Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins.

Shohei Hayashi,Tohru Dairi,Tetsuro Ujihara,Yasuharu Satoh,Yusuke Takata

doi:10.1038/srep35441

Shohei Hayashi, Tohru Dairi + Show 3 more

Open Access

https://doi.org/10.1038/srep35441

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Journal: Scientific Reports	Publication Date: Oct 18, 2016
Citations: 55	License type: CC BY 4.0

Affiliation: Hokkaido University, Kyowa Kirin (Japan)

Abstract

In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity.

Highlights

In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A
PUFA synthase activity was evaluated on the basis of PUFA productivity of recombinant E. coli expressing PUFA biosynthetic genes because PUFA synthases are huge enzyme complexes and it is difficult to prepare them as recombinants for in vitro assay
After E. coli BLR(DE3)∆fadE harbouring all the plasmids was cultured in terrific broth medium, the products were analysed by GC/MS

Summary

Introduction

In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. Alternative and sustainable sources of PUFAs are required To this end, fermentative processes have been developed using microorganisms such as microalgae, fungi, and engineered yeasts for the production of DHA, ARA, and EPA, respectively[5,6,7]. In the former pathway, which operates in plants, fungi, microalgae, and bacteria, specific desaturases and elongases catalyse individual desaturation and elongation steps to synthesize PUFAs from oleic acid (C18:1 ω9)[7] In the latter pathway, which occurs in eukaryotic microalgae and prokaryotic bacteria, PUFA synthases composed of huge enzyme complexes with multiple catalytic domains synthesize PUFAs using acetyl coenzyme A (CoA) and malonyl-CoA as starter and extender units, respectively, in a manner similar to polyketide synthases (PKSs) and fatty acid synthases (FASs)[8,9,10,11]. They possess acyltransferase (AT), malonyl-CoA transferase (MAT), ketoacyl www.nature.com/scientificreports/

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