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1.
Methods Mol Biol ; 2564: 213-222, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107344

RESUMO

Fluorescent proteins within fluorescent fusions have been reported to affect cellular growth fitness via altering native protein function and intracellular localization. Here we report in detail a procedure to analyze the growth characteristics of yeast cells expressing such fusions in comparison to unmodified parental strain. This approach can serve as an initial step in fluorescent protein characterization in vivo.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Corantes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
2.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077401

RESUMO

Ca2+ is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
3.
Elife ; 112022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36047762

RESUMO

The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2 (RagA/C in humans), and the GTPase activating complex SEAC/GATOR. However, it remains unclear if, and how, other proteins/pathways regulate TORC1 in simple eukaryotes like yeast. Here, we report that the previously unstudied GPCR-like protein, Ait1, binds to TORC1-Gtr1/2 in Saccharomyces cerevisiae and holds TORC1 around the vacuole during log-phase growth. Then, during amino acid starvation, Ait1 inhibits TORC1 via Gtr1/2 using a loop that resembles the RagA/C-binding domain in the human protein SLC38A9. Importantly, Ait1 is only found in the Saccharomycetaceae/codaceae, two closely related families of yeast that have lost the ancient TORC1 regulators Rheb and TSC1/2. Thus, the TORC1 circuit found in the Saccharomycetaceae/codaceae, and likely other simple eukaryotes, has undergone significant rewiring during evolution.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Aminoácidos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Sirolimo/metabolismo
4.
Arch Microbiol ; 204(10): 635, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127512

RESUMO

γ-Linolenic acid (GLA) is an essential n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in human and animal feed. GLA is used in many nutritional and medicinal applications, such as the treatment of cancer, inflammatory disorders, and diabetes. Currently, plant seed is the primary dietary source of GLA that is not enough to utilize on an industrial scale. To generate a sustainable novel source of GLA, the gene of delta-6 desaturase, one of the essential enzymes in the GLA production pathway, was isolated from Mucor rouxii DSM1194 and expressed in P. pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72 mg/g) of total fatty acids. GLA production of recombinant yeast was studied in a fermenter by oil waste for 5 days, and results detected 6.3 g/l lipid, and 103 mg/g GLA was produced in 72 h. The present study may provide an opportunity to develop an alternative host for manufacturing GLA on an industrial scale.


Assuntos
Pichia , Saccharomycetales , Ácido gama-Linolênico , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Ácido gama-Linolênico/metabolismo
5.
Int J Mol Sci ; 23(15)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35955874

RESUMO

Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.


Assuntos
Aspergillus oryzae , Saccharomycetales , Sequência de Aminoácidos , Aspergillus oryzae/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Saccharomycetales/metabolismo , Temperatura
6.
Appl Microbiol Biotechnol ; 106(18): 5893-5912, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36040488

RESUMO

Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins in recent years. The strong promoter AOX1, induced by methanol, with the help of the α-pre-pro signal sequence, can lead to a high expression level of extracellular protein. However, this combination was not always efficient, as protein secretion in P. pastoris involves numerous procedures mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) molecular chaperones, degradation through ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways. This article summarizes the process of protein secretion, modification, and transportation in P. pastoris. In addition, the roles played by the key proteins in these processes and the corresponding co-expression effects are also listed. It is expected to lay the foundation for the industrial protein production of P. pastoris. KEY POINTS: • Mechanisms of chaperones in protein folding and their co-expression effects are summarized. • Protein glycosylation modifications are comprehensively reviewed. • Current dilemmas in the overall protein secretion pathway of Pichia pastoris and corresponding solutions are demonstrated.


