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1.
Formation of xylitol, a byproduct from xylose fermentation, is a major limiting factor in ethanol production from xylose in
engineered Zymomonas strains, yet the postulated xylose reductase remains elusive. We report here the discovery of xylose reductase in Zymomonas mobilis and, for the first time, to associate the enzyme function with its gene. Besides xylose and xylulose, the enzyme was active
towards benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, exhibiting nearly 150-times higher affinity with
benzaldehyde than xylose. The discovery of xylose reductase paves the way for further improvement of xylose fermentation in
Z. mobilis. The enzyme may also be used to mitigate toxicity of furfural and other inhibitors from plant biomass. 相似文献
2.
Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose
mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on
an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial
xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed)
and xylose conversion efficiency of 42.8%. 相似文献
3.
Zhang B Zhang L Wang D Gao X Hong J 《Journal of industrial microbiology & biotechnology》2011,38(12):2001-2010
Kluyveromyces marxianus is thermotolerant yeast that is able to utilize a wider range of substrates and has greater thermal tolerance than most other
yeast species. K. marxianus can assimilate xylose, but its ability to produce ethanol from xylose in oxygen-limited environments is poor. In the present
study, the K. marxianus xylose reductase (KmXR) gene (Kmxyl1) was cloned and the recombinant enzyme was characterized to clarify the factors that limit xylose fermentation in K. marxianus NBRC1777. KmXR is a key enzyme in the xylose metabolism of K. marxianus, which was verified by disruption of the Kmxyl1 gene. The Km of the recombinant KmXR for NADPH is 65.67 μM and KmXR activity is 1.295 U/mg, which is lower than those of most reported
yeast XRs, and the enzyme has no activity with coenzyme NADH. This result demonstrates that the XR from K. marxianus is highly coenzyme specific; combined with the extremely low XDH activity of K. marxianus with NADP+, the limitation of xylose fermentation is due to a redox imbalance under anaerobic conditions and low KmXR activity. 相似文献
4.
Anna Haldrup Michael Noerremark Finn Thyge Okkels 《In vitro cellular & developmental biology. Plant》2001,37(2):114-119
Summary The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on
xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus
35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection
was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants
selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase
system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based
system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example
of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive.
The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic
or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that
is generally recognized as safe for use in the starch industry. 相似文献
5.
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
6.
Gabriel de Souza Colombo Isis Viana Mendes Betúlia de Morais Souto Cristine Chaves Barreto Luana Assis Serra Eliane Ferreira Noronha Nádia Skorupa Parachin João Ricardo Moreira de Almeida Betania Ferraz Quirino 《Letters in applied microbiology》2022,74(6):941-948
The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions. 相似文献
7.
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
8.
L. I. Sapunova I. O. Tamkovich A. G. Lobanok 《Applied Biochemistry and Microbiology》2010,46(4):400-404
The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject
to catabolite repression, was inhibited by xylulose, an intermediate product of xylose utilization and the final product of
its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely
repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated
with its influence on the catalytic activity of the enzyme. 相似文献
9.
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
10.
Bärbel Hahn-Hägerdal Sissi Berner Kerstin Skoog 《Applied microbiology and biotechnology》1986,24(4):287-293
Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h. 相似文献
11.
M. Y. Tucker M. P. Tucker M. E. Himmel K. Grohmann S. M. Lastick 《Biotechnology letters》1988,10(2):79-84
Summary Xylose isomerase was purified from a transformedE. coli strain (LE392-pRK248/pTXI-1) (Lastick et al., 1986) that overproduces the enzyme by induction of the strong lambda PL promotor. Kinetic data, N-terminal sequence analysis, SDS polyacrylamide gel electrophoresis, size exclusion chromatography and immunodiffusion were used to compare the overproduced enzyme with xylose isomerase purified from xylose induced, non-transformedE. coli LE392 cells; no differences between these purified enzyme preparations were found. 相似文献
12.
The thermophilic mouldMalbranchea pulchella var.sulfurea TMD-8 produced extracellular xylanases in wheat straw hemicellulose as well as wheat straw. This mould utilized xylose less efficiently than glucose. Mycelial extracts contained xylose isomerase, xylose reductase, and xylitol dehydrogenase. Xylose isomerase was less thermostable than that from other microorganisms. However, xylitol dehydrogenase and xylose reductase were relatively more thermostable in comparison with these enzymes from other microorganisms. The affinity of xylose isomerase for xylose was very high (Km 10mM), while that of xylose reductase was low (Km 23.5mM). The xylitol dehydrogenase exhibited relatively high affinity for xylitol (Km 0.02mM). The activity of this enzyme, however, declined steeply, in the alkaline range. This is the first report on the occurrence of three intracellular enzymes, xylose isomerase, xylose reductase, and xylitol dehydrogenase in a thermophilic mould, which play an important role in xylose metabolism. 相似文献
13.
Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg2+ whereas 2 mM Co2+ afforded protection against inactivation by heat. The K
m
for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose. 相似文献
14.
Shinkawa S Okano K Yoshida S Tanaka T Ogino C Fukuda H Kondo A 《Applied microbiology and biotechnology》2011,91(6):1537-1544
In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation
ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and
acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant
strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although
its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield
of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose. 相似文献
15.
Takahiro Yukawa Takahiro Bamba Mami Matsuda Takanobu Yoshida Kentaro Inokuma Jungyeon Kim Jae Won Lee Yong-Su Jin Akihiko Kondo Tomohisa Hasunuma 《Biotechnology and bioengineering》2023,120(2):511-523
To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast. 相似文献
16.
Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension
in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml)
and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated
as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production
stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer
of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first
10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of α-arabinofuranosidase induction was very similar
in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile
for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different
profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases. 相似文献
17.
18.
L Lama B Nicolaus V Calandrelli I Romano R Basile A Gambacorta 《Journal of industrial microbiology & biotechnology》2001,27(4):234-240
Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular
mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K
m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C.
Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for
its activity and thermal stability. Mn2+, Co2+ or Mg2+ were of comparable efficiency for xylose isomerase reaction, while Mg2+ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240.
Received 18 March 2001/ Accepted in revised form 03 July 2001 相似文献
19.
Sigrun D. Feldmann Hermann Sahm Georg A. Sprenger 《Applied microbiology and biotechnology》1992,38(3):354-361
The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. In cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640 mU/mg), phosphoribose isomerase (1600 mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO2 and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly.
Correspondence to: G. Sprenger 相似文献
20.
Bacillus sp. 11-IS, a strain of thermophilic acidophilic bacteria, produced an extracellular xylanase during growth on xylan. The enzyme purified from the culture supernatant solution was homogeneous on disc-gel electrophoresis. The molecular weight was calculated to be 56,000 by SDS-gel electrophoresis. The enzyme had a pH optimum for activity at 4.0, and its stability range was pH 2.0 ~ 6.0. The temperature optimum was 80°C (10-min assay); however, the enzyme retained full activity after incubation at 70°C for 15 min. The enzyme acted on carboxymethyl cellulose (CMC) and cellulose, as well as on xylan. The Michaelis constants for larchwood xylan and CMC were calculated to be 1.68 mg xylose eq/ml and 0.465 mg glucose eq/ml, respectively. The predominant hydrolysis products from larchwood xylan were xylobiose, xylotriose, and xylose; the release of arabinose from rice-straw arabinoxylan was not detected. CMC was cleaved to cellobiose and larger oligosaccharides. Thus, the enzyme is considered to be an endoenzyme which degrades the β-1,4-glycosyl linkages in xylan and cellulose. 相似文献