Publications by authors named "Hyun Ah Kang"

100 Publications

Integrated genomic and transcriptomic analysis reveals unique mechanisms for high osmotolerance and halotolerance in Hyphopichia yeast.

Environ Microbiol 2021 Apr 8. Epub 2021 Apr 8.

Molecular Systems Biology Laboratory of Life Science, Chung-Ang University, Seoul, 06974, South Korea.

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.
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http://dx.doi.org/10.1111/1462-2920.15464DOI Listing
April 2021

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha.

J Microbiol 2021 Feb 1;59(2):151-163. Epub 2021 Feb 1.

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 06974, Republic of Korea.

Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein-coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heat-shock-resistant yeast host strains.
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http://dx.doi.org/10.1007/s12275-021-0646-2DOI Listing
February 2021

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast .

J Microbiol Biotechnol 2021 Feb;31(2):272-279

Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju 28644, Republic of Korea.

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, and , were found from chromosomes 3 and 5 of KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from . SfABF51 and SfABF51 showed the highest activities on -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 -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-arabinofuranosidic 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 could potentially play important roles in the degradation and utilization of hemicellulosic biomass by .
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http://dx.doi.org/10.4014/jmb.2012.12038DOI Listing
February 2021

Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae.

Nucleic Acids Res 2021 01;49(2):745-759

School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.

Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.
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http://dx.doi.org/10.1093/nar/gkaa1221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826247PMC
January 2021

Evolutionary analysis and protein family classification of chitin deacetylases in Cryptococcus neoformans.

J Microbiol 2020 Sep 1;58(9):805-811. Epub 2020 Sep 1.

Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.

Cryptococcus neoformans is an opportunistic fungal pathogen causing cryptococcal meningoencephalitis. Interestingly, the cell wall of C. neoformans contains chitosan, which is critical for its virulence and persistence in the mammalian host. C. neoformans (H99) has three chitin deacetylases (CDAs), which convert chitin to chitosan. Herein, the classification of the chitin-related protein (CRP) family focused on cryptococcal CDAs was analyzed by phylogenetics, evolutionary pressure (d/d), and 3D modeling. A phylogenetic tree of 110 CRPs revealed that they can be divided into two clades, CRP I and II with bootstrap values (> 99%). CRP I clade comprises five groups (Groups 1-5) with a total of 20 genes, while CRP II clade comprises sixteen groups (Groups 6-21) with a total of 90 genes. CRP I comprises only fungal CDAs, including all three C. neoformans CDAs, whereas CRP II comprises diverse CDAs from fungi, bacteria, and amoeba, along with other carbohydrate esterase 4 family proteins. All CDAs have the signal peptide, except those from group 11. Notably, CDAs with the putative O-gycosylation site possess either the glycosylphosphatidylinositol (GPI)-anchor motif for CRP I or the chitin-binding domain (CBD) for CRP II, respectively. This evolutionary conservation strongly indicates that the O-glycosylation modification and the presence of either the GPI-anchor motif or the chitin-binding domain is important for fungal CDAs to function efficiently at the cell surface. This study reveals that C. neoformans CDAs carrying GPI anchors have evolved divergently from fungal and bacterial CDAs, providing new insights into evolution and classification of CRP family.
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http://dx.doi.org/10.1007/s12275-020-0288-9DOI Listing
September 2020

Genome-wide functional analysis of phosphatases in the pathogenic fungus Cryptococcus neoformans.

Nat Commun 2020 08 24;11(1):4212. Epub 2020 Aug 24.

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Korea.

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.
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http://dx.doi.org/10.1038/s41467-020-18028-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445287PMC
August 2020

Core -Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death.

mBio 2020 05 12;11(3). Epub 2020 May 12.

Department of Life Science, Chung-Ang University, Seoul, South Korea

is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of -glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of with defects in the assembly of lipid-linked -glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of (Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral -glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes , the Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of and , encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core -glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from - and -deficient mice infected with the Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core -glycans. Our results demonstrated -glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of We previously reported that the outer mannose chains of -glycans are dispensable for the virulence of , which is in stark contrast to findings for the other human-pathogenic yeast, Here, we present evidence that an intact core -glycan structure is required for pathogenicity by systematically analyzing , and strains that have defects in lipid-linked glycan assembly and in virulence. The null mutants producing truncated core -glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of , thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the -glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.
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http://dx.doi.org/10.1128/mBio.00711-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218283PMC
May 2020

Yeast synthetic biology for designed cell factories producing secretory recombinant proteins.