Assuntos
Pichia , Saccharomycetales , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas , Proteômica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
7.
Sci Rep ; 12(1): 14815, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36045277

RESUMO

Luciferin biosynthetic origin and alternative biological functions during the evolution of beetles remain unknown. We have set up a bioluminescent sensing method for luciferin synthesis from cysteine and benzoquinone using E. coli and Pichia pastoris expressing the bright Amydetes vivianii firefly and P. termitilluminans click beetle luciferases. In the presence of D-cysteine and benzoquinone, intense bioluminescence is quickly produced, indicating the expected formation of D-luciferin. Starting with L-cysteine and benzoquinone, the bioluminescence is weaker and delayed, indicating that bacteria produce L-luciferin, and then racemize it to D-luciferin in the presence of endogenous esterases, CoA and luciferase. In bacteria the p-benzoquinone toxicity (IC50 ~ 25 µM) is considerably reduced in the presence of cysteine, maintaining cell viability at 3.6 mM p-benzoquinone concomitantly with the formation of luciferin. Transcriptional analysis showed the presence of gene products involved with the sclerotization/tanning in the photogenic tissues, suggesting a possible link between these pathways and bioluminescence. The lack of two enzymes involved with the last steps of these pathways, indicate the possible accumulation of toxic quinone intermediates in the lanterns. These results and the abundance of cysteine producing enzymes suggest that luciferin first appeared as a detoxification byproduct of cysteine reaction with accumulated toxic quinone intermediates during the evolution of sclerotization/tanning in Coleoptera.


Assuntos
Besouros , Luciferina de Vaga-Lumes , Luciferases de Vaga-Lume , Quinonas , Animais , Besouros/metabolismo , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vaga-Lumes/genética , Luciferina de Vaga-Lumes/metabolismo , Luciferases/genética , Luciferases/metabolismo , Luciferases de Vaga-Lume/metabolismo , Luciferinas , Medições Luminescentes , Quinonas/metabolismo , Saccharomycetales/metabolismo
8.
J Cell Sci ; 135(18)2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35975715

RESUMO

Recent studies have revealed that the growth rate of budding yeast and mammalian cells varies during the cell cycle. By linking a multitude of signals to cell growth, the highly conserved target of rapamycin complex 1 (TORC1) and protein kinase A (PKA) pathways are prime candidates for mediating the dynamic coupling between growth and division. However, measurements of TORC1 and PKA activity during the cell cycle are still lacking. By following the localization dynamics of two TORC1 and PKA targets via time-lapse microscopy in hundreds of yeast (Saccharomyces cerevisiae) cells, we found that the activity of these pathways towards ribosome biogenesis fluctuates in synchrony with the cell cycle even under constant external conditions. Analysis of the effects of mutations of upstream TORC1 and PKA regulators suggests that internal metabolic signals partially mediate these activity changes. Our study reveals a new aspect of TORC1 and PKA signaling, which will be important for understanding growth regulation during the cell cycle.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Ciclo Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Fatores de Transcrição
9.
Biochem Biophys Res Commun ; 620: 173-179, 2022 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-35803173

RESUMO

Separase is a giant cysteine protease and has multiple crucial functions. The most well-known substrate of separase is the kleisin subunit of cohesin, the cleavage of which triggers chromosome segregation during cell division (Uhlmann et al., 1999; Kamenz and Hauf, 2016) [1,2]. Recently, separase has also been found to cleave MCL-1 or BCL-XL proteins to trigger apoptosis (Hellmuth and Stemmann, 2020) [3]. Although substrate recognition through a short sequence right upstream of the cleavage site is well established, recent studies suggested that sequence elements outside this minimum cleavage site are required for optimal cleavage activity and specificity (Rosen et al., 2019; Uhlmann et al., 2000) [4,5]. However, the sequences and their underlying mechanism are largely unknown. To further explore the substrate determinants and recognition mechanism, we carried out sequence alignments and found a conserved motif downstream of the cleavage site in budding yeast. Using Alphafold2 and molecular dynamics simulations, we found this motif is recognized by separase in a conserved cleft near the binding groove of its inhibitor securin. Their binding is mutually exclusive and requires conformation changes of separase. These findings provide deeper insights into substrate recognition and activation of separase, and paved the way for discovering more substrates of separase.


