Microbial and Enzymatic Technology Group, Bioprocess Centre, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada.
Microb Cell Fact. 2010 Sep 16;9:70. doi: 10.1186/1475-2859-9-70.
Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications.
Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases.
This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.
甲醇利用（甲基营养）细菌作为细胞工厂在利用生物量衍生甲醇生产众多产品方面具有巨大潜力。生物甲醇本质上是非食品原料，这相对于利用糖的细胞工厂具有优势。可以从甲醇中获得低价值产品以及精细化学品和先进材料。例如，包括甲基杆菌（Methylobacterium extorquens）在内的几种甲醇利用细菌可以大量生产可生物降解的聚酯聚羟基丁酸酯（PHB），这是最著名的聚羟基烷酸酯（PHA）之一。为了生产具有更高附加值的第二代 PHAs，我们探索了利用 M. extorquens 生产含有 C-C 双键的功能性 PHAs 的可行性，从而使它们能够进行未来的化学/生物化学修饰，以实现高价值应用。
我们专有的 M. extorquens ATCC 55366 在以甲醇和选定的不饱和羧酸作为次级底物进行喂食时，无法产生功能性 PHAs。然而，使用基于肉桂酸作为诱导剂（肉桂酸开关）的诱导和调控表达系统，克隆荧光假单胞菌 GK13 的 phaC1 或 phaC2 基因，产生了能够将适量 C-C 双键掺入 PHA 的重组 M. extorquens 菌株，起始物为 C6= 和/或 C8=。两种重组菌株在使用 C11=时效果不佳。含有 phaC2 基因的菌株在利用 C8=和将 C-C 双键掺入 PHA 方面表现更好。溶剂分馏表明，所产生的聚合物是 PHA 共混物，因此源自天然和重组 PHA 合酶的独立作用。
这项工作构成了代谢工程应用于构建能够生产含有 C-C 双键的功能性 PHAs 的甲醇利用细菌的一个例子。在这方面，PhaC2 合酶在利用 C8=作为 C-C 双键的来源以及将 C-C 双键掺入源自 C6=或 C8=的 PHA 方面表现优于 PhaC1 合酶。因此，M. ex-phaC2 菌株是一种很有前途的生物催化剂，可用于生成未来在各个领域具有高附加值的先进（功能性）PHA。
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