Industrial chemicals of the monoterpenoid class realised through synthetic biology and pathway engineering

化工生产中的工业生物技术是21世纪关键的使能技术之一。欧洲的化学工业是世界上最大的，生物技术将对化学品生产（散装和精细化学品）产生重大影响，因为我们利用酶的生物催化潜力来开发绿色、创新和有竞争力的化学工艺。手性分子的制造是一个特别的挑战：微生物是化合物的极其丰富的来源，也是化学工业或新药使用的代谢途径。在这项研究计划中，我们将开发新的生物催化剂来合成一组重要的有机化合物（单萜类化合物），这些化合物在食品和香料工业中具有很高的商业价值。开发最有前途的单萜类化合物的一个严重障碍是它们在自然资源中的可得性有限。这些分子的提取和蒸馏成本高，产量低，需要大量的自然资源。供应和环境条件的季节性变化也影响生产。产物外消旋混合物的形成也是传统化学合成制造单萜类化合物的一个主要缺点。这可以通过使用昂贵的手性催化剂或从手性池中提取材料来缓解，但对于萜类化合物来说，这些构建块无法从自然资源中获得足够的丰度。酶催化合成是有吸引力的：区域和立体选择性生物转化，使用“绿色”反应条件和缺乏有毒副产物相结合，使生物合成在可持续制造中具有吸引力。美国和欧洲的立法承认生物合成制造过程中的产品被认为是“天然的”。这增加了显著的市场价值，特别是在食品和调味品制造中。确定萜类化合物的酶途径很重要，因为(i)对“天然产物”的要求；（ii）不能通过化学合成轻易地获得它们；（iii）经典合成的环境影响；自然资源供应有限。我们的工作将结合新酶催化剂的知识应用和合成生物学来设计新的单萜类化合物代谢途径。我们将通过对大肠杆菌菌株的遗传操作，在全细胞合成微生物中产生单萜合成途径，以(i)上调类异戊二烯前体生物合成中的酶，（ii）结合非天然基因来创造单萜合成的新途径。我们将创造几种新的生物合成途径，即(-)-柠檬烯的生物合成；（5R）-香芹酮及其衍生物；(+) -pulegone;(+) -menthofuran;紫苏醇及其衍生物；薄荷醇化合物。我们将产生新的大肠杆菌菌株（每个菌株都含有上调的类异戊二烯前体生物合成基因），其中将引入额外的外源/工程基因，编码单萜烯合成的组分途径酶。菌株将优化酶表达、生长和单萜类生物合成，通过基于系统的分析和建模，实现高效的全细胞生物转化。工程菌株将依次用于产生单萜类化合物，而不需要酶分离或外源添加辅助因子循环系统。该方案将改变商业上重要的单萜类化合物的工业合成。它提供了BBSRC基于知识的生物经济战略重点，并利用合成/系统生物学来支持工业的下一代生物催化。

Industrial biotechnology in chemicals manufacture is one of the key enabling technologies in the 21st Century. The European chemical industry is the largest in the world and biotechnology is poised to make a substantial impact on chemicals production (both bulk and fine chemicals) as we harness the biocatalytic potential of enzymes to develop green, innovative and competitive chemical processes. The manufacture of chiral molecules is a particular challenge: microorganisms are tremendously rich sources of compounds and metabolic pathways of use to the chemicals industry or as new pharmaceuticals. In this research programme we will develop new biocatalysts to synthesise an important group of organic compounds (the monoterpenoids) that have high commercial value in the food and fragrances industries. A serious obstacle to exploiting the most promising monoterpenoids is their limited availability from natural resources. Extraction and distillation of these molecules is expensive, low-yielding and requires substantial expenditure of natural resources. Seasonal variation in supply and environmental conditions also affect production. Formation of product racemic mixtures is also a major drawback in the manufacture of monoterpenoids by classical chemical synthesis. This can be mitigated by use of expensive chiral catalysts or by starting with materials from the chiral pool, but for terpenoids these building blocks are not available in sufficient abundance from natural resources. Enzyme-catalysed synthesis is attractive: regio- and stereo-selective biotransformation, use of 'green' reaction conditions and lack of toxic by-products combine to make biosynthesis attractive in sustainable manufacturing. US and European legislation acknowledges that products from biosynthetic manufacturing processes are considered 'natural'. This adds significant market value, especially in foods and flavourings manufacture. Identifying enzymatic routes to terpenoids is important because of (i) the requirement for 'natural products'; (ii) the inability to access them readily by chemical synthesis; (iii) environmental impact of classical synthesis; (iv) limited availability from natural resources. Our work will unite knowledge-based application of new enzyme catalysts with synthetic biology to design new metabolic pathways to the monoterpenoids. We will generate pathways for monoterpenoid synthesis in whole cell synthetic microorganisms by genetic manipulation of E. coli strains to (i) upregulate enzymes in isoprenoid precursor biosynthesis and (ii) incorporate non-native genes to create novel pathways for monoterpenoid production. We will create several novel biosynthetic pathways, namely for the biosynthesis of (-)-limonene; (5R)-carvone and derivatives; (+)-pulegone; (+)-menthofuran; perillyl alcohol and derivatives; menthol compounds. We will generate novel E. coli strains (each containing upregulated isoprenoid precursor biosynthesis genes) into which additional heterologous/engineered genes encoding component pathway enzymes for monoterpene synthesis will be introduced. Strains will be optimised for enzyme expression, growth and monoterpenoid biosynthesis to enable efficient whole-cell biotransformation through systems-based analysis and modelling. Engineered strains will be used sequentially to generate monoterpenoids without need for enzyme isolation or exogenous addition of cofactor recycling systems. The programme will transform the industrial synthesis of commercially important monoterpenoids. It delivers on the BBSRC Knowledge Based Bio-Economy strategic priority and exploits synthetic/systems biology to underpin next generation biocatalysis for industry.