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The widespread use of biocatalysis in industry will require conversions to achieve high space-time yields. For many next generation bioconversions the low thermodynamic equilibrium constant of reactions currently limits their industrial implementation. This thesis aims to establish a series of generic microscale methods for the rapid evaluation of process options to enhance the yield of such equilibrium-controlled bioconversions. These are evaluated based on the asymmetric synthesis of chiral amino alcohols by the CV2025 ω-transaminase (ω-TAm) from C. violaceum DSM30191. Using 50 mM each of L-Erythrulose (Ery) as substrate and S-α-methylbenzylamine (MBA) as amino donor the standard yield of the product 2−amino−1,3,4−butanetriol (ABT) is just 26 % (mol.mol-1). This reaction produces acetophenone (AP) as a by-product which is also inhibitory to the CV2025 ω-TAm. Microscale methods to evaluate four process options for increasing the bioconversion yield were established each operating at 300 µL volume. The first option involves screening of alternative amino donors to the widely-used MBA. The second couples ω-TAm with an alcohol dehydrogenase (ADH) to convert the inhibitory AP by-product into the non-inhibitory (R)-1-phenylethanol (a glucose dehydrogenase (GDH) is also present to facilitate co-factor recycling). The third approach involves physical in-situ product removal (ISPR) of the volatile AP by operation at reduced pressure in a 96-well vacuum manifold. The final approach involves ISPR using polymeric resins, such as the AmberliteTM XAD series, for selective adsorption of AP from the bioconversion medium. For the particular reaction studied here, use of alternative amino donors such as isopropylamine (IPA) enabled a 2.8-fold increase in the reaction yield to 72 % (mol.mol-1) while the second enzyme system, though more expensive to implement, achieved a 3.8-fold increase in yield and almost quantitative conversion (98 % (mol.mol-1)). Each of the microscale methods was subsequently implemented as process options in an automated micro-scale process sequence linking biocatalyst production, bioconversion and product analysis. The high throughput potential of the methods was then illustrated using a focused library of ten novel ω-TAms from various strains. The bioconversion rates and yields of each enzyme were evaluated using a range of alternative amino donors. This highlight one novel ω-TAm, able to utilise L-α-serine as amino donor at improved yield than the CV2025 ω-TAm. This opens up the possibility of engineering whole cell TAm biocatalysts where the normally expensive amino donors are synthesised in vivo by normal amino acid metabolism starting from simple, cheap substrates. The microscale methods also enabled rapid optimisation of bioconversion conditions using the second enzyme system. This identified the possibility of reducing by 40-fold the standard concentrations of the co-factor recycling enzymes thus reducing the contribution of the enzymes to the overall Costs of Goods (CoG). Overall, this work has established an efficient and generic microscale approach to aid bioprocess design and specifically to help increase the yield of equilibrium-controlled bioconversions. The bioconversion rates and yields for the optimised reaction systems were subsequently verified for preparative scale bioconversions. |
