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Directed Evolution of Enantiocomplementary Malonate Decarboxylases.

Reference	BB/I020764/1
Principal Investigator / Supervisor	Professor Jason Micklefield
Co-Investigators / Co-Supervisors	Professor David Leys, Professor Nicholas Turner
Institution	The University of Manchester
Department	Chemistry
Funding type	Research
Value (£)	317,821
Status	Completed
Type	Research Grant
Start date	07/11/2011
End date	06/11/2014
Duration	36 months

Abstract

New biocatalysts are required for more sustainable biotechnology-based manufacture of pharmaceuticals, agrochemicals and other fine chemicals. Traditionally a limited number of established enzymes have been used for a narrow range of industrial biotransformations. However recent advances, particularly in directed evolution and high-throughput screening technologies, mean that it has become possible to engineer and optimise a wider range of enzymes for specific processes. Recently we determined the structure and mechanism of an arylmalonate decarboxylase (AMDase), which can deliver homochiral carboxylic acids from simple disubstituted malonates, which are readily available from renewable resources. The chiral carboxylic acids are valuable intermediates in the production of pharmaceuticals and other products. However, AMDases have limited substrate scope and, like most classes of enzymes, provide access to just one enantiomer, which is problematic if the opposite enantiomer is required. In this project, we aim to use structure-guided directed evolution to develop enantiocomplementary malonate decarboxylases that would provide access to either enantiomer of a much wider range of chiral carboxylic acids than is currently possible by any single existing biocatalytic or non-enzymatic chemical method. To do this we will re-engineer a 'mirror image' AMDase active site switching a key Cys residue to the opposite side of the active site. The cysteine can function as a general acid, protonating the opposite face of the planar enediolate intermediates resulting from decarboxylation of disubstituted malonates. In addition, we will also desymmetrise the active site of related Asp/Glu racemase enzymes, which possess two active site cysteines required for general acid-base catalysis of racemisation, to similarly generate enantiocomplementary malonate decarboxylases. The new enzymes will be optimised and used to prepare a range of chiral carboxylic acids.

Summary

Currently, the manufacture of pharmaceuticals, agrochemicals and other fine chemicals relies heavily on synthetic chemical methods, which use deleterious solvents, reagents and catalysts as well as non-renewable petrochemical precursors, which have serious detrimental environmental impact. Consequently, alternative biotechnology based processes are sought for the more economic and environmentally sustainable manufacture of those chemicals that are essential to maintain human health and quality of life. Central to the development of industrial biotechnologies is the availability of new enzymes, with tailored properties, that can be used to catalyse the transformation of renewable precursors into the required products under environmentally benign conditions. To date, industrial applications of enzymes have relied on a limited number of established enzymes, which catalyse a narrow range of transformations. However, recent genome sequencing has led to the discovery of a wider range of enzymes from natural sources. In addition new directed evolution technologies allow the properties of enzymes and even the reactions they catalyse to be altered and optimised for specific processes. Recently we solved the first structure and determined the detailed mechanism of a decarboxylase enzyme (AMDase) that catalyses the loss of carbon dioxide (decarboxylation) from malonic acid derivatives to generate chiral carboxylic acids. In this project, we aim to use these structural and mechanistic insights to develop more powerful decarboxylase enzymes that can provide access to a much wider range of structurally diverse carboxylic acids, which are particularly common intermediates in production of pharmaceuticals, agrochemicals and other products. The new decarboxylase enzymes are also attractive because the substrates can be generated from malonic acid, a natural precursor derived from renewable sources (fermentation). The availability of chiral carboxylic acids, which are single enantiomers (one of two possible stereoisomers that are non-superimposable mirror images) is of critical importance particularly for pharmaceutical production. Typically, enzymes only produce one of the two possible enantiomers, which is problematic if the opposite enantiomer is required. We will therefore use directed evolution technologies to develop enzymes that are enantiocomplementary. In this way, one enzyme can be used to produce one enantiomer (left-handed molecule), whilst another enzyme can produce the opposite enantiomer (right-handed molecule). Along with our industrial partners at BASF, who are the world's largest chemical manufacturers, we will tailor the new enantiocomplementary decarboxylases for production of key pharmaceutical intermediates. This includes chiral carboxylic acids used to manufacture non-steroidal anti-inflammatory drugs (from the ibuprofen family), the antiplatelet agent clopidogrel (the world's second-best selling drug) and captopril which is used to treat cardiac conditions. The family of enzymes to which the decarboxylases belong are known to be promiscuous, and can catalyse a wider range of reactions than decarboxylations, including racemisations and isomerisations. We aim to further explore the promiscuity of this enzyme family, with a view to developing alternative reactions that would also be of industrial importance.

