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Noda Institute for Scientific Research GRANT2014 Young Investigator Research GrantOutline of Research Result

Elucidation of triglyceride accumulation mechanism in an oleaginous yeastNaoya TakakuwaHokkaido Agricultural Research Center, National Agriculture and Food Research Organization

Research aims

Results

Fats (triglycerides, TGs) are essential for life and areused in both food and industrial applications. TGs are normally extracted from a variety of oil seed plants and animalsources. However, the Japanese self-sufficiency ratio forfats is only 13%, making it crucial to secure fat resources toprepare for the increasing global demand for fat materials.
The extraction of TGs from oleaginous yeasts such asYarrowia and Cryptococcus is a well-established alternativefor the manufacture of TG without relying on oil seedplants. However, these yeasts have not yet been used infood; therefore, there is a concern regarding the safety ofsuch usage. Furthermore, because fatty acids (FAs) constituting TGs of such oleaginous yeasts include an abundanceof saturated FAs, they are unsuitable for use as biodieselfuel in cold districts, such as Hokkaido.
Certain yeasts of the genus Kluyveromyces are composedof edible ingredients and their whole genome sequenceshave been decoded, which renders them ideal cellular models to elucidate mechanisms underlying TG accumulation.
In the present study, as part of our efforts to develop a technology for efficient TG production for both food and fuelapplications, we analyzed effects of varied genetic functions and culture conditions on TG production.

While the 11 gene overexpression strains did not exhibitany significant differences in cell weight compared with theparental strain, three of the overexpression strains tended tocontain increased amounts of TG (Fig. 1). Of all the strains,the DGA1 overexpression strain had the highest TG content, producing 1.4 times the volume of TG produced bythe parental strain. In addition, the TG production of LPP1and ARE overexpression strains was 1.3 times greater thanthat of the parental strain. DGA1, LPP1, and ARE are all involved in the synthetic pathway downstream of phosphatidic acid (PA), an intermediate substance of TG synthesis.
PA, which is also used in the synthesis of cell membranephospholipids (PLs), is reported to increase TG yield anddecrease PL yield in SNF2-deficient S. cerevisiae.3) Thus, itcan be predicted that in K. lactis, the overexpression ofgenes downstream of PA synthesis will reduce the activitiesof the PL synthetic pathway and, consequently, increase TGproduction.
A K. marxianus strain isolated from raw milk producedTG more efficiently than its type strain or K. lactis. An initial pH of the culture medium between 5.0 and 6.0 yieldedthe greatest amount of TGs, which was estimated to reach4.1 g/L. The constituent FAs of TGs produced by the iso-MethodsBased on the genome sequence of the yeast Saccharomyces cerevisiae, 11 genes encoding enzymes involved in thesynthetic pathway from dihydroxyacetone phosphate(DHAP) to TG were amplified via polymerase chain reaction using the genomic DNA of K. lactis. After linkage toan expression vector, as described in previous reports,1)each amplified gene was incorporated into the genomicDNA of K. lactis. The TG content of each gene overexpression strain was analyzed in accordance with previously described methods.2)To discover novel yeasts of the genus Kluyveromycesthat accumulate TG in higher concentrations compared toK. lactis, we examined the distribution of yeasts in rawmilk. Using sweet sorghum juice as a culture medium, theTG content in K. marxianus specimens isolated from rawmilk was analyzed.

Fig. 1. Effects of overexpression of the 11 genes on the growth and TGcontent of K. lactis. GPD1, glycerol-3-phosphate (G3P) dehydrogenase;GAT1 and GAT2, G3P acyltransferases; AYR1, 1-acyl DHAP reductase;SLC1 and SLC4, 1-acyl G3P acyltransferases; PAH1, DPP1 and LPP1,phosphatidate phosphatases; DGA1 and ARE, diacylglycerol acyltransferases.

36Table 1.

Fuel properties of biodiesel from K. marxianus.

Property

SG (g/cm3)

KV (mm2/s)

FP (°C)

CNIV

K. marxianus (isolate)Biodiesel standards (EU and Japan)

0.880.86–0.90

4.93.5–5.0162≥120

59≥5197≤120

SG, specific gravity; KV, kinematic viscosity; FP, flash point; CN, cetane number; IV, iodine value.

lated strain are all commonly found in edible oils. Moreover,the FA composition of TGs produced by the isolated strainconformed to all biodiesel standards that can be estimatedfrom FA composition, such as specific gravity (Table 1).
ConclusionThe results of the present study revealed that in the yeastK. lactis, TG production was improved by overexpressionof genes encoding enzymes downstream of PA in the synthetic pathway from DHAP to TG. In the yeast Y. lipolytica,TG production was accelerated through the overexpressionof genes encoding enzymes in the synthetic pathways ofFAs and disruption of genes encoding enzymes responsiblefor TG degradation.4) Therefore, it can be inferred that altering functions of such genes in K. lactis also leads to theamelioration of TG production.
Additionally, these results indicate that K. marxianus isolated from raw milk excels in producing TG, which can beused in food and as fuel. If the genes in the TG syntheticpathway are altered in this isolated strain, it may be possible to further increase TG yield.
References1) Takakuwa, N., Ohnishi, M., and Oda, Y. (2008) Significance of theKlLAC1 gene in glucosylceramide production by Kluyveromyceslactis. FEMS Yeast Res. 8: 839–845.
2) Takakuwa, N., Nagahama, S., Matsumura, H., Kinoshita, M., andOhnishi, M. (2013) Efficient conversion of crude glycerol into triacylglycerol by the yeast Pseudozyma sp. TYC-2187 for biodiesel production. J. Oleo Sci. 62: 605–612.
3) Kamisaka, Y., Noda, N., Tomita, N., Kimura, K., Kodaki, T., andHosaka, K. (2006) Identification of genes affecting lipid contentusing transposon mutagenesis in Saccharomyces cerevisiae. Biosci.
Biotechnol. Biochem. 70: 646–653.
4) Tai, M., and Stephanopoulos, G. (2013) Engineering the push andpull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica forbiofuel production. Metab. Eng. 15: 1–9.