Biotransformations Problem Statements
Problem 1: Seeking New Fluorinases
Proposal
Fluorine substitution is a valuable tool in medicinal chemistry used to improve the pharmacokinetic properties of potential drug candidates. Organofluorine compounds often exhibit improved stability and bioavailablity as compared to their non-fluorinated counterparts, and make up the active ingredient of many top-performing pharmaceuticals. The chemical incorporation of fluorine utilizes hazardous reagents, which are sometimes unselective or intolerant of functional groups. A greener and more selective methodology to effect fluorination is sought.
Exemplification of the proposal
A single fluorinase enzyme has been identified (1), characterized (2), and subsequently used in the engineering of a bacterial strain to produce a fluoro-analog of the anticancer agent salinosporamide A (3).
We seek to identify and prepare additional fluorinase enzymes for the biological production of medicinally relevant organofluorine compounds.
Expected output of the research
The development of a fluorinase kit for widespread screening by organic chemists. Enzymatic fluorination would provide a green improvement to current chemical fluorination methods.
Renewable solvents should be considered as media for these reactions, and they should demonstrate improved ‘green’ metrics versus the transformation they are replacing. A view towards broader application and industrialization should also be provided.
1. Nature, 2002, 416, 279.
2. Nature, 2004, 427, 561-565.
3. J. Nat. Prod., 2010, 73, 378-382.
Problem 2: General purpose selection methods
Biocatalysis represents a green and sustainable method for the manufacture of various APIs in the pharmaceutical industry. As the application of biocatalysis on an industrial scale expands, the need for improved biocatalysts possessing desirable properties including enhanced stability, enantio- and regio-selectivity, improved solvent and temperature tolerance, is increasing.
Protein engineering methods have undergone significant advances in past years, particularly with respect to protein design and implementation of new protein function. Irrespective to the method used for generation of mutant libraries, it is necessary to have robust high-throughput assays to evaluate the mutant variants within a short time and at low cost. Typically, high-throughput screening performed in microtiter plates are suitable only for libraries containing 103-105 variants, exceeding libraries of this size results in impractical timelines and reagent costs.
Selection, on the other hand, offers an alternative to the limitations outlined above. Many selection approaches link enzyme activity to survival of the host, however this strategy suffers from complex intracellular environments, must be “tailor-made” for the assay in question, and usually use a substrate other than the molecule of interest for the development.
To overcome these limitations, GSK are seeking general purpose selection methods which would not be limited by the size of mutant library (eg 109 and higher) to enable evaluation of much larger protein-sequence space. Any methods should preferably be independent of enzyme class and allow the use of “true” substrates.
Problem 3. In Silico Enzyme Evolution
Proposal
Biocatalysis is widely recognised as a highly useful methodology for the development of more sustainable routes to small molecule (API) synthesis. Enzyme evolution is a common method to improve natural enzyme starting points to develop greater enantioselectivity, greater activity, greater solvent tolerance etc…. However even the most capable modern methodologies for screening will only allow up to 109 mutants to be evaluated and more commonly only 103 to 105 mutants can be evaluated. These throughputs only allow a full factorial analysis of 3 to 7 amino acids across the whole sequence (203 = 8000 , 207 = 1.28E9). Improvements in screening technologies may lead to increases in throughput, but it will never be possible to full explore the effect of each natural amino acid in combination at more than a small percentage of a protein sequence (an average enzyme will have perhaps 400 aa’s, examining 5 percent of these in full generates 1026 possible mutations). The ability to identify a small number of residues to change, and/or specific amino acids to change and/or methods to iteratively explore the potential space yet still find global maxima would have a major impact on the probability of success of enzyme evolution projects. Thus developing in silico methods to allow the design of much smaller, smarter libaries would have a great impact on the area of enzyme evolution. Ideally these designs would also be able to be reduced to practice using existing molecular biology techniques, or means to develop appropriate techniques identified.
Exemplification
Software like the 3DM database (http://www.bioprodict.nl/) can be used to generate structure-based sequence alignments using protein data available in the literature to analyse amino acid distribution to suggest evolutionary favoured and forbidden amino acids. This data can be used to create smaller smarter libraries.
Expected output of the research
New algorithms capable of analysing sequence and structure data of proteins with activity data (measured or inferred) to provide more rapid access to improved enzyme characteristics.
Demonstration of software against an appropriate reaction / biocatalyst to give real world confirmation of the softwares capability.
Problem 4: Metagenomics
Problem
It is widely accepted that standard microbiological methods for culturing microorganisms from the environment cannot fully access the true extent of microbial biodiversity. For example, only 0.1% - 1.0% of soil bacteria are culturable using traditional methods. This leaves a largely untapped genetic pool that can be used for the discovery of novel genes, metabolic pathways, enzymes and natural products.
Exemplification of the problem
One of the major challenges for metagenomics is the development of methods to capture the diversity and dynamics of intricate microbial communities. One option is to explore the use of modern sequencing technologies and bioinformatic tools to better understand the complexities of microbial communities.
It is proposed to explore the development of methodologies in generating metagenomic DNA or cDNA libraries from environmental sources. This can be followed by establishing capabilities to better understand gene expression and production of functional proteins, and screening the libraries generated to identify novel enzymes and/or microbial strains for use as biocatalysts.
Expected outcome of the research
Specific focus of the research proposal may be on one or more of the following:
1. Isolation of metagenomic DNA from prokaryotic/microbial organisms
2. Isolation of metagenomic cDNA from eukaryotic organisms
3. Generation of metagenomic libraries in a commercial vector and/or a novel multi-host vector
4. Library screen via homology based PCR amplification and/or function based assays
5. Bioinformatics exploration of the metagenomes
Problem 5: Novel approach for use of lipophilic substrates and recovery of polar compounds from fermentation/whole cell mixtures
Fermentation approaches to API’s or their intermediates are considered green and sustainable ways to synthesize complex molecules. They also frequently offer orthogonal synthetic routes to those achievable by traditional synthetic chemistry approaches. However, fermentation processes are usually run at high volumes due to limited substrate solubility in aqueous media or due to toxic effects of the substrate/product on the cells. The reaction products produced during fermentation often display polar characteristics complicating their efficient recovery from the fermentation broth. This problem is further compounded when titres are low.
It is proposed to explore sustainable and practical solutions for the presentation of hydrophobic substrates to whole cell reactions and isolated enzyme reaction mixtures. In addition, solutions for the efficient recovery of polar products from fermentation broths is of considerable interest.