通过高通量测序研究了大曲中丝状真菌的物种多样性,并结合荧光定量PCR对丝状真菌进行了定量分析;此外,还通过纯培养对高温曲中的丝状真菌进行筛选,并使用简并引物对分离株的次级代谢产物合成酶基因进行扩增。结果表明,Thermomyces属的丰度随大曲温度的升高而上升,而Aspergillus属的丰度随大曲温度的升高而下降;丝状真菌在大曲中的生物量同样随大曲温度的升高而下降;利用纯培养的方法从高温大曲中分离得到50株丝状真菌,隶属于10个属(Aspergillus、Monascus、Byssochlamys、Lichtheimia、Rhizomucor、Mucor、Arthrinium、Alternaria、Thermomyces和Rasamsonia),并且这些丝状真菌含有多种次级代谢产物合成酶表达基因;大曲来源的丝状真菌具有强大的次级代谢产物合成潜力,大曲是优良的微生物和次级代谢产物生物合成的基因库,可能成为挖掘新天然产物的新来源。
This study investigated the diversity of Daqu filamentous fungi and their abilities to synthesize bioactive compounds. High-throughput sequencing was used to study the diversity of filamentous fungi followed by quantitative analysis using fluorescence real-time PCR. Further, culture-depended method was applied to isolate the fungi followed by amplifying the genes involved in secondary metabolites biosynthesis. It was found that the abundance of genus Thermomyces increased with increasing temperature of Daqu, while the abundance of genus Aspergillus decreased. Besides, the biomass of filamentous fungi decreased with temperature. There were 50 filamentous fungal isolates affiliated to 10 genera (Aspergillus, Monascus, Byssochlamys, Lichtheimia, Rhizomucor, Mucor, Arthrinium, Alternaria, Thermomyces, and Rasamsonia) obtained from high temperature Daqu, and these strains had many genes involved in producing secondary metabolites, indicating that Daqu-derived filamentous fungi had great potentials to produce multiple types of secondary metabolites. Therefore, Daqu is a superior reservoir of microorganisms and genes for secondary metabolites biosynthesis, which can be a new source for mining new natural products.
[1] ZHENG X, TABRIZI M R, NOUT M J R, et al. Daqu-a traditional Chinese liquor fermentation starter [J]. J Inst Brew, 2011, 117(1):82-90.
[2] DU H, WANG X S, ZHANG Y H, et al. Exploring the impacts of raw materials and environments on the microbiota in Chinese Daqu starter [J]. Int J Food Microbiol, 2019, 297:32-40.
[3] CHEN B, WU Q, XU Y. Filamentous fungal diversity and community structure associated with the solid state fermentation of Chinese Maotai-flavor liquor [J]. Int J Food Microbiol,2014, 179:80-84.
[4] KNIGHT R, VRBANAC A, TAYLOR B C, et al. Best practices for analysing microbiomes [J]. Nature Reviews Microbiology,2018, 16(7):410-422.
[5] 乔晓梅, 赵景龙,杜小威,等. 高通量测序法对清香大曲真菌群落结构的分析[J]. 酿酒科技,2015(4):28-31.
[6] 夏玙, 罗惠波,周平,等. 不同处理方式的大曲真菌群落差异分析[J]. 食品科学,2018, 39(22):173-179.
[7] WANG X S, DU H, ZHANG Y, et al. Environmental microbiota drives microbial succession and metabolic profiles during Chinese liquor fermentation [J]. Appl Environ Microbiol,2018, 84(4):13.
[8] WANG X S, DU H, XU Y. Source tracking of prokaryotic communities in fermented grain of Chinese strong-flavor liquor [J]. Int J Food Microbiol, 2017, 244: 27-35.
[9] ANDERSEN M R, NIELSEN J B, KLITGAARD A, et al. Accurate prediction of secondary metabolite gene clusters in filamentous fungi [J]. Proceedings of the National Academy of Sciences of the United States of America,2013, 110(1):E99-E107.
