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.
1. Ester to amide: design of a HTS and wild-type/commercial enzyme screening
2. N-alkylation via activated alcohol
3. Developing biocatalytic systems for epoxidation of alkenes.
4. Understanding / Predicting Protein folding in Escherichia coli
5. Defining operating space for novel biocatalyst classes
6. Nitrile reduction to primary amines
7. Metal Sequestering
8. 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 wildtypes 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-toamide 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), 297hydroxy 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 multipurpose 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.
O
O
serine acetyltransferase
OH
HO
NH2
O
Beta-pyrazolylalanine synthase
OAc
HO
N
HO
NH2
NH2
OH
OH
L-Mimosine synthase
O
HO
NH2
N N
+
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
1. In the first instance a better understanding of the likelihood of success for
progression of a given olefinic substrate.
2. A wider choice of biocatalytic systems giving preferentially (S)-epoxides.
3. A kit could be prepared (described via publication in the first instance).
4. 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.
5. 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 acyltRNAs. 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 Sadenosylmethionine, 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.
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.
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 oxidoreductases 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
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.
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.
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.
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).
Exemplification of the proposal
Whole cell biotransformations in the pharma industry, (e.g. 11- hydroxylation step in
GSK’s betamethasone process), frequently use slurries of poorly water-soluble substrates
and/or products, followed by an organic solvent extraction of the whole reaction liquors.
It is proposed to apply immobilised whole cell biocatalysis to such a slurry reaction
through combination of process engineering and appropriate selection of immobilisation
matrix (robust encapsulating materials could include polyesters or polyamides both of
which have examples prepared in aqueous solutions at mild temperatures).
Expected output of the research
Potential benefits would include reduction of waste and cost through reuse of the catalyst,
simplified downstream processing due to removal of the encapsulated cells, and reduced
catalyst requirements through enhanced stability.
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.