Rhizopus oryzae is a generally known as safe (GRAS) filamentous fungus, commonly associated with production of some oriental traditional foods (Londoño-Hernández et al., 2017). Rhizopus delemar has been differentiated as a species of R. oryzae producing fumaric-malic acid (Abe et al., 2007). In this study, we isolated R. delemar SSU VMBB-02 from banh men made in Daklak province, Vietnam. The R. delemar SSU VMBB-02 strain was grown on potato dextrose agar solid medium for 5 days at 25°C and its hyphal mass was collected. The genomic DNA was extracted and purified by using the modified cetyl trimethylammonium bromide method from freeze-dired hyphae (Leslie and Summerell, 2006). The whole genome sequencing was performed by Theragen Bio Institute. The library for sequencing was made by using a TruSeq DNA PCR-free library preparation kit according to the manufacturer’s instructions and sequenced on the Illumina HiSeq 2500 platform (Illumina). As a result of a sequencing, total reads of 17,026,137,418 bp were obtained, and the coverage was 447-fold. The sequence reads were assembled by de novo by SPAdes assembler (Bankevich et al., 2012), which resulted in 5,705 contigs and the whole genome size of 37.9 Mb (N50, 16 kb). The G + C content of the assembled draft genome was 34.4%.

Draft genome annotation was performed by using Funannotate pipeline v1.8.9 (Palmer and Stajich, 2017). First, 11.68% of repetitive sequences were masked by RepeatMasker (open-4.0.7) and RepeatModeler (open-1.0.11) (Tarailo-Graovac and Chen, 2004). The ab initio gene models for the contigs were predicted by using the GeneMark-ES (v4.38) (Ter-Hovhannisyan et al., 2008), GlimmerHMM (v3.0.4) (Majoros et al., 2004) and AUGUSTUS (v3.3.3) (Keller et al., 2011) programs. Evidence-based gene models were made by aligning the contigs with the protein sequence database (UniProtKB) using DIAMOND (v2.0.14) (Buchfink et al., 2021) and then polishing using Exonerate (v2.4.0) (Slater and Birney, 2005). EVidenceModeler (v1.1.1) (Haas et al., 2008) as implemented in the Funannotate pipeline was used to generate the consensus models from the ab initio and evidence-based gene models. The consensus models were functionally annotated after removing short lengths, transposable elements and gaps. A total of 12,076 gene models were used for making 97,106 valid annotations by carrying out sequence similarity searches against the Pfam (v34.0), InterPro (v79.0), BUSCO (v2.0), EggNOG (v4.5), MEROPS (v12.0) and CAZyme (v9.0) databases. We predicted 12,076 protein-coding genes. Among them, 9,149 genes have InterPro domains, of which 6,709 were categorized to Gene Ontology (GO). Further analyses of the genes encoding carbohydrate-active enzymes revealed that 325 genes were involved in CAZymes (21 auxiliary activities, 56 carbohydrate esterases, 7 carbohydrate-binding modules, 118 glycoside hydrolases, 116 glycosyl transferases, and 7 polysaccharide lyases) and 370 genes in proteolytic enzymes (36 aspartic peptidases, 78 cysteine peptidases, 109 metallo peptidases, 7 protease inhibitors, 108 serine peptidases and 32 threonine peptidases) (Table 1). In addition, 549 genes encoding the secreted transcripts were predicted by using the SignalP secretome prediction program (v4.1) (Armenteros et al., 2019). Thirty tRNA genes were predicted by using tRNAscan-SE (v2.0.9) (Lowe and Eddy, 1997). Additionally, fourteen secondary metabolite biosynthesis gene clusters (with 21 biosynthetic enzymes and 15 smCOGs) were found by using antiSMASH 5.0.0 (Blin et al., 2019), which may be involved in unknown secondary metabolisms including terpene, sidrophore and non ribosamal peptide synthesis (NRPS) (Table 2).

Table 1

Features	Value
Draft genome size, bp	37,902,973
GC content, %	34.4
Number of contigs	5,705
Number of contigs ≥ 2 kb	3,067
Contig N50, bp	16,092
Protein coding genes	12,076
Number of genes having InterPro domains	9,149
Coverage of InterPro, %	76
Number of gene ontology assigned	6,709
Number of genes involved in CAZymes	325
Number of protease genes by MEROPS	370
Number of secondary metabolite gene clusters	14

Table 2

No.	Contig number	Secondary metabolite type	Nucleotide location
			Start	End
1	ROVContig0012.1	Siderophore	3,838	16,018
2	ROVContig0078.1	Terpene	26,853	41,211
3	ROVContig0123.1	Terpene	1	16,888
4	ROVContig0129.1	Fungal-RiPP	1	34,725
5	ROVContig0291.1	Terpene	11,848	25,530
6	ROVContig0318.1	Terpene	1	16,870
7	ROVContig0413.1	NRPS	1	22,108
8	ROVContig0611.1	Terpene	1	13,711
9	ROVContig0615.1	Terpene	1	14,098
10	ROVContig0813.1	NRPS-like	1	14,269
11	ROVContig0861.1	Terpene	590	13,605
12	ROVContig1115.1	Terpene	1	10,774
13	ROVContig1162.1	NRPS-like	1	10,337
14	ROVContig2258.1	NRPS-like	1	4,049

Nucleotide sequence accession number

The draft genome sequence of R. delemar SSU VMBB-02 (KCTC 46675) has been deposited in GenBank under the accession number JAFCNC000000000.

베트남의 전통주 생산을 위한 발효 개시제인 반멘으로부터 사상성 곰팡이와 효모균을 분리하였다. Rhizopus 속은 반멘에서 분리된 Rhizopus oryzae, Rhizopus delemar 및 Rhizopus microsporus를 포함한 주요 곰팡이군이었다. 분리된 Rhizopus 25 균주 중 가수분해효소 활성이 높은 R. delemar SSU VMBB-02 (KCTC 46675) 균주를 선정하여 전장유전체 염기서열을 Illumina HiSeq 플랫폼 기술을 활용하여 해독, 조립, 분석하였다. 유전체 분석 결과 전체 유전체 크기는 37.9 Mb로 G + C 함량 34.4%, 생성된 컨티그 5,705개, 단백질 코딩 유전자 12,076개를 포함하고 있었다. 추가적인 분석을 통하여 탄수화물 분해효소와 단백질 분해효소를 코딩한 유전자의 수, 이차 대사물질 생합성 유전자 클러스터 개수 등을 밝혀냈다. 이 연구의 결과는 발효제 유래의 다른 Rhizopus 균주들과 비교해 볼 수 있는 유용한 유전체정보를 제공할 것이다.

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).

The authors have no conflict of interest to report.