Full length ArticleCo-expression of glucose-6-phosphate dehydrogenase and acyl-CoA binding protein enhances lipid accumulation in the yeast Yarrowia lipolytica
Introduction
Recently, there has been renewed interest in using microorganisms as a source of lipids synthesized from inexpensive carbon feedstocks. Of great interest are lipids enriched in certain fatty acids, including polyunsaturated fatty acids for use in food and animal feeds [1], and short-chain fatty acids for use in the chemical industry [2], as substitutes for exotic oils [3], and as substrates for the synthesis of biodiesel and biosurfactants [4].
Microorganisms that accumulate lipids to more than 20% of dry cell weight are considered oleaginous. Of these, the yeast Yarrowia lipolytica is of particular value in metabolic engineering, as a wide range of genetic markers, molecular tools, and systems for gene expression are already available [5], [6]. Indeed, Y. lipolytica strains with enhanced lipid accumulation have been engineered by over-expression of genes encoding enzymes of triacylglycerol synthesis, and by elimination of fatty acid catabolism [7], [8], [9], [10], [11], [12].
Lipids are highly reduced molecules. Consequently, triacylglycerol overproduction requires large quantities of NADPH, two of which are oxidized to NADP+ for each fatty acid elongation step, and one is consumed for each fatty acid desaturation reaction. In oleaginous microorganisms, cytosolic NADPH is generated by malic enzyme, the citrate–α-ketoglutarate redox shuttle, and the pentose phosphate pathway [13]. Y. lipolytica malic enzyme is NAD+-dependent and mitochondrial [14], [15]. Moreover, over-expression of cytosolic NADP+-dependent malic enzyme from Mortierella alpina did not improve lipid synthesis in Y. lipolytica [14]. Thus, it could be concluded that malic enzyme does not play a role in generation of cytosolic NADPH in Y. lipolytica. On the other hand, the citrate–α-ketoglutarate NADPH redox shuttle transports reducing equivalents from the mitochondrial matrix to the cytoplasm via exchange of mitochondrial citrate for cytosolic α-ketoglutarate [16]. In this system, citrate transported from the mitochondria is converted to α-ketoglutarate through oxidation of isocitrate by cytosolic NADP+-dependent isocitrate dehydrogenase, and the resulting α-ketoglutarate is shuttled back to the mitochondria to complete the cycle. However, NADP+-dependent isocitrate dehydrogenase in Y. lipolytica is also mitochondrial [17]. Finally, 13C-metabolic flux analysis revealed that a highly active pentose phosphate pathway is a major source of reduced NADPH for anabolic reactions in wild type and recombinant Y. lipolytica [18], [19].
Acyl-CoA binding protein (ACBP) is a 10 kDa cytosolic protein found in all eukaryotes [20]. ACBP maintains the acyl-CoA pool, transports acyl-CoA to membranes and organelles for oxidation or transacylation, and protects acyl-CoA from cellular acyl-CoA hydrolases. Acyl-CoA provides feedback inhibition to enzymes in triacylglycerol synthesis in the cascade, including acetyl-CoA carboxylase, fatty acid synthetase, and acyl-CoA synthetase. In vitro, ACBP strongly attenuates the inhibitory activity the long-chain acyl-CoA against acetyl-CoA carboxylase [21]. In vivo, over-expression of either bovine or yeast genes encoding ACBP in Saccharomyces cerevisiae increases intracellular acyl-CoA level, indicating that the protein relieves feedback inhibition, and thereby induces increasing synthesis of acyl-CoA [22], [23].
In this study, we evaluated the effect of over-expressing NADP+-dependent glucose-6-phosphate dehydrogenase (encoded by ZWF1), an enzyme in the pentose phosphate pathway, and acyl-CoA binding protein on lipid accumulation in Y. lipolytica yeast. The goal was to stimulate lipid production by providing a bigger pool of co-factors required for lipid synthesis, such as NADPH, and by eliminating feedback inhibition via sequestration with acyl-CoA binding protein, respectively. The manipulation of genes was performed in the PEX10 null mutant background. PEX10 gene encodes peroxin, inactivation of which abrogates peroxisome biogenesis and consumption of hydrophobic substrates as the sole carbon source [24], [25]. Co-expression of ZWF1 and ACBP genes enhanced de novo lipid production by 41% compared with wild type, thereby increasing the lipid content to 30% of dry cell weight.
Section snippets
Materials and methods
Plasmids and Y. lipolytica strains constructed in this study are listed in Table 1. Oligonucleotide primers are summarized in Supplementary Table 1. Plasmids are schematically illustrated in Fig. 1. Detailed description of materials and methods used in this study is presented in Supplementary methods. The expression cassettes were integrated via non-homologous recombination into random loci of the genome which could be transcriptionally active or, opposite, silent. Thus, pools of twenty clones
Over-expression of ZWF1 gene
We hypothesized that over-expression of ZWF1 gene, which encodes NADP+-dependent glucose-6-phosphate dehydrogenase, would boost cytosolic NADPH and thereby increase de novo lipid synthesis. To prevent lipid degradation, ZWF1 was over-expressed in the PEX10 null mutant strain W29 (Δpex10). Strain W29 (Δpex10) did not differ considerably from the parental strain W29 in the term of biomass production, although total lipid accumulation increased by 8 % (Table 2). Unexpectedly, W29 (Δpex10) produced
Acknowledgements
The work was carried out using the equipment of the Unique Scientific Facility of BRC VKPM with technical support of the Centre for Collective Use of GosNIIgenetika, and with financial support of the Ministry of Education and Science of the Russian Federation (project code RFMEFI62514X0005).
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