17-ERACoBioTech Enzyme platform for the synthesis of chiral aminoalcohols

Industrial chemical processes carried out through "classic" chemistry and catalysis often involve the use of hazardous substances, use considerable amounts of energy and generate toxic waste. In contrast, enzymes catalyze the natural "manufacture and modification" of molecules with exquisite selectivity, unparalleled rate acceleration and under mild reaction conditions. Industrial Biotechnology, the application of enzymes to catalyze industrial reactions, as a Key Enabling Technology has the potential to improve chemical processes in compliance with the concept and principles of Green Chemistry and holistically combines economics with social and environmental aspects.
Many successfully industrialized examples include the synthesis of chiral fine chemicals by means of different enzymes, as well as the use of immobilized whole-cells and biosynthetic processes for the provision of bulk chemicals. Yet, several drawbacks hinder the full potential of biotechnology for a sustainable bioeconomy, such as the lack of a sufficiently broad range of enzyme platforms and long development timelines. These restrictions are particularly relevant for the synthesis of compounds having multiple centers of chirality such as amino alcohols which is a moiety found in many bioactive natural products such as alkaloid.
Amino alcohols are highly important due to their bioactivity, and function as chiral building blocks for the synthesis of pharmaceuticals and agrochemicals. The synthesis of enantiomerically pure amino alcohols is difficult to achieve by chemical approaches and typically requires tedious routes involving uneconomical steps for protective group manipulations. Although the biocatalytic synthesis of individual target amino alcohols has been reported, no coherent strategy for a systematic access to structurally broad and stereochemically diverse sets of amino alcohols has been developed yet.
To overcome these drawbacks, the multidisciplinary consortium TRALAMINOL will focus on the development of a powerful two-step biocatalytic strategy on the basis of only two classes of reaction types: The approach makes use of key enzymes that catalyze carbon-carbon (C-C) bond formation (to create keto alcohols) followed by aminotransfer (to create amino alcohols) in highly controlled fashion due to the enzymes' chemo-, regio- and enantioselectivity.
Enzyme portfolios of each type that allow access to all possible enantiomers will be developed as a robust industrial biocatalytic platform. Further intense development of these two critical enzyme types, and the creation of an integrated reaction platform for the sustainable manufacture of multifunctional chiral building blocks, will significantly strengthen the global competitiveness of the European chemical and pharmaceutical industries and accelerate the transition from a dependence on fossil raw materials toward a sustainable bio-based economy.

C-C bond forming reactions are at the heart of industrial organic synthesis but the use of C-C bond-forming enzymes as industrial biocatalysts remains a promising, yet largely unexplored field. TRALAMINOL has C-C bond formation as the first key step in its platform using diverse C-C bond-forming enzymes to catalyze the formation of a range of hydroxy ketones and hydroxy aldehydes. The consortium has access to a range of advanced catalysts for the synthesis of aldols using aldolases, and acyloins using transketolases. These enzymes, including thermostable examples, have been engineered for broader substrate promiscuity, including that for the nucleophilic component (e.g., pyruvate, dihydroxyacetone, hydroxyacetone, acetone, ethanal, propanal, butanal etc. for aldolases; similarly, hydroxypyruvate, pyruvate, oxobutyrate etc. for transketolases). All of those components are bio-based, sustainable building blocks that will be transformed into molecules with high added value.
For the second step, transaminases will be used. In industrial applications they have a broad substrate range and high levels of regio- and stereoselectivity, although issues from unfavorable equilibrium and product inhibition remain. The TRALAMINOL will use substrate cycling and pathway engineering for coupling the product formation to the driving force of irreversible decarboxylation or oxygen-based redox conversions. Simple synthetic cascades will be built starting from readily available building blocks and sustainable, inexpensive substrates under mild reaction conditions. The reaction cascades will be designed in a way that the target structures can be defined by the choice of substrate and specificity of the enzymes involved to generate different stereoisomers in a controlled fashion.
Available enzyme collections (aldolases, transketolases, aminotransferases) and new enzymes from metagenomes will be developed and used in panels for identification of the best enzymes for building cascades.

The project will have impact in each of the areas defined by the BBSRC and RCUK: economy, society, knowledge and people.

In terms of economic impact the PI has experience of working with industry, and the commercialisation of research findings and creation of a spin-out company. The enzymes and data resulting from this project will be made available (after any need for IP protection) to the wider academic and industrial community via publication, and dissemination at conferences and meetings. Beneficiaries of this work therefore extends beyond the immediate participants. It will benefit the UK economy by sustaining high-level research and the successfully developed biocatalytic cascades and pathways will be promoted widely within the pharmaceutical and fine chemical industries.

The impact of knowledge generation from the research will arise from the communication and publication of new scientific advances and public outreach activities to schools.

The societal impact will arise from the development of available and new enzymes in multienzyme systems to generate new chiral compounds. The use of biocatalysis in the chemical and pharmaceutical industry is growing, but is limited by the types of enzymes used and the compounds generated. By identifying new enzymes and methods for assembling these to achieve sustainable synthesis, these resources will assist the UK government meet its targets on greenhouse gas emissions and help mitigate the negative environmental effects of global warming. The use of synthetic biology for pathway construction is a key part of the cross council research strategy and synthetic biology is one of the BBSRC strategic priority areas. In addition, if new biologically active compounds are identified that can be made sustainably and cheaply this will impact positively on the general public.

The impact on people will arise from training and career development of the PDRA and provision of skilled scientist in the industrial biotechnology sector.

The findings will also influence future research directions, since the successful completion of this proposal will provide the data to embed biocatalytic strategies to new compounds that has to date been little unexplored. In addition, a wide spectrum of potential end-users, which include commercial companies, charities and the health care systems may profit from the outputs of this project in the longer term. While the impact of the funded research will be global, the intellectual property and thus the commercial profits will be likely to enhance the competiveness of the UK. UCL who will hold the intellectual property rights to the enzymes and compounds generated in this proposal, will benefit from licensing and industry collaboration.