Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation

Accepted Manuscript Title: Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation Author: Qi Shen Yue Chen Danfeng Jin Hui Lin Qun Wang Yu-Hua Zhao PII: DOI: Reference:

S0944-5013(16)30179-3 http://dx.doi.org/doi:10.1016/j.micres.2016.07.005 MICRES 25923

To appear in: Received date: Revised date: Accepted date:

28-4-2016 19-7-2016 19-7-2016

Please cite this article as: Shen Qi, Chen Yue, Jin Danfeng, Lin Hui, Wang Qun, Zhao Yu-Hua.Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation Qi Shen1†, Yue Chen2†, Danfeng Jin3, Hui Lin 4, Qun Wang1, Yu-Hua Zhao1* 1

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College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, PR China Institute of Horticulture, Zhejiang Academy of Agriculture Science, Hangzhou,

Zhejiang, PR China 3

Institute of Microbiology, Jiangxi Academy of Sciences,

Nanchang, Jiangxi,

PR

China 3

Institute of Environment Resource and Soil Fertilizer, Zhejiang Academy of

Agriculture Science, Hangzhou, Zhejiang, PR China



Corresponding author. Tel: +86-571-88208557, fax: +86-571-88206995

E-mail address: [email protected] (Y. Zhao) †

: These authors contributed equally to this work

Abstract In this work, Trichosporon fermentans CICC 1368, which has been shown to accumulate cellular lipids efficiently using industry-agricultural wastes, was subjected to preliminary genome analysis, yielding a genome size of 31.3 million bases and 12,702 predicted protein-coding genes. Our analysis also showed a high degree of

gene duplications and unique genes compared with those observed in other oleaginous yeasts, with 3–4-fold more genes related to fatty acid elongation and degradation compared with those in Rhodosporidium toruloides NP11 and Yarrowia lipolytica CLIB122. Phylogenetic analysis with other oleaginous microbes suggested that the lipogenic capacity of T. fermentans was obtained during evolution after the divergence of genera. Thus, our study provided the first draft genome and comparative analysis of T. fermentans, laying the foundation for its genetic improvement to facilitate cost-effective lipid production.

Keywords: Microbial oil; Microbial genome; Trichosporon fermentans; Comparative analysis; Evolution

1. Introduction Microbial oils, commonly known as single-cell oils (SCOs), can be used as feedstock for biodiesel production (Zhu et al., 2008; Huang et al., 2009). There is an increasing need to achieve SCOs production from inexpensive raw materials, such as lignocellulosic biomass from agriculture activities; glycerol (Fakas et al., 2009), oils, fats (Papanikolaou and Aggelis, 2003), and sugars (Zhu et al., 2008) from the food industry; and sludge from wastewater treatments (Xue et al., 2008), owing to the low cost and waste recycling capacity of this technology.

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As a type of yeast belonging to the family Cryptococcus, Trichosporon fermentans is considered an “oleaginous’’ microbe. T. fermentans can also produce a large amount of extracellular lipase from olive oil or tung oil (Chen et al., 1992). Moreover, culture of T. fermentans in nitrogen-limited medium promotes accumulation of cellular lipids, contributing to over 50% of observed lipid accumulation. Members of Trichosporon fermentans have been reported to able to produce cellular lipids efficiently using industry-agricultural wastes (Zhu et al., 2008; Huang et al., 2009; Shen et al., 2013; Zhan et al., 2013). Importantly, T. fermentans has been shown to have a tolerance for inhibitory compounds in biomass hydrolysates (Huang et al., 2009). Thus, T. fermentans is a unique yeast strain with great biotechnological potential for SCOs production. Recent increases in the amount of available genomic data have accelerated our understanding of lipid metabolism at the cellular level. To date, many studies have described the genomic information for many oleaginous micro-organisms, including Nannochloropsis, Rhodococcus, Yarrowia, Mucor, and Aspergillus (Vieler et al., 2012; Morin et al., 2014; Zhao et al., 2014). However, although many studies have described the results of lipid production by T. fermentans, our understanding of the molecular basis of its microbial oleaginicity and the reduced capacity for strain engineering is limited because the genetic background of this yeast remains poorly documented. Here, we performed genomic sequencing of T. fermentans and subsequent

