Biotransformation Problem Statement

The following ideas could form the basis of postdoctoral or graduate research in various areas of chemistry which are poorly addressed from a sustainability perspective.

Ideas 1-5 are preferred from GSKs perspective, however the subsequent proposals (6-8) also hold merit and we would be interested in progressing in the event of strong academic interest.

Ester to amide: design of a HTS and wild-type/commercial enzyme screening

N-alkylation via activated alcohol

Developing biocatalytic systems for epoxidation of alkenes.

Understanding / Predicting Protein folding in Escherichia coli

Defining operating space for novel biocatalyst classes

Nitrile reduction to primary amines

Metal Sequestering

Immobilised whole cells to catalyse slurry reactions

For reference:

ACS GCI pharmaceutical roundtable wish list for better technologies (Green Chem., 2007, 411)

1. Ester to amide: design of a enzyme kit

Problem

Amide formation from esters typically proceeds in a 3 stage procedure:

1. Hydrolysis of the ester to a carboxylic acid.

2. Activation of the carboxylic acid

3. displacement of the activating group.

The second of these stages is itself a process with poor credentials from a green perspective (eg activation as an acyl chloride using reagents like thionyl chloride, or with toxic, poor atom-efficient coupling reagents like dicyclohexylcarbodiimide etc..).

A green transformation going directly from esters to amides without the use of toxic materials is considered by many to be one of the most important, fundamental functional group transformations from a sustainable perspective as this synthetic transformation is so widely used in organic chemistry (within in the Pharma industry in particular – see ACSGCI roundtable wishlist).

Exemplification of Problem

It is known that CAL B, a particularly promiscuous enzyme in terms of substrate specificity, can perform this transformation. A wider search of commercial and wild-types enzymes will certainly lead to other valuable leads.

Expected Output of Research

1. It is highly likely that creating an ‘enzymatic ester-to-amide’ kit would lead to much greater uptake of this technology by practicing organic chemists. This ester-to-amide kit is the primary objective of this proposal.

2. To optimise this technology across a broad range of esters and amine nucleophiles to allow more sterically congested products to be prepared will require an enzyme evolution project. It is likely that input from specialist biotech CROs would be required to prepare this second generation kit. However, before this follow-up technology could be used a general purpose, easy to follow screening process (eg colorimetric) would be required that is quick, reliable and easy to use would be required. Therefore, a secondary objective for this proposal (if time permits) will be defining screening conditions suitable for use in a HTS.

Examples of CAL-B catalysed enantioselective aminolysis of esters:

Canadian Journal of Chemistry (2002), 80(6), 608-612 (enantioselective aminolysis of an isoxazolylacetate alkyl ester).

Revista de la Sociedad Quimica de Mexico (2004), 48(4), 300-304 (amidation of amino esters in organic solvents)

Applied Microbiology and Biotechnology (2007), 75(2), 297-302 (synthesis of chiral  -hydroxy amides by two sequential enzymatic catalyzed reactions).

2. N-Alkylation via activated alcohol

Problem

OH activation for nucleophilic substitution is one of the key areas where greener methodologies are being sought to avoid the use of alkylating agents and minimise waste (see ACSGCI roundtable wishlist). Ideally the alcohol would be displaced directly.

Exemplification of the problem

A number of enzymes transfer nitrogen heterocycles (and aliphatic thiols) to acetyl serine and the methodology has been used industrially (Wacker). These synthases retain activity on isolation, do not require cofactors and so could potentially be readily used in multi-purpose kit and are not limited to methyl transfer.

Refs: J. Biosci Bioeng. 2004, 97, 322; PTC Appl, 2004, US 6,756,216 B2; Chem. Biol. 2009, 16, 277 and refs therein.

We would like to investigate whether enzymes with similar activity towards simple acetylated alcohols exist in nature or if existing ones can be modified – perhaps by testing microbial strains, metagenomics or directed evolution.

Note that a parallel exists for progressing this project with the development of MAO’s by Nick Turner. Prior to his work, little precedent for microbial MAO’s that accept amine substrates was available.

