ArticlePDF Available

An effective and rapid method for RNA preparation from non-conventional yeast species


Abstract and Figures

The increased use of high-throughput RNA-based analysis has spurred the demand for rapid and simple preparation of high quality RNA. RNA preparation from non-conventional yeasts having diverse cell wall and morphological characteristics is often inefficient using current methods adapted for the model yeast, Saccharomyces cerevisiae. We report a simple RNA preparation method based on glass bead-mediated breakage in a formamide/EDTA solution. High quality RNA is generated within 15 min from various non-conventional yeasts species. The obtained RNA can be directly used for experimentation without further RNA purification and buffer exchange.
Content may be subject to copyright.
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage:
An eective and rapid method for RNA preparation from non-conventional
yeast species
Dong Wook Lee
, Chang Pyo Hong
, Hyun Ah Kang
Molecular Systems Biology Laboratory, Department of Life Science, Chung-Ang University, Seoul, 06974, South Korea
TheragenEtex Bio Institute, Suwon, 16229, South Korea
RNA extraction
Non-saccharomyces yeasts
Cell wall
The increased use of high-throughput RNA-based analysis has spurred the demand for rapid and simple pre-
paration of high quality RNA. RNA preparation from non-conventional yeasts having diverse cell wall and
morphological characteristics is often inecient using current methods adapted for the model yeast,
Saccharomyces cerevisiae. We report a simple RNA preparation method based on glass bead-mediated breakage in
a formamide/EDTA solution. High quality RNA is generated within 15 min from various non-conventional yeasts
species. The obtained RNA can be directly used for experimentation without further RNA purication and buer
Advanced RNA-based analysis technologies, such as micro-array,
RNA sequencing, and quantitative real-time polymerase chain reaction
(qRT-PCR), require simple and rapid RNA preparation with minimal
exposure to unintended conditions to obtain sucient quantities of
non-degraded, high quality RNA. Saccharomyces cerevisiae has been
used as a model yeast to study many characteristics of eukaryotes [1],
as an industrial microbe for many fermented beverages and bioethanol
production [2], and as the host strain for the production of useful
medicinal recombinant proteins and metabolites [3]. In recent decades,
non-conventional yeasts, or non-Saccharomyces yeasts species, have
drawn increased attention for diverse biotechnological applications [4].
Various non-conventional yeasts species are responsible for avor de-
velopment in fermented foods like traditional alcoholic beverages [5],
and have unique characteristics, such as thermo/osmo-tolerance, that
are suitable for sustainable bioprocesses, such as simultaneous sac-
charication and fermentation for bioethanol production [6]. Several
non-conventional yeasts have been developed as a host system for
production of metabolites and recombinant proteins with industrial
potential [7]. Development of traditional molecular genetic techniques
and more advanced synthetic biology tools in non-conventional yeasts
species is expected to expand and diversify their impact on bio-
technology [8]. For a better understanding of the unique genomic and
functional characteristics of non-conventional yeasts species, whole-
genome sequencing and transcriptome analysis have become essential
to provide comprehensive information on physiological activities and
regulation of gene expression in metabolic pathways with potential
biotechnological importance. Comparative transcriptome proling data
under diverse culture conditions will facilitate the identication of key
targets for metabolic engineering [9].
There are numerous genus and species of yeasts. They have very
dierent morphological characteristics that include capsule structure of
Cryptococcus neoformans [10], multi-polar hyphae of Saccharomycopsis
buligera [11],and pseudo-hyphae of Yarrowia lipolytica [12]. Isolation
of high quality total RNA from non-conventional yeasts with diverse
cell wall structure and morphological characteristics is often inecient
using current methods and RNA extraction kits, which are adapted for
S. cerevisiae. Current methods to obtain RNA from S. cerevisiae include
acid hot-phenol extraction [13], a water bath method [14], the
RNAsnapprotocol [15], and one-step hot formamide extraction [16].
Although RNAs are eciently obtained from S. cerevisiae using those
methods, the preparation of high quality RNA is often challenging in
other yeasts species that have mycelial formation with hyphae, thick
cell wall, and carbohydrate capsule structure. For total RNA extraction
from the multi-polar mycelial yeast S. buligera [11] and the en-
capsulated yeast C. neoformans [17], frozen yeast cells were ground in
liquid nitrogen using a mortar and a pestle before total RNA extraction.
However, grinding requires large amounts of cells and the RNA ob-
tained from the same quantity of yeast cells can vary markedly due to
the dierences of physical force. We recently tried to prepare total RNA
samples from the non-conventional dimorphic pseudo-hyphal forming
yeast Hyphopichia burtonii [18] using several current RNA extraction
methods, including hot-acid phenol, grinding-combined with RNA
Received 7 May 2019; Received in revised form 19 August 2019; Accepted 26 August 2019
Corresponding author.
