Optimization of Hydroperoxide Lyase Production for Recombinant Lipoxygenase Pathway Cascade Application

Cis-3-hexenal and its more stable isomer, trans-2-hexenal, are highly valued chemicals used in the food and perfume industries. They are produced by the plant lipoxygenase pathway, where two enzymes, lipoxygenase (LOX) and hydroperoxide lyase (HPL), are involved. However, the application of this pathway is limited, especially due to the instability of HPL. This enzyme belongs to the cytochrome P450 enzyme family and needs heme as a prosthetic group. Its synthesis must be effectively performed by a host organism in order to produce an active protein. In this work, Pseudomonas aeruginosa LOX was expressed in Escherichia coli BL21(DE3), and whole cells were used for the synthesis of 13(S)-hydroperoxy-(Z,E,Z)-9,11,15-octadecatrienoic acid (13-HPOT) as a substrate for HPL. Expression of Psidium guajava HPL was carried out by recombinant E. coli JM109(DE3) in autoinduction media, and the influence of the addition of heme precursors delta-ALA and FeII+ was studied. Specific activity of whole cells expressing HPL was measured by the direct use of a synthesized 13-HPOT solution (2.94 mM of total hydroperoxides, 75.35% of 13-HPOT (2.22 mM)) and increased 2.6-fold (from 61.78 U & BULL;mg(-1) to 159.95 U & BULL;mg(-1)) with the addition of 1 mM FeII+ to the autoinduction media. Productivity and activity were further enhanced by an increase in the expression temperature, and a total of 3.30 & BULL;10(5) U & BULL;dm(-3) of culture media was produced in the optimized process.

Automated summary

Cis-3-hexenal and trans-2-hexenal are important chemicals in the food and perfume industries, produced through the plant lipoxygenase pathway involving enzymes like LOX and HPL. This study successfully expressed Pseudomonas aeruginosa LOX and Psidium guajava HPL in E. coli, enhancing activity and productivity through the addition of prosthetic groups and optimization of culture conditions.