共查询到20条相似文献,搜索用时 15 毫秒
1.
Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol 总被引:1,自引:0,他引:1
Schoondermark-Stolk SA Tabernero M Chapman J Ter Schure EG Verrips CT Verkleij AJ Boonstra J 《FEMS yeast research》2005,5(8):757-766
Branched-chain amino acids (BCAAs) are key substrates in the formation of fusel alcohols, important flavour components in fermented foods. The first step in the catabolic BCAA degradation is a transaminase step, catalyzed by a branched-chain amino acid transaminase (BCAAT). Saccharomyces cerevisiae possesses a mitochondrial and a cytosolic BCAAT, Bat1p and Bat2p, respectively. In order to study the impact of the BCAATs on fusel alcohol production derived from the BCAA metabolism, S. cerevisiae BCAAT-deletion mutants were constructed. The BCAA l-leucine was exogenously supplied during cultivations with mutants of S. cerevisiae. BAT1 deletion is not essential for fusel alcohol production, neither under glucose nor under ethanol growth conditions. The 3-methyl-1-butanol production rate of bat1Delta-cells on ethanol was decreased in comparison with that of wild-type cells, but the cells were still able to produce 3-methyl-1-butanol. However, drastic effects in fusel alcohol production were obtained in cells lacking BAT2. Although the constructed bat2Delta-single deletion strain and the bat1Deltabat2Delta-double deletion strain were still able to produce 3-methyl-1-butanol when grown on glucose, they were incapable of producing any 3-methyl-1-butanol when ethanol was the sole carbon source available. In the circumstances used, gene expression analysis revealed a strong upregulation of BAT2 gene activity in the wild type, when cells grew on ethanol as carbon source. Apparently, the carbon metabolism is able to influence the expression of BCAATs and interferes with the nitrogen metabolism. Furthermore, analysis of gene expression profiles shows that the expression of genes coding for other transaminases present in S. cerevisiae was influenced by the deletion of one or both BCAATs. Several transaminases were upregulated when a BCAAT was deleted. Strikingly, none of the known transaminases was significantly upregulated when BAT2 was deleted. Therefore we conclude that the expression of BAT2 is essential for 3-methyl-1-butanol formation on the non-fermentable carbon source, ethanol. 相似文献
2.
Schoondermark-Stolk SA Jansen M Veurink JH Verkleij AJ Verrips CT Euverink GJ Boonstra J Dijkhuizen L 《Applied microbiology and biotechnology》2006,70(2):237-246
Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved.
To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening
and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of
the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol
concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation,
a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions).
A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven
genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e.
BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification
of interesting target genes for improvement of yeast strains used in industrial processes. 相似文献
3.
Gemma Beltran Nicolas Rozès Albert Mas José M. Guillamón 《World journal of microbiology & biotechnology》2007,23(6):809-815
The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen
regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth
rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less
ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced
from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity
and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent
causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable
interest for wine making. 相似文献
4.
Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant
to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type
or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was
much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells. 相似文献
5.
Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae 总被引:5,自引:0,他引:5
Lindahl AL Olsson ME Mercke P Tollbom O Schelin J Brodelius M Brodelius PE 《Biotechnology letters》2006,28(8):571-580
The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression
vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the
yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed
for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while
plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced
600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation.
Revisions requested 14 November 2005; Revisions received 17 January 2006 相似文献
6.
7.
Err-Cheng Chan Peter P. Ueng Karri L. Eder Li Fu Chen 《Journal of industrial microbiology & biotechnology》1989,4(6):409-417
Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively. 相似文献
8.
Summary As steroid hormones are known to influence amino acid metabolism we tested the hypothesis that ovariectomy should lead to significant changes in this system.We found that after ovariectomy serum alanine was significantly decreased (p = 0.0006) in contrast to serum glycine and branched chain amino acids (BCAA). The ratio of glycine/BCAA, a parameter for anabolism or catabolism was not changed after ovariectomy. If, however, the amino acid alanine as the link to carbohydrate and lipid metabolism was introduced the alanine/BCAA ratio was significantly altered (p = 0.01).Although serum cholesterol was altered as well (increased,p = 0.03), no significant correlation with alanine was found. We can therefore assume that there are two independent mechanisms for lipid and amino acid changes after ovariectomy.The most prominent finding was that estradiol replacement after ovariectomy restored increased cholesterol levels but did not restore alanine levels. Other ovarial hormones must be incriminated for the regulation of alanine metabolism. The anabolic effects of estradiol as decreasing glycine and BCAA were noticed which rules out insufficient estradiol replacement. 相似文献
9.