E-mail address: (H.A. Kang).
Analytical Biochemistry 586 (2019) 113408
Available online 27 August 2019
0003-2697/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
Fig. 1. Yeast RNA extraction method based on
FE/glass bead breakage. (A) Outline of the FE/
glass bead breakage method for small-scale RNA
extraction from yeast cells. (B) Gel analysis of RNA
samples in FE solution. H. burtonii and H. pseudo-
burtonii were cultivated in YPD with 1 M NaCl for
the indicated times (030 min) to impose an osmotic
stress. RNAs were extracted from the yeast cells
with total OD
2.0 and equal volumes of each
samples were separated by 1.2% agarose MOPS/
formaldehyde gel electrophoresis. (C) RNA sample
preparation from various non-conventional yeasts
species. Sf:S. buligera,Yl:Y. lipolytica,Wa:W.
anomalus,Ws:W. subpelliculosus,Op:O. para-
polymorpha, and Ca:C. albicans. Yeast cells were
cultivated in YPD and total RNAs were extracted
(total OD
2.0). (D) Optimization of glass bead
volume. Dh:D. hansenii,Cn:C. neoformans,Cn
Δcap59:C. neoformans acapsulular strain. Dierent
volumes of glass beads (0, 50, 100 μl) were added to
the yeast cells (OD
2.0) suspended in 100 μlFE
solution. RNA samples in FE solution were directly
mixed with 2 x RNA sample loading buer (R1386-
1VL, Sigma-Aldrich) and SafePinky nucleotide
staining solution (S1001-025, GenDEPOT).
Fig. 2. Application of the RNA samples prepared by FE/glass bead breakage to downstream enzymatic reactions. Relative expression levels of H. burtonii and
H. pseudoburtonii genes (ENA5A and ERG11) upon 1 M NaCl treatment, as analyzed by qRT-PCR and RNA-Seq. The expression levels of genes by RNA-Seq were
quantied using TopHat and Cuinks [24] with the value of fragments per kilobase of exon per million fragments mapped (FPKM), and dierential expressions
between control (0 min) and other conditions (5, 15, or 30 min) were analyzed using Cudiwith two replicates with the cutoat p-value < 0.01. For qRT-PCR,
each sample was analyzed in duplicate and normalized by endogenous control genes HbATP4 and HpCYS3, respectively, which showed no apparent expression
changes at the transcript level in the presence of 1 M NaCl. The amplication eciency of the primers of targets and control genes used for qRT-PCR was validated
very similar by analyzingC
values with serially diluted cDNA.
D.W. Lee, et al. Analytical Biochemistry 586 (2019) 113408
isolation kit (Qiagen RNeasy Mini Kit, 74104), and one-step hot for-
mamide extraction. We failed to obtain high quality RNA (see Sup-
porting Fig. S1 A, B, and C in the supplementary material).
Here, we report a simple RNA extraction method based on glass
bead-mediated breakage in formamide/ethylenediaminetetraacetic
acid (EDTA) solution (FE) at room temperature (RT) (Fig. 1A). The
procedure is performed in microcentrifuge tubes and takes only 15 min.
1. Protocol
For RNA sample preparation, yeast cells are cultivated overnight in
23 ml of yeast extract peptone dextrose (YPD) medium (1% yeast ex-
tract, 2% peptone and 2% glucose) at 28 °C with shaking at 220 rpm.
