转录组测序分析3种不同链长脂肪酸对酿酒酵母基因转录水平的影响
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摘要
将不同链长的脂肪酸己酸(C_6)、十二烷酸(C_12)及十六烷酸(C_16)添加到对数期酿酒酵母中,通过转录组测序分析其对酿酒酵母基因转录水平的影响。结果表明,在不同链长脂肪酸存在条件下,酿酒酵母基因的转录数量、基因转录水平均有所差异,其中短链脂肪酸C_6对酵母基因转录水平的影响最为显著,C_16对酿酒酵母的影响最小。此外,通过转录组测序数据分析对不同脂肪酸存在时的转录因子及转运蛋白进行预测,其中包括17条转录因子及40条转运蛋白基因。最后分析不同链长脂肪酸存在对脂肪酸合成途径的关键酶基因转录水平的影响,为进一步分析酿酒酵母中脂肪酸生物合成过程中的基因表达及调控机制提供支持。
In this study, we focused on the influence of fatty acids with different chain lengths(hexanoic, lauric and hexadecanoic acid) on gene expression at the transcriptomic level in Saccharomyces cerevisiae at the logarithmic phase.The results demonstrated that the number of transcribed genes and transcription levels changed during logarithmic growth in the presence of each of the three fatty acids; hexanoic acid had the greatest impact on gene transcription levels while the impact of hexadecanoic was the lowest. Furthermore, by analysis of the transcriptomic sequencing data, we predicted that there might be 40 transporter protein genes and 17 transcript factors. Finally, we analyzed the transcription levels of the key enzyme genes involved in the fatty acid biosynthesis pathway that were affected by the fatty acids with different chain lengths. This research provides theoretical support for a better understanding of gene expression and the underlying regulatory mechanism in fatty acid biosynthesis by S. cerevisiae.
In this study, we focused on the influence of fatty acids with different chain lengths(hexanoic, lauric and hexadecanoic acid) on gene expression at the transcriptomic level in Saccharomyces cerevisiae at the logarithmic phase.The results demonstrated that the number of transcribed genes and transcription levels changed during logarithmic growth in the presence of each of the three fatty acids; hexanoic acid had the greatest impact on gene transcription levels while the impact of hexadecanoic was the lowest. Furthermore, by analysis of the transcriptomic sequencing data, we predicted that there might be 40 transporter protein genes and 17 transcript factors. Finally, we analyzed the transcription levels of the key enzyme genes involved in the fatty acid biosynthesis pathway that were affected by the fatty acids with different chain lengths. This research provides theoretical support for a better understanding of gene expression and the underlying regulatory mechanism in fatty acid biosynthesis by S. cerevisiae.
引文
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[2]LIAN J,MISHRA S,ZHAO H.Recent advances in metabolic engineering of Saccharomyces cerevisiae:new tools and their applications[J].Metabolic Engineering,2018,50:85-108.DOI:10.1016/j.ymben.2018.04.011.
[3]ZHOU Y J,BUIJS N A,ZHU Z,et al.Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories[J].Nature Communications,2016,7:11709.DOI:10.1038/ncomms11709.
[4]GUO W,SHENG J,ZHAO H,et al.Metabolic engineering of Saccharomyces cerevisiae to produce 1-hexadecanol from xylose[J].Microbial Cell Factories,2016,15:24.DOI:10.1186/s12934-016-0423-9.
[5]JONG B W,SHI S,VALLE-RODRíGUEZ J O,et al.Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration[J].Journal of Industrial Microbiology&Biotechnology,2015,42:477-486.DOI:10.1007/s10295-014-1540-2.
[6]CHEN Y,LI F,GUO J,et al.Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1[J].Journal of Industrial Microbiology&Biotechnology,2014,41:563-572.DOI:10.1007/s10295-013-1390-3.
[7]L E B E R C,P O L S O N B,F E R N A N D E Z-M O Y A R,e t a l.Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae[J].Metabolic Engineering,2015,28:54-62.DOI:10.1016/j.ymben.2014.11.006.
[8]GAJEWSKI J,BUELENS F,SERDJUKOW S.Engineering fatty acid synthases for directed polyketide production[J].Nature Chemical Biology,2017,13:363-365.DOI:10.1038/nchembio.2314.
[9]LEBER C,DA SILVA N A.Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids[J].Biotechnology and Bioengineering,2014,111:347-358.DOI:10.1002/bit.26288.
[10]ZHU Z,ZHOU Y J,KANG M K,et al.Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast[J].Metabolic Engineering,2017,44:81-88.DOI:10.1016/j.ymben.2017.09.007.