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comparative genomics analysis to investigate candidate orthologous genes and enzymes related to oleaginicity among oleaginous microbes. Our results provided important insights into the genetic basis of oleaginous fungi and are expected to facilitate the engineering of single cell oils producers. 2. Materials and Methods 2.1 Fungal strain

T. fermentans (CICC 1368) (China Center of Industrial Culture Collection), which is known to be able to be cultured on PDA and YPD plates, was selected for genome sequencing. T. fermentans was precultured in YEPD medium (20 g/L glucose, 10 g/L peptone, and 10 g/L yeast extract) at 30°C and 180 rpm for 48 h. Yeast cells were checked for intracellular lipid bodies indicative of lipid accumulation by Nile Red (NR) fluorescence staining (Kimura et al., 2004). Microscopy was performed with a Nikon Eclipse 80i light microscope, equipped with a digital camera using a 465–495 nm excitation filter, a 505 nm diachronic mirror, and a 515–535 nm barrier filter.

2.2 DNA extraction

Genomic DNA of T. fermentans CICC1368 was extracted using a Yeast DNA Kit (Omega) according to the manufacturer’s instructions. The quality and quantity of extracted DNA were measured using a Qubit 2.0 fluorometer (Life Technologies).

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The extracted DNA (≥500 ng/µL) was used for whole genome sequencing and PCR verification. All DNA samples were stored in distilled H2O at –20°C.

2.3 Genome sequencing and assembly

The genome of T. fermentans was sequenced on a HiSeq™ 2000 system (Illumina, CA, USA) at BIOMARKER (Beijing, China). Assembly was performed using SOAPdenovo software (Li et al., 2009).

2.4 Gene prediction and annotation

In order to reach high accuracy, the gene structures of T. fermentans were predicted using the EVidenceModeler (EVM) algorithm. PseudoPipe was selected with default settings for pseudogene identification. Predicted genes were annotated by BLAST searches against protein databases with an E-value of 1E-5 as follows: NR (www.ncbi.nlm.nih.gov), KOGs and COGs (Tatusov et al., 2003), GO (Ashburner et al., 2000), KEGG (Kanehisa et al., 2004), and Swiss-Prot (Wu et al., 2006), and gapped BLAST and PSI-BLAST (Altschul et al., 1997) were used for gene function annotation. Pathway mapping was conducted by associating EC assignment and KO assignment with KEGG metabolic pathways based on BLAST search results.

2.5 Orthology and phylogenomic analysis

Predicted proteins of T. fermentans were compared with the predicted proteins of

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nine other sequenced fungi. A comparison of all proteins was performed by BLASTP search against all other proteins in these genomes. Sequences with E-values of 1E-5 or less and at least 40% sequence identity over 60% were considered homologous sequences. In total, 1,441 orthologs were obtained and aligned with MAFFT (Katoh and Standley, 2013). Amino acid sequences were input into the RAxML program to create a maximum likelihood tree. The divergence time between the compared species was estimated by the PL method with r8s version 1.8 (http://loco.biosci.arizona.edu/r8s/) using the calibration against the origin of Ascomycota at 500–600 million years ago (Lucking et al., 2009). Multigene family analysis was performed for predicted genes in the T. fermentans genome and other sequenced fungal genomes using OrthoMCL (Li et al., 2003) with an E-value of 1E20.