The pre-cultured yeast cells are inoculated in fresh YPD medium at an
optical density at 600 nm (OD
) of 0.30.6, grown to an OD
approximately 1.02.0, which is nearly the early phase of exponential
Yeast cells (total 2 OD
unit) are transferred to 1.7 ml micro-
centrifuge tubes and harvested (13,000 rpm, 1 min, RT) using a tabletop
microcentrifuge. The supernatant is completely removed by pipetting
(an important step, since residual can aect the eciency and quality
of the RNA extract) and the cells are washed by resuspension in 1 ml of
distilled water (DW) and centrifugation. The washed cells can be stored
at 80 °C after freezing with liquid nitrogen. When required, the cell
pellet is suspended in 100 μl of FE (98% formamide, 0.01 M EDTA),
prepared by mixing formamide (99.5%, F9037; Sigma-Aldrich) and
EDTA (0.5 M, pH 8.0, ML005-01; WelGENE). For optimal results, the FE
solution volume is increased in proportion to the volume of the cell
pellet (50 μl per 1.0 OD
of cell suspension). A 50 μl volume of RNase-
free glass beads (500 μm, GB05-RNA; Nextadvance) is added and the
cell suspension is ground by vortexing for three cycles of 30 s (total
90 s) at 6500 rpm at RT using a Pre-cellys 24 homogenizer (Bertin
Technologies). If a dierent homogenizer is used, such as an MT-360
TOMY (Seiko Corporation), the vortex time may need to be optimized
(see Supporting Fig. S1D,Fig. S1E, and Fig. S2). The optimal vortexing
time was dened as the shortest time generating the maximum quantity
of RNA with the 28S:18S ratio above 1.8, which was chosen as 90 s with
Pre-cellys 24 homogenizer in our study. The homogenized sample is
centrifuged at 13,000 rpm for 1 min at RT. The supernatant is trans-
ferred to a new 1.7 ml microcentrifuge tube. The extracted but un-
puried RNA in this FE solution can be directly assessed by RNA gel
electrophoresis in 10% 3-morpholinopropane-1-sulfonic acid (MOPS)/
0.75% formaldehyde running buer. This RNA can also be directly used
for sequential DNase treatment or cDNA synthesis reaction. To avoid
interference of the enzyme reaction by formamide, it is critical to dilute
the RNA samples in FE solution to less than 5% of the total solution
[19]. If the RNA concentration in FE solution is higher than 100 ng/μl,
the RNA sample can be directly used for RNA-Seq analysis without any
further treatment. But, when the RNA concentration is not high enough,
ethanol precipitation is recommended to concentrate RNA. As an op-
tion, the RNA in FE solution can be diluted in diethyl pyrocarbonate
(DEPC)-treated water (C-9030; Bioneer). The sample can be stored at
20 °C or 80 °C at this step.
The method yielded high quality RNA samples from the mycelial
yeast H. burtonii KJJ43 and H. pseudoburtonii KJS14 isolated from
Korean Nuruk fermentation starter (Supporting Table S1). The RNA
samples in FE solution were directly subjected to RNA formaldehyde-
agarose gel electrophoresis, which revealed non-degraded large rRNA
and low molecular weight tRNA and 5S rRNA (Fig. 1B). We further
applied this method to the preparation of RNA samples from various
non-conventional yeasts species, including S. buligera KJJ81, Y. lipo-
lytica PolF, Wickerhamomyces anomalus Y4, Wickerhamomyces sub-
pelliculosus SMY-04, Ogataea parapolymorpha DL1-L, Candida albicans
ATCC32354, Debaryomyces hansenii KD-2, Cryptococcus neoformans H99,
and C. neoformans acapsular mutant Δcap59 (Fig. 1C and D).In the case
of D. hansenii, which forms biolms [20], and C. neoformans, which
possesses a thick extracellular capsule, we tested dierent volumes of
glass beads. The use of 50 μl was generally appropriate for the pre-
paration of RNAs from the most yeasts species tested in this study. An
increased amount of glass beads could produce more ecient cell lysis,
but could result in more degradation of RNA. Compared to the wild
type C. neoformans strain, RNA was extracted more eciently from the
acapsular strain C. neoformans Δcap59, indicating that the capsule
structure might lessen yeast cell breakage by glass beads.
The quality of the RNA samples of H. burtonii and H. pseudoburtonii
cultivated with 1 M NaCl to impose osmotic stress was further assessed
by several quality control analyses after ethanol precipitation pur-
ication of the RNA samples in FE solution. The concentration and RNA
integrity number (RIN) of RNA samples prepared from small-scale yeast
cell suspensions (total OD
2.0) were sucient for RNA-Seq analysis
(Supporting Tables S2A and B). In the case of H. burtonii, the nal yields
of total RNA from the same total cell OD were variable among the
samples, due to the physiological change of this hyphae forming yeast
under salt stress conditions. Despite such dierences in nal yields
among the RNA samples from H. burtonii cells cultivated under dierent
osmotic conditions, such as cultivation in the presence of 1 M NaCl, 1 M
KCl, and 1 M sorbitol, the quality of RNA samples prepared by FE/glass
bead breakage method was mostly good enough for RNA-Seq analysis
with high ratio of 28S/18S and RIN values (Supporting Table S3). For
RNA-Seq analysis, the qualied RNA (1 μg) in each sample was sub-
jected to poly(A) mRNA enrichment by using magnetic beads with oligo
(dT) and then sheared into short fragments. Using reverse transcriptase
and random hexamer primers, the rst strand cDNA of mRNA frag-
ments was synthesized, and the second strand cDNA was then synthe-
sized using DNA Polymerase I and RNase H. The synthesized cDNA was
subjected to end-repair and poly(A) tailing and connected with se-
quencing adapters using a TruSeq Stranded mRNA Sample Prep Kit
(Illumina). The proper cDNA fragments, puried by a BluePippin in-
strument (Sage Science), according to the manufacturer's instructions,
were selected for further PCR amplication. Subsequently, the libraries
were subjected to paired-end sequencing with a 100 bp read length
using an Illumina HiSeq 2500 platform. The feasibility of the RNA
samples of H. burtonii and H. pseudoburtonii for polymerase chain re-
action (PCR) analysis was assessed. The genes chosen for PCR were H.
