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1.
Artigo em Inglês | MEDLINE | ID: mdl-35538374

RESUMO

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha in metabolic engineeringare expounded. • Three promoter engineering strategies are introduced in order to expand the promoter toolbox.

2.
Nat Chem Biol ; 18(5): 520-529, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35484257

RESUMO

Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-L-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l-1) and ferulic acid (3.8 ± 0.3 g l-1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.


Assuntos
Produtos Biológicos , Saccharomyces cerevisiae , Produtos Biológicos/metabolismo , Ácidos Cafeicos/metabolismo , Hidroxibenzoatos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
3.
Sheng Wu Gong Cheng Xue Bao ; 38(2): 760-771, 2022 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-35234396

RESUMO

Fatty acids (FA) are widely used as feed stocks for the production of cosmetics, personal hygiene products, lubricants and biofuels. Ogataea polymorpha is considered as an ideal chassis for bio-manufacturing, due to its outstanding characteristics such as methylotroph, thermal-tolerance and wide substrate spectrum. In this study, we harnessed O. polymorpha for overproduction of fatty acids by engineering its fatty acid metabolism and optimizing the fermentation process. The engineered strain produced 1.86 g/L FAs under the optimized shake-flask conditions (37℃, pH 6.4, a C/N ratio of 120 and an OD600 of seed culture of 6-8). The fed-batch fermentation process was further optimized by using a dissolved oxygen (DO) control strategy. The C/N ratio of initial medium was 17.5, and the glucose medium with a C/N ratio of 120 was fed when the DO was higher than 30%. This operation resulted in a titer of 18.0 g/L FA, indicating the potential of using O. polymorpha as an efficient cell factory for the production of FA.


Assuntos
Ácidos Graxos , Saccharomycetales , Meios de Cultura , Fermentação , Engenharia Metabólica , Saccharomycetales/genética , Saccharomycetales/metabolismo
4.
Synth Syst Biotechnol ; 7(1): 498-505, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34977394

RESUMO

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (P PDH , P PYK , P FBA , P PGM , P GLK , P TRI , P GPI , P ADH1 , P TEF1 and P GCW14 ) were obtained with the activity range of 13%-130% of the common promoter P GAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which P PDH and P GCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced P ICL1 , rhamnose-induced P LRA3 and P LRA4 , and a bidirectional promoter (P Mal -P Per ) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter P LRA3 by 4.7-10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.

5.
Appl Microbiol Biotechnol ; 105(23): 8761-8769, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34748038

RESUMO

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.


Assuntos
Engenharia Metabólica , Metanol , Pichia/genética , Regiões Promotoras Genéticas , Saccharomycetales
6.
Synth Syst Biotechnol ; 6(2): 63-68, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33869812

RESUMO

Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy, making it an ideal host for bio-manufacturing. However, the lack of gene integration sites limits its systems metabolic engineering, in particular construction of genome-integrated pathway. We here screened the genomic neutral sites for gene integration without affecting cellular fitness, by genomic integration of an enhanced green fluorescent protein (eGFP) gene via CRISPR-Cas9 technique. After profiling the growth and fluorescent intensity in various media, 17 genome positions were finally identified as potential neutral sites. Finally, integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3, enabled the production of 4.5 mg/L fatty alcohols, indicating that these neutral sites can be used for streamline metabolic engineering in O. polymorpha. We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.

7.
iScience ; 24(3): 102168, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33665582

RESUMO

Methanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes. Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in O. polymorpha. Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%-30% to 60%-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the production of fatty alcohols in O. polymorpha. These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of O. polymorpha as an attractive cell factory for industrial application.

8.
Se Pu ; 37(8): 798-805, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642249

RESUMO

Construction of microbial cell factories is a feasible strategy for the sustainable production of chemicals, biofuels, and pharmaceutical molecules. However, the complex metabolism and rigid regulation of microbes hinders the efficient synthesis of the products of interest. Proteomics and metabolomics analyze enzymes and metabolites in terms of systems biology, thus unraveling complex biological systems and providing significant clues for microbial metabolic engineering. Here, the applications of proteomics and metabolomics in microbial metabolic engineering were reviewed, including the construction of genome-scale metabolic models, optimization of microbial product synthesis, guidance of microbial stress tolerance engineering, and prediction of rate-limiting steps. In addition, proteomics and metabolomics could be employed to explore secondary metabolic pathways in plants, to reveal novel genes or pathways for microbial synthesis of natural products. Finally, the development of bio-big data was discussed.


