Organic Chemistry via Biocatalysis
Bernard WITHOLT*, Marcel WUBBOLTS, and Andrew SCHMID
Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, ETH-Honggerberg HPT, 8093 Zurich, Switzerland
Pseudomonads and other bacteria that grow on hydrocarbons are potential biocatalysts for the oxidation of aliphatic and aromatic compounds such as medium and long chain length alkanes, fatty acids, toluene and styrene derivatives, and substituted 2-phenols. Oxidation pathways begin with mono- or di-oxygenases that generally have a wide substrate range, are regiospecific and often stereospecific. The oxidation reactions carried out by these enzymes are difficult or impossible with conventional chemistry. Hence they appeal to biotechnologists. Monooxygenases incorporate oxygen atoms into specific substrate methyl or methylene groups to produce aliphatic alcohols, to aromatic rings to produce phenols, and add oxygen atoms to double bonds to form epoxides. Monooxygenase reactions require reduced cofactors (NADH, NADPH) for the reduction of the oxygen atom which is not incorporated into the substrate and are therefore carried out in vivo, in whole cell systems, where these complex enzyme systems perform best, and cofactor regeneration occurs through normal metabolism.
All of the systems we study have been cloned in E. coli hosts, where products can be synthesized without being metabolized further. This is important so that products can accumulate in the medium. Since monooxygenase substrates and products are typically apolar, they must be supplied to growing cells either in limiting amounts, in the vapor phase or dissolved in a bulk liquid phase. We have explored these approaches, with a focus on the cell physiology during growth in two-liquid phase systems, containing 10 - 20% (v/v) apolar solvent. The effect of solvents on cell function depends on the strain in question, the solvent used, and the culture conditions. For effective biocatalysis, cells must survive despite solvent toxicity and cells must catalyze the required bioconversion in the presence of solvents.
We have maximized cell growth in such media, reaching cell densities of 40 g/1 (dry mass) for E. coli recombinants and 100 g/1 (dry mass) for Pseudomonas strains. In continuous cultures, cell densities between 3 - 20 g/1 (dry mass) can be maintained. We have produced terminal alkanols, alkanoic acids, ω-hydroxy and oxo-fatty acids, chiral epoxyalkanes, substituted chiral styrene-epoxides, and a series of 2,3- dihydroxybiphenyl derivatives, in the g to kg scale.
For industrial applications, several questions remain. What are the maximum space-time yields attainable in two-liquid phase fermentations under practical conditions? To what extent can oxygen enriched air be used for aeration? Do increased pressures, useful in increasing dissolved oxygen and permitting work with volatile compounds, improve the performance and range of biooxidations? What safety regimes should be considered in working with flammable organic solvents, oxygen enriched air, and elevated pressure? To answer these questions, we have recently developed a high-pressure EX-proof bioreactor system with Bioengineering AG (Wald, Switzerland), which will be used to explore the systems described above under pilot conditions.