burtonii ATP4 (HbATP4),encoding a mitochondrial ATP synthase sub-
unit, and H. pseudoburtonii CYS3 (HpCYS3),which codes for cy-
stathionine gamma-lyase in the cysteine assimilation pathway. These
genes were chosen because they were shown to be expressed con-
stitutively regardless of salt stress conditions in our RNA-Seq data (GEO
submission number GSE130141). The sequences of PCR primers are
presented in Supporting Table S4. When we conducted PCR with the
primer sets of HbATP4 and HpCYS3 using the RNA samples in FE so-
lution as templates, we did not observe any DNA bands amplied by
PCR before cDNA synthesis (Supporting Figs. S3A and B). The absence
of amplication of PCR products from the RNA samples without DNase
treatment indicated the lack of contamination by chromosomal DNA of
the RNA prepared by the FE/glass bead breakage method. For cDNA
synthesis, the RNA samples in FE solution were used directly without
buer exchange and DNase treatment. After cDNA synthesis, we con-
rmed the amplication of HbATP4 and HpCYS3 DNA fragments by
PCR from the synthesized cDNAs (Supporting Figs. S3C and D).
To examine the consistency of expression patterns of H. burtonii and
H. pseudoburtonii genes between RNA-Seq data and other RNA analysis
data, we carried out qRT-PCR analysis with the gene specic primers
for ENA5A (encoding an ATPase sodium pump) and ERG11 (encoding
lanosterol 14-alpha-demethylase in the ergosterol biosynthesis
pathway) of H. burtonii and H. pseudoburtonii, using the synthesized
cDNAs as templates (Fig. 2).The activity of the sodium pump family is
important for osmotolerance [21], and the expression levels of genes
involved in the ergosterol pathway are decreased by osmotic stress in S.
cerevisiae [22]. Our RNA-Seq data revealed the increased expression of
ENA5A and the decreased expression of ERG11 in both H. burtonii and
D.W. Lee, et al. Analytical Biochemistry 586 (2019) 113408
H. pseudoburtonii, as expected. For cDNA synthesis, 1 μl of RNA in FE
buer was diluted with 15 μl of DEPC-treated water, mixed with 4 μlof
SuperiorScript III Master Mix (Enzynomics) for cDNA synthesis reaction
at 42 °C for 45 min. The concentration of synthesized cDNA was ad-
justed to have Cq values within the range of 2035 in qRT-PCR, which
was carried out with CFX96 Optical Module (Biorad) using TB Green
Premix Ex Taq (Takara). Gene expression values were calculated by the
method [23] using HpCYS3 and HbATP4 as internal control,
respectively, for H. burtonii and H. pseudoburtonii genes in duplicated
experiments. The relative expression patterns of ENA5A and ERG11
detected by qRT-PCR were consistent with those based on RNA-Seq
data, although the fold change values were generally lower in the qRT-
PCR data compared to those in the RNA-Seq data (Fig. 2A and B), in-
dicating that this method ensures good quality of RNA samples, which
is important for experimentally reliable and repeatable results.
The RNA preparation method presented in this paper is a rapid
(within 15 min) and simple procedure that can be done at RT.
Compared to the widely used hot-acid phenol method and the recently
developed one-step hot formamide extraction method, this method does
not employ hot temperature and takes much less time to obtain high
quality RNA samples (Supporting Fig. S1F). With this method, a very
small amount of cell mass (OD
2.0) is sucient to generate RNA
samples required for downstream applications, such as RNA-Seq and
qRT-PCR analysis. The method can be easily adapted for high-
throughput analysis of multiple RNA samples generated from diverse
yeasts species. With the increasing attention on non-conventional yeasts
species in both academic and industrial sectors, we anticipate that our
RNA isolation method will be useful for diverse RNA-based analyses for
a variety of yeasts species having diverse morphologies and cell wall
This work was supported by the National Research Foundation of
Korea, Grant No. NRF-2017M3C1B5019295 (STEAM Research Project)
and by the Korean Ministry of Agriculture, Food, and Rural Aairs,
Grant No. 918010042HD030 (Strategic Initiative for Microbiomes in
Agriculture and Food).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
[1] T. Bilinski, A. Bylak, R. Zadrag-Tecza, The budding yeast Saccharomyces cerevisiae as
a model organism: possible implications for gerontological studies, Biogerontology
18 (2017) 631640.
