Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase.

Huiya Gu,Robert E. Jinkerson,Philip E. Schneider,Fiona K. Davies,Matthew C. Posewitz,Lyle A. Sisson

doi:10.3389/fpls.2016.00690

Abstract

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions.

Highlights

Derived diesel from water-oxidizing, photosynthetic microorganisms (PSMs) is considered an efficient and promising next-generation technology for the production of renewable fuels (Radakovits et al, 2010; Work et al, 2013)
To identify organisms and enzymes capable of facilitating medium-chain fatty acids (MCFAs) production in algae and cyanobacteria, we surveyed an algal collection isolated from Great Salt Lake (GSL) for organisms with natively high levels of MCFA (Figure 1A)
Growth under different light intensities indicated that cells grew to a slightly higher density at lower light intensities (20 μmol photosynthetically active radiation (PAR) m−2 s−1 and 60 μmol PAR m−2 s−1) relative to a higher light intensity (120 μmol PAR m−2 s−1), where the final cell concentrations were decreased relative to cultures at lower light

Summary

Introduction

Derived diesel from water-oxidizing, photosynthetic microorganisms (PSMs) is considered an efficient and promising next-generation technology for the production of renewable fuels (Radakovits et al, 2010; Work et al, 2013). Research efforts have focused on areas ranging from optimizing photosynthetic yields to performing scalability assessments (Larkum et al, 2012; Ho et al, 2014; Quinn and Davis, 2015). These efforts can be combined with advances in next-generation DNA sequencing and metagenomic analysis to characterize the diversity of phototrophic life at the enzyme/molecular level, which facilitates the discovery of genetic “parts” that can be. These features lead to poor coldflow temperature properties (longer chain lengths) and oxidative instability (higher unsaturation) (Knothe, 2005, 2008)

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Results

Conclusion