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

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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.