[2] D.Q. Zheng, P.M. Wang, J. Chen, K. Zhang, T.Z. Liu, X.C. Wu, Y.D. Li, Y.H. Zhao,
Genome sequencing and genetic breeding of a bioethanol Saccharomyces cerevisiae
strain YJS329, BMC Genomics 13 (2012) 479491.
[3] H. Kim, S.J. Yoo, H.A. Kang, Yeast synthetic biology for the production of re-
combinant therapeutic proteins, FEMS Yeast Res. 15 (2015) 116.
[4] R. Riley, S. Haridas, K.H. Wolfe, M.R. Lopes, C.T. Hittinger, et al., Comparative
genomics of biotechnologically important yeasts, Proc. Natl. Acad. Sci. U.S.A. 113
(2016) 98829887.
[5] A. Gamero, R. Quintilla, M. Groenewald, W. Alkema, T. Boekhout, L. Hazelwood,
High-throughput screening of a large collection of non-conventional yeasts reveals
their potential for aroma formation in food fermentation, Food Microbiol. 60 (2016)
[6] S. Rebello, A. Abraham, A. Madhavan, R. Sindhu, P. Binod, K.B. Arun, E.M. Aneesh,
A. Pandey, Non-conventional yeast cell factories for sustainable bioprocesses, FEMS
(Fed. Eur. Microbiol. Soc.) Microbiol. Lett. 365 (2018) 110.
[7] V.T. Luu, H.Y. Moon, J.Y. Hwang, B.K. Kang, H.A. Kang, Development of re-
combinant Yarrowia lipolytica producing virus-like particles of a sh nervous ne-
crosis virus, J. Microbiol. 55 (2017) 655664.
[8] J.M. Wanger, H.S. Alper, Synthetic biology and molecular genetics in non-con-
ventional yeasts: current tools and future advances, Fungal Genet. Biol. 89 (2016)
[9] M. Bellasio, A. Peymann, M.G. Steiger, M. Valli, M. Sipiczki, M. Sauer, A.B. Graf,
H. Marx, D. Mattanovich, Complete genome sequence and transcriptome regulation
of the pentose utilizing yeast Sugiyamaella lignohabitans, FEMS Yeast Res. 16 (2016)
[10] J.R. Perfect, Cryptococcus neoformans: a sugar-coated killer with designer genes,
FEMS Immunol. Med. Microbiol. 45 (2005) 395404.
[11] J.H. Choo, C.P. Hong, J.Y. Lim, J.A. Seo, Y.S. Kim, D.W. Lee, S.G. Park, G.W. Lee,
E. Carroll, Y.W. Lee, H.A. Kang, Whole-genome de novo sequencing, combined with
RNA-Seq analysis, reveals unique genome and physiological features of the amy-
lolytic yeast Saccharomycopsis buligera and its interspecies hybrid, Biotechnol.
Biofuels 9 (2016) 246268.
[12] J.C. Torres-Guzmán, A. Domínguez, HOY1, a homeo gene required for hyphal for-
mation in Yarrowia lipolytica, Molecular Cell Biology 17 (1997) 62836293.
[13] M.E. Schmitt, T.A. Brown, B.L. Trumpower, A rapid and simple method for pre-
paration of RNA from Saccharomyces cerevisiae, Nucleic Acids Res. 18 (1990)
[14] J. Li, J. Liu, X. Wang, L. Zhao, Q. Chen, W. Zhao, A waterbath method for pre-
paration of RNA from Saccharomyces cerevisiae, Anal. Biochem. 384 (2009)
[15] M.B. Stead, A. Agrawal, K.E. Bowden, R. Nasir, B.K. Mohanty, R.B. Meagher,
S.R. Kushner, RNAsnap: a rapid, quantitative and inexpensive, method for isolating
total RNA from bacteria, Nucleic Acids Res. 40 (2012) 19.
[16] D. Shedlovskiy, N. Shcherbik, D.G. Pestov, One-step hot formamide extraction of
RNA from Saccharomyces cerevisiae, RNA Biol. 12 (2016) 17221726.
[17] S.A. Cheon, E.J. Thak, Y.S. Bahn, H.A. Kang, A novel bZIP protein, Gsb1, is required
for oxidative stress response, mating, and virulence in the human pathogen
Cryptococcus neoformans, Sci. Rep. 7 (2017) 115.
[18] M. Groenewald, M.T. Smith, Re-examination of strains formerly assigned to
Hyphopichia burtonii, the phylogeny of the genus Hyphopichia, and the description of
Hyphopichia pseudoburtonii sp. nov, Int. J. Syst. Evol. Microbiol. 60 (2010)
[19] P. Chomczynski, Solubilization in formamide protects RNA from degradation,
Nucleic Acids Res. 20 (1992) 37913792.
