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世纪的关键使能技术之一。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(包括散装和精细化学品)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、创新和具有竞争力的化学工艺。手性分子的制造是一个特别的挑战：微生物是非常丰富的化合物来源和代谢途径，可以用于化学工业或作为新的药物。在这个研究计划中，我们将开发新的生物催化剂来合成一类在食品和香料工业中具有很高商业价值的重要有机化合物(单萜类)。开发最有前景的单萜类化合物的一个严重障碍是它们从自然资源中获得的有限。提取和蒸馏这些分子是昂贵的、低产量的，并且需要大量的自然资源。供应和环境条件的季节性变化也会影响生产。产物外消旋混合物的形成也是传统化学合成法生产单萜类化合物的主要缺点。这可以通过使用昂贵的手性催化剂或从手性池中的材料开始缓解，但对于萜类化合物来说，这些构建单元从自然资源中得不到足够的丰富。酶催化合成很有吸引力：区域和立体选择性的生物转化，使用绿色的反应条件，以及没有有毒副产物，这些结合在一起，使生物合成在可持续制造中具有吸引力。美国和欧洲的法律承认，来自生物合成制造过程的产品被认为是“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方面。由于(1)对“天然产物”的要求；(2)不能通过化学合成轻易地获得它们；(3)经典合成对环境的影响；(4)从自然资源中获得的有限，因此确定获得萜类化合物的酶途径很重要。我们的工作将把基于知识的新型酶催化剂的应用与合成生物学相结合，以设计单萜类化合物的新代谢途径。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建立单萜类化合物合成的途径，以(I)上调类异戊二烯前体生物合成中的酶，(Ii)整合非天然基因，为单萜类化合物的合成创造新的途径。我们将创建几个新的生物合成途径，即(-)-柠檬烯的生物合成；(5R)-香芹酮及其衍生物的生物合成；(+)-普利酮的生物合成；(+)-薄荷呋喃的生物合成；紫苏醇及其衍生物的生物合成；薄荷醇化合物的生物合成。我们将产生新的大肠杆菌菌株(每个菌株都含有上调的异戊二烯前体生物合成基因)，其中将引入额外的异源/工程基因，编码单萜合成的组分途径酶。将对菌株的酶表达、生长和单萜类生物合成进行优化，以通过基于系统的分析和建模实现高效的全细胞生物转化。工程菌株将被顺序使用来产生单萜类化合物，而不需要酶分离或辅因子循环系统的外源添加。该计划将改变具有重要商业意义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优先事项，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.