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Draft genome sequence of Rhizopus microsporus SSU VM08-07 isolated from alcohol fermentation starter
Korean J. Microbiol. 2021;57(3):235-237
Published online September 30, 2021
© 2021 The Microbiological Society of Korea.

Jae Yun Lim and Jeong-Ah Seo*

School of Systems Biomedical Science, Soongsil University, Seoul 06978, Republic of Korea
Correspondence to: E-mail: sja815@ssu.ac.kr; Tel.: +82-2-820-0449; Fax: +82-2-824-4383
Received July 14, 2021; Revised September 14, 2021; Accepted September 17, 2021.
Abstract
Rhizopus microsporus SSU VM08-07 (KCTC 56757) was isolated from banh men, a traditional Vietnamese alcoholic fermentation starter and identified as a major fungus. The draft genome sequence of R. microsporus SSU VM08-07 was generated by using Illumina HiSeq platform. Genome assembly resulted in 1,592 contigs with a total size of 27.6 Mb and 40.1% of G + C content. The annotated genomic sequence contains 10,544 protein coding genes. The genome information of R. microsporus SSU VM08-07 will provide much more applicable feature on production of the traditional alcoholic beverage.
Keywords : Rhizopus microsporus, alcohol fermentation, banh men, draft genome, Illumina HiSeq
Body

Banh men is generally considered as a rich source for the isolation and selection of microorganisms that can be utilized in the food industry (Thanh et al., 2008). Four species of filamentous fungi in the family Mucoraceae, Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, and Amylomyces sp., were commonly found in banh men. These species are strong amylase producers and are frequently found in amylolytic Asian fermentation starters (Hesseltine et al., 1988). The genus Rhizopus was generally found in the microbial community associated with banh men. Specifically, R. microsporus has been known as the most frequently isolated fungal species from banh men (Lee and Fujio, 1999). In this study, out of 17 R. microsporus strains isolated from banh men, we selected the R. microsporus SSU VM08-07 strain that has a high protease activity to generate the whole genome sequence data.

Rhizopus microsporus SSU VM08-07 was cultured on potato dextrose agar (PDA) solid medium for 5 days at 25°C. Mycelia from the solid culture were collected and used for genomic DNA isolation using the modified cetyl trimethylammonium bromide (CTAB) method (Leslie et al., 2006). Genomic DNA was sequenced based on NGS technology by the Theragen Bio Institute. A genomic library was constructed using a TruSeq DNA PCR-free library preparation kit according to the manufacturer’s instructions and sequenced on the Illumina HiSeq platform (Illumina) to generate 202 bp paired-end reads. By a whole-genome shotgun sequencing technology, TruSeq paired-end libraries were generated and sequenced on a HiSeq 2500 platform resulting in 39,276,880 paired-end reads (~15.9 Gb) which represent a 519 fold estimated genome coverage. The sequence reads were de novo assembled with SPAdes v. 3.11.0 (Bankevich et al., 2012). We removed the shorter contigs than 500 bp and mitochondrial or other contaminated contigs. The assembled genome of R. microsporus SSU VM08-07 consists of 1,592 contigs with a total size of 27,620,702 bp (N50, 39 kb; G + C content, 40.1%).

Repetitive sequence masking, gene prediction and gene annotation were performed using Funannotate pipeline v1.7.4 (Palmer and Stajich, 2018). The genome contains 8.13% repetitive sequences masked by RepeatMasker v.open-4.0.7 using a custom repeat library generated by RepeatModeler v.open-1.0.11 (Chen, 2004). Gene prediction with Funannotate was performed running ab initio predictors Augustus v3.3.3, GeneMark-ES v.4.38 and GlimmerHMM v3.0.4 using soft-masked genome sequence. Consensus gene models were generated by Funannotate running EVidenceModeler v.1.1.1. Funannotate assigned putative gene products by searches to the UniProtKB, InterPro, eggnog, MEROPS, and dbCAN databases. The tRNA genes were identified by using tRNAscan-SE (v2.0.7) (Lowe and Eddy, 1997). The secondary metabolite gene clusters were analyzed by antiSMASH 5.0.0 (Weber et al., 2015). Out of 10,544 predicted genes, while 6,439 genes were into GO categories, 8,685 genes have InterPro domains. Among them, 262 genes have CAZymes and 385 have proteolytic enzymes. In addition, we annotated and further analyzed fifteen genomes of R. microsporus strains to compare with R. microsporus SSU VM08-07. Conclusively this strain has more genes involved in degradation of both carbohydrate and protein associated with fermentation than other strains except few strains (Table 1). Twenty secondary metabolite gene clusters were also found on the genome of this fungus. Much genome information of R. microsporus SSU VM08-07 will provide important clues for a role of this fungus in traditional alcohol fermentation process.

Genome featres of Rhizopus microsporus SSU VM08-07

Features Value
Genome size (bp) 27,620,702
G + C content (%) 40.1
Number of contigs 1,592
Number of contigs ≥ 2 kb 1,105
Contig N50 (bp) 39,368
Protein coding genes 10,544
Number of genes having InterPro domains 8,685
Coverage of InterPro (%) 82
Number of Gene Ontology assigned 6,439
Number of genes involved in CAZymes 262
Number of protease genes 385
Number of secondary metabolite gene clusters 20


Nucleotide sequence accession number

The draft genome sequence of R. microsporus SSU VM08-07 (KCTC 56757) has been deposited in GenBank under the accession number JAFCND000000000.

적 요

Rhizopus microsporus SSU VM08-07 (KCTC 56757)은 베트남 전통주 제조 발효제인 반멘으로부터 분리된 가장 주요한 곰팡이로 동정되었다. Rhizopus microsporus SSU VM08-07의 유전체 염기 서열은 Illumina HiSeq platform을 활용하여 해독하였고, 유전체 분석을 통하여 총 크기는 27.6 Mb이고 G + C 함량은 40.1%인 1,592개의 컨티그로 조립하였다. 주석이 달린 유전체 염기서열은 10,544개의 단백질 코딩 유전자를 포함하고 있으며 NCBI에 등록된 15개의 동종 곰팡이 유전체를 추가 분석한 결과, 발효 특성과 관련된 탄수화물, 단백질 분해효소 코딩 유전자가 상대적으로 많음을 확인하였다. Rhizopus microsporus SSU VM08-07 균주의 유전체정보는 전통주 알코올 발효 과정에서 이 곰팡이의 역할에 대한 중요한 정보를 제공할 수 있을 것이다.

Acknowledgments

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agricultural Microbiome R&D Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA-918010-4).

Conflict of Interest

The authors have no conflict of interest to report.

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