2.6 Protein family classification and evolution analysis

Protein families were classified using InterproScan analysis in order to identify genes that descended from a common ancestor (Li et al., 2006). Putative enzymes involved in carbohydrate utilization were identified by BLAST search against the Carbohydrate-Active enZYmes database (http://www.cazy.org/). Protease protein families were classified by BLAST search against the MEROPS database. Additionally, G-protein coupled receptors, protein kinases, and transcription factors

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were identified using database for sequences of GPCDB 7 transmembrane helices (http://www.cbs.dtu.dk/services/TMHMM/) as well as KinBase (http://kinase.com/) and the Fungal Transcription Database (http://ftfd.snu.ac.kr/).

3. Results and discussion

3.1. Lipid accumulation by T. fermentans during fermentation

Nile red (NR) is highly fluorescent in hydrophobic organic solvents and when it comes in contact with lipid bodies (Krishnamoorthy and Ira, 2001). It has therefore been utilized to monitor the accumulation of neutral lipids in the form of lipid globules in oleaginous unicellular algae (Cooksey et al., 1987), yeasts and fungi (Kimura et al., 2004). In this study, a various oval and ellipsoidal lipid bodies were observed in yeast cells using NR staining and fluorescence microscopy (Fig. 1), demonstrating that T. fermentans could accumulate lipid substantially. According to Xu et al., R. toruloides and Y. lipolytica are high-lipid yeasts, which can both achieve lipid contents over 50% under certain cicrumstances (Xu et al., 2013). The lipid accumulation capacity of T. fermentans was also confirmed lately. Zhu (Zhu et al., 2008) reported that addition of various sugars to pretreated molasses efficiently enhanced lipid accumulation in T. fermentans by as much as 50%. The high lipid accumulation capacity of T. fermentans was also tested in our laboratory, and we found that this organism could ferment on glycerol medium. Furthermore, addition of sweet potato vine hydrolysate led to a 4.34-fold increase in the lipid yield from T. 7

fermentans on glycerol medium (Shen et al., 2013). In summary, T. fermentans was found to be a high-lipid-value yeast, showing high SCOs productivity with respect to its biomass, and sequencing of this robust producer of SCOs would be highly beneficial.

3.2. General features of the genome

T. fermentans is attractive owing to its capacity to produce abundant amounts of lipids using biomass hydrolysates and its high tolerance to inhibitory compounds. To date, over 100 fungal genomes have been sequenced and are publicly available. However, T. fermentans has not been extensively investigated genetically or genomically. In this study, the genome of T. fermentans was sequenced using an Illumina sequencer platform. The sequence was deposited in public genome databases (DDBJ/EMBL/GenBank accession LFTX00000000, first version LFTX01000000). The assembly contained a total contig length of 31.3 Mb with a GC content of 57.97% (Table 1). Annotation of the assembled genome sequence generated 12,702 gene models with an average transcript length of 1.8 kb. The gene-coding regions were quite dense, with an average of 2.7 kb per gene. Approximately 16% of the predicted genes encoded proteins has no homologs in the NR protein databases. Among the genes with homologs in other organisms, 5,649 genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Table 1). The 12,702 predicted genes could be classified into 25 functional categories, and approximately

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4% of the predicted genes belonged to the lipid transport and metabolism category (Fig. 2).

3.3. Phylogenetic relationships in oleaginous microbes

Many oleaginous microbes have been identified in genera such as Nannochloropsis, Rhodococcus, Yarrowia, Mucor, and Aspergillus, and the lipid contents of these organisms are all over 20%, with some as high as 50% (Beopoulos et al., 2011; Li et al., 2011). To determine the possible evolutional relationship between oleaginous fungi (Rhodosporidium toruloides NP11, Yarrowia lipolytica CLIB122, Rhodotorula glutinis ATCC 204091, Rhodococcus opacus PD630, Mucor circinelloides CBS277.49, Aspergillus oryzae RIB40, Phaeodactylum tricornutum CCAP 1055/1 and Nannochloropsis gaditana CCMP526), orthologous proteins were identified among nine genera and used for phylogenetic analysis with phylogenetic estimation using maximum likelihood (PhyML) in this study. The phylogeny segregated fungal species of Ascomycota, Mucoromycotina, and Basidiomycota. Rhodococcus opacus is a bacterium species in the genus and is moderately chemolithotrophic. Furthermore, nine oleaginous microbes were placed into five clusters (Fig. 3A). Three Basidiomycota genera were placed in a genus, and Y. lipolytica and A. oryzae were also grouped. M. circinelloides was a singleton in a separated branch. Our yeast showed a closer evolutionary relationship with Y.

