Cloning, heterologous expression and functional characterization of a ∆9-desaturase of Pseudomonas putida A (ATCC 12633).?

ROMINA MARISA HEREDIA; LUCCHESI, GLORIA INÉS

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

CLONING, HETEROLOGOUS EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF A ∆9-DESATURASE OF Pseudomonas putida A (ATCC 12633). Adaptive response of P. putida A (ATCC 12633) to tetradecyltrimethylammonium (TTAB) involve the immediate fluidification of cell membranes (Polarization value decreased from 0.12±0.01 to 0.08±0.01) with changes in the content of fatty acid (FA) of the phospholipids membrane: the levels of unsaturated FA (16:1Δ9 and 18:1Δ11) decreased while the amount of saturated FA (15:0 and 16:0) increased. Therefore, the effect of TTAB was an increase in the fluidity of the membrane while P. putida cells seemed to respond decreasing the degree of UFA in order to counteract the action of this compound. The decrease in the content of 16:1Δ9 and the concomitant increase in the level of 16:0 could be explained by inhibition of a Δ9desaturase enzyme. In this work, a gene coding for a putative Δ9 fatty acid desaturase-like protein was isolated from P. putida A (ATCC 12633), cloned and heterologously expressed in Escherichia coli BL21, a ∆9 desaturase deficient organism. The gene, named mf539821, has an open reading frame of 1185 bp, codes for 394 amino acids with a predicted molecular weight of 45 kDa and showed 85% sequence identity to ∆9 desaturase gene of P. putida KT2440, P. putida DOT-T1E and P. fluorescens PICF7. To determine the functional activity in vivo of the recombinant desaturase, E coli cells were cultured in LB medium with palmitic acid [1-14C] and induced at 28 ºC, with 1% lactose. The cultures were collected and their FA were analysed by TLC. Compared to the negative control, E coli cells overexpressing the putative desaturase gene increased by twice the amount of monounsaturated fatty acids formed from exogenous palmitic acid [1-14C].Thus, we conclude that mf539821 codes for a functional desaturase enzyme of P. putida A (ATCC 12633). The successful expression of the enzyme will be used to determine if its inhibition is or not involved in the adaptative response of P. putida to cationic surfactants.