Aspergillus niger, a member of ‘generally regarded as safe’ (GRAS) organism, is used in industry for enzyme production such as glucoamylase and cellulase (Cairns et al., 2018). In our previous study, enzymatic activities of twenty-one strains of A.niger isolated from wheat-based fermentation starter of Korea were measured (Jeong and Seo, 2022). Among them, A. niger KJC3 (KCTC 56813) and KYF3 (KCTC 56814) showed high β-glucosidase activities about three times higher than that of CBS 513.88 (40.5 Unit/g), a reference strain of A. niger (Pel et al., 2007). Therefore, the genomes of the two Korean strains were sequenced and analyzed to compare with the reference strain.

Two strains were cultured on 5 ml of complete media (Seo et al., 2006) and incubated at 25°C for 2 days by shaking at 180 rpm. The genomic DNA was extracted from the cultures using modified CTAB method (Cota-Sánchez et al., 2006). Long-read libraries were prepared using PacBio Single-Molecule Real-Time (SMRT) library method. SMRT sequencing was performed using PacBio Sequel System (PacBio) and resulted in a total of 1.84 Gb of filtered data of KJC3 (about 45x coverage; mean length, 11.3 Kb; N₅₀ 16.0 Kb) and 2.28 Gb of KYF3 (about 60x coverage; mean length, 9.8 Kb; N₅₀ 13.4 Kb). As a result of de novo assembly using HGAP4 pipeline (Chin et al., 2016), the genome of KJC3 was assembled into 15 contigs (40.27 Mb; N₅₀, 5.10 Mb; GC content, 49.50%), and the genome of KYF3 was assembled into 10 contigs (37.46 Mb; N₅₀, 4.68 Mb; GC content, 49.50%) (Table 1).

Table 1 Draft genome features of Aspergillus niger KJC3 and KYF3 compared with the reference strain CBS 513.88

Features	KJC3 (KCTC 56813)	KYF3 (KCTC 56814)	CBS 513.88
Genome size, Mb	40.27	37.46	33.98
GC content, %	49.50	49.50	50.40
Number of contigs	15	10	19
N50, Mb	5.10	4.68	2.53
Number of chromosomes	8	8	8
Number of unplaced contigs	7	2	-
Number of protein coding genes	13,876	11,526	10,978
Number of InterPro	10,834	9,340	8,888
Number of CAZymes	537	492	475
Number of protease genes	405	376	353
Number of secondary metabolite clusters	88	90	99
BUSCO completeness, %	93.15	98.69	94.66
GenBank accession number	GCA_029783905	GCA_029783925	GCA_000002855
Reference	This study	This study	Pel et al. (2007)

Through the homology search with genome sequence of A. niger CBS 513.88, the eight large contigs out of total contigs were constructed into eight chromosomes without a gap in both KJC3 and KYF3 (Fig. 1). Eight chromosomes were completed by telomere-to-telomere assembly except for N-terminal of chromosome 6. Seven contigs of KJC3 (1.43 Mb) and two contigs of KYF3 (1.34 Mb) were not placed to chromosome of A. niger CBS 513.88. The percentage of gene density was calculated from the total number of genes within 2.5 Kb. We find low density regions on chromosome 3, 6 and contigs of KJC3 and chromosomes 6, 8 and contigs of KYF3. These could be caused by gene shuffling, horizontal gene transfer or insertion of foreign genes (Kjærbølling et al., 2020).

Fig. 1. Chromosome maps of Aspergillus niger KJC3 and KYF3.
The gray-colored bar represents the eight chromosomes and the unplaced contigs of two strains. Location of AT-rich centromere regions is indicated as the red-colored bar. The deep gray-colored bar indicates the region where the percentage of gene density is greater than 50%.

Gene annotation was performed using Funannotate pipeline v.1.8.9 (Palmer and Stajich, 2020), and CAZyme and protease genes of three strains were searched from the CAZyme (dbCAN v.9.0.) and MEROPS database (MEROPS v.12.0.), respectively. KJC3 had 13,876 protein coding genes, of which 10,834 had InterPro domain (Table 1). KYF3 had 11,526 protein coding genes, of which 9,340 had InterPro domain. The number of CAZymes and protease genes of KJC3 and KYF3 was higher than that of A. niger CBS 513.88. Among the CAZymes, the number of ‘auxiliary activity family 3’ genes related to cellulolytic activity was higher in two Korean strains than CBS 513.88; 96 for KJC3, 101 for KYF3, and 91 for CBS 513.88. The quality of genome assemblies and annotations was evaluated using BUSCO v.5.2.2 (Simão et al., 2015), and genomes of KJC3 and KYF3 were 93.15%, and 98.69% complete, respectively. The genomic information of KJC3 and KYF3, which have high cellulolytic activity, is meaningful in that Korean A. niger strains showed useful properties and potential as enzyme producers in industry.

Nucleotide sequence accession number

The draft genome sequences of A. niger KJC3 (KCTC 56813) and KYF3 (KCTC 56814) have been deposited to the NCBI GenBank database under the accession numbers JAPVRD 000000000 (KJC3) and JAPVRE000000000 (KYF3). Genome sequences of two strains were also deposited to the National Agricultural Biotechnology Information Center (NABIC) under accession numbers NG-1540-000001 – NG-1540-000015 (KJC3) and NG-1541-000001 – NG-1541-000010 (KYF3).

국내 발효 개시제에서 분리된 Aspergillus niger KJC3와 KYF3는 A. niger의 참조 균주인 CBS 513.88보다 3배 이상 높은 셀룰로오스 분해 활성을 보였다. KJC3와 KYF3의 전장 유전체는 PacBio Sequel 플랫폼을 사용하여 해독되었고 8개의 염색체와 각각 7개, 2개의 균주 특이적인 컨티그로 조립되었다. 유전자 주석 분석 결과 두 균주 모두 CBS 513.88보다 더 많은 CAZyme, protease와 같은 효소 관련 유전자를 가지고 있었다. 셀룰로오스 분해 활성이 높은 KJC3와 KYF3의 유전체 정보는 국내에서 분리한 A. niger 균주의 유용한 특성과 다양한 산업에서 효소 생산자로서의 가능성을 이해하는 데 도움이 될 것이다.

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (RS-2023-00230782)” Rural Development Administration, Republic of Korea.

The authors declare that there is no conflict of interest.