[20] F. Zhang, Y. Tang, Y. Ren, K. Yao, Q. He, Y. Wan, Y. Chi, Microbial composition of
spoiled industrial-scale Sichuan paocai and characteristics of the microorganisms
responsible for paocai spoilage, Int. J. Food Microbiol. 275 (2018) 3238.
[21] M. Rep, M. Krantz, J.M. Thevelein, S. Hohmann, The transcriptional response of
Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required
for the induction of subsets of high osmolarity glycerol pathway-dependent genes,
J. Biol. Chem. 275 (2000) 82908300.
[22] F.M. Montanes, A.P. Ahuir, M. Proft, Repression of ergosterol biosynthesis is es-
sential for stress resistance and is mediated by the Hog1 MAP kinase and the Mot3
and Rox1 transcription factors, Mol. Microbiol. 79 (2011) 10081023.
[23] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using
RealTime quantitative PCR and the 2
method, Methods 25 (2001) 402408.
[24] C. Tranpnell, A. Roberts, L. Go, G. Pertea, D. Kim, et al., Dierential gene and
transcript expression analysis of RNA-seq experiments with TopHat and Cuinks,
Nat. Protoc. 7 (2012) 562578.
D.W. Lee, et al. Analytical Biochemistry 586 (2019) 113408
... A linearized vector reference sequence of pTO128 (pPZP-Natca) was generated from the circular vector sequence and 150 bp of sequence from the opposite border was added to each border of the linearized sequence. Reads were mapped to the linear pTO128 (pPZP-NATca) reference sequence using the Burrows-Wheeler Aligner with maximum exact matches (BWA-MEM) 64 configured with default parameters except for minimum seed length = 50 and band width = 2. Mapped reads were visualized using IGV 62 and sorted based on position and sequences that extended beyond the left and right boundaries of the tDNA was extracted. Consensus sequences of the extracted reads were mapped to the C. auris B8441 reference genome (GCA_002759435.2) using NCBI Blast. ...
... RNA extraction. RNA extraction was performed as described previously 64 . Briefly, cells were grown to mid-exponential phase at 30°C in YPD and harvested by centrifugation. ...
Full-text available
Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome. Some isolates of the emerging fungal pathogen Candida auris can form cellular aggregates or filaments. Here, Santana and O’Meara use Agrobacterium-mediated transformation and a CRISPR-Cas9 system to identify several genes that regulate C. auris morphogenesis.
Full-text available
Candida albicans is a frequent colonizer of human mucosal surfaces as well as an opportunistic pathogen. C. albicans is remarkably versatile in its ability to colonize diverse host sites with differences in oxygen and nutrient availability, pH, immune responses, and resident microbes, among other cues. It is unclear how the genetic background of a commensal colonizing population can influence the shift to pathogenicity. Therefore, we undertook an examination of commensal isolates from healthy donors with a goal of identifying site-specific phenotypic adaptation and genetic variation associated with these phenotypes. We demonstrate that healthy people are reservoirs for genotypically and phenotypically diverse C. albicans strains, and that this genetic diversity includes both SNVs and structural rearrangements. Using limited diversity exploitation, we identified a single nucleotide change in the uncharacterized ZMS1 transcription factor that was sufficient to drive hyper invasion into agar. However, our commensal strains retained the capacity to cause disease in systemic models of infection, including outcompeting the SC5314 reference strain during systemic competition assays. This study provides a global view of commensal strain variation and within-host strain diversity of C. albicans and suggests that selection for commensalism in humans does not result in a fitness cost for invasive disease.
The yeast species Hyphopichia is common in nature and strongly competitive under harsh environmental conditions. Here, we characterized Hyphopichia burtonii KJJ43 and H. pseudoburtonii KJS14, which exhibit strong halotolerance, using genomic and transcriptomic analyses. The genomes of H. burtonii and H. pseudoburtonii comprised eight chromosomes with 85.17% nucleotide identity and significant divergence in synteny. Notably, both Hyphopichia genomes possessed extended gene families of amino acid permeases and ATP‐binding cassette (ABC) transporters, whose dynamic expression patterns during osmotic stress were revealed using transcriptome profiling. Intriguingly, we found unique features of the HOG pathway activated by Hog1p even under non‐osmotic stress conditions and the upregulation of cytosolic Gpd1p protein during osmotic stress. Associated with hyperfilamentation growth under high osmotic conditions, a set of genes in the FLO family with induced expression in response to NaCl, KCl, and sorbitol supplementation were identified. Moreover, comparative transcriptome analysis reveals the NaCl‐specific induction of genes involved in amino acid biosynthesis and metabolism, particularly BAT2. This suggests the potential association between oxoacid reaction involving branched‐chain amino acids and osmotolerance. The combined omics analysis of two Hyphopichia species provides insights into the novel mechanisms involved in salt and osmo‐stress tolerance exploited by diverse eukaryotic organisms. This article is protected by copyright. All rights reserved.