FEMS Yeast Res 2020 03;20(2)

Laboratory of Molecular Systems Biology, Department of Life Science, Chung-Ang University, Seoul 06974, South Korea.

Yeasts are prominent hosts for the production of recombinant proteins from industrial enzymes to therapeutic proteins. Particularly, the similarity of protein secretion pathways between these unicellular eukaryotic microorganisms and higher eukaryotic organisms has made them a preferential host to produce secretory recombinant proteins. However, there are several bottlenecks, in terms of quality and quantity, restricting their use as secretory recombinant protein production hosts. In this mini-review, we discuss recent developments in synthetic biology approaches to constructing yeast cell factories endowed with enhanced capacities of protein folding and secretion as well as designed targeted post-translational modification process functions. We focus on the new genetic tools for optimizing secretory protein expression, such as codon-optimized synthetic genes, combinatory synthetic signal peptides and copy number-controllable integration systems, and the advanced cellular engineering strategies, including endoplasmic reticulum and protein trafficking pathway engineering, synthetic glycosylation, and cell wall engineering, for improving the quality and yield of secretory recombinant proteins.
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http://dx.doi.org/10.1093/femsyr/foaa009DOI Listing
March 2020

Mec1 Modulates Interhomolog Crossover and Interplays with Tel1 at Post Double-Strand Break Stages.

J Microbiol Biotechnol 2020 Mar;30(3):469-475

Department of Life Sciences, Chung-Ang University, Seoul 06974, Republic of Korea.

During meiosis I, programmed DNA double-strand breaks (DSBs) occur to promote chromosome pairing and recombination between homologs. In , Mec1 and Tel1, the orthologs of human ATR and ATM, respectively, regulate events upstream of the cell cycle checkpoint to initiate DNA repair. Tel1 and Mec1 are required for phosphorylating various meiotic proteins during recombination. This study aimed to investigate the role of Tel1 and Mec1 in meiotic prophase via physical analysis of recombination. Tel1 cooperated with Mec1 to mediate DSB-to-single end invasion transition, but negatively regulated DSB formation. Furthermore, Mec1 was required for the formation of interhomolog joint molecules from early prophase, thus establishing a recombination partner choice. Moreover, Mec1 specifically promoted crossover-fated DSB repair. Together, these results suggest that Tel1 and Mec1 function redundantly or independently in all post-DSB stages.
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http://dx.doi.org/10.4014/jmb.1909.09020DOI Listing
March 2020

Short bZIP homologue of sulfur regulator Met4 from Ogataea parapolymorpha does not depend on DNA-binding cofactors for activating genes in sulfur starvation.

Environ Microbiol 2020 01 19;22(1):310-328. Epub 2019 Nov 19.

Laboratory of Molecular Systems Biology, Department of Life Science, Chung-Ang University, Seoul, 06974, Korea.

The acquisition of sulfur from environment and its assimilation is essential for fungal growth and activities. Here, we describe novel features of the regulatory network of sulfur metabolism in Ogataea parapolymorpha, a thermotolerant methylotrophic yeast with high resistance to harsh environmental conditions. A short bZIP protein (OpMet4p) of O. parapolymorpha, displaying the combined structural characteristics of yeast and filamentous fungal Met4 homologues, plays a key role as a master regulator of cell homeostasis during sulfur limitation, but also its function is required for the tolerance of various stresses. Domain swapping analysis, combined with deletion analysis of the regulatory domains and genes encoding OpCbf1p, OpMet28p, and OpMet32p, indicated that OpMet4p does not require the interaction with these DNA-binding cofactors to induce the expression of sulfur genes, unlike the Saccharomyces cerevisiae Met4p. ChIP analysis confirmed the notion that OpMet4p, which contains a canonical bZIP domain, can bind the target DNA in the absence of cofactors, similar to homologues in other filamentous fungi. Collectively, the identified unique features of the O. parapolymorpha regulatory network, as the first report on the sulfur regulation by a short yeast Met4 homologue, provide insights into conservation and divergence of the sulfur regulatory networks among diverse ascomycetous fungi.
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http://dx.doi.org/10.1111/1462-2920.14849DOI Listing
January 2020

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

Anal Biochem 2019 12 27;586:113408. Epub 2019 Aug 27.

