A simplified process design for P450 driven hydroxylation based on surface displayed enzymes

Abstract

New production routes for fine and bulk chemicals are important to establish further sustainable processes in industry. Besides the identification of new biocatalysts and new production routes the optimization of existing processes in regard to an improved utilization of the catalysts are needed. In this paper we describe the successful expression of P450BM3 on the surface of E. coli cells with the Autodisplay system. The successful hydroxylation of palmitic acid by using surface-displayed P450BM3 was shown. Besides optimization of surface protein expression, several cofactor regeneration systems were compared and evaluated. Afterwards, the development of a suitable process for the biocatalytic hydroxylation of fatty acids based on the re-use of the catalysts after a simple centrifugation was investigated. It was shown that the catalyst can be used for several times without any loss in activity. By using surface-displayed P450s in combination with an enzymatic cofactor regeneration system a total turnover number of up to 54,700 could be reached, to the knowledge of the authors the highest value reported for a P450 monooxygenase to date. Further optimizations of the described reaction system can have an enormous impact on the process design for more sustainable bioprocesses. Biotechnol. Bioeng. 2016;113: 1225-1233. © 2015 Wiley Periodicals, Inc.

A tailor‐made, self‐sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase

“…Taking into account only the first 10 rounds of reaction in which substrate conversion was 95% or higher, a turnover number of 10,000 was calculated for Z_P450 BM3. This falls into the TTN range of P450 systems, immobilized enzymes included, showing the highest operational performance (Dong et al, ; Ströhle et al, ; Whitehouse et al, ). After 20 rounds of reaction, the TTN of the immobilized Z_P450 BM3 was ∼18,000.…”

“…A space‐time yield of 8 mM/hr was attained in the first 2 hr of reaction. An operational half‐life for the heterogeneous catalyst after 18 repeated batches was determined from these experiments, an outstanding value employing an immobilized P450 (Ströhle et al, ; Tan et al, ). In each round, NADPH was regenerated at least 3.5 times.…”

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

“…Also for E. coli whole cell catalyst expressing a monooxygenase, the benefits of such systems have been demonstrated by Strö hle et al [66]. Because of the facilitated recovery and reusability of the catalyst, a remarkable turnover number (TON) of >54,000 was reached, representing the highest TON ever reported for P450 monooxygenase according to the authors [66]. Sü hrer et al proposed a different strategy enabling almost 28,000 fusion proteins per cell, by fusing galactosidase to an inner membrane anchor from rabbit liver cytochrome b 5 .…”

“…As an example, Candida antarctica lipase B was displayed on Pichia pastoris, with the additional result of increased solvent and thermal stability [65]. Also for E. coli whole cell catalyst expressing a monooxygenase, the benefits of such systems have been demonstrated by Strö hle et al [66]. Because of the facilitated recovery and reusability of the catalyst, a remarkable turnover number (TON) of >54,000 was reached, representing the highest TON ever reported for P450 monooxygenase according to the authors [66].…”

“…(b) Coexpression of membrane-integrated transport proteins for active influx and efflux [63 ,64]. (c) Fusion protein of enzyme and membrane anchors to display enzymes on the cell surface [65,66].…”