Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

Abstract

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.

Figure 1.

Synteny map of the contigs of A. niger ATCC 1015 to the supercontigs of A. niger CBS 513.88. The coloring of the chromosomes shows syntenic regions in A. niger CBS 513.88. Arabic numerals show the number of the supercontig in A. niger CBS 513.88. Gray areas show regions not found in the CBS 513.88 genome sequence (Pel et al. 2007). (Filled black circles) Proposed locations of centromeric regions. Sequenced telomeres are marked with a T. Zeroes mark the first base of the contigs. A black line underneath a section of the chromosomes denotes inverted sequence. Black histograms show SNPs per kilobase (number of single nucleotide polymorphisms/kilobase) between the sequences of the two strains (y-axis: 0–160 SNPs/kb). Gaps between contigs and centromeres are not to scale. The alignment demonstrates almost complete synteny between the two strains, with the exception of a cross-over event between the left arms of chromosomes III and VII. An overview of the genes found in the gaps unique to ATCC 1015 can be found in Supplemental Table 2.

Figure 2.

Horizontal gene transfer of alpha-amylase genes from A. oryzae to A. niger CBS 513.88. (A) Unmatched region identified for the left arm of chromosome III spanning 65 kb for ATCC 1015 encoding 30 predicted genes and 85 kb for CBS 513.88 encoding 24 predicted genes. The unmatched region is flanked on one side by a small local inversion of three predicted genes (in green). (B) The 12.4-kb HGT region is part of an identical 12.7-kb region present in A. oryzae RIB40 supercontigs SC113 and SC023. In A. niger CBS 513.88, the transferred region is enclosed by a 203-bp inverted repeat (red arrow). An12g07000 (blue) is a putative transposase and identical to the A. oryzae transposon Aot1 tnpA gene. Similarly, the A. niger CBS 513.88 alpha-amylase encoding gene An12g06930 (orange) is identical to A. oryzae annotated genes AO090120000196 (SC113) and AO090023000944 (SC023). (C) Proposed duplication–recombination event between supercontig 12 and supercontig 05 of the 12-kb HGT region. Breakpoints are indicated with dotted lines. The fragment encoding alpha-amylase An05g02200 is identical to An12g06930 (orange). The breakpoints are flanked with additional copies of the 203-bp repeat region (red arrow). The region encoding genes An12g06940 to An12g06970 is identical to the region encoding An05g02210 to An05g02130. The downstream breakpoint occurred in the predicted gene coding region of An12g06970, thereby deleting the original start codon and upstream region.

Figure 3.

(A) Phylogenetic relationship of several black Aspergilli based on partial sequencing of beta-tubulin. The tree was rooted to Aspergillus aculeatus CBS 172.66. (B) Phylogenetic relationship of seven strains of A. niger based on sequencing of 1-kb variable regions from four chromosomes. The tree was rooted to the sequence obtained from Aspergillus carbonarius IMI 388653. Clades based on the exo-metabolomic groupings of Table 2 are shown. For both trees, bootstrap values above 80% of the 1000 performed reiterations are shown.