Assuntos
Engenharia Metabólica , Metabolômica , Proteômica , Biocombustíveis , Redes e Vias Metabólicas
9.
Microb Cell Fact ; 18(1): 63, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940138

RESUMO

Non-conventional yeasts are playing important roles as cell factories for bioproduction of biofuels, food additives and proteins with outstanding natural characteristics. However, the precise genome editing is challenging in non-conventional yeasts due to lack of efficient genetic tools. In the past few years, CRISPR-based genome editing worked as a revolutionary tool for genetic engineering and showed great advantages in cellular metabolic engineering. Here, we review the current advances and barriers of CRISPR-Cas9 for genome editing in non-conventional yeasts and propose the possible solutions in enhancing its efficiency for precise genetic engineering.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Biotecnologia , Sistemas CRISPR-Cas , Saccharomyces cerevisiae/metabolismo
10.
Microb Cell Fact ; 18(1): 27, 2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30711013

RESUMO

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.


Assuntos
Aciltransferases/metabolismo , Dipeptídeos/biossíntese , Pichia/genética , Aciltransferases/genética , Enzimas , Glutamina/metabolismo , Microbiologia Industrial/métodos , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sphingobacterium/enzimologia , Sphingobacterium/genética
11.
Methods Enzymol ; 617: 83-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30784416

RESUMO

Microbial synthesis represents an alternative approach for the sustainable production of chemicals, fuels, and medicines. However, construction of biosynthetic pathways always suffers from side reactions, toxicity of intermediates, or low efficiency of substrate channeling. Subcellular compartmentalization may contribute to a more efficient production of target products by reducing side reactions and toxic effects within a compact insular space. The peroxisome, a type of organelle that is involved in catabolism of fatty acids and reactive oxygen species, has attracted a great deal of attention in the construction of eukaryotic cell factories with little impact on essential cellular function. In this chapter, we will systematically review recent advances in peroxisomal compartmentalization for microbial production of valuable biomolecules. Additionally, detailed experimental designs and protocols are also described. We hope a comprehensive understanding of peroxisomes will promote their application in metabolic engineering and synthetic biology.


Assuntos
Engenharia Metabólica/métodos , Peroxissomos/metabolismo , Leveduras/metabolismo , Vias Biossintéticas , Carotenoides/genética , Carotenoides/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Microbiologia Industrial/métodos , Penicilinas/metabolismo , Peroxissomos/genética , Biologia Sintética/métodos , Leveduras/genética
12.
Microb Cell Fact ; 16(1): 181, 2017 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084541

RESUMO

BACKGROUND: Multiple lignocellulose-derived inhibitors represent great challenges for bioethanol production from lignocellulosic materials. These inhibitors that are related to the levels of intracellular reactive oxidative species (ROS) make oxidoreductases a potential target for an enhanced tolerance in yeasts. RESULTS: In this study, the thioredoxin and its reductase from Kluyveromyces marxianus Y179 was identified, which was subsequently achieved over-expression in Saccharomyces cerevisiae 280. In spite of the negative effects by expression of thioredoxin gene (KmTRX), the thioredoxin reductase (KmTrxR) helped to enhance tolerance to multiple lignocellulose-derived inhibitors, such as formic acid and acetic acid. In particular, compared with each gene expression, the double over-expression of KmTRX2 and KmTrxR achieved a better ethanol fermentative profiles under a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. At last, the mechanism that improves the tolerance depended on a normal level of intracellular ROS for cell survival under stress. CONCLUSIONS: The synergistic effect of KmTrxR and KmTRX2 provided the potential possibility for ethanol production from lignocellulosic materials, and give a general insight into the possible toxicity mechanisms for further theoretical research.