Expected output of research

By coupling the synthase with an enzyme capable of converting alcohols to acetates (such as lipases) in the same pot, this methodology could provide a versatile and green method of transforming alcohols to amines.

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.

3. Developing biocatalytic systems for epoxidation of alkenes.

Problem

Biocatalytic stereoselective epoxidation of olefins has been conducted for some time and there is a large body of literature to support this. There are currently 2 key issues which, if addressed, would encourage considerable greater take-up within the scientific / manufacturing community of this green technology:

1. a majority of the epoxides formed from bacteria catalysed systems give the (R) configuration for the epoxide products.

2. there is no general methodology which would give a researcher a good understanding on whether a given biocatalytic system will in fact result in the desired epoxidation without actually conducting an experiment.

Exemplification of the problem

A screen of peroxidase containing bacteria, (ideally cultured in the presence of suitable olefins), against a range of different olefinic substrates will create a basic understanding of the desired biocatalytic epoxidation reaction space. Those bacteria which show reactivity against a range of substrates (ie some promiscuity towards substrates) would form the basis of an biocatalytic epoxidation kit.

Expected output of the research

In the first instance a better understanding of the likelihood of success for progression of a given olefinic substrate.
A wider choice of biocatalytic systems giving preferentially (S)-epoxides.
A kit could be prepared (described via publication in the first instance).
The most wide-ranging systems wrt substrate specificity (especially those with a preference for the production of (S) configurations) could be used as a starting point for an evolution project(s) to develop mutants with even greater scope against various olefins.
The mutants could form the basis of a commercial kit (with appropriate partnership from a biotech 3rd party).

4. Understanding / Predicting Protein folding in Escherichia coli.

Problem

Genetically Modified Microorganisms (GMMs) are becoming ubiquitous as miniature factories to produce a range of desired proteins in safe, well characterised host strains under controlled growth conditions. Escherichia coli is the most widely used host in the production of recombinant enzymes but, despite a 30 year history of use and many specific improvements to the process, for a small but significant proportion of proteins it remains difficult to express these in soluble, catalytically active form.

These more challenging proteins include many unique enzymes sourced from plants, actimomycetes and extremophilic microorganisms which are highly desirable as potential biocatalysts. In these cases the codon usage of the native DNA is often so different from the host that successful gene expression is difficult due to lack of the required acyl-tRNAs. Current low-cost DNA synthesis allows the “reformatting” of such genes to optimised E. coli codon usage, yet some proteins remain insoluble.

Exemplification of the problem

Solubility, and indeed catalytic activity, require correct folding of the protein and it is believed that misfolding and subsequent aggregation of insoluble protein are the result of rapid translation. A recent publication* has demonstrated that protein solubility may be enhanced by the presence of a low proportion of rare codons and it is hypothesised that pauses during translation due to low abundance of rare tRNAs may provide time for correct folding of the protein. If this were true, it would be possible to specify sequences for optimum solubility where proteins are accessed through gene synthesis.

It is proposed to explore the expression of a range of synthetic genes using varying proportions of rare codons to encode the same protein, evaluating the expressed protein under standard conditions and comparing with physiological controls such as expression temperature and transcriptional controls such as promoter strength.

Expected output of the research

The importance of understanding protein production is reflected in the 2009 Nobel prize for chemistry being based around ribosome understanding. More predictable expression of xenobiotic proteins in fully active and soluble form would enhance yields, reduce time lost in seeking alternative hosts and expression conditions and ultimately reduce the cost of access to novel biocatalysts resulting in a greater uptake of synthetic processes using greener, biocatalytic methods.

* Microbial Cell Factories 2009, 8:41

5. Defining operating space for novel biocatalyst classes

Problem

GSK has targeted a number of gaps in the “biotransformation toolbox”, where no third part solutions currently exist or are likely to be developed. In these cases we are developing small kits of enzymes which, it is hoped, will provide the desired reaction chemistry for a diverse range of potential substrates.

