"Cloning, heterologous expression and functional characterization of a delta9 desaturase of Pseudomonas putida A (ATCC 12633)"

ROMINA MARISA HEREDIA; GLORIA INÉS LUCCHESI

Buenos Aires

Congreso; Reuniòn Conjunta de sociedades de Biociencias; 2017

Adaptive response of P. putida A (ATCC 12633) to tetradecyltrimethylammonium(TTAB) involve the immediate fluidificationof cell membranes (Polarization value decreased from 0.12±0.01to 0.08±0.01) with changes in the content of fatty acid (FA) of thephospholipids membrane: the levels of unsaturated FA (16:1Δ9 and18:1Δ11) decreased while the amount of saturated FA (15:0 and16:0) increased. Therefore, the effect of TTAB was an increase inthe fluidity of the membrane while P. putida cells seemed to responddecreasing the degree of UFA in order to counteract the action ofthis compound. The decrease in the content of 16:1Δ9 and the concomitantincrease in the level of 16:0 could be explained by inhibitionof a Δ9desaturase enzyme. In this work, a gene coding for aputative Δ9 fatty acid desaturase-like protein was isolated from P.putida A(ATCC 12633), cloned and heterologously expressed inEscherichia coli BL21, a Δ9 desaturasedeficient organism. The gene,named mf539821, has an open reading frame of 1185 bp, codes for394 amino acids with a predicted molecular weight of 45 kDa andshowed 85% sequence identity to Δ9 desaturase gene of P. putidaKT2440, P. putida DOT-T1E and P. fluorescens PICF7.To determinethe functional activity in vivoof the recombinant desaturase, E colicells were cultured in LB medium with palmitic acid [1-14C] and inducedat 28 ºC, with1% lactose. The cultures were collected andtheir FA were analysed by TLC. Compared to the negative control,E coli cells overexpressing the putative desaturase gene increasedby twice the amount of monounsaturated fatty acids formed fromexogenous palmitic acid [1-14C].Thus, we conclude that mf539821codes for a functional desaturase enzyme of P. putida A (ATCC12633).The successful expression of the enzyme will be used to determineif its inhibition is or not involved in the adaptative responseof P. putida to cationic surfactants.