Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3.

Lawal Garba,Raja Noor Zaliha Raja Abd Rahman,Siti Nurbaya Oslan,Mohd Shukuri Mohamad Ali

doi:10.1371/journal.pone.0160681

Lawal Garba, Raja Noor Zaliha Raja Abd Rahman + Show 2 more

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https://doi.org/10.1371/journal.pone.0160681

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Journal: PloS one	Publication Date: Aug 5, 2016
Citations: 13	License type: CC BY 4.0

Affiliation: Universiti Putra Malaysia, Gombe State University

Abstract

Fatty acid desaturase enzymes play an essential role in the synthesis of unsaturated fatty acids. Pseudomonas sp. A3 was found to produce a large amount of palmitoleic and oleic acids after incubation at low temperatures. Using polymerase Chain Reaction (PCR), a novel Δ9- fatty acid desaturase gene was isolated, cloned, and successfully expressed in Escherichia coli. The gene was designated as PA3FAD9 and has an open reading frame of 1,185 bp which codes for 394 amino acids with a predicted molecular weight of 45 kDa. The activity of the gene product was confirmed via GCMS, which showed a functional putative Δ9-fatty acid desaturase capable of increasing the total amount of cellular unsaturated fatty acids of the E. coli cells expressing the gene. The results demonstrate that the cellular palmitoleic acids have increased two-fold upon expression at 15°C using only 0.1 mM IPTG. Therefore, PA3FAD9 from Pseudomonas sp.A3 codes for a Δ9-fatty acid desaturase-like protein which was actively expressed in E. coli.

Highlights

Fatty acid desaturase enzymes catalyse the desaturation reactions that introduce double bonds into fatty acyl chains
The previous study has confirmed the ability of Pseudomonas sp., A3 to produce monounsaturated fatty acids at low temperature [14]
To investigate the production of unsaturated fatty acids in response to temperature changes, the bacterium was cultured at different incubation conditions and analysed its fatty acids using GCMS (Table 1)

Summary

Introduction

Fatty acid desaturase enzymes catalyse the desaturation reactions that introduce double bonds into fatty acyl chains. The enzymes are broadly divided into two phylogenetically unrelated groups of soluble acyl-acyl carrier protein desaturases and a more widespread group of membrane-bound desaturases, which are made up of acyl-lipid and acyl-coA desaturases [1,2]. Desaturase enzymes have been reported in all members of prokaryotes and eukaryotic organisms [3,2]. There are differences in substrate specificity and cellular location, desaturase enzymes utilise the same reaction mechanisms, which require an electron transport system of either ferredoxin-NADP+ oxidoreductase and ferredoxin, or cytochrome b5 reductase, cytochrome b5, NAD (P)H, and molecular oxygen [3,4]. The Δ9-desaturase enzymes insert the first double bond into saturated fatty acyl chains, initiating the first step of polyunsaturated fatty acids (PUFAs) production, as most other.

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