Assuntos
Kluyveromyces/enzimologia , Lignina/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Ácido Acético/química , Ácido Acético/metabolismo , Técnicas de Cultura Celular por Lotes , Etanol/metabolismo , Formiatos/química , Formiatos/metabolismo , Furaldeído/química , Furaldeído/metabolismo , Lignina/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/classificação , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/classificação , Tiorredoxinas/genética
13.
FEMS Yeast Res ; 17(6)2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28922844

RESUMO

Inulinase from Kluyveromyces marxianus (KmINU1) has wide application in industrial biotechnology, and it is supposed that its expression is regulated at the transcriptional level via the promoter. Therefore, in this study, the KmINU1 promoter, fused to the reporter EGFP gene, was analyzed to determine its fundamental regulatory mechanisms in Saccharomyces cerevisiae S288C (ATCC 204508). Induction by inulin and repression by high glucose concentrations of KmINU1 promoters are reported, and the promoter with a length of 353 bp was shown to have the highest strength with the weakest responses to high glucose concentration. Responses of the KmINU1 promoter to carbon source were shown to be related to the Mig1 binding site extending from -496 to -485 bp. Hence, we propose clear regulation mechanisms of the inulinase promoter, which may provide a powerful theoretical reference for its application in protein production.


Assuntos
Regulação Fúngica da Expressão Gênica , Glicosídeo Hidrolases/genética , Kluyveromyces/enzimologia , Kluyveromyces/genética , Regiões Promotoras Genéticas , Sítios de Ligação , Biotecnologia , Repressão Catabólica , Perfilação da Expressão Gênica , Genes Reporter , Glucose/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Microbiologia Industrial , Inulina/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Transcricional
14.
Sheng Wu Gong Cheng Xue Bao ; 33(6): 923-935, 2017 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-28895354

RESUMO

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.


Assuntos
Arabinose/biossíntese , Fermentação , Kluyveromyces/metabolismo , Xilose/biossíntese , Etanol , Microbiologia Industrial
15.
Bioresour Technol ; 245(Pt B): 1603-1609, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28624247

RESUMO

In this study, the optimum induction and reaction conditions, and fermentation process of producing Ala-Gln by E. coli Origami 2 overexpressing α-amino acid ester acyltransferase (OPA) were investigated. Besides, the Ala-Gln synthesis by OPA achieved the maximum molar yield of 94.7% and productivity of 1.89g/L/min due to the extremely high enzyme activity. On this basis, repeated-cycle batch fermentation to produce Ala-Gln indicated that OPA could maintain high Ala-Gln yields and enzyme stabilities after several cell recycling. Consequently, the cost-efficient and environmentally friendly approach for Ala-Gln production by recycling OPA makes a great contribution to further industrial-scale applications.


Assuntos
Aciltransferases , Escherichia coli , Glutamina , Aminoácidos , Dipeptídeos
16.
Sci Rep ; 7: 44190, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28367963

RESUMO

The microbial contamination of central air conditioning system is one of the important factors that affect the indoor air quality. Actual measurement and analysis were carried out on microbial contamination in central air conditioning system at a venue in Dalian, China. Illumina miseq method was used and three fungal samples of two units were analysed by high throughput sequencing. Results showed that the predominant fungus in air conditioning unit A and B were Candida spp. and Cladosporium spp., and two fungus were further used in the hygrothermal response experiment. Based on the data of Cladosporium in hygrothermal response experiment, this paper used the logistic equation and the Gompertz equation to fit the growth predictive model of Cladosporium genera in different temperature and relative humidity conditions, and the square root model was fitted based on the two environmental factors. In addition, the models were carried on the analysis to verify the accuracy and feasibility of the established model equation.


Assuntos
Ar Condicionado , Microbiologia do Ar , Candida , Cladosporium , Umidade , Candida/classificação , Candida/isolamento & purificação , China , Cladosporium/classificação , Cladosporium/isolamento & purificação
17.
Biotechnol Biofuels ; 10: 79, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28360937

RESUMO

BACKGROUND: Bioethanol from lignocellulosic materials is of great significance to the production of renewable fuels due to its wide sources. However, multiple inhibitors generated from pretreatments represent great challenges for its industrial-scale fermentation. Despite the complex toxicity mechanisms, lignocellulose-derived inhibitors have been reported to be related to the levels of intracellular reactive oxygen species (ROS), which makes oxidoreductase a potential target for the enhancement of the tolerance of yeasts to these inhibitors. RESULTS: A typical 2-Cys peroxiredoxin from Kluyveromyces marxianus Y179 (KmTPX1) was identified, and its overexpression was achieved in Saccharomyces cerevisiae 280. Strain TPX1 with overexpressed KmTPX1 gene showed an enhanced tolerance to oxidative stresses. Serial dilution assay indicated that KmTPX1 gene contributed to a better cellular growth behavior, when the cells were exposed to multiple lignocellulose-derived inhibitors, such as formic acid, acetic acid, furfural, ethanol, and salt. In particular, KmTPX1 expression also possessed enhanced tolerance to a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. The maximum glucose consumption rate and ethanol generation rate in KmTPX1-expressing strain were significantly improved, compared with the control. The mechanism of improved tolerance to FAF depends on the lower level of intracellular ROS for cell survival under stress. CONCLUSION: A new functional gene KmTPX1 from K. marxianus is firstly associated with the enhanced tolerance to multiple lignocellulose-derived inhibitors in S. cerevisiae. We provided a possible detoxification mechanism of the KmTPX1 for further theoretical research; meanwhile, we provided a powerful potential for application of the KmTPX1 overexpressing strain in ethanol production from lignocellulosic materials.