Assuntos
Saccharomyces cerevisiae , Saccharomycetales , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Endopeptidases/metabolismo , Simulação de Dinâmica Molecular , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Securina/química , Securina/genética , Securina/metabolismo , Separase/genética
10.
Proc Natl Acad Sci U S A ; 119(29): e2201711119, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35858340

RESUMO

Methanol-based biorefinery is a promising strategy to achieve carbon neutrality goals by linking CO2 capture and solar energy storage. As a typical methylotroph, Pichia pastoris shows great potential in methanol biotransformation. However, challenges still remain in engineering methanol metabolism for chemical overproduction. Here, we present the global rewiring of the central metabolism for efficient production of free fatty acids (FFAs; 23.4 g/L) from methanol, with an enhanced supply of precursors and cofactors, as well as decreased accumulation of formaldehyde. Finally, metabolic transforming of the fatty acid cell factory enabled overproduction of fatty alcohols (2.0 g/L) from methanol. This study demonstrated that global metabolic rewiring released the great potential of P. pastoris for methanol biotransformation toward chemical overproduction.


Assuntos
Ácidos Graxos não Esterificados , Engenharia Metabólica , Metanol , Saccharomycetales , Reatores Biológicos , Biotransformação , Ácidos Graxos não Esterificados/biossíntese , Metanol/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
11.
Methods Mol Biol ; 2513: 271-290, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35781211

RESUMO

Genome-scale metabolic models (GEMs) provide a useful framework for modeling the metabolism of microorganisms. While the applications of GEMs are wide and far reaching, the reconstruction and continuous curation of such models can be perceived as a tedious and time-consuming task. Using RAVEN, a MATLAB-based toolbox designed to facilitate the reconstruction analysis of metabolic networks, this protocol practically demonstrates how researchers can create their own GEMs using a homology-based approach. To provide a complete example, a draft GEM for the industrially relevant yeast Hansenula polymorpha is reconstructed.


Assuntos
Redes e Vias Metabólicas , Saccharomycetales , Genoma Fúngico , Redes e Vias Metabólicas/genética , Modelos Biológicos , Saccharomycetales/genética , Saccharomycetales/metabolismo
12.
Yeast ; 39(8): 440-448, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35811458

RESUMO

Efficient bioconversion of methanol, which can be generated from greenhouse gases, into valuable resources contributes to achieving climate goals and developing a sustainable economy. The methylotrophic yeast Ogataea methanolica is considered to be a suitable host for efficient methanol bioconversion because it has outstanding characteristics for the better adaptive potential to a high methanol environment (i.e., greater than 5%). This capacity represents a huge potential to construct an innovative carbon-neutral production system that converts methanol into value-added chemicals under the control of strong methanol-induced promoters. In this review, we discuss what is known about the regulation of methanol metabolism and adaptation mechanisms for 5% methanol conditions in O. methanolica in detail. We also discuss about the potential to breed "super methylotrophic yeast," which has potent growth characteristics under high methanol conditions.


Assuntos
Metanol , Saccharomycetales , Regulação Fúngica da Expressão Gênica , Metanol/metabolismo , Pichia/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Leveduras/metabolismo
13.
Sheng Wu Gong Cheng Xue Bao ; 38(7): 2594-2605, 2022 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-35871627

RESUMO

Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A (CSA), we constructed a recombinant Pichia pastoris GS115 strain capable of synthesizing chondroitin (Ch) from glycerol by introducing the Ch synthase coding genes kfoC, kfoA and UDP-glucose dehydrogenase coding gene tuaD into the P. pastoris chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase (C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant P. pastoris cells. Eventually, controllable synthesis of 0-40% CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.