Molecular Systems Biology Laboratory, Department of Life Science, Chung-Ang University, Seoul, 06974, South Korea. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ab.2019.113408DOI Listing
December 2019

Perception of Child Abuse and Child Disciplinary Practice among Adults Abused as Children: Comparison to General Population.

Soa Chongsonyon Chongsin Uihak 2019 Mar;30(2):57-65

Division of Child Welfare & Studies, College of Human Ecology, Sookmyung Women's University, Seoul, Korea.

Objectives: The aim of this study was to compare differences in perception and knowledge of child abuse and child disciplinary practices according to the history of child abuse victimization.

Methods: A questionnaire survey on child abuse was conducted with 491 adults raising children. We compared the perception and knowledge of child abuse and child disciplinary practices between two groups of adults with and without a history of childhood abuse victimization.

Results: The group with a history of childhood abuse had lower levels of knowledge of child abuse (F=6.990, p<0.01) and engaged in more negative disciplinary practices (F=5.974, p<0.05) than those without. However, no differences in the perception of child abuse were observed between the two groups.

Conclusion: The results suggest that adults with a history of childhood abuse have lower levels of knowledge of child abuse and use more negative disciplinary practices in raising their children. This highlights the need to administer not only educational but also more direct hands-on interventions to vulnerable parents in order to foster healthy parenting and disciplinary practices.
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http://dx.doi.org/10.5765/jkacap.180032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289501PMC
March 2019

Screening and Selection of Production Strains: Secretory Protein Expression and Analysis in Hansenula polymorpha.

Methods Mol Biol 2019 ;1923:133-151

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, Republic of Korea.

The thermotolerant methylotrophic yeast Hansenula polymorpha has been used as a host for the high-level production of recombinant proteins from industrial enzymes to therapeutic proteins. Despite favorable characteristics of the H. polymorpha-based platform for application to heterologous gene expression, several problems and limitations, such as over-glycosylation and proteolytic degradation, can be encountered in the development of production strains for secretory proteins. Here, H. polymorpha genetic tools and host strains, developed for authentic processing and modification of secretory recombinant proteins, are introduced with the analytical protocols.
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http://dx.doi.org/10.1007/978-1-4939-9024-5_5DOI Listing
June 2019

Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression.

Appl Microbiol Biotechnol 2019 Mar 31;103(5):2277-2293. Epub 2019 Jan 31.

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 06974, South Korea.

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants P-OCH1 and P-CHS3 and the double mutant P-OCH1/P-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the P-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.
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http://dx.doi.org/10.1007/s00253-019-09614-4DOI Listing
March 2019

Dissection of differential vanadate sensitivity in two Ogataea species links protein glycosylation and phosphate transport regulation.

Sci Rep 2018 11 6;8(1):16428. Epub 2018 Nov 6.

Bach Institute of Biochemistry, Research Center of Biotechnology RAS, Moscow, Russian Federation.

The closely related yeasts Ogataea polymorpha and O. parapolymorpha differ drastically from each other by sensitivity to the toxic phosphate analog vanadate. Search for genes underlying this difference revealed two genes, one designated as ABV1 (Alcian Blue staining, Vanadate resistance), which encodes a homologue of Saccharomyces cerevisiae Mnn4 responsible for attachment of mannosylphosphate to glycoside chains of secretory proteins, and the other designated as its S. cerevisiae homologue PHO87, encoding the plasma membrane low affinity phosphate sensor/transporter. The effect of Pho87 on vanadate resistance was bidirectional, since it decreased the resistance on phosphate-depleted medium, but was required for pronounced protection against vanadate by external phosphate. This highlights the dual function of this protein as a low affinity phosphate transporter and an external phosphate sensor. Involvement of Pho87 in phosphate sensing was confirmed by its effects on regulation of the promoter of the PHO84 gene, encoding a high affinity phosphate transporter. The effect of Abv1 was also complex, since it influenced Pho87 level and enhanced repression of the PHO84 promoter via a Pho87-independent pathway. Role of the identified genes in the difference in vanadate resistance between O. polymorpha and O. parapolymorpha is discussed.
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http://dx.doi.org/10.1038/s41598-018-34888-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219546PMC
November 2018

Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus.

