Draft Genome Sequence of the Yeast Pichia manshurica YM63, a Participant in Secondary Fermentation of Ishizuchi-Kurocha, a Japanese Fermented Tea

Pichia manshurica is common in fermentation; however, genome analysis has never been reported for the species. This yeast plays a role in the secondary fermentation of Ishizuchi-kurocha, a traditional Japanese fermented tea. This paper presents the draft genome sequence of P. manshurica YM63, isolated from the leaves of fermented tea.

P ichia manshurica (phylum Ascomycota, subphylum Saccharomycotina) is a common yeast found in fermented animal feed and foods such as fermented maguey juice (1), a traditional beverage of Burkina Faso (2), wines (3), and silages (4). Ishizuchikurocha is a traditional fermented tea in Shikoku Island, Japan (5). The yeast species Pichia manshurica naturally occurs during the production process of Ishizuchi-kurocha. The yeast species has important roles in fermentation and rotting plant materials. The genome sequences of Pichia kudriavzevii (6,7) and Pichia membranifaciens (8), which are closely related to P. manshurica, and their comparative analysis (9) have been presented. Genome analysis of P. manshurica, however, has not been reported.
We used strain YM63, isolated from leaves in the secondary fermentation step of Ishizuchi-kurocha production. The strain was confirmed to be the correct species by 28S rRNA sequencing. YM63 was cultured in yeast extract-peptone-dextrose broth at 30°C for 14 h with shaking at 130 rpm. We prepared genomic DNA from the YM63 strain using a blood and cell culture DNA minikit (Qiagen, Inc.). Genome sequencing was performed using a MiSeq platform (Illumina, Inc.) and GridION with flow cell type R9.4.1 (Oxford Nanopore Technologies). For MiSeq sequencing, genomic DNA was sheared with a Covaris S2 sonicator (Covaris, Inc.) to obtain ϳ500-bp DNA fragments. A library was prepared from 200 ng of fragmented DNA using a preparation kit (Kapa HyperPrep kit; Kapa Biosystems) and an adapter kit (FastGene adapter kit; Nippon Genetics Co. Ltd.). After quantification and qualification, the prepared library was sequenced with MiSeq technology to produce 2 ϫ 151-bp paired-end reads. A total of 2,327,748 reads with a Q30 of 83.4% were obtained by MiSeq sequencing. For GridION analysis, a library was prepared using a ligation sequence kit (Oxford Nanopore Technologies). By GridION analysis, 749,822 reads (average length, 16,203 bp) were obtained. MiSeq reads were trimmed using the parameters -q 20 -l 127 with Sickle v1.33 (10). A total of 2,170,013 filtered reads were used for a subsequent assembly. Adaptor sequences in the reads from GridION were trimmed with Porechop v0.2.3, and the trimmed reads were quality filtered using the parameters -min_mean_q 80.05 -min_length 1000 with Filtlong v0.2.0. Error-prone read data from GridION were processed using Canu v1.8 (11). A total of 668,602 reads was used for the subsequent assembly. Finally, assembly was done using MaSuRCA v3.2.8 (12), and polishing was done using Bowtie 2 v2.3.4.1 (13) and Pilon v1.22 (14).
The draft genome includes eight contigs with a total size of 12,405,322 bp. The GC content is 42.3%. The N 50 contig and the longest contig sizes are 2,862,201 bp and 3,465,259 bp, respectively. The total contig size of P. membranifaciens KS47-1 is 11.4 Mb (8). The genome assembly size of P. membranifaciens NRRL Y-2026 is 11.5 Mb (9), which is slightly smaller than that of P. manshurica YM63. According to BLAST homology analysis, the scf7180000000032 contig (GenBank accession no. BJFO01000006) is estimated to be a mitochondrial genome. Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 (15,16) analyses using Ascomycota and Saccharomycetales data sets showed 83% and 80.5% complete BUSCO, respectively.
Data availability. The draft genome sequence of Pichia manshurica YM63 was deposited in DDBJ/ENA/GenBank under accession no. BJFO01000001 to BJFO01000008. The raw sequencing reads were submitted to the DRA under accession no. DRA008229.

ACKNOWLEDGMENTS
This work was partly supported by intramural funding from the Obihiro University of Agriculture and Veterinary Medicine and a support project for Ishizuchi-kurocha production and branding in Ehime Prefecture.
We thank the Social Welfare Corporations Peace (Saijo, Ehime, Japan), Satsuki-Kai (Saijo, Ehime, Japan), and Visee (Saijo, Ehime, Japan) for kindly providing the Ishizuchikurocha, as well as the governments of Ehime Prefecture and Saijo City for assistance in this research. We thank Bioengineering Lab. Co., Ltd. for sequencing and bioinformatic analysis of the whole genome. We also thank Hiroki Takahashi for comments and helpful discussion on the manuscript.