18.
J Biol Chem ; 292(7): 2660-2669, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28057755

RESUMO

In eukaryotic cells, two conserved protein kinases, Gcn2 and TOR complex 1 (TORC1), couple amino acid conditions to protein translation. Gcn2 functions as an amino acid sensor and is activated by uncharged tRNAs that accumulate when intracellular amino acids are limited. Activated Gcn2 phosphorylates and inhibits eukaryotic initiation factor-2α (eIF2α), resulting in repression of general protein synthesis. Like Gcn2, TORC1 is also involved in sensing amino acid conditions. However, the underlying mechanism remains unclear. In the present study, we show that TORC1 is a direct target of Gcn2 kinase in the yeast Saccharomyces cerevisiae In response to amino acid starvation, Gcn2 binds to TORC1 and phosphorylates Kog1, the unique regulatory subunit of TORC1, resulting in down-regulation of TORC1 kinase activity. In the absence of Gcn2, TORC1 signaling activity increases and becomes unresponsive to amino acid starvation. Our findings demonstrate that TORC1 is an effector of Gcn2 in amino acid signaling, hence defining a novel mechanism by which TORC1 senses amino acid starvation.


Assuntos
Aminoácidos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação para Baixo , Ligação Proteica
19.
Sheng Wu Gong Cheng Xue Bao ; 31(5): 670-81, 2015 May.
Artigo em Chinês | MEDLINE | ID: mdl-26571688

RESUMO

To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.


Assuntos
Glicosídeo Hidrolases/química , Kluyveromyces/enzimologia , Glicosídeo Hidrolases/genética , Microbiologia Industrial , Inulina , Kluyveromyces/genética , Pichia , Sacarose , Temperatura
20.
Biotechnol Biofuels ; 8: 115, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26273319

RESUMO

BACKGROUND: Ethanol production from non-crop materials, such as Jerusalem artichokes, would make a great contribution to the energy industry. The non-conventional yeast, Kluyveromyces marxianus, is able to carry out ethanol fermentation of sugar molecules obtained from inulin-containing materials by consolidated bioprocessing. Lower inulin concentrations and micro-aeration can lead to a relatively fast and ideal fermentation process; however, it is unclear what causes the inhibition of higher concentrations of inulin and the promotion effect of aeration. RESULTS: Next-generation sequencing technology was used to study the global transcriptional response of K. marxianus Y179 under three fermentation conditions, including 120 g/L inulin without aeration (120-N), 230 g/L inulin without aeration (230-N), 230 g/L inulin with aeration by ORP controlling at -130 mV (230-130mV). A total of 35.55 million clean reads were generated from three samples, of which 4,820 predicted that open reading frames were annotated. For differential expression analysis, 950 and 1,452 differentially expressed genes were discovered under the conditions of 230-130mV and 120-N, respectively, and the sample 230-N was used as the control. These genes are mainly associated with the pathways of central carbon metabolism and ethanol formation. Increased expression of inulinase and the low activity of the autophagy-related gene, ATG8, ensured fast and ideal fermentation processes. CONCLUSIONS: Despite being reported as the "crabtree-negative" species, K. marxianus Y179 could achieve favorable ethanol fermentation profiles under micro-aeration and high inulin concentrations. K. marxianus Y179 cells responded to inulin concentrations and micro-aeration that is involved in the whole ethanol metabolism network. These results will serve as an important foundation for further exploration of the regulatory mechanisms involved in ethanol fermentation from inulin by consolidated bioprocessing and also provide a valuable reference for future studies on optimization and reconstruction of the metabolism network in K. marxianus.

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