Aspergillus neoniger is one of the 26 species of Aspergillus section Nigri and is morphologically very similar to Aspergillus niger and Aspergillus tubingensis (Varga et al., 2011). Aspergillus neoniger is known to have high catalytic activity such as α-glucosidase (Kumar and Mutturi, 2020). In our previous study, the enzyme activities of 28 strains of Aspergillus section Nigri from a wheat-based fermentation starter in Korea was measured, and A. neoniger KCN5 (= KCTC 56924) with high cellulolytic activity was obtained (Jeong and Seo, 2022). Aspergillus neoniger KCN5 showed 566.3 unit/g of β-glucosidase activity which is more than five times higher than that of A. niger N402 (144.6 unit/g) (Jeong and Seo, 2022), previously reported for high cellulose degrading ability (Delmas et al., 2012). Therefore, the genome of A. neoniger KCN5 was sequenced and compared to A. niger N402.

Aspergillus neoniger KCN5 was cultured on 5 ml of complete media (Seo et al., 2006) under shaking condition (180 rpm) at 25°C for 2 days. The genomic DNA was extracted from the culture using modified CTAB method (Cota-Sánchez et al., 2006). The whole genome sequencing of A. neoniger KCN5 was performed using an Illumina HiSeq 2500 platform and a paired-end strategy (Illumina). The Illumina reads were sequenced to an average coverage of 100-fold. De novo assembly was performed using SPAdes assembler v3.14.1 (Prjibelski et al., 2020) and resulted in the 464 scaffolds with a total size of 37.9 Mb (N₅₀, 2.95 Mb; GC content, 48.7%) (Table 1).

Table 1 Draft genome feature of Aspergillus neoniger KCN5 compared with Aspergillus niger N402

Features	Aspergillus neoniger KCN5	Aspergillus niger N402
Genome size, bp	37,911,110	35,570,168
GC content, %	48.70	49.60
Number of scaffolds	464	19
N50, bp	2,949,984	2,899,611
Number of protein-coding genes	13,680	11,236
Number of InterPro	10,342	9,237
Number of CAZymes-related genes	504	485
Number of protease genes	383	363
Number of secondary metabolite clusters	101	107
BUSCO completeness, %	94.1	98.8
GenBank accession number	GCA_029783945	GCA_900248155
Reference	Jeong and Seo (2022)	Delmas et al. (2012)

Through mapping on the reference sequence of A. niger CBS 513.88 (GCF_000002855; Pel et al., 2007), the eight large scaffolds of A. neoniger KCN5 were obtained containing 2 to 5 contigs. The telomeric repeats were detected in all terminal regions of chromosomes, except for the C-terminal end of chromosome 3. Therefore, it appears that these scaffolds correspond to individual chromosomes. The number of unplaced contigs were 456 with a total size of 2.03 Mb.

Average nucleotide identity (ANI) values between A. neoniger KCN5 and 9 representative strains of Aspergillus section Nigri (Varga et al., 2011) were calculated using OrthoANI (Lee et al., 2016). The genome of A. neoniger KCN5 showed about 87–95% identity with 9 representative strains of Aspergillus section Nigri. ANI-based phylogenetic tree showed that the genome of A. neoniger KCN5 had 95.1% identity with A. neoniger CBS 115656 (Fig. 1).

Fig. 1. Average nucleotide identity (ANI)-based phylogenetic tree of A. neoniger KCN5.
Phylogenetic tree was constructed using hclust function in R package based on the ANI values of A. neoniger KCN5 and 9 strains of Aspergillus section Nigri. Whole genome sequences of 9 strains were obtained from GenBank. ANI values between ten genomes were calculated using OrthoANI.

Gene annotation was performed using Funannotate pipeline v.1.8.9 (Palmer and Stajich, 2020) and the results of A. neoniger KCN5 and A. niger N402 were compared (Table 1). Aspergillus neoniger KCN5 had 13,680 protein-coding genes, 2,444 more genes than A. niger N402 had. As a result of searching the CAZyme database (dbCAN v.9.0.), the number of CAZyme genes of A. neoniger KCN5 was higher than that of A. niger N402. In A. neoniger KCN5, more genes and isoforms belonging to glycoside hydrolases (GHs) were found compared to A. niger N402. Aspergillus neoniger KCN5 had 27 more isoform genes in GH13 α-amylase family and 20 more isoform genes in GH43 cellulase family.

Nucleotide sequence accession number

The draft genome sequence of A. neoniger KCN5 (= KCTC 56924) has been deposited to the NCBI GenBank database under the accession number JAPVRF000000000. Genome sequence of A. neoniger KCN5 was also deposited to the National Agricultural Biotechnology Information Center (NABIC) under accession number NG-1542-000001 – NG-1542-000464.

국내 발효 개시제에서 분리한 Aspergillus neoniger KCN5는 Aspergillus section Nigri의 참조 균주 중 셀룰로오스 분해능이 높은 것으로 알려진 A. niger N402보다 약 5배 이상 높은 베타-글루코시데이스 활성을 보였다. Aspergillus neoniger KCN5의 전장 유전체는 Illumina HiSeq 2500 플랫폼을 사용하여 해독되었고 8개의 염색체가 확인되었다. Aspergillus neoniger KCN5와 Aspergillus section Nigri의 참조 균주 간의 염기서열 평균 유사도를 계산하였고 A. neoniger KCN5의 염기서열은 A. neoniger CBS 115656의 염기서열과 95.1%의 유사도를 보였다. 유전자 주석 분석 결과 A. neoniger KCN5는 A. niger N402보다 단백질 코딩 유전자가 2,444개, CAZyme 관련 유전자가 19개 많았고 CAZyme 중 셀룰라아제 계열 GH43 유전자와 동형 유전자의 개수가 27개 더 많았다. 셀룰로오스 분해 활성이 높은 A. neoniger KCN5의 유전체 정보는 국내에서 분리한 A. neoniger 균주의 효소 생산자로서의 유용한 특성을 보여주고 있다.

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.