Full-text available
Two genes encoding the probable α-L-arabinofuranosidase (EC isozymes with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from the chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, the amylolytic yeast isolated from Korean wheat-based Nuruk, respectively. Each open reading frame consists of 1,551 nucleotides which encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% of amino acid sequence identities with eukaryotic ABFs from the filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45°C and pH 7.0 in sodium phosphate buffer and at 50°C and pH 6.0 in sodium acetate buffer, respectively. These exo-acting enzymes belonging to the Glycoside Hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)- and α-(1,3)-L-arabino furanosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)- and α-(1,3)-double substituted substrates than the single substituted ones. These hydrolases are supposed to play the important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.
Full-text available
The non-conventional yeasts Kluyveromyces lactis, Yarrowia lipolytica, Ogataea polymorpha and Pichia pastoris have been developed as eukaryotic expression hosts because of their desirable growth characteristics, including inhibitor and thermo-tolerance, utilization of diverse carbon substrates, and high amount of extracellular protein secretion. These yeasts already have established in the heterologous production of vaccines, therapeutic proteins, food additives, and bio-renewable chemicals, but recent advances in genetic tool box have the potential to greatly expand and diversify their impact on biotechnology. The diversity of these yeasts includes many strains possessing highly useful, and in some cases even uncommon, metabolic capabilities potentially helpful for bioprocess industry. This review outlines the recent updates of non-conventional yeast in sustainable bioprocesses. Key words: Bioprocess; Non-conventional; Yeast; Recombinant proteins
Full-text available
Current methods for isolating RNA from budding yeast require lengthy and laborious steps such as freezing and heating with phenol, homogenization with glass beads, or enzymatic digestion of the cell wall. Here, extraction with a solution of formamide and EDTA was adapted to isolate RNA from whole yeast cells through a rapid and easily scalable procedure that does not require mechanical cell lysis, phenol, or enzymes. RNA extracted with formamide-EDTA can be directly loaded on gels for electrophoretic analysis without alcohol precipitation. A simplified protocol for downstream DNase treatment and reverse transcription reaction is also included. The formamide-EDTA extraction of yeast RNA is faster, safer, and more economical than conventional methods, outperforms them in terms of total yield, and greatly increases throughput.
Full-text available
The human pathogen Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, normally faces diverse stresses in the human host. Here, we report that a novel, basic, leucine-zipper (bZIP) protein, designated Gsb1 (general stress-related bZIP protein 1), is required for its normal growth and diverse stress responses. C. neoformans gsb1Δ mutants grew slowly even under non-stressed conditions and showed increased sensitivity to high or low temperatures. The hypersensitivity of gsb1Δ to oxidative and nitrosative stresses was reversed by addition of a ROS scavenger. RNA-Seq analysis during normal growth revealed increased expression of a number of genes involved in mitochondrial respiration and cell cycle, but decreased expression of several genes involved in the mating-pheromone-responsive MAPK signaling pathway. Accordingly, gsb1Δ showed defective mating and abnormal cell-cycle progression. Reflecting these pleiotropic phenotypes, gsb1Δ exhibited attenuated virulence in a murine model of cryptococcosis. Moreover, RNA-Seq analysis under oxidative stress revealed that several genes involved in ROS defense, cell-wall remodeling, and protein glycosylation were highly induced in the wild-type strain but not in gsb1Δ. Gsb1 localized exclusively in the nucleus in response to oxidative stress. In conclusion, Gsb1 is a key transcription factor modulating growth, stress responses, differentiation, and virulence in C. neoformans.
