Breeding of a 5-Fluorouridine-resistant Mutant with Increased Cellulose Production from Acetobacter xylinum subsp. nonacetoxidans

UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.     

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).     

Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities

Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml⁻¹, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran.     

Photosynthetic Capacity and Quantum Requirement of Three Secondary Mutants of Scenedesmus obliquus with Deletions in Carotenoid Biosynthesis*

Photosynthetic capacity and quantum requirement of photosynthesis were evaluated in three secondary mutants of Scenedesmus obliquus possessing specific blocks in carotenoid biosynthesis. These were derived from the mutant, C-2 A', a strain which develops chlorophyll only in the light. All strains were capable of a normal and rapid production of chlorophyll and the development of a functional photosynthetic apparatus when exposed to light. LHC-II levels in the mutants lacking the β,ε-carotenoids (strains C-2 A'-34,1 and C-2 A'-67, 3b) were considerably reduced in fully greened cells but normal in the β,β-epoxycarotenoid deficient-only strain (C-2 A'-67,1) (Bishop, 1996). The maximum photosynthetic capacity and the quantum efficiency (at λ=682nm) of photosynthesis of cells of C-2A'-34,1 and C-2 A'-67,1 showed no significant changes when compared to the original parent strain, C-2 A'. However, the quantum requirement of the strain lacking both types of carotenoids, C-2 A'-67,3b, was slightly increased. It is of particular interest that both strains lacking the β,ε-carotenoids also show a preferential loss of the oligomeric form of the LHC and its associated chlorophylls. The potential requirement of the different carotenoids in maintenance of photosynthetic efficiency in Scenedesmus is discussed.     

60Coγ射线诱变褐色高温单孢菌及其在粪便发酵中的应用研究

本试验以褐色高温单孢菌(Therm om onospora fusca)为出发菌株,通过60Coγ射线诱变孢子悬液,采用透明圈法初筛和摇瓶培养复筛的方法,获得了一株纤维素酶高产菌株AV5,与出发菌株相比,其产酶能力提高1.8倍。接种牛粪发酵后,牛粪中粗纤维含量降低率为32.95%,是接种出发菌株相对降解率的1.7倍。     

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.     

Heavy‐ion mutagenesis significantly enhances enduracidin production by Streptomyces fungicidicus

To improve fermentative production of enduracidin, heavy‐ion beams generated by the Heavy Ion Research Facility in Lanzhou (HIRFL), China, were employed for the first time to generate mutations in Streptomyces fungicidicus. Initial screening detected 44 positive mutants with larger inhibition zone, which were subsequently tested based on flask fermentation. Finally, 20 mutants showed 20% increase in enduracidin production, when compared with the original strain. Among them, enduracidin production by the three mutants (M13, M30, and M34) was significantly higher than that by the original strain. In particular, mutant M30 exhibited highest enduracidin production, which was 114% higher than that obtained with the original strain. Following culture optimization, the maximal enduracidin yield obtained by M30 reached 918.5 mg/L in 10 days, which was 34% higher than that noted in the control.     

角蛋白降解菌的筛选及其UV诱变选育

从长年堆积废羽毛的土壤,屠鸡场下水道、池塘底污泥中分离出一株角蛋白降解菌C-1,摇瓶发酵测得其角蛋白酶活达35.6U,经形态观察和生理生化鉴定该菌株属于芽孢杆菌属.对菌株进行Uv诱变,在照射时间为100 s下得到一株产角蛋白酶活较高突变株C-1-4,其酶活比原始出发菌株提高了122%,且遗传性状稳定.     

Biosynthèse de cellulose bactérienne à pertir de glycérol sélectivement deutérié en position 1, 2, ou 3: Etude par résonance magnétique nucléaire

Glycerol specifically deuterated at C-1, C-2, or C-3 was prepared and used for the biosynthesis of bacterial cellulose with Acetobacter xylinum.The material obtained were converted into glucitol hexaacetate and analyzed by 250-MHz nuclear magnetic resonance and mass spectrometry. The spectra indicated that the protons of the C-3 position of the starting glycerol were incorporated as substituents of the C-6 and C-1 positions of the cellulose. Similarly, protons of the C-2 and C-5 positions of the cellulose came essentially from water and the protons bonded at the C-3 and C-4 positions of the cellulose from protons bonded to C-1 of the starting glycerol.     

新一代糖化酶高产菌株的选育及其工业应用

【目的】建立对糖化酶生产菌种黑曲霉随机突变文库进行筛选的方法,以获得糖化酶酶活提高的突变菌株。【方法】以一株可产糖化酶的黑曲霉菌株Aspergillus niger X1为出发菌株,经硫酸二乙酯诱变获得突变文库,采用葡萄糖的结构类似物——2-脱氧葡萄糖进行筛选,并在筛选过程中逐渐提高2-脱氧葡萄糖浓度,定向选育具有2-脱氧葡萄糖抗性、高产糖化酶的突变株。【结果】获得的高产突变菌株DG36摇瓶发酵糖化酶产量比出发菌株A.niger X1提高22.2%–33.8%,经工业水平50 m~3罐发酵测试,突变株DG36发酵128 h糖化酶活可达49094 U/m L,在相同发酵时间内,其酶活较出发菌株A.niger X1提高32.8%,发酵时间缩短16.9%。【结论】本研究开发了一种以2-脱氧葡萄糖为抗性标记选育高产糖化酶突变株的方法,所得突变株DG36遗传性状稳定,与出发菌相比具有菌丝粗壮、产酶期提前、糖化酶活高、发酵时间短、有利于发酵后处理的优点。     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.     

