4.2 Production of PCL precursors
The biocatalytic production of PCL or its precursors has been heavily investigated over the last years (Table 3). Approaches based on isolated enzymes, [15, 40-44] as well as whole cells,[16, 45-47] have successfully been established. However, most of these approaches relied on cyclohexanol as a substrate[41-47], which needs to be synthesized from cyclohexane employing an ecologically critical process[48]. Additionally, inhibition of CHMO by cyclohexanol or substrate inhibition necessitated the development of suitable reaction concepts, e.g., two-liquid phase[40] or fed-batch systems[43]. The highest productivity of 1.87 g L-1 h-1 was obtained with isolated enzymes by employing an appropriate feeding strategy for the complete conversion of 283 mM cyclohexanol to 6HA[43] (Table 3). The CHMO from Acinetobacterheterologously expressed in E. coli showed the highest total turnover number (TTN) with almost 70,000 molε-CLmolCHMO-1 [46]. In general, whole-cell approaches show lower yields on biocatalyst, as target enzymes constitute only about 1-10% (w/w) of cells, but avoid the enormous effort to purify the enzymes.
Compared to cyclohexanol, cyclohexane is an even more challenging substrate due to its high volatility and toxicity. In comparison to solvent-sensitive E. coli employed to convert cyclohexanol to 6HA[47], we obtained a 10-fold higher specific whole-cell activity and a similar yield on biocatalyst (Table 3).P. taiwanensis VLB120 is known to tolerate low-logP solvents and can, therefore, be considered suitable for the biotransformation of the more toxic substrate cyclohexane [49, 50]. Possible prolongation of the reaction with an appropriate substrate feeding and the application of a high-cell density setup hold big potential to further improve the product titer and the volumetric productivity.
This study, for the first time, demonstrates a whole-cell approach directly converting cyclohexane to the PCL precursor 6HA. The biotransformation to ε-CL presented by Karande et al.[16] could be optimized by enhancing the conversion, yield on biocatalyst, TTN, and specific activity (Table 3). The use of isolated enzymes to convert cyclohexane to ε-CL suffered from low conversion and TTN, which can be attributed to mass transfer limitations or inherent instability of P450 monooxygenases[7, 15]. The cellular environment allows for more stable catalytic activities with superior productivities. Efficient cyclohexane mass transfer without cell toxification will constitute a major future challenge and may be solved by cyclohexane feeding potentially via the gas phase. The achieved increase in whole-cell activity and conversion is a huge step forward towards the establishment of an economically viable process [51].