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1.
Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulatexyl operon expression on diauxic growth and expression of axylA-lacZ fusion.xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation ofxylR yields a two-fold increase in expression ofxylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. WhenxylR andcre are inactivated together a residual two-fold repression ofxylA is found. Inactivation ofxylR affects diauxic growth by shortening the lag phase from 70 to 40 min. In-frame deletion ofccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivatedcre site inxylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously. 相似文献
2.
Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing
cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi
and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant
amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced
BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation.
Although xylitol inhibited in vitro BsXI activity significantly (K
I = 5.1 ± 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when
xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation
by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro. 相似文献
3.
4.
Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40?min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously. 相似文献
5.
Thierry Marcel Daniel Drocourt Gérard Tiraby 《Molecular & general genetics : MGG》1987,208(1-2):121-126
Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis. 相似文献
6.
We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation.
The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient)
strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism
approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore
holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates. 相似文献
7.
Summary Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase (xylA), xylulokinase (xylB), and regulatory (xylR) or transport (xylT) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10–15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp BglII fragment and a 2.6 kbp HindIII-SalI fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene. 相似文献
8.
Xylose/glucose isomerases are important industrial enzymes that are most widely used in food industries; however, their previously reported expression levels do not meet the requirements for industrial application. Here, an antibiotic resistance marker (ARM)‐free system driven by ribosomal RNA (rRNA) promoters is developed to obtain high‐level xylose/glucose isomerase (XI/GI) expression in Streptomyces rubiginosus (S. rubiginosus). The rRNA promoter rrnD yields the highest glucose isomerase production titer of XIs/GIs, which is eight times higher than that of ermEp* and 2.6 times higher than that of kasOp*. The integrated ARM gene is removed by further introduction of the Cre plasmid with a temperature‐sensitive replicon. The production titer of XIs/GIs is further improved by replacing the xylR gene with an additional expression glucose isomerase cassette at the xylR locus. Ultimately, the glucose isomerase activity reaches up to 79.7 ± 7.5 U mL?1 at 96 h. The results support the robustness and stability of XI/GI production with this ARM‐free system using optimal ribosomal promoters in S. rubiginosus, demonstrating strong potential in large‐scale industrial applications. Besides, the results imply that rRNA promoters are strong promoters that can be used for protein engineering or metabolic engineering. 相似文献
9.
Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry 总被引:20,自引:0,他引:20
A new method for the selection of transgenic plants has been developed. It is based upon selection of transgenic plant cells
expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose. The xylose isomerase selection system was tested in potato
and the transformation frequency was found to be approximately ten fold higher than with kanamycin selection. The level of
enzyme activity in the transgenic plants selected on xylose was 5- to 25-fold higher than the enzyme activity in control plants.
Potato transformants were stable over two generations in Southern blotting analysis. This novel selection system is more efficient
than the traditionally used kanamycin-based selection systems. In addition, the xylose isomerase system is independent of
antibiotic or herbicide resistance genes, but depends on an enzyme that is generally recognized as safe for use in the starch
industry and which is already being widely utilized in specific food processes.
Received: 13 August 1997 / Revision received: 26 November 1997 / Accepted: 15 December 1997 相似文献
10.
Gopinath V Meiswinkel TM Wendisch VF Nampoothiri KM 《Applied microbiology and biotechnology》2011,92(5):985-996
Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants
expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived
from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or
when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations
and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and
xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates
were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production
and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The
ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate
as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock
for large-scale amino acid production. 相似文献
11.
Anjali Madhavan Sriappareddy Tamalampudi Kazunari Ushida Daisuke Kanai Satoshi Katahira Aradhana Srivastava Hideki Fukuda Virendra S. Bisaria Akihiko Kondo 《Applied microbiology and biotechnology》2009,82(6):1067-1078
The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in
Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family
II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular
mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the
enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of
the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose. 相似文献
12.
Harhangi HR Akhmanova AS Emmens R van der Drift C de Laat WT van Dijken JP Jetten MS Pronk JT Op den Camp HJ 《Archives of microbiology》2003,180(2):134-141
The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min–1mg protein-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials. 相似文献
13.
Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase. 总被引:8,自引:4,他引:4
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G C Saari A A Kumar G H Kawasaki M Y Insley P J O''''Hara 《Journal of bacteriology》1987,169(2):612-618
The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp. strain 3876, a gram-positive bacterium, has been determined. A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain. One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety. There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase. The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides. The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons. The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids. The xylose isomerase from Ampullariella sp. strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E. coli, Bacillus subtilis, and Streptomyces violaceus-ruber. In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase. We present evidence suggesting that in Ampullariella sp. strain 3876 these genes are similarly arranged. 相似文献
14.
B. Christien Lokman Pieter van Santen Jan C. Verdoes Jaap Krüse Rob J. Leer Mark Posno Peter H. Pouwels 《Molecular & general genetics : MGG》1991,230(1-2):161-169
Summary A cluster of three genes involved in d-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis
d-xylose isomerase (68% and 77%, respectively), and to E. coli
d-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment d-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of d-xylose isomerases of different bacteria suggests that L. pentosus
d-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli
d-xylose isomerase and not to a second similarity group comprising d-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5-xylR (1167 bp, repressor) — xylA (1350 bp, D-xylose isomerase) — xylB (1506 bp, d-xylulose kinase) — 3 is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB. 相似文献
15.
Byoung-Chan Kim Sun Nyoung Yu Kwang Youn Kim Jung Sook Lee Yu-Ryang Pyun Soon Cheol Ahn 《Biotechnology letters》2010,32(7):929-933
The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95°C. The enzyme required divalent cations including Zn2+ for its maximal activity and thermostability. 相似文献
16.
Aims: The production of aureofuscin is very low in the wild‐type strain. We attempt to increase the production of aureofuscin by over‐expression of a controlling gene in the wild‐type strain. Methods and Results: The aurj3M gene was PCR‐amplified from Streptomyces aureofuscus SYAU0709, ligated into vector pMD19 and sequenced. The predicted translation of the 579‐bp cloned fragment was 97% similar to pimM from Streptomyces natalensis, which has an N‐terminal PAS domain and a LuxR‐type C‐terminal helix–turn–helix. Recombinant bacterial strains were constructed by transforming SYAU0709 with an expression plasmid (pBJJ3M) that contained aurj3M, thereby increasing the number of aurj3M gene copies. Conclusions: Bioassays for the antibiotic compound aureofuscin indicated that the recombinant bacteria had greater antifungal activity than the wild‐type strain. Specifically, the recombinant strain produced approx. 600% more aureofuscin, as quantified by high‐performance liquid chromatography analysis. Significance and Impact of the Study: To our knowledge, this approach has not been attempted in S. aureofuscus before and few genes in the aureofuscin pathway have been cloned and characterized. 相似文献
17.
Monia Mezghani Mohamed Ali Borgi Radhouane Kammoun Hedi Aouissaoui Samir Bejar 《Enzyme and microbial technology》2005,37(7):735-738
In order to over express the xylA gene of Streptomyces sp. SK strain, it was cloned under the control of the constitutive ermE-up promoter. This construct was integrated through site-specific recombination process into the chromosome of a Streptomyces violaceoniger glucose isomerase deficient strain using the non-replicative vector pTS55. The resulting CBS4 strain shows a perfect stability in the absence of selection pressure. Its glucose isomerase activity was about four and nine-fold greater, than that obtained from Streptomyces sp. SK, respectively fully induced or not by xylose. 相似文献
18.
19.
The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivations on single carbon sources, it was demonstrated that xylose acted as a carbon catabolite repressor (xylose cultivations), while the enzymes in the xylose utilisation pathway were also subject to repression in the presence of glucose (glucose cultivations). In the wild type strain growing on the sugar mixture, glucose repression of xylose utilisation was observed; with xylose utilisation occurring only after glucose was depleted. This phenomenon was not seen in the creA deleted strain, where glucose and xylose were catabolised simultaneously. Measurement of key metabolites and the activities of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression had been relieved. Xylose was both a repressor and an inducer of xylanases at the same time. The creA mutation seemed to have pleiotropic effects on carbohydratases and carbon catabolism. 相似文献