Assuntos
Sulfatos de Condroitina , Saccharomycetales , Animais , Técnicas de Cultura Celular por Lotes , Sulfatos de Condroitina/metabolismo , Pichia/genética , Pichia/metabolismo , Polissacarídeos , Proteínas Recombinantes/genética , Saccharomycetales/metabolismo
14.
Bioprocess Biosyst Eng ; 45(9): 1515-1525, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35881246

RESUMO

Komagataella phaffii (K. phaffii) is a famous microbial cell of heterologous protein and value-added chemicals production because of its strict and strong promoter (alcohol oxidase 1 promoter, PAOX1). Formate is an attractive substitute of traditional inducer methanol because methanol is toxic and explosive. To obtain high level of Aspergillus niger ATCC1015 xylanase as a model of heterologous protein by K. phaffii at formate induction, insertion of three-copy cis-acting element W3A into PAOX1 additionally, and co-expression of transcription factor Mit1 under another PAOX1 were carried out separately and simultaneously. The yield of xylanase increased by 41% at formate induction when Mit1 was co-expressed. Furtherly, the yield of xylanase increased by 42% using sorbitol as supplemental carbon source with the result of 408.3 × 103 U‧L-1 xylanase. Therefore, a non-methanol needed and inducible heterologous protein expression system of Komagataella phaffii was developed successfully.


Assuntos
Endo-1,4-beta-Xilanases , Saccharomycetales , Endo-1,4-beta-Xilanases/biossíntese , Formiatos , Regiões Promotoras Genéticas , Saccharomycetales/genética , Saccharomycetales/metabolismo
15.
Mol Biol Cell ; 33(9): ar85, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35704464

RESUMO

Chromosome biorientation is promoted by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore-microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore, and spindle microtubules. Here we show that a small domain of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This domain, the single alpha helix (SAH), is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of its three locations through targeted mutations and fusion constructs, we determined their individual contributions to chromosome biorientation. We find that only the inner centromere localization is sufficient for cell viability on its own. However, when combined, the inner kinetochore and microtubule binding activities are also sufficient to promote accurate chromosome segregation. Furthermore, we find that the two pathways target the CPC to different kinetochore attachment states, as the inner centromere-targeting pathway is primarily responsible for bringing the complex to unattached kinetochores. We have therefore discovered that two parallel localization pathways are each sufficient to promote CPC activity in chromosome biorientation, both depending on the SAH domain of INCENP/Sli15.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Aurora Quinase B/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Fosforilação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
16.
Bioengineered ; 13(5): 13860-13871, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35726822

RESUMO

Due to its bacteriolytic activity, hen egg white lysozyme (HEWL) is widely used in the feed, food, and pharmaceutical industries. However, its application is hindered by low protein expression levels in microbial expression systems. In this work, a novel fusion protein expression strategy was proposed for increasing the expression level of HEWL. First, HEWL, fused with a highly expressed fusion protein partner xylanase XynCDBFV, is expressed in Pichia pastoris. Secondly, a linker including endogenous protease cleavage sites was introduced between two fusion proteins in order to separate them directly during the secretion process. Finally, the results show that the supernatant of XynCDBFV-HEWL has a higher HEWL expression level and activity compared with HEWL only. It should be noted that the expression of HEWL reaches to about 3.5 g/L, and the activity of HEWL against Micrococcus lysodeikticus reaches to 1.50 × 105 U/mL in a fed-batch fermentation, which is currently the highest level of recombinant expression of an egg white-derived lysozyme. Taken together, we acquired bioactive HEWL for large-scale recombinant production in Pichia pastoris using a novel fusion protein expression strategy, which could then be used for a variety of applications.


Assuntos
Muramidase , Saccharomycetales , Clara de Ovo , Muramidase/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes , Saccharomycetales/metabolismo
17.
Commun Biol ; 5(1): 561, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35676418

