Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum.

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.     

Temperature-dependent dimorphism of the yeastArxula adeninivorans Ls3

Arxula adeninivorans Ls3 is described as an ascomycetous, arthroconidial, anamorphic, xerotolerant yeast, which was selected from wood hydrolysates in Siberia. By using minimal salt medium or yeast-extract-peptone-medium with glucose or maltose as carbon source it was shown that this yeast is able to grow at up to 48° C. Increasing temperatures induce changes in morphology from the yeast phase to mycelia depending on an altered programme of gene expression. This dimorphism is an environmentally conditioned (reversible) event and the mycelia can be induced at a cultivation temperature of 45° C. Depending on the morphology of strain Ls3 (yeast phase or mycelia) the secretion behaviour as well as the spectrum of polypeptides accumulated in the culture medium changed. The activities of the accumulated extracellular enzymes glucoamylase and invertase were 2 to 3 times higher in cultures grown at 45° C than in those grown at 30° C. While the level of the glucoamylase protein secreted from mycelia between 45 and 70 hours did not change, biochemical activity decreased after a cultivation time of 43 hours. It was shown that this effect depended on both the catabolic repression of the glucoamylase by glucose and the thermal inactivation of this enzyme in media without or with low concentrations of starch or maltose.     

Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.     

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 )h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.     

黑曲霉糖化酶基因启动子功能鉴定

从糖化酶工业生产菌株Aspergillus nigerCICIM F0410基因组DNA中扩增糖化酶基因启动子(PglaA),并将该启动子替换质粒pRS303K上KmR基因启动子,构建成糖化酶基因启动子功能检测质粒pRS-PglaA-KmR。将pRS-PglaA-KmR转入E.coliJM109中,得到重组菌E.coli(pRS-PglaA-KmR)。通过对重组菌的氨基糖苷磷酸转移酶基因活性检测,表明PglaA在E.coli中具有驱动KmR基因表达的活性。采用不同诱导物进行培养发现,葡萄糖、蔗糖、乳糖、麦芽糖或玉米淀粉,可以不同程度增强PglaA的强度。     

Influence of carbohydrates and polyols on l-lactic acid production and fatty acid formation by Rhizopus arrhizus

Growth and l-lactic acid production on 24 different carbohydrates and polyols (glycerol, mannitol and sorbitol) by Rhizopus arrhizus CCM 8109 were determined. The d- but not the l-forms of xylose, fructose, galactose, mannose, glucose, cellobiose, maltose and sucrose and partially hydrolysed starch were converted to l-lactic acid. Changes in lipid formation and fatty acid composition were detected in biomass grown on the different sugars. In the presence of polyols, growth and considerable production of lipids were observed with little or no lactate production. Invertase was mainly associated with the mycelium during growth on sucrose, whereas glucoamylase and -amylase were produced extracellularly during growth on starch.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Polytechnical University, Radlinského 9, SK-812 37 Bratislava, Slovak Republic     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

The distribution of carbohydrate side chains along the polypeptide chain of glucoamylase

Glucoamylase is a starch-hydrolyzing enzyme with a glycoprotein structure, used industrially for the conversion of starch to glucose, citric acid, corn syrups, and high-fructose sweeteners. This enzyme possesses an unusual type of structure in which many carbohydrate side chains are linked O-glycosidically to serine and threonine residues of the polypeptide chain. The carbohydrate side chains may be single monosaccharide residues or oligosaccharides of mannose, glucose, galactose, and in some cases N-acetylglucosamine. New data from experiments on the CNBr fragmentation of glucoamylase followed by chemical and immunological characterization of the fragments show that the carbohydrate side chains are distributed randomly along the polypeptide chain. Such a structure is appropriately termed a random model reprensentation for the glucoamylase molecule.     

α-Glucosidase synthesis in batch and continuous culture ofSaccharomyces cerevisiae

Glucose prevented maltose utilization in batch culture ofSaccharomyces cerevisiae whereas in a mixed carbohydrate-limited system, maltose and glucose were consumed simultaneously. The specific activity of -glucosidase depended on the dilution rate as well as the proportion of maltose in the mixture. The chemostat provides a way of reaching the low residual concentrations of glucose in the broth that are necessary to release catabolite repression and permit maltose induction of -glucosidase.     

Effect of maltose on glucoamylase formation by Aspergillus niger

Low levels of glucoamylase are produced when Aspergillus niger is grown on sorbitol, but substitution of the latter by glucose, maltose, or starch results in greater formation of glucoamylase as measured by enzymatic activity. Both glucoamylase I and glucoamylase II are formed in a yeast extract medium; however, glucoamylase I appears to be the only form produced when ammonium chloride is the nitrogen source. Maltose or isomaltose (1.4 x 10(-4)m), but no other disaccharides or monosaccharides, dextrins, dextrans, or starches, stimulated glucoamylase formation when added to mycelia pregrown on sorbitol-ammonium salts. The induction of glucoamylase by maltose was independent of sulfate concentration but showed a dependency on low pH and the absence of utilizable carbon sources.     

Expression of α-amylase in cultured callus of french bean

α-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression. After subculture of the calli, α-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on the enzyme expression. Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose and maltose, to the medium repressed expression. Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced higher α-amylase expression as compared to addition of 3% mannitol. This result suggests that osmotic stress enhances α-amylase activity in the calli. Furthermore, high concentrations of agar in the medium increased α-amylase activity in the calli. It is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the α-amylase activity in the calli.     

Physiological characterization of starch-utilizing <Emphasis Type="Italic">Saccharomyces</Emphasis> sp. SK0704 isolated from kimchi

Saccharomyces sp. SK0704 (further defined as SK0704) isolated from long-term-ripening kimchi was identified by a biochemical method with an API kit; its physiology was found to be very similar to that of S. cerevisiae ATCC 26603 (further defined as ATCC 26603), except in terms of starch utilization. SK0704 did not excrete extracellular glucoamylase, but utilized starch as a sole carbon source under only aerobic conditions. Crude enzyme excreted from SK0704 catalyzed the saccharification of starch to glucose, but ATCC 26603 did not. The PCR product obtained using the chromosomal DNA of SK0704 and the primers designed on the basis of the extracellular glucoamylase-coding gene of S. diastaticus was homologous with the intracellular sporulation-specific glucoamylase of S. cerevisiae. SDS-PAGE pattern of soluble protein extracted from yeast cells grown on glucose was greatly different from that on starch. From these results, we proposed that the SK0704 may have a specific physiological function for starch catabolism such as membrane transport system and intracellular sac-charification of starch.     

Sugar uptake in a 2-deoxy-d-glucose resistant mutant ofSaccharomyces cerevisiae

Summary The non-metabolizable and toxic glucose analogue 2-deoxy-d-glucose (2-DOG) has been widely employed to screen for regulatory mutants which lack catabolite repression. A number of yeast mutants resistant to 2-DOG have recently been isolated in this laboratory. One such mutant, derived from aSaccharomyces cerevisiae haploid strain, was demonstrated to be derepressed for maltose, galactose and sucrose uptake. Furthermore, kinetic analysis of glucose transport suggested that the high affinity glucose transport system was also derepressed in the mutant strain. In addition, the mutant had an increased intracellular concentration of trehalose relative to the parental strain. These results indicate that the 2-DOG resistant mutant is defective in general glucose repression.