Draft Genome Sequences of Itaconate-Producing Ustilaginaceae

Abstract

Some smut fungi of the family Ustilaginaceae produce itaconate from glucose. De novo genome sequencing of nine itaconate-producing Ustilaginaceae revealed genome sizes between 19 and 25 Mbp. Comparison to the itaconate cluster of U. maydis MB215 revealed all essential genes for itaconate production contributing to metabolic engineering for improving itaconate production.

GENOME ANNOUNCEMENT

The members of the family of Ustilaginaceae, belonging to the phylum Basidiomycota, are known to naturally produce many different industrially interesting compounds, such as organic acids, lipids, and polyols (1–6). Previous bioprospecting utilizing glucose or glycerol identified several itaconate-producing Ustilaginaceae (1, 7). The investigated species and strains varied in their product spectra and the amounts of product. Among the species Ustilago maydis, individual strains differed highly in their ability to produce organic acids, although the product spectra did not differ. Some of the species investigated, for example Ustilago vetiveriae CBS 131474, produced itaconate only with glycerol as carbon source (1, 7). Also, the pH dependency of itaconate production varied a lot. While in U. maydis itaconate production is only possible in the pH-range of 5 to 7, Ustilago cynodontis strains can tolerate pH values as low as 3 (1). To gain a deeper understanding of the sequence-function relationship between itaconate genes (8) and itaconate production, the genomes of 12 Ustilaginaceae with different itaconate production levels were sequenced. The itaconate clusters of Sporisorium iseilematis-ciliati BRIP 60887 a, Pseudozyma tsukubaensis NBRC 1940, Pseudozyma hubeiensis NBRC 105055, Ustilago vetiveriae CBS 131474, and Ustilago maydis strains AB33P5ΔR, ATCC 22892, ATCC 22899, ATCC 22901, and ATCC 22904, were compared, as well as Ustilago xerochloae CBS 131476 and Ustilago cynodontis NBRC 9727 and CBS 131467 (data not shown).

Here, we present the draft genome sequences of these Ustilaginaceae. Genomic DNA (gDNA) was isolated by standard phenol-chloroform extraction (9). Eurofins Genomics (Ebersberg, Germany) did the library creation using an NEBNext Ultra DNA Library prep kit for Illumina (Art no. E7370) and sequencing by using an llumina HiSeq2500 machine with TruSeq SBS kit v3, both according to manufacturer’s instructions. Sequencing mode was 1 × 100 and the software used was HiSeq Control Software 2.0.12.0 RTA 1.17.21.3 bcl2fastq-1.8.4. Quality check of the sequence data was performed with FastQC (Version 0.11.2). The SPAdes-3.7.0-Linux pipeline was used for de novo genome assembly of single-read libraries and read error or mismatch correction, including BayesHammer, IOnHammer, SPAdes, MismatchCorrector, dipSPAdes, and truSPAdes. The k-mer size was determined to 55 using VelvetOptimiser Version 2.2.5. Parameters of the resulting sequences are summarized in Table 1.

TABLE 1 .

Genome sequence parameters

Strain	Reference or source	NCBI GenBank accession no.a	Sequence size (bp)	No. of large contigs (>300 bp in size)	G+C content (%)	Avg contig sequence size N50 (bp)	Max contig sequence size (bp)
Ustilago maydis AB33P5ΔR	11	LZQU00000000	19,929,430	1,978	50	111,545	354,120
Sporisorium iseilematis-ciliati BRIP 60887 a	Culture collection of the Queenslad Plant Pathology Herbarium (BRIP), Australia	MJEU00000000	23,207,148	15,412	54	128,139	422,913
Pseudozyma tsukubaensis NBRC 1940	12	MAIP00000000	23,769,677	12,840	52	161,943	632,715
Pseudozyma hubeiensis NBRC 105055	13	MAIO00000000	21,322,328	9,793	54	260,601	1,046,041
Ustilago vetiveriae CBS 131474	13	MAIM00000000	19,606,533	5,932	52	160,125	620,444
Ustilago maydis ATCC 22892	1	LYOO00000000	20,622,051	4,767	50	104,350	527,495
Ustilago maydis ATCC 22899	1	LYZD00000000	20,208,930	2,938	51	109,028	306,704
Ustilago maydis ATCC 22901	1	LZNJ00000000	20,063,391	2,505	51	102,739	354,117
Ustilago maydis ATCC 22904	1	LZQT00000000	20,132,962	2,695	49	120,656	551,297

^aVersions described are the first versions.

By comparison to the itaconate gene cluster of U. maydis MB215 (8) (GenBank Accession Number KT852988), the complete itaconate cluster was identified in all sequenced organisms except Pseudozyma tsukubaensis NBRC 1940, which did not contain rdo1 and cyp3, which encode a putative dioxygenase and a monooxygenase involved in OH-paraconate production, respectively (10). The synteny of the itaconate cluster is preserved in the investigated Ustilaginaceae. The sequences will foster research on the biology of Ustilaginaceae and increase the list of tools for metabolic engineering of itaconate production by Ustilaginaceae.

Accession number(s).

The whole-genome sequences have been deposited in DDBJ/ENA/GenBank. Their accession numbers and version numbers described in this paper are listed in Table 1.