[10] IMHOFF J F. Natural products from marine fungi—Still an underrepresented resource [J]. Mar Drugs, 2016, 14(1):19.
[11] JALGAONWALA R E, MOHITE B V, MAHAJAN R T. A review: Natural products from plant associated endophytic fungi [J]. Journal of Microbiology and Biotechnology Research,2017, 1(2):21-32.
[12] YU Z, ZHANG B, SUN W, et al. Phylogenetically diverse endozoic fungi in the South China Sea sponges and their potential in synthesizing bioactive natural products suggested by PKS gene and cytotoxic activity analysis [J]. Fungal Diversity, 2013, 58(1):127-141.
[13] 杜海, 杜小威,赵景龙,等. 贮存过程中大曲原核微生物多样性及土味素含量变化规律[J]. 食品与发酵工业,2017, 43(4):1-6.
[14] 王雪山, 杜海,徐岩. 清香型白酒发酵过程中微生物种群空间分布[J]. 食品与发酵工业,2018, 44(9):1-8.
[15] EDGAR RC. Search and clustering orders of magnitude faster than BLAST [J]. Bioinformatics,2010, 26(19):2 460-2 461.
[16] KLJALG U, LARSSON K H, ABARENKOV K, et al. Unite: A database providing web-based methods for the molecular identification of ectomycorrhizal fungi [J]. New Phytol,2005, 166(3):1 063-1 068.
[17] ROUSK J, BTH E, BROOKES P C,et al. Soil bacterial and fungal communities across a pH gradient in an arable soil [J]. The ISME Journal,2010, 4(10):1 340-1 351.
[18] KRISHNAN Y, BONG C P C, AZMAN N F,et al. Co-composting of palm empty fruit bunch and palm oil mill effluent: microbial diversity and potential mitigation of greenhouse gas emission [J]. Journal of Cleaner Production,2017, 146:94-100.
[19] WHITE T J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics [J]. PCR Protocols: a guide to methods and applications,1990, 18:315-322.
[20] TAMURA K, STECHER G, PETERSON D,et al. MEGA6: molecular evolutionary genetics analysis version 6.0 [J]. Mol Biol Evol,2013, 30(12):2 725-2 729.
[21] LETUNIC I, BORK P. Interactive tree of life v2: Online annotation and display of phylogenetic trees made easy [J]. Nucleic Acids Res,2011,39(2):W475-W478.
[22] KHALDI N, SEIFUDDIN F T, TURNER G,et al. SMURF: Genomic mapping of fungal secondary metabolite clusters [J]. Fungal Genet Biol,2010, 47(9):736-741.
[23] CHEN B, WU Q, XU Y. Filamentous fungal diversity and community structure associated with the solid state fermentation of Chinese Maotai-flavor liquor [J]. Int J Food Microbiol,2014, 179:80-84.
[24] SINGH S, MADLALA A M, PRIOR B A. Thermomyces lanuginosus: properties of strains and their hemicellulases [J]. FEMS Microbiol Rev,2003, 27(1):3-16.
[25] ZHAO J, YANG N, ZENG R Y. Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in Antarctic sediment [J]. Extremophiles,2008, 12(1):97-105.
[26] SCANLAN P D, MARCHESI J R. Micro-eukaryotic diversity of the human distal gut microbiota: Qualitative assessment using culture-dependent and-independent analysis of faeces [J]. The ISME Journal,2008, 2(12):1 183-1 193.
[27] LIN X, HUANG Y J, ZHENG Z H, et al. Endophytes from the pharmaceutical plant, Annona squamosa: isolation, bioactivity, identification and diversity of its polyketide synthase gene [J]. Fungal Diversity,2010, 41(1):41-51.
[28] PIEL J, HUI D, FUSETANI N, et al. Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured bacterial consortia [J]. Environ Microbiol,2004, 6(9):921-927.
[29] KHALDI N, COLLEMARE J, LEBRUN M H, et al. Evidence for horizontal transfer of a secondary metabolite gene cluster between fungi [J]. Genome biology,2008, 9(1):R18.
