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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2023; 51(4): 551-554

https://doi.org/10.48022/mbl.2311.11004

Received: November 7, 2023; Accepted: December 6, 2023

Genome Sequence of the Yeast Strain Sporobolomyces phaffii RJAF-17, Which Produces the Lipoamino Acid Surfactants

Parthiban Subramanian1, Jeong-Seon Kim2, Jun Heo2, and Yiseul Kim2*

1National Agrobiodiversity Center, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea
2Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea

Correspondence to :
Yiseul Kim,         dew@korea.kr

Abstract

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We report the draft genome sequence of Sporobolomyces phaffii RJAF-17, a basidiomycetous yeast strain producing lipoamino acid surfactants, N-palmitoyl leucine and N-parmitoleyl glutamine. The annotation and classification of protein-coding genes provided the basic information for the genome of strain RJAF-17, including prediction of abundant genes as well as detection of genes involved in the biosynthesis of lipoamino acids. With the molecular importance of lipoamino acids as promising alternatives to chemical surfactants, the genomic information of strain RJAF-17 can help us understand the role of biomolecules in yeasts and explore possibilities of large-scale synthesis for industrial applications.

Keywords: Yeast, Sporobolomyces phaffii, biosurfactant, genome

Body

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In response to high demand for environmental and industrial applications of microorganisms, efforts have been made to explore microorganisms with biotechnological potential. Among several useful products derived from microorganisms, biosurfactants have a significant importance as they are widely used around the world for a variety of purposes. According to a recent survey, the global market size of biosurfactants is projected to reach a compound annual growth rate of 5.5% from 2020 to 2026, while the market size of chemical surfactants is estimated to grow at around 5.3% from 2020 to 2027 (https://www.alliedmarketresearch.com). Adverse effects of climate change and surge of overall pollution urge the need to look for environmentally friendly biosurfactants for everyday applications. With this in mind, wild flowers were collected in Gwangyang-si, Jeollanamdo, Republic of Korea for isolation of yeast strains, of which Sporobolomyces phaffii RJAF-17 from Prunus mume for. alphandii was selected for further analyses. Briefly, approximately 3 g of the sample was suspended in 10 ml of sterile saline. The suspension was serially diluted and 0.1 ml of each dilution was spread onto Yeast Malt Agar (Difco). After incubation at 25℃ for 3−4 days, colonies with different colors and morphologies were isolated. Analysis of the internal transcribed spacer region of strain RJAF-17 exhibited the highest similarity value to Sporobolomyces phaffii in the phylum Basidiomycota. Characterization of the compounds produced by strain RJAF-17, including measurement of surface tension and determination of chemical structure revealed its capability to synthesize lipoamino acid surfactants (patent application number 10-2023-0149087). A genomic investigation of strain RJAF-17 would assist in identifying the genetic sources which can be developed later for industrial applications. Hence, we present here the genomic information of biosurfactant-producing yeast strain RJAF-17.

The genome of strain RJAF-17 was sequenced using a combination of HiFi sequencing (Sequel II System) and Illumina HiSeq X-ten (Illumina, USA) platforms provided by Macrogen, Republic of Korea. High quality de novo genome assemblies were generated based on HiFi reads by using genome assembly application from PacBio. First, PanCake 1.1.2 [1] was used to overlap the reads and Nighthawk to phase the overlapped reads. Following the removal of chimeras and duplicates from the overlapped reads, a string graph was constructed and primary contigs as well as haplotigs were generated. The primary contigs and haplotigs were polished using Racon 1.5.0 [2]. To remove haplotype duplications in the primary contig set, purge_dups was used for retrieving potential haplotype duplications and move them to the haplotig set. Following this, the genome assembly generated was subjected to further analyses. The raw sequencing data was submitted to the National Center for Biotechnology Information (NCBI) under the Bio-Project PRJNA1005338 with the accession number SRX21362516 and SRX21362517.

All further analyses were carried out on the Galaxy Web platform (https://usegalaxy.org). Assessments of assembly completeness and repetitive elements were conducted with BUSCO 4.1.4 [3] using the dothideomycetes_ odb10 lineage dataset and with RepeatMasker 4.1.5., respectively. For quality assessment of gene prediction, annotations of strain RJAF-17 were performed using AUGUSTUS and MAKER software with Neurospora crassa as a model for training [4]. A comparison of the annotations using the two different tools are provided in Table 1. The functional annotation was carried out based on publicly available databases, including Clusters of Orthologous Groups (COG), Gene Ontology (GO), Carbohydrate-Active Enzymes (CAZy), and Pfam using eggNOG-mapper 2 [5]. Additionally, the genome was screened for Kyoto Encyclopedia of Genes and Genomes and Protein Analysis THrough Evolutionary Relationships using KOBAS 2.0 to study functional metabolism of genes [6]. Prediction of secondary metabolite gene clusters was accomplished with the fungal version of antiSMASH 7.0 (https://fungismash.secondarymetabolites.org/#!/start). Average nucleotide identity (ANI) was calculated by ANI calculator (http://enve-omics.ce.gatech.edu/ani/index).

