Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum

Abstract

Renewable diesel surrogates made from shorter chain length fatty acids have improved cold flow properties. Acyl-ACP thioesterases specific for shorter chain length fatty acids are therefore of considerable interest in the genetic engineering of biofuel producing organisms, both for their ability to increase the production of shorter fatty acids, and for their involvement in fatty acid secretion in bacterial systems. Here we show that the heterologous expression of two thioesterases, biased towards the production of lauric (C12:0) and myristic acid (C14:0), causes increased accumulation of shorter chain length fatty acids in the eukaryotic microalga Phaeodactylum tricornutum. Accumulation of shorter chain length fatty acids corresponds to transgene transcript levels. We achieved levels of C12:0 of up to 6.2% of total fatty acids and C14:0 of up to 15% by weight. Unlike observations in cyanobacteria, no significant secretion of fatty acids was observed. Instead, we found that 75–90% of the shorter chain length fatty acids produced was incorporated into triacylglycerols. Our results demonstrate that overexpression of thioesterases is a valid way to improve the biofuel production phenotype of eukaryotic microalgae.

Introduction

Fatty acids in algae are most commonly long or very long chain fatty acids with varying degrees of unsaturation. Biodiesel made from saturated short or medium chain length fatty acids has a relatively low cloud point and is resistant to oxidation (Stournas et al., 1995). Previously, shorter chain length specific acyl-ACP thioesterases have been transgenically overexpressed in vascular plants, bacteria and cyanobacteria. In vascular plant seeds, this resulted in the enhanced accumulation of shorter chain fatty acids (Voelker et al., 1992, Voelker et al., 1996). In bacteria, thioesterase overexpression increases the production of total free fatty acids (Liu et al., 2010, Lu et al., 2008, Voelker and Davies, 1994), and in the case of cyanobacteria it also causes significant fatty acid secretion (Roessler et al., 2009). Consequently, the transgenic expression of thioesterases is of great interest in many lipid based biofuel production models. Here we provide the first report of transgenic overexpression of shorter chain length specific thioesterases in a eukaryotic microalga. Algae, due to their high photoautotrophic biomass production rates and naturally occurring high lipid levels are of significant interest in next generation biofuel production systems (Chisti, 2007, Hu et al., 2008, Radakovits et al., 2010), and the genetic engineering of algae is likely to become a crucial component in lowering the costs of algal biofuel production (Radakovits et al., 2010, Sheehan et al., 1998). Despite this fact, genetic engineering of algae to improve fuel feedstock production phenotypes is still in its infancy. Most advances in the genetic engineering of carbon storage pathways in algae have been achieved in Chlamydomonas reinhardtii (Li et al., 2010a, Li et al., 2010b, Wang et al., 2009, Work et al., 2010), but molecular and genetic methods have been developed for other algal model species; including Phaeodactylum tricornutum (Apt et al., 1996, Bowler et al., 2008, De Riso et al., 2009, Saade and Bowler, 2009, Siaut et al., 2007, Zaslavskaia et al., 2000). We have now succeeded in transgenically overexpressing two different shorter chain length fatty acid acyl-ACP thioesterases, one from Cinnamomum camphora (Yuan et al., 1995) and the other from Umbellularia californica (Voelker et al., 1992), in P. tricornutum, a diatom that is an attractive model alga for biodiesel production (Kroth, 2007b, Saade and Bowler, 2009), since it grows well photoautotrophically in minimal sea water medium while producing significant amounts of triacylglyceride (TAG) (Alonso et al., 2000, Rodolfi et al., 2009). In this study, we describe the results of these genetic manipulations, which include an increased production of shorter chain length fatty acids and an increase in the amount of total lipid produced per cell. We further show that the amount of shorter chain length fatty acids produced corresponds to the amount of thioesterase transcript. In addition, we found that no significant secretion of fatty acids occurs and that the majority of the shorter chain length fatty acids are incorporated into TAG.