Investigation of terminal alkene formation by polyketide synthases and the application toward sustainable alpha-olefin production

拟议标题：特定聚酮合成酶的特征及其在生物生产高度可取的短链烯烃方面的应用。最近对以石油为基础的燃料和商品化学品的供应和成本的关切引起了人们对开辟这些重要碳氢化合物的替代路线的兴趣。尤其是α-烯烃，这是一类目前从石油中提取的化合物，是许多工业生产的塑料、短链脂肪酸、表面活性剂的前体，最近被吹捧为潜在的燃料替代品。合成商品和特种化学品的一个潜在途径是改造微生物，将廉价的可再生糖转化为所需的产品。不幸的是，可用于产生不同化学官能团的生物转化的范围不足，因此限制了我们取代所有石油衍生商品化学品的能力。为了生产这一重要的化学物质，加州大学伯克利分校的科学家们已经确定了由一类聚酮合成酶(PKS)末端模块组成的酶组分，这些模块通过一种独特的结构特征产生α-烯烃。了解这一神秘反应的机理将为未来酶生物催化剂的设计，特别是为特定终端烯烃生产而设计的新的PKS组件铺平道路。有关PKS模块的研究和工程工作将在获奖者Jay D.Keasling教授(加州大学伯克利分校)的监督下进行，由来自广泛社会经济和教育背景的不同群体的学生进行。特别是，通过iCLEM计划以及与加州大学伯克利分校扩展计划的合作，Keasling和他的团队旨在让高中和本科生参与到正规大学要求之外的教育(例如通过副学士学位和证书计划)，以获得宝贵的实践经验。这项建议的目的是了解这些独特的，保守的末端硫代转移酶硫酯酶(ST-TE)的PKS双域是如何发挥功能来产生α-烯烃的。该项目的具体目标包括1)表征四个末端烯烃形成PKS模块的总体底物耐受性和基本动力学参数；2)对CurM TE进行深入的机理分析，以更好地理解这一关键转变；以及3)利用这些信息来指导可持续生产C5(戊烯)和C6(己烯)末端烯烃以及相关的芳香烯-苯乙烯的两种嵌合合成酶的产生。PKS蛋白将在大肠杆菌或本地烯烃生产商(如蓝藻聚球藻PCC7002)中表达。聚合结果将被用来设计专门用于生产这些烯烃化合物的高活性催化剂。嵌合PKS将在大肠杆菌和聚球藻PCC7002中进行评估，以更好地探索PKS活性和在自然和非自然宿主中的烯烃形成。本发明的定义这种独特的终止机制和应用该发现来创造新的酶将提供一条通往末端烯烃的生物路线，末端烯烃最终可能包含新的官能团(例如，卤素、胺等)。这可以用来创造具有改变性质的新的可交叉连接的聚合物，或者具有改进的燃料特性的化合物，所有这些都来自低成本的可再生资源。

Proposed Title: Characterization of specific polyketide synthases and application toward the biological production of highly-desirable, short-chain olefins. Recent concerns as to the supply and cost of petroleum-based fuels and commodity chemicals have sparked an interest in creating alternative routes to these important hydrocarbon compounds. In particular the alpha-olefins, a class of compounds currently derived from petroleum, serve as precursors to many industrially produced plastics, short chain fatty acids, surfactants and recently have been touted as potential fuel alternatives. One potential route for synthesizing commodity and specialty chemicals is to engineer microorganisms to transform inexpensive, renewable sugars to desired products. Unfortunately, the scope of biological transformations available to generate differing chemical functional groups is lacking, thus limiting our ability to replace all petroleum-derived commodity chemicals. In an effort to produce this important set of chemicals, scientists at the University of California, Berkeley have identified enzyme components comprising a class of polyketide synthase (PKS) terminal modules known to generate alpha-olefins by way of a unique structural feature. Understanding the mechanism of this enigmatic reaction will pave the way for future enzyme biocatalyst design, in particular, new designer PKS assemblies for specific terminal alkene production. Studies and engineering efforts concerning the PKS modules will be executed under the supervision of the awardee, Prof. Jay D. Keasling (UC, Berkeley), by a diverse group of students from a wide range of socioeconomic and educational backgrounds. In particular, through the iCLEM program and partnership with the University of California, Berkeley Extension Program, Keasling and his group aim to involve high school and undergraduate students pursuing education outside the formal university requirements (e.g. through Associate Degrees and certificate programs) to gain valuable hands-on experience. The goal of this proposal is to understand how these unique, conserved terminal sulfotransferase-thioesterase (ST-TE) didomains of PKS function to produce alpha-olefins. The specific project aims include 1) to characterize the overall substrate tolerance and fundamental kinetic parameters of four terminal olefin-forming PKS modules; 2) to perform an in-depth mechanistic analysis of the CurM TE to better understand this pivotal transformation; and 3) to use this information to guide the generation of two chimeric synthases for the sustainable production of C5 (pentene) and C6 (hexene) terminal olefins, as well as the related aromatic alkene, styrene. PKS proteins will be expressed in Escherichia coli or a native olefin producer, such as cyanobacteria Synechococcus PCC 7002. Aggregate results will be leveraged to design highly active catalysts specific for producing these olefinic compounds. Chimeric PKSs will be evaluated in both E. coli and Synechococcus PCC 7002 to better explore PKS activity and olefin formation in both natural and unnatural hosts. Definition of this this unique termination mechanism and application of the findings to create novel enzymes will provide a biological route to terminal alkenes, which may ultimately contain novel functional groups (e.g., halogens, amines, etc.) that can be used to create new cross-linkable polymers with altered properties, or compounds with improved fuel characteristics, all from low-cost renewable resources.