Table 1 . Statistics of gene prediction using the different programs.

AttributeAUGUSTUSMAKER
Contigs1313
Number of genes predicted5,8617,864
Number of transcripts predicted5,8617,864
Complete BUSCOs1,4681,658
Missing BUSCOs21288
Number of selected queries by4,3626,015
EggNOG-mapper(74.4%)(76.4%)
Pfam hits*4,0865,618
GO hits*2,6563,582
EC hits*1,2371,680
CAZy hits*80117

*Number of predicted genes that contain at least one Pfam domain, one GO term, one enzyme, and one CAZy hit.

The genome assembly was 19.7 Mb (19,710,391 bp) in size and consisted of 13 scaffolds. The largest scaffold was 3,769,252 bp long and the shortest was 463,633 bp long with N50 value of 1.9 Mb (1,875,694 bp). The GC content was estimated to be 57.5%. Completeness of the genome assembly was 92.2%. Analysis of repetitive elements exhibited very few repeat elements in the genome (2.86%). The predicted genes using AUGUSTUS (5,861 genes) and MAKER (7,864 genes) were submitted to eggNOG-mapper for scanning (Table 1). Although there is currently no ANI threshold range for yeast species demarcation, calculation of ANI was performed between strain RJAF-17 and three available genomes of the genus Sporobolomyces at NCBI. The ANI results indicated that strain RJAF-17 showed the highest values with Sporobolomyces salmonicolor (GCA 001373355.1, 76.7%), followed by Sporobolomyces pararoseus (GCA 010758995.1, 76.4%) and Sporobolomyces roseus (GCA 016617785.1, 76.3%).

Functional annotation using outputs from both AUGUSTUS and MAKER elucidated functional traits of the coding sequences (Fig. 1). In terms of COG categories, strain RJAF-17 contained many genes related with “post-translational modification, protein turnover, chaperones” followed by “translation, ribosomal structure and biogenesis”. Analysis using fungal antiSMASH resulted in identification of non-ribosomal peptide synthetase (NRPS domain, contigs 1 and 3), terpene synthesis domain (contigs 2 and 4), and betalactone synthesis domain (contig 2) (Table 2). As mentioned earlier, strain RJAF-17 was observed to produce surfactant molecules namely N-palmitoyl leucine and N-parmitoleyl glutamine, which are categorized as lipoamino acids (patent application number 10-2023-0149087). These lipoamino acids have been well established for their surfactant property [7, 8]. Formed by the association of a polar amino acid and a non-polar long-chain compound, these molecules have high surface activity resulting in surfactant characteristics. In bacteria, this acylation reaction is reported to be carried out by two enzymes, an Nacetyltransferase and an O-acetyltransferase of which the former catalyzes the initial conjugation of the amino acid to a beta hydroxy fatty acid followed by conjugation of a second fatty acid to the lysolipid by the later [9]. In this study, we found five genes for N-acetyltransferase in the annotated genome of strain RJAF-17 but not for O-acetyltransferases. As Sporobolomyces spp. have been reported for their ability to reduce animal wastes and biosurfactant activity [10], further experimental and genomic analyses can help identify biosynthetic pathway for the lipoamino acids.

Table 2 . Secondary metabolite gene clusters determined by the fungal version of antiSMASH.

ContigRegionTypeFromToMost similar known clustersSimilarity
Contig 11.1NRPS1,464,7621,510,798Nonribosomal peptide synthase of yeast Rhodotorula toruloides54.9%
Contig 22.1Terpene56,42178,302Hypothetical protein from Rhodotorula paludigena involved in ergosterol biosynthetis70.3%
2.2Betalactone1,585,0391,617,9966-Coumarate-CoA ligase of Rhodotorula toruloides NP1168.3%
Contig 33.1NRPS-like438,470483,419L-Aminoadipate-semialdehyde dehydrogenase of Sporobolomyces salmonicolor81.3%
Contig 44.1Terpene75,068102,257Squalene cyclase (SQCY) domain gene of Sporobolomyces salmonicolor66.0%
4.2Terpene572,343593,838Lycopene cyclase/phytoene synthase of Sporobolomyces pararoseus71.3%


Figure 1.Functional annotation of the genome of strain RJAF-17 indicating top 15 COG categories.

Acknowledgment

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This study (Project No. 015675) was carried out with the support of National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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