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lipolytica and A. oryzae than with the other species, particularly Rhodosporidium toruloides and Rhodotorula glutinis. The above results suggested that oleaginous microbes may have acquired their lipogenic capacity during evolution before the divergence of Ascomycota, Bacterium, Basidiomycota, Heterokontophyta, and Mucoromycota. Then, we analyzed the distribution of genes among the nine microbes. As shown in Fig. 3B, the total gene number of T. fermentans was the highest among the microorganisms examined in this analysis; this was also true for the numbers of special genes. Thus, we concluded that the wide substrate spectrum and tolerance to inhibitory compounds observed in T. fermentans may due to its genetic diversity.

3.4. Comparative analysis of T. fermentans with two other yeasts exhibiting high lipid yields

As shown in Table 2, T. fermentans had a larger genome size than the oleaginous yeasts Rhodosporidium toruloides NP11 and Y. lipolytica CLIB122. The GC content of T. fermentans was 57.97%, which was between the values reported for R. toruloides NP11 and Y. lipolytica CLIB122 as traditional fungi. The genome was predicted to have 12,702 protein-coding genes, a value that was higher than those found for R. toruloides NP11 (8,171) and Y. lipolytica CLIB122 (7,357), as shown in Table 2. Then, we analyzed the distribution of genes among the three fungi. As shown in Fig. 4A, the total gene number of T. fermentans was higher than those of the other

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two yeasts; this trend was also observed for the number of special genes, multicopy genes, and unclustered genes. The number of single-copy genes in the T. fermentans genome was less than those in the R. toruloides and Y. lipolytica genomes, while the number of other genes was similar to that R. toruloides but slightly higher than that of Y. lipolytica (Frankham, 2005). These results reconfirmed the genetic diversity of T. fermentans and provided genetic information supporting the wide substrate spectrum of this organism and its tolerance to inhibitory compounds. This analysis also confirmed the close evolutionary proximity among yeasts exhibiting high lipid production (Fig. 4B) and yielded 1,713 genes that were exclusive to T. fermentans, which corresponded to 36% of the total gene repertoire in T. fermentans; this was higher than the fraction of unique genes in R. toruloides NP11 and Y. lipolytica CLIB122. Of all predicted genes, 84% of the translated proteins (representing 10,695 genes) showed sequence similarity to proteins in known databases, and 37% (4,719 genes) could be annotated into different functional categories by gene ontology (GO; Fig. 5A). Compared with the other two oleaginous yeasts, T. fermentans encoded a larger number of proteins involved in fatty acid elongation and degradation, as shown by KEGG pathway analysis (Fig. 5B). These results may explain the high lipid productivity and lipid profiles of T. fermentans observed in our assays (Shen et al., 2013; Zhan et al., 2013).

