHA production processes at low cost levels,and the development of PHA with better properties is necessary. PHA has been intensively studied for seventy years since the first PHA, poly(3-hydroxybutyrate)(PHB),was discovered in Bacillus megaterium by the French scientist Lemoigne in 1926.In the following years,research on purification of PHB granules from the cell and other forms of PHAs e.g.PHBV's production,and in succession investigations with other monomers,genes,microorganisms,metabolic pathways and the potential use of these biopolymers was realized.Recent research has focused on the use of alternative substrates,novel extraction methods,genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive.
When we engineered Escerichia coli to produce different kind of PHAs,we found that Escerichia coli could not only accumulate approximate 90%PHB of the cell dry weight without any harmful effect on cell growth but also produce succinic acid and other organic acid.Thus we were devoted to investigate the mechanism that PHB can benefit the host Escerichia coli for survival under stress conditions such as cold shock, pH and cell density.
A wide variety of microorganisms are able to accumulate polyhydroxyalkanoates (PHAs) as intracellular carbon/ energy storage compounds or reducing power for coping with changing,often oligotrophic environments.Various PHAs,as well as the best-known poly 3-β-hydroxybutyrate(PHB),were found to be accumulated and degraded as required under environmental conditions by most natural PHAs producing bacteria.When the environment is sufficient with carbon source or the C/ N ratio is quite high(＞20),the PHAs accumulation is much faster than degradation. While facing different stresses,such as low nutrient availability and detrimental physical,chemical,or biological factors,these bacteria begin to mobilize PHAs to conquer those unfavorable environments.This paper we reported for the first time that a stress-induced system enabled E.coli,a non-PHB producer,to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.In this study,the successful expression of PHB biosynthesis and PHB depolymerase genes in E.coli was confirmed by PHB production and 3-hydroxybutyrate secretion.To confirm the capability of 3HB utilization by E.coli,an in vitro experiment of 3-hydroxybutyral-CoA synthetase activity measurement was performed.The consumption of 3HB as carbon and energy source when CoA and ATP existed in the reaction mixture indicated that the complete PHB mobilization in engineered E.coli was realized and 3HB can serve as an energy material.
Starvation experiment demonstrated that the complete PHB mobilization system in E.coli served as an intracellular energy and carbon storage system,which increased the survival rate of the host when carbon resources were limited.Stress tolerance experiment indicated that E.coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.This engineered E.coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation.
So far,PHAs with more than 150 types of monomers have been synthesized, ranging from stiff plastics to flexible elastomers,which properties are mainly depending on the monomer composition and molecular weight.PHB is a highly crystalline material which is stiffer and more brittle than synthetic plastics, Medium-chain-length(mcl)-PHAs are elastic polyesters but hard to manipulate, therefore,their industrial applications are limited.The copolymers consisting of both scl and mcl-PHAs greatly improve the flexibility and toughness owing to its diverse monomers.To a considerable extent,the substrate specificity of the PHA synthases determines the composition of the accumulated PHA.
This section is aimed at obtain a broad substrate specificity hybrid PHA synthase which will posseses the substrate specificity of both parent PHA synthases and thus can incorporate both scl and mcl-hydroxyalkanyl-CoA precursors into copolymers. This paper introduced an incremental truncation hybrid enzyme library to create a broad substrate specificity hybrid enzyme.For hybrid or chimeric enzyme construction,it is difficult to predict exactly which fusion-points in domain swapping will produce an active hybrid enzyme.Thus,Incremental truncation was thought as a powerful strategy in the engineering of novel biocatalysts.In this study,we created a hybrid library with PHA synthase gene from Ralstonia eutropha as C-terminus and the gene from Pseudomonas aeruginosa as N-terminus using incremental truncation method.
First,a recombinant strain harboring two PHA synthase genes was constructed,the polymer accumulated in this strain is a blend of PHB(about 90%) and PHA(about 10%).Then the incremental truncation was used to create a phaCl_(pa)-phbC_(Re) hybrid protein library.As revealed by PHB production in recombinant E.coli,25 hybrids with different length were functional.The truncated mutants of PhbC_(Re) showed a gradually decreased in vivo enzyme activity trends along with the increased truncation degree of PhbC_(Re).High degree truncation of PhbC_(Re) at N-terminus can be complemented by N-terminus of typeⅡPHA synthase-PHA synthase from Pseudomonas aeruginosa.Importantly,three of the hybrids were found to have altered product specificity.They can produce P(3HB-co-3HA) with different monomer composition,which will broaden the variation of engineered PHA synthase. Accordingly,these results suggested that the N-terminal sequence of PHA synthase contributed to both enzyme activity and product specificity.The incremental truncation provides us a novel method to generate functional PHA synthase with desired properties and to study the reaction mechanism of the enzyme.
PHB synthase from Ralstonia eutropha purified from recombinant E.coli cells exists in aqueous solution in both monomeric(single subunit) and homodimeric(two subunits) forms in equilibrium.Several lines of evidence suggest that the homodimer is the active form of the synthase.The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site.The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis,but a second thiol group on the same protein molecule has not yet been identified.
In this paper,we confirmed the presence of synthase dimer in reaction by using complement GFP method based on reassembly of dissected fragments of green fluorescent protein fused to interacting proteins.The system used in this study consists of two plasmid vectors for indenendent expression of fusions with N-and C-terminal fragments of GFP,and allows for simple visual detection of protein-protein interactions.We demonstrate that a dimer synthase that has initiated the polymerization reaction(primed synthase) when the precursor is present,which means the dimer form of PHB synthase is significantly more stable against dissociation than the unprimed(unreacted) dimer synthase.Further study should be carried out to detect whether PHB granule formation begins at the inner site of the cytoplasmic membrane which is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.
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