Full-text available
Background Genomic studies on fungal species with hydrolytic activity have gained increased attention due to their great biotechnological potential for biomass-based biofuel production. The amylolytic yeast Saccharomycopsis fibuligera has served as a good source of enzymes and genes involved in saccharification. Despite its long history of use in food fermentation and bioethanol production, very little is known about the basic physiology and genomic features of S. fibuligera. ResultsWe performed whole-genome (WG) de novo sequencing and complete assembly of S. fibuligera KJJ81 and KPH12, two isolates from wheat-based Nuruk in Korea. Intriguingly, the KJJ81 genome (~38 Mb) was revealed as a hybrid between the KPH12 genome (~18 Mb) and another unidentified genome sharing 88.1% nucleotide identity with the KPH12 genome. The seven chromosome pairs of KJJ81 subgenomes exhibit highly conserved synteny, indicating a very recent hybridization event. The phylogeny inferred from WG comparisons showed an early divergence of S. fibuligera before the separation of the CTG and Saccharomycetaceae clades in the subphylum Saccharomycotina. Reconstructed carbon and sulfur metabolic pathways, coupled with RNA-Seq analysis, suggested a marginal Crabtree effect under high glucose and activation of sulfur metabolism toward methionine biosynthesis under sulfur limitation in this yeast. Notably, the lack of sulfate assimilation genes in the S. fibuligera genome reflects a unique phenotype for Saccharomycopsis clades as natural sulfur auxotrophs. Extended gene families, including novel genes involved in saccharification and proteolysis, were identified. Moreover, comparative genome analysis of S. fibuligera ATCC 36309, an isolate from chalky rye bread in Germany, revealed that an interchromosomal translocation occurred in the KPH12 genome before the generation of the KJJ81 hybrid genome. Conclusions The completely sequenced S. fibuligera genome with high-quality annotation and RNA-Seq analysis establishes an important foundation for functional inference of S. fibuligera in the degradation of fermentation mash. The gene inventory facilitates the discovery of new genes applicable to the production of novel valuable enzymes and chemicals. Moreover, as the first gapless genome assembly in the genus Saccharomycopsis including members with desirable traits for bioconversion, the unique genomic features of S. fibuligera and its hybrid will provide in-depth insights into fungal genome dynamics as evolutionary adaptation.
Full-text available
Significance The highly diverse Ascomycete yeasts have enormous biotechnological potential. Collectively, these yeasts convert a broad range of substrates into useful compounds, such as ethanol, lipids, and vitamins, and can grow in extremes of temperature, salinity, and pH. We compared 29 yeast genomes with the goal of correlating genetics to useful traits. In one rare species, we discovered a genetic code that translates CUG codons to alanine rather than canonical leucine. Genome comparison enabled correlation of genes to useful metabolic properties and showed the synteny of the mating-type locus to be conserved over a billion years of evolution. Our study provides a roadmap for future biotechnological exploitations.
The microorganisms of spoiled industrial-scale Sichuan paocai (ISSP) were isolated using six types of media, and 16S rRNA and 26S rRNA gene sequence analyses were used to identify the isolates. Meanwhile, the microbial composition was investigated using a culture-independent method through 16S rRNA and ITS sequencing on an Illumina MiSeq platform. The results obtained by these two methods were compared. Furthermore, characteristics of the isolated microorganisms responsible for ISSP spoilage were studied. Sixty-two strains belonging to twenty-three species, including three ammonia-producing genera, two gas-producing genera, two pectinase-producing genera, two cellulase-producing genera, three film-producing genera and one slime-producing genus, were isolated. Lactobacillus, Bacillus, Debaryomyces and Kazachstania were the dominant genera as confirmed through both culture-dependent and culture-independent methods. Bacillus, Paenibacillus, Pichia and Debaryomyces could be the main microorganisms responsible for ISSP spoilage. Bac. licheniformis was closely correlated with the off-flavour of ISSP. Pae. peoriae, Bac. stratosphericus, Bac. toyonensis and Bac. cereus were responsible for tissue softening, and Bac. subtilis, Bac. methylotrophicus, Pic. membranifaciens and Deb. hansenii caused film formation.
Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.
Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species.
Efficient conversion of hexoses and pentoses into value-added chemicals represents one core step for establishing economically feasible biorefineries from lignocellulosic material. While extensive research efforts have recently provided advances in the overall process performance, the quest for new microbial cell factories and novel enzymes sources is still open. As demonstrated recently the yeast Sugiyamaella lignohabitans (formerly Candida lignohabitans) represents a promising microbial cell factory for the production of organic acids from lignocellulosic hydrolysates. We report here the de novo genome assembly of S. lignohabitans using the Single Molecule Real-Time platform, with gene prediction refined by using RNA-seq. The sequencing revealed a 15.98 Mb genome, subdivided into four chromosomes. By phylogenetic analysis, Blastobotrys (Arxula) adeninivorans and Yarrowia lipolytica were found to be close relatives of S. lignohabitans. Differential gene expression was evaluated in typical growth conditions on glucose and xylose and allowed a first insight into the transcriptional response of S. lignohabitans to different carbon sources and different oxygenation conditions. Novel sequences for enzymes and transporters involved in the central carbon metabolism, and therefore of potential biotechnological interest, were identified. These data open the way for a better understanding of the metabolism of S. lignohabitans and provide resources for further metabolic engineering.
The production of recombinant therapeutic proteins is one of the fast growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic posttranslational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several non-conventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.This article is protected by copyright. All rights reserved.