Limited research and lack of commercial application means that the operating space, the range of applicable substrates and degree of selectivity, remains unexplored for these enzymes.

Exemplification of this proposal

It is proposed that a systematic survey be made of the range of “simplified” substrate molecules accepted with a view to predicting which enzymes may be applicable for reactions with more complex molecules. Currently, GSK has two lead candidates for such a project, offering generic, “green” approaches to phosphorylation and methylation respectively with potential for unprecedented selectivity.

The reverse reaction of phosphatases is unexploited because commercial phosphatases using cationic cofactors are unable to perform reverse hydrolysis. Using a third party specialist to generate novel recombinant proteins, we are developing a small collection of alternative enzymes. Substrate specificity of phosphatases may be very diverse, as recently established for the hydrolysis reaction (JBC 281, [2006], 36149).

The biocatalytic potential of methyltransferases is also unexplored because whole cell systems are required to provide and recycle the unstable biochemical methyl donor S-adenosylmethionine, and such systems are not viable as a commercial product. In addition, many of these enzymes exist in plants alone, and only recent technology allows rapid, cost effective expression in microbial cells.

Expected output of the research

The development of kits capable of performing phosphorylation, dephosphorylation and methylation on a wide range of substrates. This would provide a substantial ‘green’ improvement in these poorly addressed functional group transformations – most especially methylation which while procedurally simple to perform chemically in a laboratory is a particularly non-sustainable process.

6. Nitrile Reduction

Proposal

Nitrile reduction can provide a useful method of access to amines that are ubiquitous in pharmaceutical compounds. However, this transformation often requires the use of stoichiometric amounts of hazardous reducing agents or heavy metal catalysts. Green methodology is being sought to apply to these processes.

Exemplification of the proposal

Recently, a new class of bacterial NADPH dependent enzyme known as nitrile oxido-reductases has been identified, cloned and isolated (PNAS, 2005, 4264, US Patent No: US7364882 B1).

We could obtain / prepare a range of these enzymes (by screening and by using bioinformatics to identify other reductases via sequence similarity) and test them against a panel of appropriate nitriles (could include GSK specific substrates).

Expected output of the research

This would allow for the creation of a nitrile reductase kit which could have widespread use by organic chemists,. This would provide a substantial ‘green’ improvement in this poorly addressed functional group transformation

7. Metal Sequestering

Problem

The removal of low levels of precious or heavy metals from effluents (or indeed the environment in general) is a continuing area of interest to the entire chemical community.

Exemplification of the proposal

Bioremediation is slowly becoming recognised as an effective method for controlling metal levels and encoding a metallothionein onto the cell surface of Ralstonia eutropha, a natural inhabitant of soil, has been shown to sequester cadmium (Nature Biotechnology (2000), 18(6), 661-665).

From a pharma perspective a high number of Pd/Pt/Ru/Rh catalysed hydrogenations, cross-couplings and ring closing metathesis reactions are performed. It is essential to obtain drug products with very low, highly controlled levels of these precious metals.

This proposal could have 2 foci: the first being a screen of wild type organisms which could have an affinity for precious metals; the second would be designing (genetically modified microorganisms) to overexpress those proteins/enzymes which are known to have sequestering potential, to isolate and test these enzymes (as crude proteins) and finally to immobilise onto an inert support.

Expected output of research

A set of immobilised microorganisms and enzymes that can effectively sequester precious metals for recycling. These immobilised organisms/enzymes could be used to provide a general purpose kit for use in the fine chemical / pharma industries for screening and then easily scaled-up for manufacturing.

8. Immobilised whole cells to catalyse slurry reactions

Problem

Immobilisation is a common procedure to extend the stability, activity and operational life-cycle of whole cell biocatalysts. Compared with free microbial cultures, immobilised cells are readily removed from, and reused in, the target reaction, enhancing process sustainability and reducing costs. This technology established with wild type microorganisms is finding wider application with recent innovations such as the engineering of yeast cell surfaces to express biocatalytically useful enzymes (e.g. applications in biofuels production: Biochemical Engineering Journal 44 (2009), 2).