[11]BRAULT G,SHARECK F,HURTUBISE Y,et al.Short-chain flavor ester synthesis in organic media by an E.coli whole-cell biocatalyst expressing a newly characterized heterologous lipase[J].PLoS ONE,2014,9(3):e91872.DOI:10.1371/journal.pone.0091872.
[12]SU L,HONG R,GUO X,et al.Short-chain aliphatic ester synthesis using Thermobifida fusca cutinase[J].Food chemistry,2016,206:131-136.DOI:10.1016/j.foodchem.2016.03.051.
[13]于怀龙,马永昆,张荣,等.不同品种桑椹香气成分的主成分分析[J].食品工业科技,2016,37(10):62-66.DOI:10.13386/j.issn1002-0306.2016.10.003.
[14]李秋棉,罗均,李雪萍,等.果实香气物质的合成与代谢研究进展[J].广东农业科学,2012,39(19):104-107.DOI:10.16768/j.issn.1004-874x.2012.19.061.
[15]LIAN J,ZHAO H.Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites[J].Journal of Industrial Microbiology&Biotechnology,2015,42:437-451.DOI:10.1007/s10295-014-1518-0.
[16]TEHLIVETS O,SCHEURINGER K,KOHLWEIN S D.Fatty acid synthesis and elongation in yeast[J].Biochimica et Biophysica Acta,2007,1771:255-270.DOI:10.1016/j.bbalip.2006.07.004.
[17]SARRIA S,KRUYER N S,PERALTA-YAHYA P.Microbial synthesis of medium-chain chemicals from renewables[J].Nature Biotechnology,2017,35:1158-1166.DOI:10.1038/nbt.4022.
[18]GAJEWSKI J,PAVLOVIC R,FISCHER M,et al.Engineering fungal de novo fatty acid synthesis for short chain fatty acid production[J].Nature Communications,2017,8:14650.DOI:10.1038/ncomms14650.
[19]DAHL R H,ZHANG F,ALONSO-GUTIERREZ J,et al.Engineering dynamic pathway regulation using stress-response promoters[J].Nature Biotechnology,2013,31:1039-1046.DOI:10.1038/nbt.2689.
[20]ZHANG F,CAROTHERS J M,KEASLING J D.Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids[J].Nature Biotechnology,2012,30:354-359.DOI:10.1038/nbt.2149.
[21]TORELLA J P,FORD T J,KIM S N,et al.Tailored fatty acid synthesis via dynamic control of fatty acid elongation[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110:11290-11295.DOI:10.1073/pnas.1307129110.
[22]XU P,LI L,ZHANG F,et al.Improving fatty acids production by engineering dynamic pathway regulation and metabolic control[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111:11299-112304.DOI:10.1073/pnas.1406401111.
[23]TEIXEIRA P G,FERREIRA R,ZHOU Y J,et al.Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae[J].Microbial Cell Factories,2017,16:45.DOI:10.1186/s12934-017-0663-3.
[24]LOMAKIN I B,XIONG Y,STEITZ T A.The crystal structure of yeast fatty acid synthase,a cellular machine with eight active sites working together[J].Cell,2007,129:319-332.
[25]DENIC V,WEISSMAN J S.A molecular caliper mechanism for determining very long-chain fatty acid length[J].Cell,2007,130:663-677.DOI:10.1016/j.cell.2007.03.013.
[26]SIBIRNY A A.Yeast peroxisomes:structure,functions and biotechnological opportunities[J].FEMS Yeast Research,2016,16(4).DOI:10.1093/femsyr/fow038.
[27]LEGRAS J L,ERNY C,LE JEUNE C,et al.Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids[J].Applied and Environmental Microbiology,2010,76:7526-7535.DOI:10.1128/AEM.01280-10.
[28]BORRULL A,LOPEZ-MARTINEZ G,POBLET M,et al.New insights into the toxicity mechanism of octanoic and decanoic acids on Saccharomyces cerevisiae[J].Yeast,2015,32:451-460.
[29]ANDRISICL,COLLINSONEJ,TEHLIVETSO,etal.Transcriptional and antioxidative responses to endogenous polyunsaturated fatty acid accumulation in yeast[J].Molecular and Cellular Biochemistry,2015,399:27-37.DOI:10.1007/s11010-014-2229-6.
[30]JANSURIYAKUL S,SOMBOON P,RODBOON N,et al.The zinc cluster transcriptional regulator Asg1 transcriptionally coordinates oleate utilization and lipid accumulation in Saccharomyces cerevisiae[J].Applied Microbiology and Biotechnology,2016,100:4549-4560.