Appl Microbiol Biotechnol 2018 Apr 1;102(8):3723-3737. Epub 2018 Mar 1.

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 156-756, Republic of Korea.

Pyruvate decarboxylase (Pdc) is a cytosolic enzyme located at the branch point between fermentative and respiratory sugar catabolism. Here, we identified and functionally characterized KmPDC1 and KmPDC5 encoding two homologs of Pdc in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555. Despite the conservation of important Pdc domains, a few amino acid sequences essential for enzymatic activity are not conserved in KmPdc5p. Deletion of KmPDC1 alone eliminated most of Pdc activity, but the growth of the Kmpdc1Δ strain on glucose was comparable to that of the wild type (WT) strain under aerobic conditions. In contrast to the WT, Kmpdc1Δ could not grow on glucose under oxygen-limited conditions. The KmPDC5 deletion did not generate any apparent change in Pdc activity or growth patterns under several tested conditions. Whereas the expression of KmPDC1 was enhanced by glucose, the basic expression levels of KmPDC5 were very low, without a detectable difference between glucose and nonfermentable carbon sources. Moreover, KmPDC5 overexpression was unable to complement the growth defect of Kmpdc1Δ in the presence of antimycin A, and the purified recombinant KmPdc5p was inactive in Pdc activity assay, supporting the notion that KmPdc5p may lack Pdc enzymatic activity. Notably, compared to the WT, Kmpdc1Δ single and Kmpdc1Δpdc5Δ double mutants produced significantly less glycerol, acetate, and ethanol while accumulating pyruvate. Altogether, our data indicate that a single deletion of KmPDC1 is sufficient in Crabtree-negative K. marxianus strains to generate a starting host strain for engineering of production of high-value biomaterials derived from pyruvate without byproduct formation.
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http://dx.doi.org/10.1007/s00253-018-8862-3DOI Listing
April 2018

Evaluation of analytical similarity between trastuzumab biosimilar CT-P6 and reference product using statistical analyses.

MAbs 2018 May/Jun;10(4):547-571. Epub 2018 Mar 14.

a Biotechnology Research Institute, R&D Division, Celltrion Inc. , Incheon , Korea.

The evaluation of analytical similarity has been a challenging issue for the biosimilar industry because the number of lots for reference and biosimilar products available at the time of development are limited, whilst measurable quality attributes of target molecule are numerous, which can lead to potential bias or false negative/positive conclusions regarding biosimilarity. Therefore, appropriate statistical analyses are highly desirable to achieve a high level of confidence in the similarity evaluation. A recent guideline for the risk-based statistical approaches recommended by the US Food and Drug Administration provides useful tools to systematically evaluate analytical similarity of biosimilar products compared with reference products. Here, we evaluated analytical similarity of CT-P6, a biosimilar product of trastuzumab, with the reference products (EU-Herceptin® or US-Herceptin®) following these statistical approaches. Various quality attributes of trastuzumab were first ranked based on the clinical impact of each attribute and subsequently adjusted to one of three tiers (Tier 1, Tier 2 and Tier 3) considering the characteristics of the assay, the level of attribute present and the feasibility of statistical analysis. Two biological activities with highest potential clinical impact were evaluated by an equivalent test (Tier 1), and other bioactivities and structural/physicochemical properties relevant to the clinical impact were evaluated by a quality range approach (Tier 2). The attributes with low risk ranking or qualitative assay were evaluated by visual comparison (Tier 3). Analytical similarity assessment analyzed by the three tiers clearly demonstrated that CT-P6 exhibits highly similar structural and physicochemical properties, as well as functional activities, compared with the reference products. There were small differences observed in a few quality attributes between CT-P6 and the reference products, but the differences were very minor, and unlikely to impact on clinical outcome. The recently reported equivalent clinical efficacy of CT-P6 with the reference product further supports that CT-P6 is highly similar compared with the reference product in the view of totality-of-evidence.
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http://dx.doi.org/10.1080/19420862.2018.1440170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973688PMC
August 2019

Analytical similarity assessment of rituximab biosimilar CT-P10 to reference medicinal product.

MAbs 2018 04 6;10(3):380-396. Epub 2018 Mar 6.

a Biotechnology Research Institute, R&D Division, Celltrion Inc. , Incheon , Korea.

