Presentation Information
[P04-473]Activation Mechanism of the Transcription Factor LsSpt23p Regulating Lipid Biosynthesis in the Oleaginous Yeast Lipomyces starkeyi
○Hiroaki Takaku1, Rikako Sato1, Harutake Yamazaki1, Yosuke Shida2, Wataru Ogasawara2 (1. Niigata Univ. of Phar. & Med. & Life Sci. (Japan), 2. Nagaoka Univ. of Tech. (Japan))
Keywords:
Oleaginous yeast,Lipomyces starkeyi,Lipid,Transcription Factor
With the continuous growth of the global population and economic development, global energy demand is rapidly increasing. Fossil fuels remain the primary energy source worldwide; however, their use produces greenhouse gases that contribute to global warming. Biofuels such as biodiesel have therefore attracted considerable attention as renewable alternatives. Microbial oils produced by oleaginous microorganisms are particularly promising feedstocks for biodiesel production because they do not compete with food resources and can accumulate large amounts of lipids. The oleaginous yeast Lipomyces starkeyi is an attractive host for microbial oil production because it can accumulate lipids up to 80% of its dry cell weight. In addition, this yeast can utilize a wide range of carbon sources including glucose, xylose, arabinose, cellobiose, starch, and glycerol. Unlike other oleaginous yeasts such as Yarrowia lipolytica and Rhodosporidium toruloides, L. starkeyi secretes amylolytic enzymes, enabling direct utilization of starch-based substrates.
In this study, we investigated the transcription factor LsSpt23p, identified through comparative genome analysis between wild-type and lipid-hyperproducing mutants (K13, E15-11, E15-15, and E15-25). Functional analyses revealed that LsSpt23p directly binds to promoter regions of genes involved in the acyl-CoA synthesis pathway and the Kennedy pathway, including SLC1 and PAH1, thereby activating their transcription and redirecting carbon flux from phospholipid synthesis toward TAG biosynthesis. Overexpression of a mutant LsSPT23 allele using a strong promoter resulted in approximately a four-fold increase in lipid productivity compared with the wild-type strain, demonstrating that activation of LsSpt23p strongly enhances lipid biosynthesis.
Furthermore, the ORF mutation in LsSPT23 significantly enhanced transcription of downstream lipid biosynthesis genes and resulted in approximately two-fold higher lipid productivity even when expressed under the native promoter. These results suggested that the mutation alters the functional activity of LsSpt23p. To investigate the activation mechanism, FLAG-tagged LsSpt23p strains were constructed for protein analysis. Two forms of LsSpt23p were detected: a full-length form and a truncated form. Notably, the mutant strain contained a higher proportion of the truncated protein compared with the wild-type strain.These results suggest that the truncated LsSpt23p represents the active transcription factor that enhances expression of lipid biosynthesis genes and increases TAG production. This study provides new insights into transcriptional regulation of lipid metabolism in L. starkeyi and highlights LsSpt23p as a promising engineering target for improving microbial oil production.
This work was supported in part by the JSPS Program for Forming Japan’s Peak Research Universities (J-PEAKS) (Grant No. JPJS00420240017).
In this study, we investigated the transcription factor LsSpt23p, identified through comparative genome analysis between wild-type and lipid-hyperproducing mutants (K13, E15-11, E15-15, and E15-25). Functional analyses revealed that LsSpt23p directly binds to promoter regions of genes involved in the acyl-CoA synthesis pathway and the Kennedy pathway, including SLC1 and PAH1, thereby activating their transcription and redirecting carbon flux from phospholipid synthesis toward TAG biosynthesis. Overexpression of a mutant LsSPT23 allele using a strong promoter resulted in approximately a four-fold increase in lipid productivity compared with the wild-type strain, demonstrating that activation of LsSpt23p strongly enhances lipid biosynthesis.
Furthermore, the ORF mutation in LsSPT23 significantly enhanced transcription of downstream lipid biosynthesis genes and resulted in approximately two-fold higher lipid productivity even when expressed under the native promoter. These results suggested that the mutation alters the functional activity of LsSpt23p. To investigate the activation mechanism, FLAG-tagged LsSpt23p strains were constructed for protein analysis. Two forms of LsSpt23p were detected: a full-length form and a truncated form. Notably, the mutant strain contained a higher proportion of the truncated protein compared with the wild-type strain.These results suggest that the truncated LsSpt23p represents the active transcription factor that enhances expression of lipid biosynthesis genes and increases TAG production. This study provides new insights into transcriptional regulation of lipid metabolism in L. starkeyi and highlights LsSpt23p as a promising engineering target for improving microbial oil production.
This work was supported in part by the JSPS Program for Forming Japan’s Peak Research Universities (J-PEAKS) (Grant No. JPJS00420240017).
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