RUI: Genetic and Functional Analysis of the Nocardia corallina Polyhydroxyalkanoate Synthase

MCB-9514100丹尼斯聚羟基烷酸酯(PHAs)是许多细菌在营养压力时期积累的储能聚合物。巧合的是，这些聚合物也可以用作可生物降解的塑料。塑料的物理性能由单体单元中碳链的长度决定，其中C4均聚物相对脆弱，而C8均聚物为弹性。共聚物往往具有介于其单体单元之间的物理性质。介导聚合的酶，PHA合成酶，根据底物特异性和分子结构被分为三个不同的类别。I型PHA合成酶具有单一的多肽链，主要形成C4(3-羟基丁酸酯；3HB)单体单位。II型PHA合成酶也有一个单一的多肽链，但它们形成C4到C12(3-羟基十二酸；3HDD)单体单位，其中CG(3-羟基辛酸；3HO)和C10(3-羟基十二酸；3HD)是优势单位。III型PHA合成酶由2条多肽链组成，主要形成3HB。然而，这一类的一些成员能够将大部分3HV并入P(3HB-co-3HV)共聚物。珊瑚核杆菌的PHA合成酶(PhaCNc)是一种利用葡萄糖和果糖等无关碳源产生大量3-HV的细菌，已被克隆。初步数据表明，该PHA合酶并不完全符合这三类合酶中的任何一种。它只有一条多肽链，在基因上与II型PHA合成酶最相似，但其底物专一性似乎主要在3HV和3HH范围内，尽管其总底物范围从3HB到3HHp(3-羟基庚酸)。这相当令人兴奋，原因有几个。首先，phaCNc代表了一类潜在的新的PHA合成酶。其次，它是一种PHA合成酶，可以合成还很少被研究的新型聚羟基烷酸酯。在S恶臭假单胞菌中高效表达，在产碱杆菌和产气性克雷伯菌中合理表达，在大肠杆菌中弱表达。第四，它是一种PHA合成酶，由于它参与长链和中链脂肪酸的代谢，但只形成3HB和3HHx(3-羟基己酸酯)或3HV(3-羟基戊酸酯)和3HHp的共聚酯，因此可能有能力用于植物结构。这项研究继续在两个特定领域对phaCNc进行分析。首先，我们将分析phaCNc在上述四种不同重组宿主中的表达。将进行酶活性分析、phaCNc转录本的量化和PhaCNc水平的量化。这些数据将与每个重组宿主中聚合物的表达水平相关联。此外，将通过选择特定的密码子，将其遗传改变为更普遍的密码子，然后检查这种改变对酶活性、phaCNc转录水平、蛋白质水平和聚合物水平的结果，来分析每个宿主中罕见密码子使用的影响。最后，将phaCNc置于一个强大的、可调节的启动子的控制下的效果将得到确定。该项目这一阶段的预期结果是了解限制或促进phaCNc表达的因素。另一个结果是构建了在重组宿主中良好表达的phaCNc基因。在该项目的第二阶段，将开始研究，重点是确定介导底物专一性的phaCNc区域。使用的第一种策略是随机诱变phaCNc，并筛选PhaCNc的底物特异性改变。这将利用我们实验室开发的一种新的筛选方法进行酶促反应。表现出底物特异性改变的phaCNc基因将被分析其DNA序列的特异性变化。用于识别底物特异性调控区域的第二种策略是从不同的PHA合成酶基因构建嵌合体，并分析构建的酶活性和聚合物组成。尽管已经克隆了许多(约19种)不同的PHA合成酶，但只有一种得到了严格的研究，这就是产碱杆菌的PHA合成酶，这些研究没有扩展到本文提出的水平，因为phaCNc-phaCNc与大多数其他PHA合成酶基因相比具有优势，因为它的底物专一性属于更有可能具有商业价值的范围。这里提出的研究不仅应该适用于phaCNc，也应该适用于其他PHA合成酶基因。这项研究适用于低成本的可生物降解塑料的生产。此外，它还完成了对本科生分子生物学技术及其在基础和应用兴趣问题中的应用的培训。***

MCB-9514100 Dennis Polyhydroxyalkanoates (PHAs) are energy-reserve polymers that are accumulated by many bacterial species in times of nutritional stress. Coincidentally, these polymers may also be utilized as biodegradable plastic. The physical properties of the plastic are dictated by the length of the carbon chain in the monomer unit with C4 homopolymers being relatively brittle and C8 homopolymers being elastomeric. Copolymers tend to have physical properties intermediate between their monomer units. The enzymes that mediate the polymerization, PHA synthases, are grouped into three distinct categories based on their substrate specificity and their molecular structure. Type I PHA synthases have a single polypeptide chain and form predominately C4 (3-hydroxybutyrate; 3HB) monomer units. Type II PHA synthases also have a single polypeptide chain, but they form C4 to C12 (3-hydroxydodecanoate; 3HDD) monomer units, with Cg (3-hydroxyoctanoate; 3HO) and C10 (3-hydroxydecanoate; 3HD) being the preponderant units. Type III PHA synthase are composed of 2 polypeptide chains and form largely 3HB. However, some members of this class are able to incorporate mostly 3HV into P(3HB-co-3HV) copolymer. The PHA synthase from N. corallina (phaCNc), a bacteria which makes a substantial amount of 3-HV from unrelated carbon sources such as glucose and fructose has been cloned. Preliminary data suggests that this PHA synthase does not completely meet the specifications for any of the three classes of synthases. It has a single polypeptide chain and is genetically most similar to Type II PHA synthases, yet its substrate specificity appears to be largely in the 