CT-P10 (Truxima™) was recently approved as the world's first rituximab biosimilar product in the European Union (EU) and South Korea. To demonstrate biosimilarity of CT-P10 with the reference medicinal product (RMP), extensive 3-way similarity assessment has been conducted between CT-P10, EU-Rituximab and US-Rituximab, focusing on the physicochemical and biological quality attributes. A multitude of state-of-the-art analyses revealed that CT-P10 has identical primary and higher order structures compared to the original product. Purity/impurity profiles of CT-P10 measured by the levels of aggregates, fragments, non-glycosylated form and process-related impurities were also found to be comparable with those of RMPs. In terms of the post-translational modification, CT-P10 contains slightly less N-terminal pyro-glutamate variant, which has been known not to affect product efficacy or safety. Oligosaccharide profiling has revealed that, although CT-P10 contains the same conserved glycan species and relative proportion with the RMPs, the content of total afucosylated glycan in CT-P10 was slightly higher than in EU- or US-Rituximab. Nevertheless, the effect of the observed level of afucosylation in CT-P10 drug product on Fc receptor binding affinity or antibody-dependent cell-mediated cytotoxicity was found to be negligible based on the spiking study with highly afucosylated sample. Arrays of biological assays representative of known and putative mechanisms of action for rituximab have shown that biological activities of CT-P10 are within the quality range of RMPs. Recent results of clinical studies have further confirmed that the CT-P10 exhibits equivalent clinical efficacy and safety profiles compared to EU- and US-Rituximab. The current 3-way similarity assessment together with clinical study results confidently demonstrate that CT-P10 is highly similar with EU- and US-Rituximab in terms of physicochemical properties, biological activities, efficacy, and safety for its final approval as a biosimilar product.
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http://dx.doi.org/10.1080/19420862.2018.1433976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916562PMC
April 2018

A Novel Mitochondrial Serine O-Acetyltransferase, OpSAT1, Plays a Critical Role in Sulfur Metabolism in the Thermotolerant Methylotrophic Yeast Ogataea parapolymorpha.

Sci Rep 2018 02 5;8(1):2377. Epub 2018 Feb 5.

Department of Life Science, Chung-Ang University, Seoul, 06974, Korea.

In most bacteria and plants, direct biosynthesis of cysteine from sulfide via O-acetylserine (OAS) is essential to produce sulfur amino acids from inorganic sulfur. Here, we report the functional analysis of a novel mitochondrial serine O-acetyltransferase (SAT), responsible for converting serine into OAS, in the thermotolerant methylotrophic yeast Ogataea parapolymorpha. Domain analysis of O. parapolymorpha SAT (OpSat1p) and other fungal SATs revealed that these proteins possess a mitochondrial targeting sequence (MTS) at the N-terminus and an α/β hydrolase 1 domain at the C-terminal region, which is quite different from the classical SATs of bacteria and plants. Noticeably, OpSat1p is functionally interchangeable with Escherichia coli SAT, CysE, despite that it displays much less enzymatic activity, with marginal feedback inhibition by cysteine, compared to CysE. The Opsat1Δ-null mutant showed remarkably reduced intracellular levels of cysteine and glutathione, implying OAS generation defect. The MTS of OpSat1p directs the mitochondrial targeting of a reporter protein, thus, supporting the localization of OpSat1p in the mitochondria. Intriguingly, the OpSat1p variant lacking MTS restores the OAS auxotrophy, but not the cysteine auxotrophy of the Opsat1Δ mutant strain. This is the first study on a mitochondrial SAT with critical function in sulfur assimilatory metabolism in fungal species.
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http://dx.doi.org/10.1038/s41598-018-20630-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799214PMC
February 2018

Emerging roles of inositol pyrophosphates as key modulators of fungal pathogenicity.

Authors:
Hyun Ah Kang

Virulence 2018 01;9(1):563-565

a Department of Life Science , Chung-Ang University , Seoul , Korea.