Impact Summary

WHO WILL BENEFIT: Our industrial partners BASF are the world's largest chemical manufacturers and they are committed to developing biotechnology-based processes that are more efficient and sustainable than existing petrochemical-based processes. BASF will therefore benefit through the availability of new biocatalysts, which they can further develop and implement in their expanding portfolio of biotechnological processes. BASF have many manufacturing sites in the UK and elsewhere in the world, which produce chemicals including chiral intermediates that are widely used in the production of pharmaceuticals, herbicides, fungicides, insecticides, and other optically active fine chemicals that are of major benefit to human health and wellbeing and can contribute to wealth creation and employment in the UK. For example BASF sell a wide range of optically active alcohols, epoxides, amines and carboxylic acids under the ChiPros label to pharmaceutical and agrochemical companies in the UK and elsewhere, who would benefit through the availability of new chiral carboxylic acids and other products that could be prepared using the biocatalysts that we will develop in this project. In addition, chiral carboxylic acids have been used in the preparation of flavours (e.g. high-potency sweetners) and fragrances, as well as chiral liquid crystals or chiral dopents (for LCD screens etc) and other materials for chiral chromatography and sensors. BASF also have partnerships with companies such as Sigma-Aldrich who could supply enzymes or chiral carboxylic acids developed in this project to the academic and wider research communities. HOW WILL THEY BENEFIT: We will work closely with University KT staff and the BASF IP office to secure intellectual property rights for all new inventions we discover. Having secured IP, future development work can take place, and several routes to commercialisation can be explored. To enable this to happen, we will demonstrate how the enantiocomplementarymalonate deacarboxylases we engineer can provide access to either enantiomer of a diverse range of chiral carboxylic acid products. BASF could then develop the biocatalysts for production of selected chiral carboxylic acids, for which a market is established, and these would be included in the ChiPros library of compounds. In addition to this, BASF would also contact its extensive list of industrial costumers, to advertise the new biocatalysts and chiral products. For example, BASF produces customised chiral intermediates for many pharmaceutical companies, providing gram quantity samples in the first instance, followed by the development of kilo-scale through to commercial scale production at BASF. In addition to the chiral intermediates, the large library of biocatalysts we will deliver (and precursor plasmids) could be sold to customers, to screen against their own target substrates, with a view to licensing specific biocatalytic processes for implementation within their own company. For example, a number of leading pharmaceutical companies have their own biocatalysis groups, who may prefer to join in a partnership and to utilise their own in-house expertise to develop a specific biocatalytic process. In addition to BASF, NJT is the director and JM and DL are members of the UK Centre of Excellence in Biocatalysis (CoEBio3), which includes industrial affiliates from 15 pharmaceutical and biotech companies who have specific interest in the industrial applications of enzymes. CoEBio3 can therefore open additional pathways for exploiting the research we undertake. Finally, having secured IP, we will actively seek to communicate our scientific findings to the wider research community through scientific meetings and scholarly publications.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Industrial Biotechnology, Pharmaceuticals, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Industrial Partnership Award (IPA)

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