Fiki AE Metabteb GE Bellebna C Wartmann T Bode R Gellissen G Kunze G 《Applied microbiology and biotechnology》2007,74(6):1292-1299
The yeast Arxula
adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins.
In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence
of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native
strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature
optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for
the expression of a human serum albumin (HSA) reporter gene. 相似文献
10.
Sulin Cheng Petri Wiklund Reija Autio Ronald Borra Xiaowei Ojanen Leiting Xu Timo T?rm?kangas Markku Alen 《PloS one》2015,10(10)
Background
Fatty liver is a major cause of obesity-related morbidity and mortality. The aim of this study was to identify early metabolic alterations associated with liver fat accumulation in 50- to 55-year-old men (n = 49) and women (n = 52) with and without NAFLD.Methods
Hepatic fat content was measured using proton magnetic resonance spectroscopy (1H MRS). Serum samples were analyzed using a nuclear magnetic resonance (NMR) metabolomics platform. Global gene expression profiles of adipose tissues and skeletal muscle were analyzed using Affymetrix microarrays and quantitative PCR. Muscle protein expression was analyzed by Western blot.Results
Increased branched-chain amino acid (BCAA), aromatic amino acid (AAA) and orosomucoid were associated with liver fat accumulation already in its early stage, independent of sex, obesity or insulin resistance (p<0.05 for all). Significant down-regulation of BCAA catabolism and fatty acid and energy metabolism was observed in the adipose tissue of the NAFLD group (p<0.001for all), whereas no aberrant gene expression in the skeletal muscle was found. Reduced BCAA catabolic activity was inversely associated with serum BCAA and liver fat content (p<0.05 for all).Conclusions
Liver fat accumulation, already in its early stage, is associated with increased serum branched-chain and aromatic amino acids. The observed associations of decreased BCAA catabolism activity, mitochondrial energy metabolism and serum BCAA concentration with liver fat content suggest that adipose tissue dysfunction may have a key role in the systemic nature of NAFLD pathogenesis. 相似文献11.
Traditional healthful fermented products of Japan 总被引:2,自引:0,他引:2
A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan. 相似文献
12.
Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds. 相似文献
13.
RecombinantSaccharomyces cerevisiae expression systems were developed to produce a novel human anti-angiogenic protein called LK8, an 86 amino-acid kringle fragment
protein with three disulfide linkages. Galactose-inducible LK8 expression plasmid was constructed, and LK8 production levels
by fourS. cerevisiae strains were compared in order to select an optimal host strain.S. cerevisiae 2805 was the most efficient among the strains tested. Elevating the LK8 gene copy number through multiple integration using
δ-sequences as target sites resulted in more than a two-fold increase in the LK8 production level compared with the plasmid-based
expression system. The maximum LK8 protein concentration of 25 mg/L was obtained from batch cultivation of the yeast transformant
that harbors 16 copies of the LK8 gene. In conclusion, the strain integrated with the multiple LK8 gene secreted the protein
with relatively high yield, although, the increased LK8 gene dosage over 11 copies did not lead to further enhancement in
batch cultivations. 相似文献
14.
Marui J Matsushita-Morita M Tada S Hattori R Suzuki S Amano H Ishida H Yamagata Y Takeuchi M Kusumoto K 《Applied microbiology and biotechnology》2012,93(2):655-669
The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This
enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The
chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor
PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible
for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen
or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously.
Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement
of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible
for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9. 相似文献
15.
K. B. Taylor M. J. Beck D. H. Huang T. T. Sakai 《Journal of industrial microbiology & biotechnology》1990,6(1):29-41
Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by13C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-13C]xylose or [2-13C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-13C]xylose or [1-13C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-13C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions. 相似文献
16.