Inositol pyrophosphates (PP-IPs) are energy-rich small molecules that are omnipresent in eukaryotic cells, from yeast to mammals, playing central roles in overall cellular homeostasis as a diverse and multifaceted class of intracellular messengers. Recent studies of the metabolic pathways and physiological roles of PP-IPs in the human pathogenic fungus Cryptococcus neoformans have revealed that the PP-IP (IP) is a key metabolite essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity. This suggests the PP-IP biosynthesis pathway, comprising phospholipase C1 (Plc1) and a series of sequentially acting inositol polyphosphate kinases (IPKs), as a new virulence-related signaling pathway in C. neoformans. Given that fungal species have a reduced array of the kinases required for the synthesis of PP-IPs and that the homology between human and fungal IPKs is restricted to a few catalytically important residues, identification of IPK inhibitors specifically targeting the kinases of pathogenic fungi has emerged as a desirable and achievable strategy for antifungal drug development.
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http://dx.doi.org/10.1080/21505594.2017.1421832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000206PMC
January 2018

Structural analysis of N-/O-glycans assembled on proteins in yeasts.

J Microbiol 2018 Jan 4;56(1):11-23. Epub 2018 Jan 4.

Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.
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http://dx.doi.org/10.1007/s12275-018-7468-xDOI Listing
January 2018

Functional analysis of Mpk1-mediated cell wall integrity signaling pathway in the thermotolerant methylotrophic yeast Hansenula polymorpha.

J Microbiol 2018 Jan 4;56(1):72-82. Epub 2018 Jan 4.

Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.

Understanding the characteristics and regulation mechanisms of cell wall integrity (CWI) in yeast is important not only for basic research but also in biotechnological applications. We found significantly different CWIs in two representative strains of the thermotolerant methylotrophic yeast Hansenula polymorpha. Compared to the A16 strain (classified as Ogataea polymorpha), the DL1-L strain (classified as Ogataea parapolymorpha) has a thinner cell wall that was found to be more fragile following long-term cultivation and more sensitive to zymolyase. To gain a deeper insight into this difference, we compared the characteristics of the Mpk1pmediated CWI signaling pathway in the two strains. While a DL1-L mutant deficient in Mpk1p (mpk1Δ) showed severe growth retardation at both normal and high growth temperatures and in the presence of cell-wall disrupting agents, the A16 mpk1Δ mutant displayed only a mild defect in cell growth. Sorbitol effect on rescuing growth retardation was different in the two mpk1Δ strains, which could partly be ascribed to subtle differences in the activation of HOG pathway. Among the cell wall disruptors evaluated, only caffeine clearly increased phosphorylation of Mpk1p in DL1-L, but not in A16. A transcriptome analysis of the DL1-L strain revealed that caffeine significantly increased the expression of a subset of cell-wall related genes in an Mpk1p-dependent manner, but not the expected Rlm1-target genes. Taken together, our data support an essential role for Mpk1p in maintaining CWI in H. polymorpha, although the requirement for Mpk1p and its regulation under diverse stress conditions varies depending on the strain background.
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http://dx.doi.org/10.1007/s12275-018-7508-6DOI Listing
January 2018

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus.

J Microbiol 2017 Aug 28;55(8):655-664. Epub 2017 Jul 28.

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 06974, Republic of Korea.

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.
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http://dx.doi.org/10.1007/s12275-017-7218-5DOI Listing
August 2017

A novel bZIP protein, Gsb1, is required for oxidative stress response, mating, and virulence in the human pathogen Cryptococcus neoformans.

Sci Rep 2017 06 22;7(1):4044. Epub 2017 Jun 22.

Department of Life Science, Chung-Ang University, Seoul, 06974, Korea.

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.
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http://dx.doi.org/10.1038/s41598-017-04290-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481450PMC
June 2017

Oral vaccination through voluntary consumption of the convict grouper Epinephelus septemfasciatus with yeast producing the capsid protein of red-spotted grouper nervous necrosis virus.

Vet Microbiol 2017 May 24;204:159-164. Epub 2017 Apr 24.