3HV and 3HH range, though its total substrate range is from 3HB to 3HHp (3-hydroxyheptanoate). This is quite exciting for several reasons. First, phaCNc represents a potentially new class of PHA synthase. Second, it is a PHA synthase which makes new types of polyhydroxyalkanoates that have been little-characterized. Third, it is well-expressed in Pseudomona s putida, reasonably expressed in Alcaligenes eutrophus and Klebsiella aerogenes, and poorly expressed in Escherichia coli. Fourth, it is a PHA synthase that may have the capacity to be utilized in plant constructs because it accesses long- and medium-chain fatty acid metabolism, yet only forms copolyesters of 3HB and 3HHx (3-hydroxyhexanoate) or 3HV (3-hydroxyvalerate) and 3HHp. This research continues the analysis of phaCNc in two specific areas. In the first, expression of phaCNc will be analyzed in the four different recombinant hosts named above. Analyses will be conducted for enzymatic activity, quantitation of phaCNc transcripts, and quantitation of PhaCNc levels. This data will be correlated with the levels of expression of polymer in each recombinant host. In addition, the effect of rare codon usage in each host will be analyzed by selecting specific codons, genetically altering them to more prevalent codons, and then examining the result of this alteration on enzymatic activity, phaCNc transcript levels, protein levels, and polymer levels. Finally, the effect of placing phaCNc under the control of a strong, regulatable promoter will be determined. The expected result of this phase of the project is an understanding of the factors which limit or facilitate phaCNc expression. An additional result is the construction of phaCNc genes which are well-expressed in recombinant hosts. In the second phase of the project studies will be initiated that focus on identifying regions of phaCNc which mediate substrate specificity. The first strategy used will be to randomly mutagenize phaCNc and screen for altered substrate specificity of PhaCNc. This will done enzymatically utilizing a novel screening assay developed in our laboratory. phaCNc genes which exhibit altered substrate specificity will be analyzed as to the specific change in their DNA sequence. The second strategy used to to identify regions that regulate substrate specificity will be to construct chimeras from different PHA synthase g enes and analyze the construct for enzymatic activity and polymer composition. Though many (approximately 19) different PHA synthases have been cloned, only one has been rigorously studied, the Alcaligenes eutrophus PHA synthase, and these studies did not extend to the level proposed here for phaCNc- phaCNc has advantages over most other PHA synthase genes in that its substrate specificity falls into a range that is more likely to be of commercial interest. Studies proposed here should be applicable not only to phaCNc, but to other PHA synthase genes as well. %%% This research has application to the inexpensive production of biodegradable plastics. In addition, it accomplishes the training of undergraduate students in the techniques of molecular biology and their applications to problems of both basic and applied interests. ***