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.

BackgroundLipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli.ResultsThe lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents.ConclusionsCo-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic activity appears not to be hindered by surface display. Furthermore, the cells and the membrane preparations appeared to be stable enough to endure a European standard laundry test and show efficient fat removal properties herein.

The following study depicts the development of a whole cell biocatalyst for the prenylation of indole derivatives. For this purpose the prenyltransferase FgaPT2 from Aspergillus fumigatus was displayed on the surface of Escherichia coli cells by using Autodisplay. The presence of the prenyltransferase in the outer membrane was detected by using SDS‐PAGE and Western Blot after the proteins of the outer membrane were isolated. The orientation of the prenyltransferase towards the outside of the cells was investigated by accessibility testing with externally added proteases. The FgaPT2 whole cell biocatalyst converted up to 250 μM of indole‐3‐propionic acid, approximately 25 % of the substrate used in the assay (100 μL sample, OD578=40). Another indole substrate, L‐β‐homotryptophan was also investigated and a conversion of 13 % was determined. By optimizing the assay conditions the conversion rate could be raised to approximately 30 % of indole‐3‐propionic acid during a 24 h incubation time at 20 °C. The whole cell biocatalyst endured a storage period of one month at 8 °C without any detectable loss in activity. Reusability was confirmed by recycling the biocatalyst. After three cycles of consecutive use, the whole cell biocatalyst retained a conversion rate of 46 % of indole‐3‐propionic acid and 23 % of L‐β‐homotryptophan after the third cycle.

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