Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

Lipomyces tetrasporous NRRL Y-64009 is an oleaginous yeast with the ability to utilize plant-based sugars, glycerol, starch, and organic acids (1 – 3). Because of its high lipid accumulation (over 60%), this yeast has been considered an appealing host for industrial-scale lipid production (4). It can withstand low pH levels and detoxify inhibitors found in lignocellulosic hydrolysates (5, 6). We sequenced the genome and transcriptome of L. tetrasporous NRRL Y-64009 to further investigate its physiology and metabolism to produce biofuels and bioproducts.

The cells were grown for 2 days at 30°C and 250 rpm in YPG medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) and harvested OD_600nm of 10. Genomic DNA and total RNA were extracted using the Dr. GenTLE (from yeast) High Recovery Kit (Takara Bio Inc., Shiga, Japan) and the RNeasy Mini Kit (Qiagen, Hilden, Germany), respectively (7, 8).

Libraries larger than 10 kb for PacBio sequencing were prepared using 5 ug of genomic DNA (9, 10). The sheared DNA was treated with exonuclease, followed by end repair and blunt adapter ligation with the SMRTbell Template Prep Kit 1.0. Blue Pippin (Sage Science) was used to select the library at an 8-kb cutoff and purified with AMPure PB beads. The prepared SMRTbell template libraries were then sequenced on a Pacific Biosystems Sequel II sequencer with 1 × 900 bp sequencing movie run times using v3 sequencing primer, 8M v1 SMRT cells, and Version 2.0 sequencing chemistry. To remove sequencing artifacts, BBduk and BBMerge from BBTools 36.63 (https://sourceforge.net/projects/bbmap/) were used (11).

The Illumina Truseq Stranded mRNA Library Prep Kit was used to create stranded cDNA libraries (11). For library amplification, 1 ug RNA per sample and eight cycles of PCR were used. The libraries were quantified using a Roche LightCycler 480 real-time PCR instrument and a KAPA Biosystems Next-Generation Sequencing Library qPCR Kit. The flow cell was sequenced on an Illumina NovaSeq sequencer with NovaSeq XP V1 reagent kits and an S4 flow cell. The Illumina NovaSeq S4 was used to build an Illumina library and sequenced 2 × 151. BBDuk (version 38.79) (http://bbtools.jgi.doe.gov) was used to remove contaminants, trim reads containing adapter sequences, and right quality trim reads with quality less than 6. Reads mapped to common contaminants and ribosomal RNA reads using BBMap were removed. Filtered Fastq files were used as input for de novo RNA contig assembly. Trinity v2.8.5 was used to assemble consensus sequences from reads that had been filtered and trimmed for quality and contamination (12). Transcript-based gene predictors ESTmap and CombEST were used to incorporate RNA-Seq into the annotation. A genomic mapping and alignment program v2007-local-branch and the BLAST-like alignment tool were used to align RNA-Seq to the genome assembly (13, 14).

The 20.78 Mb genome assembly was composed of 18 contigs (N ₅₀ = 5 Mb), with a sequencing read coverage depth of 267.54× and a GC content of 48.12%. The Joint Genome Institute (JGI) Annotation pipeline (13, 14) was used to predict 8,004 protein-coding genes from the genome.

This material is based upon the work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number(s) DE-SC0018420. The work (proposal: 10.46936/10.25585/60001206) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author (s) and do not necessarily reflect the views of the U.S. Department of Energy. Microbial strains used in this work were provided by the USDA-ARS Culture Collection (NRRL).