3.5. Evolutionary analysis of proteins associated with lipid accumulation 11

In the biochemical process of lipid production in microbes, the channeling of carbon flux to lipid biosynthesis by nutrient imbalance or using nitrogen-limited conditions has been reported (Ratledge and Wynn, 2002).This process accomplished by the cooperative function of key enzymes, including malic enzyme (ME), ATPcitrate lyase (ACL), acetyl-CoA carboxylase (ACC), and fatty acid synthetase (FAS), which generate the precursor pools required for fatty acid synthesis. ME is an enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 and produces NAD(P)H. The universal presence of the ME gene in organisms confirms the importance of this enzyme in cell metabolism-related processes, including anaerobic growth (Boles et al., 1998; Li et al., 2005; Encheva et al., 2009), acetate utilization (McCullough and Roberts, 1974), salt tolerance (Liu et al., 2007), and lipid biosynthesis (Frenkel, 1975; Hodnett et al., 1996; Wynn et al., 1997). ME consists of three types: NAD+-dependent, NAD(P) +-dependent, and NADP+-dependent. Usually, ME is NADP+-specific in fungi, and both NAD+- and NADP+-dependent ME forms are found in bacteria. In oleaginous microorganisms, NADP+-dependent ME plays a key role in lipid production (Wynn et al., 1999). A total of 10 genes encoding ME were found in the T. fermentans genome; this was more than those in the other genomes examined (Fig. 6A) except for the A. oryzae and R. opacus genomes. In most microorganisms, the key enzymes in the elongation of fatty acids is ACC and FAS. ACC catalyzes acetyl-CoA to malonyl-CoA. Heterologous expression of

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ACC from the oleaginous fungus Mucor rouxii in the non-oleaginous yeast Hansenula polymorpha has been shown to achieve only a 40% increase in total fatty acid content (Ruenwai et al., 2009). In addition, the expression of ACC in A. oryza also did not result in a significant increase in fatty acid production relative to the parental strain (An and Oh, 2013). In eukaryotes, the complex regulation of ACC may restrict improvements in lipid production. However, Y. lipolytica is an exception in eukaryotic microbes, as Tai et al. (Tai and Stephanopoulos, 2013) reported that overexpression of ACC results in a 2-fold increase in lipid content in this organism. In the T. fermentans genome, there were two genes encoding the ACC enzyme; in contrast, the other genomes only had zero or one genes encoding this enzyme (Fig. 6B). These ACC genes in T. fermentans had high amino acid similarity with their homologs in R. toruloides, Y. lipolytica, A. oryzae, and M. circinelloides genomes. FASs are typically classified into two variants, the dissociated type II system and the integrated type I multi-enzyme. In fungi, FASs are usually composed of eight distinct domains and organized into two subunits or one polypeptide (Jenni et al., 2007). Liang et al. (Liang and Jiang, 2013) speculated that the subunits of FAS are challenging targets for metabolic engineering for enhancement of fatty acid metabolism, owing to the nature of FAS as a multi-enzymatic complex that contains subunits whose activities depend on one another. In this study, eight genes were found to encode FAS (Fig. 6C); this was more than the numbers of FAS-encoding genes in other oleaginous microorganism genomes examined in this study. Notably, these

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genes showed low amino acid similarity with their homologs. ACL catalyzes the conversion of citrate to acetyl-CoA and oxaloacetate and is a key enzyme in lipid accumulation in mammals and oleaginous fungi. Dramatic increases in ACL-enhanced production of fatty acids (1.7-fold) and TAG (1.9-fold) were observed in A. oryzae compared with those of the parental strain (Tamano et al., 2013). In T. fermentans, eight genes were found to encode ACL (Fig. 6D), similar to the results in A. oryzae and R. opacus genomes, i.e., six and seven genes, respectively. Genes in the T. fermentans genome shared low amino acid similarity with their homologs in the other genomes. In summary, all four key enzymes related to SCO production were present in multiple copies in the T. fermentans genome. Specifically, we found 10 genes encoding ME and eight genes coding FAS. Combined with the high lipid productivity of T. fermentans, we concluded that lipid biosynthesis did not depend on one ratelimiting enzyme, such as ACC, but rather was controlled by a number of component enzymes of the fatty acid biosynthetic machinery.

