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

RUI：珊瑚诺卡氏菌聚羟基脂肪酸合酶的遗传和功能分析

• 批准号: 9514100
• 负责人: Douglas Dennis
• 金额: $ 26.3万
• 依托单位: James Madison University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9514100&HistoricalAwards=false
• 关键词: RUI; Genetic; Functional; Analysis; Nocardia

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. ***

MCB-9514100丹尼斯多羟基烷烃（PHAS）是能量储存的聚合物，在营养应激时期，许多细菌物种积累。巧合的是，这些聚合物也可以用作可生物降解的塑料。塑料的物理特性取决于单体单元中碳链的长度，而C4均聚物相对脆性，C8均聚物是弹性的。共聚物倾向于在单体单元之间具有物理特性。基于其底物特异性及其分子结构，将介导聚合的酶（PHA合酶）分为三个不同的类别。 I型PHA合酶具有单个多肽链，主要形成C4（3-羟基丁酸； 3HB​​）单体单位。 II型PHA合成酶也具有单个多肽链，但它们形成C4至C12（3-羟基多二烷酸酯； 3HDD）单体单位，带有CG（3-羟基二链霉菌; 3HO）和C10（3HO）和C10（3-羟基甲酸盐; 3hdhdecanoate; 3HdHd）是前独立的。 III型PHA合酶由2个多肽链组成，主要形成3HB。但是，该类的一些成员能够将3HV大部分纳入P（3HB-CO-3HV）共聚物中。 来自Corallina N. Corallina（PHACNC）的PHA合酶是一种细菌，从无关的碳源（例如葡萄糖和果糖）产生了大量3-HV。初步数据表明，该PHA合酶不完全满足三类合成酶中的任何一个。它具有单个多肽链，并且在遗传上与II型PHA合酶最相似，但其底物特异性似乎在很大程度上在3HV和3HH范围内，尽管其总底物范围为3HB至3HHP（3-羟基烷酸盐）。由于几个原因，这非常令人兴奋。首先，PHACNC代表了一种潜在的新类PHA合酶。其次，它是一种PHA合酶，它使新型的多羟基烷酸盐量很少被征兆。第三，它在伪虫的伪虫中表达了很好的表达，在藻素的浓度和克雷伯菌中合理表达，在大肠杆菌中表达不佳。第四，它是一种PHA合酶，可能具有在植物构建体中使用的能力，因为它可以访问长和中链脂肪酸代谢，但仅形成3HB和3HHX和3HHX（3-羟基己酸）（3-羟基己酸）或3HV（3HV）或3HV（3-Hydroxyvalerate）和3HHP的共溶性。 这项研究继续在两个特定领域对PHACNC进行分析。 首先，将在上述四个不同的重组宿主中分析PHACNC的表达。将进行分析，以进行酶活性，PHACNC转录本的定量和PHACNC水平的定量。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，将它们更改为更普遍的密码子，然后检查这种改变酶活性，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效果。最后，将确定将PHACNC置于强，可调节启动子的控制之下的效果。该项目阶段的预期结果是了解限制或促进PHACNC表达的因素。另一个结果是在重组宿主中表达良好表达的PHACNC基因的构建。 将在项目研究的第二阶段开始，着重于识别介导底物特异性的PHACNC区域。使用的第一种策略将是随机诱变PHACNC和筛选以改变PHACNC的底物特异性。这将在我们的实验室开发的新型筛选测定法上使用酶促进行。在其DNA序列的特定变化方面，将分析显示出改变底物特异性的PHACNC基因。用于识别调节底物特异性区域的第二种策略是从不同的PHA合酶G ENES构造嵌合体，并分析构建体以获得酶活性和聚合物组成。 尽管已克隆了许多（大约19个）不同的PHA合酶，但仅对Alcaligenes Eutrophus Pha合酶进行了严格的研究，这些研究并未扩展到这里提出的水平，因为PHACNC-PHACNC具有比大多数其他PHA合成酶基因的优势，因为它的基础特定范围更可能涉及该范围的范围。这里提出的研究不仅适用于PHACNC，还适用于其他PHA合酶基因。 %%％这项研究适用于廉价生产可生物降解的塑料。 此外，它还完成了本科生在分子生物学技术中的培训及其在基本和应用利益的问题上的应用。 ***