Selective Chemical Transformation of Organic Substrates by Microorganisms

	PROJECT DESCRIPTION Students at a Further Education College use timetabled 'free time' to investigate products arising from microbial fermentation. Some have their work accredited at A Level.	AREAS OF SCIENCE Astronomy Chemistry Computing Engineering / Technology Environment Interdisciplinary Life Science / Medicine Physics	WHERE School Other institution Field / Expedition WHEN In curriculum Extra curricular Work experience	SCHOOL TYPE Primary Secondary to 16 Post 16 Independent Maintained Sixth Form College Further Education
	PROJECT ORGANISERS Dr Paul Smith, Filton College, Bristol. Professor Tom Simpson, School of Chemistry, University of Bristol, advises.

Project Participants

Currently 11 students aged 16-18 are involved in the project. The activities take place in students' timetabled 'free time' (two or three hours a week), involving about one sixth of the two year groups studying Chemistry full time at Filton College. The project work covers many topics typical of most A Level syllabuses in Biology and Chemistry with associated data logging and processing. Some students have opted to present their research work for accreditation in part fulfilment of the requirements of the UCLES Modular Sciences Syllabus (extended laboratory study component).

Science Programme

The case of Thalidomide exemplifies the need for stereo-chemically pure pharmaceutical formulations. The reason this sedative drug caused malformation of the limbs of the foetus when taken by mothers in early pregnancy is now ascribed to the fact that it was marketed as a racemic mixture; the pure enantiomer is believed to be free of these tragic side effects.

Fermentation of organic substrates, utilising bakers' yeast (Saccharomyces cerevisiae) under aerobic conditions, yields a wide range of stereochemically useful products, and is providing an excellent field for student chemical research. The microbial reduction of b-ketoesters to the corresponding chiral secondary alcohols is probably the most thoroughly explored of these transformations. The configuration of the product is, however, critically dependent on: (i) the structure of the b-ketoester substrate; (ii) the strain of yeast used and (iii) the fermentation conditions (cell density, nutritional status, pH, temperature).

This project is directed principally at the systematic investigation of the effects of these variables on enantiomeric purity, together with an exploration of the enzymic basis of this behaviour using cell-free extracts. The project is entirely student led with regular research seminars in which priorities are identified, experiments planned, materials ordered and results discussed.

Personal Development

An extended practical investigation of this type is invaluable as a vehicle for science education offering opportunities to use many of the theoretical ideas covered by A Level syllabi to solve 'real' problems. Practical skills are developed in searching the literature, in the chemical synthesis of substrates and racemic products, in microbiological techniques, in process control and in analytical chemistry. Not least, the project provides numerous opportunities for students to come to grips with the rigours of project management, including time management, rostering, control of chemical stock/ordering of materials, record keeping, financial control and the transfer of skills as new students join the project.

Outcomes

Students feed beta-ketoesters to a suspension of yeast cells in a small fermenter

Initial effort has been directed towards defining a standard set of conditions for fermenter runs. In the team's hands, yeast reduction of ethyl 3-oxobutanoate, resulted in an essentially quantitative conversion to (S)-(+)-ethyl 3-hydroxybutanoate. Polarimetric analysis of the redistilled product gave [a]²⁰_D = +33^o (c = 2.83, CHCl₃) which indicates a minimum enantiomeric excess (e.e.) of 76.4% (where e.e. = |(R-S)/(R+S)|). This is in agreement with literature values. Project participants have shown that the rate of formation of this compound approximately doubles when the pH of the culture medium is lowered by 1 pH-unit over the range pH 3-7, although the affect on optical purity remains to be established.

The ethyl 3-hydroxybutanoate fragment is found in a number of natural products, including (S)-(+)-sulcatol and (R,R)-(-)-grahamimycin A, a broad spectrum antibiotic isolated from Cytospora sp. and so may be a useful intermediate in their synthesis.

Under similar conditions, yeast reduction of ethyl 3-oxohexanoate gives, in contrast, the other enantiomer (R)-(-)-ethyl 3-hydroxyhexanoate, which illustrates well the effect of structural variation of the substrate on the stereochemical course of the reaction.

Preliminary research findings of the project were given as a poster presentation 'The Need for Stereochemically Pure Formulations' at the Wellcome Trust/Clifton Scientific Trust conference 'Experiments in Science Education: Pupils as Scientists', London, October 1993.

Project Origin

The project developed out of informal discussions between Dr Smith and Professor Simpson and was launched in March. Dr Eric Albone, Clifton Scientific Trust, provided much helpful encouragement and advice during the start-up period.

Resources

The research programme is centred on the college laboratories, with occasional visits to the University of Bristol, School of Chemistry (mainly for recording mass, IR and NMR spectra and for polarimetric analysis) and to industrial production plants (eg the antibiotic production facilities at SmithKline Beecham Pharmaceuticals, Worthing).
The principal problem is the provision of staff supervision time which is becoming increasingly difficult within the increasingly commercial ethos of the Further Education sector. For the future, much depends on the view college management takes of scientific project work of this kind. Currently the project will have to progress within the leader's unpaid time. Laboratory supervision is, of course, particularly important on safety grounds alone for such chemical work.
Students use the 2-3 hours per week currently timetabled 'free' in which to contribute to project objectives. Project members enjoy the use of a chemistry laboratory equipped with a 6m fume cupboard and gas chromatography facilities.
The University of Bristol has allowed the long term loan of a Hewlett Packard 5710A gas chromatograph, while BASF plc made a generous gift of two 'Minilab' semi-microscale glassware kits.
Funding is obtained through the Scientific Research in Schools Scheme.

Contact

Dr Paul J Smith, Filton College, Filton Avenue, Bristol BS12 7AT.
Tel: +44 (0) 117 9694217.