Laboratory of Virology, College of Pharmacy, Chung-Ang University, 84 Heukseok-Ro, Dongjak-Gu, Seoul 06974, South Korea. Electronic address:

Nervous necrosis viruses (NNV) cause serious economic losses in marine fish cultivation. The red-spotted grouper NNV (RGNNV) is the most common species of NNV worldwide. There have been many efforts to develop prophylactic NNV vaccines, and various types of vaccine candidate have been suggested. However, most were designed as injectable vaccines, which are not suitable for large-scale vaccination and cause too much stress to the fish. Oral vaccination through voluntary feeding is an ideal way to provide protective immunity to fish. In the present study, recombinant Saccharomyces cerevisiae producing RGNNV capsid protein was used as oral vaccine. The recombinant yeast was prepared in freeze-dried form after disruption. Convict groupers were divided into three groups, control, and oral and parenteral vaccination groups, each consisting of 700 fishes. The control group received no treatment, the parenteral group received one intraperitoneal injection of RGNNV virus-like particles, and the oral vaccination group consumed feed containing the lysed recombinant yeast; voluntary intake was allowed four times at one-week intervals. Both vaccination groups produced serum RGNNV neutralizing antibody titers of >10 (log 2, 9.96), sustained for at least 95days post-immunization. In addition, in response to challenge with RGNNV both groups suffered significantly reduced mortality and had reduced brain RGNNV titers. These results indicate that recombinant yeast-based oral fish vaccines have great potential for large-scale vaccination.
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http://dx.doi.org/10.1016/j.vetmic.2017.04.022DOI Listing
May 2017

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.

Appl Microbiol Biotechnol 2017 Apr 18;101(7):2979-2989. Epub 2017 Jan 18.

Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea.

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.
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http://dx.doi.org/10.1007/s00253-017-8101-3DOI Listing
April 2017

Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast and its interspecies hybrid.

Biotechnol Biofuels 2016 11;9:246. Epub 2016 Nov 11.

Department of Life Science, Chung-Ang University, Seoul, 06974 South Korea.

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 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 .

Results: We performed whole-genome (WG) de novo sequencing and complete assembly of KJJ81 and KPH12, two isolates from wheat-based 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 before the separation of the CTG and clades in the subphylum . 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 genome reflects a unique phenotype for clades as natural sulfur auxotrophs. Extended gene families, including novel genes involved in saccharification and proteolysis, were identified. Moreover, comparative genome analysis of 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 genome with high-quality annotation and RNA-Seq analysis establishes an important foundation for functional inference of 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 including members with desirable traits for bioconversion, the unique genomic features of and its hybrid will provide in-depth insights into fungal genome dynamics as evolutionary adaptation.
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http://dx.doi.org/10.1186/s13068-016-0653-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5106798PMC
November 2016

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

J Microbiol 2016 Oct 30;54(10):667-74. Epub 2016 Sep 30.

Department of Life Science, College of Natural Science, Seoul, 06974, Republic of Korea.

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.
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http://dx.doi.org/10.1007/s12275-016-6401-4DOI Listing
October 2016

A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

J Biotechnol 2016 Sep 10;233:190-9. Epub 2016 Jul 10.

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, South Korea; Bio-Integration Research Center for Nutra-Pharmaceutical Epigenetics, Chung-Ang University, Seoul 156-756, South Korea. Electronic address:

The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.
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http://dx.doi.org/10.1016/j.jbiotec.2016.07.006DOI Listing
September 2016

Sir2 phosphorylation through cAMP-PKA and CK2 signaling inhibits the lifespan extension activity of Sir2 in yeast.

Elife 2015 Sep 2;4. Epub 2015 Sep 2.

Department of Microbiology and Molecular Biology, College of Bioscience and Biotechnology, Chungnam National University, Daejeon, Republic of Korea.

Silent information regulator 2 (Sir2), an NAD(+)-dependent protein deacetylase, has been proposed to be a longevity factor that plays important roles in dietary restriction (DR)-mediated lifespan extension. In this study, we show that the Sir2's role for DR-mediated lifespan extension depends on cAMP-PKA and casein kinase 2 (CK2) signaling in yeast. Sir2 partially represses the transcription of lifespan-associated genes, such as PMA1 (encoding an H(+)-ATPase) and many ribosomal protein genes, through deacetylation of Lys 16 of histone H4 in the promoter regions of these genes. This repression is relieved by Sir2 S473 phosphorylation, which is mediated by active cAMP-PKA and CK2 signaling. Moderate DR increases the replicative lifespan of wild-type yeast but has no effect on that of yeast expressing the Sir2-S473E or S473A allele, suggesting that the effect of Sir2 on DR-mediated lifespan extension is negatively regulated by S473 phosphorylation. Our results demonstrate a mechanism by which Sir2 contributes to lifespan extension.
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http://dx.doi.org/10.7554/eLife.09709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586308PMC
September 2015