Relationship between Sulfaguanidine Resistance and Increased Cellulose Production in Acetobacter xylinum BPR3001E

The mechanism of the increased cell growth and cellulose production of Acetobacter xylinum subsp. sucrofermentans BPR3001E, a sulfaguanidine (SG)-resistant mutant, was investigated. We found that adding p-aminobenzoic acid (PABA) to cultures of the parent strain, BPR2001, led to increased levels of intracellular adenosine-related purine compounds and increased cellulose production. Furthermore, adding ATP increased the cellulose production by permeabilized BPR2001 cells. On the other hand, the intracellular levels of PABA and adenosine-related purine compounds in BPR3001E cells were higher than those in BPR2001 cells. These results suggest that SG resistance increases enhance cellulose production through increased levels of intracellular high-energy compounds caused by increased PABA biosynthesis, reflecting the promoted supply of cellulose precursors.     

Crystalline cellulose degradation by a strain of Cellulomonas and its mutant derivatives

Summary Mutant derivatives of a strain of Cellulomonas (CS1-1) were shown to be able to degrade crystalline cellulose (cotton wool) more efficiently compared to the parent strain. These mutants were also more effective in the accumulation of reducing sugar in the growth medium under certain environmental conditions. Differences between the mutant derivatives and CS1-1 were reflected by assay of the amount and distribution of various cellulolytic enzymes.     

定向进化Rho1提高酿酒酵母的乙醇耐受性和超高浓度乙醇发酵性能

燃料乙醇发酵过程中酿酒酵母细胞活性被高浓度乙醇严重抑制而导致发酵提前终止，生产强度严重降低，因此构建同时具有高耐受性、高发酵性能的菌株一直是发酵工业追求的目标。选取酿酒酵母细胞形态调节关键基因小GTP酶家族成员Rho1,构建易错PCR产物文库，以酿酒酵母S288c为出发菌株采取“富集-自然生长-复筛”的筛选策略，成功筛选得到两株乙醇胁迫耐受性与发酵性能均提高的突变株M2和M5。测序发现突变株过表达的Rho1序列出现了3～5个氨基酸的突变和大片段的缺失突变。以300 g/L起始葡萄糖进行乙醇发酵，72 h时，M2和M5的乙醇滴度比对照菌株分别提高了19.4%和22.3%,超高浓度乙醇发酵能力显著提高。本研究为利用蛋白定向进化方法改良酵母菌复杂表型提供了新的作用靶点。     

Induction of Pleiotropic Mutation in Streptomyces griseus by Incubation under Stress Conditions for Mycelial Growth

When Streptomyces griseus strain 2247 was cultivated under stress conditions for growth, such as growth in media containing toxic compounds, pleiotropic mutants were obtained at a high frequency. These mutants have lost simultaneously streptomycin productivity, streptomycin resistance, spore-forming ability, and pigment productivity, although the genes for streptomycin biosynthesis and A-factor production are proficient.     

纳豆芽孢杆菌产VK2菌株的NTG与N＋注入复合诱变选育

以纳豆芽孢杆菌BN-2-6为出发菌株,利用亚硝基胍（NTG）和N＋注入复合诱变选育产维生素K2的突变株。经过NTG诱变后得到突变株BN-N30—1,其维生素K2的产量提高了53％,继而采用低能N＋注入技术进行处理得到突变株BN-P15—11-1,维生素K2的产量比BN—N30—1提高了96％,比原始菌株提高了166％。结果表明,对纳豆芽孢杆菌BN-2-6进行NTG和低能N＋注入复合诱变的效果明显,突变菌株维生素K2的产量显著提高。     

Biological Pretreatment with Two Bacterial Strains for Enzymatic Hydrolysis of Office Paper

The cellulose-hydrolyzing strains, Sphingomonas paucimobilis MK1 and Bacillus circulans MK2, were separated from soil and were grown together in a single culture plate. Growth B. circulans MK2 in liquid culture required symbiosis with S. paucimobilis MK1. Biological pretreatment with the combined strain suspension after the liquid culture improved enzymatic hydrolysis of office paper from municipal wastes. Sugar recovery by S. paucimobilis MK1 (51%) was 1.4 times higher than that of the untreated sample (30%) and in the strain combination with B. circulans MK2, recovery was further improved by 2.5 times (75%). The sugar recovery in maximum condition was enhanced up to 94% for office paper. Furthermore, biological pretreatment effects were confirmed for more than 1 day less time. In X-ray diffraction patterns for the crystallinity of cellulose in office paper changed after biological pretreatment, the crystallinity was increased in comparison to that in untreated paper. The mechanism of biological pretreatment effect was explained by the fact that the strain acted as an endoglucanase, which hydrolyzes amorphous areas randomly.     

Stepwise Introduction of Regulatory Genes Stimulating Production of α-Amylase into Bacillus subtilis : Construction of an α-Amylase Extrahyper Producing Strain

The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.

These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain.     

离子注入麦角甾醇酵母选育及发酵工艺

通过离子注入诱变筛选到产麦角甾醇酵母高产菌,得率较出发菌提高55％～60％。对高产菌进行发酵条件优化,发现其对发酵通氧量有更高的要求,培养基中添加10％的糖或0．1％的Ca（NO3）2对麦角甾醇发酵有明显促进作用。