4. Conclusions

This study firstly reported the genomic analyses of T. fermentans, one of the most widely used oleaginous yeasts. We developed the fundamental genomic and molecular resources for characterization of T. fermentans and performed comparative analyses that revealed the relationship between T. fermentans and other oleaginous

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microbes. The current phylogenic results suggested that the evolutionary history of lipid metabolic genes was largely dominated by vertical inheritance. Based on the existence of a large repertoire of genes encoding key enzymes in fatty acid production, we concluded that T. fermentans should have the potential for high, efficient lipid production through genetic engineering.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (41271335; 31470191), the Major State Basic Research Development Program of China (973 Program) (2015CB150502).

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Figure Legends

Figure 1 The morphology of T. fermentans during cell growth. (A) Microscopic image of yeast under optical microscopy. (B) Microscopic image of Nile red staining of yeast under blue light (wavelength 420–490 nm).

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Figure 2 Distribution of predicted T. fermentans proteins among functional groups. The predicted proteins were categorized into four main categories: cellular process and signaling (blue), information storage and processing (red), metabolism (yellow) and poorly characterized (green). X-axis indicates the number of genes in a specific functional cluster. The right-side legend shows a description of the 24 functional categories.

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Figure 3 Phylogenetic analysis and comparison of T. fermentans and eight other oleaginous microorganisms. (A) Phylogenetic analysis of T. fermentans and other oleaginous microorganisms. Cladogram based on unique genes in the genomes. (B) Comparison of the numbers of different types of genes in T. fermentans and other oleaginous microbes.

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Figure 4 Comparisons of T. fermentans, R. toruloides NP11, and Y. lipolytica CLIB122. (A) Comparison of the numbers of different types of genes in T. fermentans, R. toruloides NP11, and Y. lipolytica CLIB122. (B) Venn diagram representation of shared/unique genes in T. fermentans and comparison with those in R. toruloides NP11 and Y. lipolytica CLIB122.

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Figure 5 Comparisons of genes and proteins in T. fermentans, R. toruloides NP11, and Y. lipolytica CLIB122. (A) Comparison of the numbers of GO terms in T. fermentans, R. toruloides NP11, and Y. lipolytica CLIB122 genomes in the various GO categories. Each circle represents the relative fraction of genes represented in each of the categories for each genome. The gene numbers are also shown. (B) Comparison of proteins involved in pathways related to fatty acid metabolism in T. fermentans, R. toruloides NP11, and Y. lipolytica CLIB122 by KEGG pathway analysis.

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Figure 6 Unrooted phylogenetic trees showing differences in gene expansion in oleaginous microbes. (A) ME; (B) ACC; (C) FAS; (D) ACL. (Red circles identify genes that are present only in the T. fermentans genome; orange circles indicate Aspergillus oryzae RIB40; yellow circles indicate Mucor circinelloides CBS277.49; green circles indicate Nannochloropsis gaditana CCMP526; cyan circles indicate haeodactylum tricornutum CCAP 1055/1; blue circles indicate Rhodotorula glutinis ATCC 204091; purple circles indicate Rhodococcus opacus PD630; black circles indicate Rhodosporidium toruloides NP11; brown circles indicate Yarrowia lipolytica CLIB122).

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Table 1 General features of the T. fermentans genome. General features

Contents

Size of assembled genome (Mb)

31.3

Scaffold/contig

3824

Contig N50 (bp)

13698

Contig N90 (bp)

45808

Mean (bp)

8183.7

Max (bp)

85446

G+C content (%)

57.97

Protein-coding gene models

12702

Exons

34116

Introns

22314

Properties of predicted gene models

No. of genes

NR alignment

10695

KEGG alignment

5649

KOG/COG assignment

5737

Swissprot assignment

7018

GO assignment

4719

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Table 2 Comparison of the primary genome features between T. fermentans, Rhodosporidium toruloides NP11, and Y. lipolytica CLIB122. Feature

T. fermentans

Rhodosporidium

Y. lipolytica CLIB122

toruloides NP11 Sequencing platform

Illumina

Illumina GA

-

Size (Mbp)

31.3

20.2

20.55

G+C content (%)

57.97

62

48.98

Protein-coding genes

12702

8171

7357

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