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

丹尼斯聚羟基烷酸酯（PHAs）是一种能量储备聚合物，是许多细菌在营养压力下积累的。巧合的是，这些聚合物也可以用作可生物降解的塑料。塑料的物理性能是由单体单元中碳链的长度决定的，C4均聚物相对脆，而C8均聚物具有弹性。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，根据它们的底物特异性和分子结构分为三种不同的类别。I型PHA合酶具有单肽链，主要形成C4（3-羟基丁酸酯；3HB）单体单位。II型PHA合酶也有单肽链，但它们形成C4到C12（3-羟基十二酸酯；3HDD）单体单元，其中Cg（3-羟基辛酸酯；3HO）和C10（3-羟基癸酸酯；3HD）是优势单元。III型PHA合酶由2条多肽链组成，主要形成3HB。然而，该类别的一些成员能够将大部分3HV纳入P（3HB-co-3HV）共聚物中。一种从不相关的碳源如葡萄糖和果糖中产生大量3-HV的细菌——N. corallina （phaCNc）的PHA合成酶已被克隆。初步数据表明，这种PHA合酶不完全符合这三类合酶中的任何一类的规格。它具有单一多肽链，在遗传上与II型PHA合成酶最相似，但其底物特异性似乎主要在3HV和3HH范围内，尽管其总底物范围从3HB到3HHp（3-羟基庚酸）。出于几个原因，这是相当令人兴奋的。首先，phaCNc代表了一类潜在的新PHA合酶。其次，它是一种PHA合成酶，它可以产生新的类型的聚羟基烷酸盐，这种酶很少被表征。三是在恶臭假单胞菌中表达良好，在富营养化钙菌和产气克雷伯菌中表达合理，在大肠杆菌中表达不良。第四，它是一种PHA合成酶，可能有能力在植物结构中被利用，因为它参与长链和中链脂肪酸代谢，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。本研究继续在两个特定领域分析phaCNc。首先，我们将分析phaCNc在上述四种不同重组宿主中的表达。将对酶活性、phaCNc转录本的定量和phaCNc水平的定量进行分析。该数据将与每个重组宿主中聚合物的表达水平相关。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转录物水平、蛋白质水平和聚合物水平的影响，来分析每个宿主中罕见密码子使用的影响。最后，将phaCNc置于一个强的、可调节的启动子控制下的效果将被确定。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表达良好的phaCNc基因。在项目的第二阶段，研究将集中于确定介导底物特异性的phaCNc区域。第一种策略是随机诱变phaCNc并筛选改变的phaCNc底物特异性。这将通过酶学方法利用我们实验室开发的一种新型筛选试验来完成。对于底物特异性改变的phaCNc基因，将根据其DNA序列的具体变化进行分析。用于确定调节底物特异性区域的第二种策略是构建不同PHA合成酶基因的嵌合体，并分析其酶活性和聚合物组成。虽然已经克隆了许多（大约19种）不同的PHA合成酶，但只有一种被严格研究，即Alcaligenes富营养化PHA合成酶，这些研究并没有扩展到本文对phaCNc提出的水平- phaCNc比大多数其他PHA合成酶基因具有优势，因为它的底物特异性在一个更有可能具有商业价值的范围内。本文提出的研究不仅适用于phaCNc，也适用于其他PHA合酶基因。这项研究可应用于廉价生产生物可降解塑料。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上的应用的培训。＊＊＊

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. ***