RESUMO

Expression of secreted recombinant proteins burdens the protein secretion machinery, limiting production. Here, we describe an approach to improving protein production by the non-conventional yeast Komagataella phaffii comprised of genome-wide screening for effective gene disruptions, combining them in a single strain, and recovering growth reduction by adaptive evolution. For the screen, we designed a multiwell-formatted, streamlined workflow to high-throughput assay of secretion of a single-chain small antibody, which is cumbersome to detect but serves as a good model of proteins that are difficult to secrete. Using the consolidated screening system, we evaluated >19,000 mutant strains from a mutant library prepared by a modified random gene-disruption method, and identified six factors for which disruption led to increased antibody production. We then combined the disruptions, up to quadruple gene knockouts, which appeared to contribute independently, in a single strain and observed an additive effect. Target protein and promoter were basically interchangeable for the effects of knockout genes screened. We finally used adaptive evolution to recover reduced cell growth by multiple gene knockouts and examine the possibility for further enhancing protein secretion. Our successful, three-part approach holds promise as a method for improving protein production by non-conventional microorganisms.


Assuntos
Saccharomycetales , Técnicas de Inativação de Genes , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Fluxo de Trabalho
18.
EMBO J ; 41(15): e110271, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35735140

RESUMO

Nuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm, and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyltransferases (KATs) promote transcription through acetylation of chromatin-associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX-2) complex, to the nuclear basket. The Esa1-mediated nuclear export of mRNAs in turn promotes entry into S phase, which is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism is not only limited to G1/S-expressed genes but also inhibits the expression of the nutrient-regulated GAL1 gene specifically in daughter cells. Overall, these results reveal how acetylation can contribute to the functional plasticity of NPCs in mother and daughter yeast cells. In addition, our work demonstrates dual gene expression regulation by the evolutionarily conserved NuA4 complex, at the level of transcription and at the stage of mRNA export by modifying the nucleoplasmic entrance to nuclear pores.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Acetilação , Transporte Ativo do Núcleo Celular/fisiologia , Ciclo Celular , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
19.
Methods Mol Biol ; 2528: 145-157, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35704190

RESUMO

It has recently been demonstrated that budding yeast telomeres are transcribed into TERRA, a long noncoding RNA. Due to the G-rich nature of the coding strand, TERRA has a tendency to form DNA-RNA hybrids and potentially R-loops, which in turn, promote repair at short telomeres. Here, we report two methods to detect DNA-RNA hybrids at yeast telomeres, namely, DRIP, which employs the S9.6 hybrid-recognizing antibody, and R-ChIP, which takes advantage of a catalytic dead form of RNase H1 (Rnh1-cd). We use cross-linked material for both protocols as we have found that this does not negatively affect recovered material, and furthermore allows the precipitation of other proteins from the identical cross-linked material. Although both methods are successful in terms of detecting DNA-RNA hybrids at telomeres, the R-ChIP method yields an approximately ten-fold increased enrichment.


Assuntos
RNA , Saccharomycetales , DNA/genética , RNA/genética , RNA/metabolismo , Ribonuclease H/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Telômero/genética , Telômero/metabolismo
20.
Food Res Int ; 156: 111349, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35650978

RESUMO

The purpose of this work was to reveal the relationship between the microbial diversity and flavor profiles of traditional dry-cured duck from a metabolomic perspective. Enterococcus, Psychrobacter, Macrococcus, Salinivbrio, and Staphylococcus were the dominant bacterial genera, while Trichophyton, Kurtzmaniella, Blumeria, Cladosporium, Lysurus, Aspergillus, Starmerella and Debaryomyces were the dominant fungal genera of dry-cured duck. The results showed that aldehydes, alcohols, furan, and ketone compounds were the main volatile flavor compounds of dry-cured duck. Moreover, the identified metabolites of dry-cured duck were classified and included amino acids, amines, polypeptides, amino acid derivatives, polyols, fatty acids, organic acids, flavonoids and isoflavones. Heatmap analysis was used to illuminate the relationships between the microbial diversity and flavor profiles, as well as metabolites. These results will provide an effective theoretical reference for the standardization and modernization of dry-cured duck production.


Assuntos
Patos , Saccharomycetales , Aminoácidos/metabolismo , Animais , Patos/metabolismo , Ácidos Graxos , Manipulação de Alimentos/métodos , Metabolômica , Saccharomycetales/metabolismo
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