Screening and Mutagenesis of Aspergillus niger for the Improvement of Glucose-6-Phosphate Dehydrogenase Production

The Aspergillus niger strain ZBY-7 was selected as the original strain of glucose-6-phosphate dehydrogenase production. After mutagenesis of the strain by means of UV irradiation and nitrosoguanidine, mutants of Aspergillus niger resistant to a certain metabolic inhibitor were obtained. Five of the mutants showed increased glucose-6-phosphate dehydrogenase production. The mutant resistant to antimycin A (Aspergillus niger AM-23) produced the highest level of glucose-6-phosphate dehydrogenase (695.9% of that produced by the original strain).     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger

Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.     

Selective isolation of Aspergillus niger mutants with enhanced glucose oxidase production

After mutagenization and selection, mutant Aspergillus niger strains resistant to certain agents were obtained. Seven of the mutants showed increased extracellular glucose oxidase (GOD), the level for individual cases ranged widely from 8.8 to over 138.5% in comparison with the parental strain. Studies of the relationship between method of selection and frequency of mutation showed that the highest frequency of positive mutations (15.8% and 17.3%) was obtained from mutants resistant to ethidium bromide (1 mmol 1^-1) and sodium gluconate (45%), respectively. The time course of growth and enzyme production by the most active mutant AM-11 showed intra- and extracellular GOD activities to have increased about 2.2- and 2.4-fold, respectively, compared with the parental strain.     

Beta-fructofuranosidase production by 2-deoxyglucose resistant mutants of aspergillus niger in submerged and solid-state fermentation

Aspergillus niger produces extracellular beta-fructofuranosidase under submerged (SmF) and solid state fermentation (SSF) conditions. After UV mutagenesis of conidiospores of A. niger, 2-deoxyglucose (10 g/l) resistant mutants were isolated on Czapek's minimal medium containing glycerol as a carbon source and the mutants were examined for improved production of beta-fructofuranosidase in SmF and SSF conditions. One of such mutant DGRA-1 overproduced beta-fructofuranosidase in both SmF and SSF conditions. In SmF, the mutant DGRA-1 showed higher beta-fructofuranosidase productivity (110.8 U/l/hr) than the wild type (48.3 U/l/hr). While in SSF the same strain produced 322 U/l/hr of beta-fructofuranosidase, 2 times higher than that of wild type (154.2 U/l/hr). In SmF, both wild type and mutants produced relatively low level of beta-fructofuranosidase in medium containing sucrose with glucose than from the sucrose medium. However in SSF, the DGRA-1 mutant grown in sucrose and sucrose+ glucose did not show any difference with respect to beta-fructofuranosidase production. These results indicate that the catabolite repression of beta-fructofuranosidase synthesis is observed in SmF whereas in SSF such regulation was not prominent.     

Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism.

Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis.     

N+注入选育黑曲霉益生菌及其突变菌株产酶条件的研究

以益生菌株黑曲霉AN01为材料,经N 多次诱变得突变益生菌株AN03。结果表明,出发益生菌株AN01酸性蛋白酶、纤维素酶和果胶酶的酶活分别由原来的71.6Ug、141.7Ug和264.8Ug相继提高到996.5Ug、940.4Ug和906.5Ug。突变益生菌株AN03经传5代培养,产酶特性稳定。试验还研究了变突变益生菌株AN03最佳产酶条件,培养基为每升含麸皮105g,玉米芯105g,豆粕105g,氯化铵16g,pH5.0。30℃培养4d。     

制霉菌素抗性筛选高活性苎麻脱胶菌诱变菌株

利用制霉菌素抗性筛选高渗透性突变株,提高黑曲霉菌对苎麻纤维的脱胶能力,使微生物脱胶能用于工业生产实践之中.分别用紫外线、硫酸二乙酯、亚硝酸作为诱变剂对黑曲霉3.0.2菌株进行诱变处理.以制霉菌素抗性为遗传标记,从突变菌株中定向筛选得到一株高活性苎麻脱胶菌黑曲霉3.0.2-26.在以未经刮制的苎麻韧皮为主要碳源,0.7%(NH_4)_2SO_4为氮源,添加00.5%KCl;00.5%MgSO_4;0.1%K_2HPO_4; 0.1%酵母膏;Tween80 0.1%的培养液中,接入黑曲霉3.0.2-26,置30℃下,150 r/min处理30 h左右,脱胶苎麻纤维的残胶率平均为14.43%.     

Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase.

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate-accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn2+ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.     

Ultraviolet mutagenesis of the cellulase producer Aspergillus niger using protoplasts

Abstract Ultraviolet mutagenesis of Aspergillus niger strain O97 was achieved using protoplasts. Protoplasts of A. niger O97 showed the same ultraviolet killing kinetics as intact cells. After mutagenesis, several mutants were found in regenerated collonies. These mutants differ from the original strain in spore colour and cellulase-producing ability. The most active strain, designated 97V3-3 has an altered spore colour, and its carboxymethylcellose-hydrolysing, filter-paper-degradation, cotton-de-gradation and β-glucosidase activities were increased by 45.4%, 19.1%, 28.2% and 18.2%, respectively.     

Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.     

High-level production of the low-calorie sugar sorbitol by Lactobacillus plantarum through metabolic engineering

Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both l- and d-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed.     

Strain improvement of Aspergillus niger for enhanced lipase production

The enhancement of lipase production from Aspergillus niger was attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants were selected on media containing bile salts. Nitrous acid mutants exhibited increased efficiency for lipase production when compared with UV mutants in submerged fermentation. The hyperproducing UV and nitrous acid mutants were further subjected to a second step of mutagenesis to devise an economical and ecofriendly technique for lipase production by the effective use of hydrocarbons. One percent kerosene was found to be optimal for lipase production, and one of the mutant strains NAII exhibited 2.53 times more increased lipase activity than the parental strain did. This investigation indicates a possible role for the A. niger mutant strains in the biodegradation of oil-polluted environments for the development of ecofriendly technologies.     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。     

黑曲霉组成型表面展示南极假丝酵母脂肪酶B及其发酵调控

黑曲霉表面展示南极假丝酵母脂肪酶B(CALB)可有效应用于食品、化妆品、医药等行业。以黑曲霉Aspergillus niger SH-1为宿主细胞构建诱导型糖化酶基因启动子表面展示CALB,在较高浓度葡萄糖碳源的发酵中CALB表达会受到抑制,发酵后期菌体容易出现菌丝断裂和展示酶活力下降等问题。采用组成型3-磷酸甘油醛脱氢酶基因启动子替代诱导型糖化酶基因启动子的细胞表面展示CALB黑曲霉菌株可有效解决上述问题,该菌株不但可以利用葡萄糖,而且还能利用木糖为发酵碳源,以木糖为碳源发酵在144 h展示酶水平达到1 100.28 U/g。文中探讨了甘蔗渣水解液发酵生产黑曲霉表面展示CALB,初步达到预期的结果,为甘蔗渣的综合利用提供了新途径。     

Effect of modified glucose catabolism on xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 μmol g⁻¹ (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied by an increase in xanthan production of up to 2.23 g l⁻¹ (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of glucose remained approximately the same.     

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.     

13C-NMR analysis of glucose metabolism during citric acid production by Aspergillus niger

The effect of glucose concentration on glycolytic metabolism under conditions of citric acid accumulation by Aspergillus niger was studied with 13C-labelled glucose. The results show that during cultivation at high glucose (14%, w/v), most of the label in citric acid is in C-2/C-4, and is thus due to the pyruvate carboxylase reaction. However, a significant portion is also present in C-1/C-5, whose origin is less clear but most likely due to reconsumption of glycerol and erythritol. Formation of trehalose and mannitol is high during the early phase of fermentation and declines thereafter. The early fermentation phase is further characterized by a high rate of anaplerosis from oxaloacetate to pyruvate, which also decreases with time. At low glucose concentrations (2%, w/v), which lead to a significantly reduced citric acid yield and formation rate, labelling of citrate in C-2/C-4 is decreased and C-l/C-5 labelling increased. Growth on 2% glucose is also characterized by an appreciable scrambling of mannitol and considerable backflux from mannitol to trehalose (indicating tight glycolytic control at the fructose-6-phosphate step) and an increased anaplerotic formation of pyruvate from oxaloacetate. These data indicate that cultivation on high sugar concentrations shifts control of glycolysis from fructose-6-phosphate to the glyceraldehyde-3-phosphate dehydrogenase step.