Ferri S Miura S Sakaguchi A Ishimura F Tsugawa W Sode K 《Marine biotechnology (New York, N.Y.)》2004,6(6):625-632
The gene encoding the fructosyl-amine oxidase (FAOD) from the marine yeast Pichia sp. N1-1 was cloned and expressed in Escherichia coli. Partial amino acid sequence analysis of the Pichia sp. N1-1 FAOD allowed the design of oligonucleotide primers for the amplification of the gene by inverse polymerase chain reaction. The FAOD gene was found to be devoid of introns and to encode a 48-kDa protein composed of 429 amino acid residues. The FAD-binding consensus sequence GXGXXG and the FAD covalent attachment-site cysteine residue have been identified within the predicted amino acid sequence. Comparisons with the amino acid sequences of other eukaryotic FAODs showed only 30% to 40% identities, establishing that the isolated Pichia N1-1 gene encodes a unique FAOD. Recombinant FAOD expression levels in E. coli reached 0.48 U/mg of soluble protein, which is considerably greater than native expression levels by inducing Pichia sp. N1-1 with fructosyl-valine (f-Val). The kinetic properties of the recombinant enzyme were almost indistinguishable from those of the native enzyme. We previously reported on the construction of a number of effective Pichia sp. N1-1 FAOD-based biosensors for measuring f-Val, a model compound for glycated hemoglobin. The further development of these biosensor systems can now greatly benefit from protein engineering and recombinant expression of the FAOD from Pichia N1-1.Note: The previous online version (January 20, 2005) of this article appeared with the legends of Figures 1 and 2 transposed. This version contains the figures with their appropriate legends. 相似文献
17.
The branched chain amino acids (BCAAs), l-valine, l-leucine, and l-isoleucine, have recently been attracting much attention as their potential to be applied in various fields, including animal
feed additive, cosmetics, and pharmaceuticals, increased. Strategies for developing microbial strains efficiently producing
BCAAs are now in transition toward systems metabolic engineering from random mutagenesis. The metabolism and regulatory circuits
of BCAA biosynthesis need to be thoroughly understood for designing system-wide metabolic engineering strategies. Here we
review the current knowledge on BCAAs including their biosynthetic pathways, regulations, and export and transport systems.
Recent advances in the development of BCAA production strains are also reviewed with a particular focus on l-valine production strain. At the end, the general strategies for developing BCAA overproducers by systems metabolic engineering
are suggested. 相似文献
18.
19.
Ae-Young Mo Seung-Moon Park Yun-Sik Kim Moon-Sik Yang Dae-Hyuk Kim 《Biotechnology and Bioprocess Engineering》2005,10(6):576-581
Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus
pollution of animal waste. We have engineered the cell surface of the yeast,Saccharomyces cerevisiae by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional
functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) ofAspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast α-agglutinin,
a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced
intoS. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate
assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin
of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium
when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast
and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be
16,000 molecules per cell. 相似文献
20.
S. Sakamoto I. Terada M. Iijima H. Matsuzawa T. Ohta 《Applied microbiology and biotechnology》1994,42(4):569-574
Escherichia coli TG1, transformed with an expression plasmid pAQN carrying the aqualysin I (AQI) gene derived from Thermus aquaticus YT-1 under the control of the tac promoter, was cultivated under various conditions in order to find fermentation conditions for the efficient production of the thermophilic protease, AQI. The amount of AQI produced was closely related to the growth phase at the time of isopropyl--d-thiogalactopyranoside (IPTG) induction, and the highest production was obtained when it was added during the exponential growth phase. The addition of yeast extract had a greater effect on AQI production than did Polypeptone or casamino acids, and AQI productivity increased from 1.1 × 103 kU/g to 2.7 × 103 kU/g cells when 2 g/l yeast extract was supplied. Furthermore, the specific growth rate improved from 0.35 h–1 to 0.89 h–1 when 5 g/l yeast extract was supplied. The culture temperature also affected AQI gene expression. When the temperature was shifted from 37°C to 34°C at the time of IPTG induction, 19 kU/ml enzymatically active AQI was obtained, corresponding to a 28% increase over the amount produced in a batch culture without a shift. This is about a 44-fold higher yield than was obtained from the original strain, T. aquaticus YT-1. 相似文献