Optimizing the Production of Recombinant Hydroperoxide Lyase in Escherichia coli Using Statistical Design

Sophie Vincenti,Jacques Maury,Eva Faillace,Virginie Brunini-Bronzini De Caraffa,Liliane Berti,Magali Mariani,Jessica Croce

doi:10.3390/catal11020176

Abstract

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their “fresh green” aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box–Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L−1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L−1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported.

Highlights

Hydroperoxide lyase (HPL) is a membrane-bound enzyme mainly distributed in higher plants, algae, and fungi [1,2,3]
HPLs are heme enzymes belonging to the cytochrome P450 family and are divided into two subfamilies by their substrate specificities, namely CYP74B for 13-HPLs and CYP74C
Vegetal oils are hydrolyzed by lipase, and the polyunsaturated fatty acids (PUFAs) released are converted by LOX and HPL into green note aldehydes [23,33,34,35,36]

Summary

Introduction

Hydroperoxide lyase (HPL) is a membrane-bound enzyme mainly distributed in higher plants, algae, and fungi [1,2,3]. HPL is involved in the lipoxygenase (LOX) pathway, which is a lipid oxidation pathway activated in response to biotic or abiotic stress, or during various plant stages development, to produce phyto-oxylipins acting as defense and signaling molecules [4,5,6,7,8,9,10,11,12] In this pathway, LOX first catalyzes the oxygenation of polyunsaturated fatty acids (PUFAs), such as linoleic or α-linolenic acids, to produce corresponding hydroperoxides [4,13,14]. Given the high demand for such natural flavors and the low yield of extracting the compounds directly from plants, efficient biocatalytic processes using the enzymes of the LOX pathway are developed [19,32] In these processes, vegetal oils are hydrolyzed by lipase, and the PUFAs released are converted by LOX and HPL into green note aldehydes [23,33,34,35,36]

Methods

Results

Discussion

Conclusion