Effect of furfural addition on validamycin-A production in fermentation of Streptomyces hygroscopicus 5008

Furfural is one of main inhibitors in hemicellulose hydrolysates such as xylose mother liquor, but its positive effect on the production of validamycin-A (VAL-A), a widely used agricultural antibiotic, was interestingly found in fermentation of Streptomyces hygroscopicus 5008. The furfural level in medium up to 1 g/L was effectively converted to furfuryl alcohol and furoic acid by the microorganism. Both intracellular H₂O₂ level and ValG enzyme activity of the cells were enhanced by furfural addition. Xylose mother liquor medium with supplementation of about 1 g/L furfural could enhance the VAL-A titer by 39 %. This work is helpful to VAL-A fermentation using the hemicellulose hydrolysate.     

Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium

Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.     

A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp. EC 10

beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form of carbon catabolite repression by glycerol. beta-Xylosidase and xylose isomerase copurified strictly, and the preparation was found homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A. Streptomyces sp. produced apparently a bifunctional beta-xylosidase-xylose isomerase enzyme. The molecular weight of the enzyme was measured to be 163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits. The Streptomyces sp. beta-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5 and 45 degrees C. The xylose isomerase optimal temperature was 70 degrees C and maximal activity was observed in a broad range pH (5-8). Enhanced saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action. The first 35 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced by other microorganisms but not with other published N-terminal sequences of beta-xylosidases.     

Comparison of beta-Glucosidase Activities in Different Streptomyces Strains

Cellobiase (beta-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of beta-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the beta-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.     

Discovery and characterization of a xylose reductase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> ZM4

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.     

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O₂ limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O₂ supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O₂ supply.Correspondence to: T. Yoshida     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.     

The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures

The kinetics in batch culture of the acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The fastest initial growth and transition from an acid to a solvent metabolism occurs on glucose, with a final 62 g/L glucose conversion. On xylose, an initial slower growth rate and a longer metabolic transition result in higher cellular and acids concentration, thus in a level of fermented sugar limited to 47 g/L. Batch fermentations on mixtures of glucose and xylose show that both sugars can be fermented, with a higher rate for glucose. However, xylose fermentation is inducible and inhibited at glucose level above 15 g/L. Mixtures of glucose and xylose yield the highest amount of fermented sugars, up to 68 g/L, as a result of both a fast metabolic transition on glucose and a strong acid reconsumption on xylose. In all cases, solvent production is triggered at a total acid concentration between 4 and 5 g/L, whereas the final inhibition of the fermentation takes place at a total butanol and acid concentration between 18 and 20 g/L.     

Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli

The production of ethanol from xylose by ethanologenic Escherichia coli strain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of native E. coli alcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.     

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD?-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l?1 h?1 xylose consumption rate, 0.25 g l?1 h?1 ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.     

Production of Glucose Isomerase by Streptomyces flavogriseus

A microorganism that produces glucose isomerase was isolated from soil and identified as a strain of Streptomyces flavogriseus. The organism produced a large quantity of glucose isomerase when grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of ryegrass straw. The organism produced glucose isomerase both intra- and extra-cellularly. The highest level of intracellular glucose isomerase (3.5 U/ml) was obtained in about 36 h by a culture grown on straw hemicellulose; the extracellular enzyme (1.5 U/ml) appeared in cultures grown for about 72 h. About equal levels of enzyme were produced in cultures grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of straw, but production of the enzyme was drastically reduced when the organism was grown on other carbon sources. As a nitrogen source, corn steep liquor produced the best results. Soy flour extract, yeast extract, and various peptones also were adequate substrates for glucose isomerase production. Addition of Mg²⁺, Mn²⁺, or Fe²⁺ to the growth medium significantly enhanced enzyme production. The organism, however, did not require Co²⁺, which is commonly required by microorganisms used in the production of glucose isomerase.     

Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.     

Production of transglutaminase by Streptomyces isolates in solid-state fermentation

Aims:  To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates.
Methods and Results:  Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50°C. Molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size (∼37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities.
Conclusions:  Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase.
Significance and Impact of the Study:  Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.     

7号淀粉酶链霉菌M1033木糖异构酶基因序列分析

测定了来自海南的7号淀粉酶M1033木糖异构酶(Ⅺ)基困的DNA序列。：该酶的结构基因由l161bp组成,相当于387个氨基酸残基。其GC含量为72．1克分子％,密码子第三位的Gc利用率达98克分子％。在氨基酸序列上,M1033的木糖异构酶与其它放线菌菌株的相比具有较高的同源性;特别是与3种链霉菌菌株的同源性高达90%左右。     

Characterization of Bioethanol Production from Hexoses and Xylose by the White Rot Fungus Trametes versicolor

Bioethanol production by white rot fungus (Trametes versicolor), identified from fungal mixture in naturally decomposing wood samples, from hexoses and xylose was characterized. Results showed that T. versicolor can grow in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and only xylose. Xylose was efficiently fermented to ethanol in media containing mixtures of hexoses and xylose, such as MBMC and G11XY11 media (Table?1), yielding ethanol concentrations of 20.0 and 9.02?g/l, respectively, after 354?h of hypoxic culture. Very strong correlations were found between ethanolic fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) after 354?h in culture in MBMC medium. In a medium (G11XY11) containing a 1:1 glucose/xylose ratio, fermentation efficiency of total sugars into ethanol was 80% after 354?h.     

Metabolic engineering of the initial stages of xylose catabolism in yeasts for construction of efficient producers of ethanol from lignocelluloses

Plant biomass possesses a huge potential as a source for biofuel production. The main components of biomass are glucose and five-carbon sugar xylose. The yeast Saccharomyces cerevisiae that is used for industrial ethanol production from glucose is unable to xylose fermentation. Therefore a microorganism capable for efficient fermentation of both glucose and xylose has to be found in nature or constructed for economically feasible biomass conversion to ethanol. The active xylose fermentation could be performed by increasing the efficiency of initial stages of xylose metabolism. In this review the enzymes of initial stages of xylose metabolism in yeasts (xylose reductase, xylitol dehydrogenase, xylulokinase) and bacteria (xylose isomerase and xylulokinase) are characterized. The ways for construction of yeast strains capable of efficient alcoholic xylose fermentation are discussed.     

Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry

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