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。     

提高展青霉(Penicillium patulum)产灰黄霉素效价的诱变育种研究

为进一步筛选高产灰黄霉素的工业生产菌株,分别对前期采用紫外线-氯化锂（UV-LiCl）、半导体激光（LD laser）及CO2激光（CO2laser）对展青霉FS80-1复合诱变获得三株高产菌株进行液体发酵和固体培养比较。结果表明,通过UV-LiCl复合诱变获得突变菌株GM120-43的液体发酵产灰黄霉素效价11 982μg/mL,比出发菌株提高37.52%,固体培养效价为89 496μg/g（干重）,比出发菌株提高80.04%。;半导体激光诱变获得突变株LD100-1的液体发酵效价9 440μg/mL,固体培养效价119 766μg/g干重,比出发菌株FS80-1提高了140%;两个突变株的生物学特性均发生不同程度的变化,突菌株GM120-43适合于液体发酵生产,突变株LD100-1适合于固体发酵培养。     

阿维链霉菌的诱变育种

以阿维链霉菌(Streptomyces avermitilis)76-12为出发菌株,采用亚硝基胍、吖啶橙、紫外线和氯化锂分别对其孢子和原生质体进行诱变,经抗代谢物理性筛选,获得一系列高产突变株,其中N-1-2高产突变株的发酵单位是出发菌株的2.47倍。实验中同时获得了只产阿维菌素a组分的突变株G-32、Bla组分含量高的Ave8菌株和产蓝绿色孢子的突变株UA-G等。     

曲酸生产菌的诱变选育

Aspergillusoryzae2336经两次紫外诱变后筛选出突变菌株UV21012-1。该菌株曲酸产量比出发菌株提高了1．68倍;遗传性状稳定,传代5次曲酸产量基本不下降,而且发酵周期也比出发菌株明显缩短。     

胞苷生产菌的选育

目的：筛选得到胞苷发酵单位较高的菌株,并对发酵过程作初步研究。方法：以胞苷脱氨酶缺失枯草芽孢杆菌DOS7为出发菌株,对其进行紫外诱变、5-氟胞苷（5FCR＋）和2-杂氮尿嘧啶抗性（2AU＋）抗性筛选。结果：通过紫外诱变和抗性筛选得到突变株DOS7-2-1000-15,抗5-氟胞苷和2-杂氮尿嘧啶的临界浓度分别为800mg/L和1 000mg/L。同时检测了抗5-氟胞苷突变株中CTP合成酶的活性,比原始菌株提高了12.4%,突变株DOS7-2-1000-15发酵过程结果为：36℃发酵72h能积累胞苷最高为3.5g/L。结论：筛选得到的突变株DOS7-2-1000-15的遗传稳定性较好,可稳定发酵。     

3-羟基丙酸高产菌株的筛选及诱变选育

【目的】3-羟基丙酸是一种重要的化学平台化合物,期望得到一株能够高产3-羟基丙酸的菌株。【方法】从土壤及粪便筛选并对得到的菌株进行鉴定和复合诱变。【结果】得到了一株能够利用丙酸发酵生产3-羟基丙酸的酵母Y-11,经生理生化鉴定及18S rDNA序列分析确定其为Candida sp.(假丝酵母)。以Y-11为出发菌株,经紫外-亚硝基胍-60Coγ复合诱变得到了突变性状稳定且可遗传的高产菌株5-13B,其3-羟基丙酸的产量为11.78 g/L,是出发菌株的2.46倍。【结论】对出发菌株和突变株的发酵特性进行了比较,结果表明突变株的3-羟基丙酸产量、对底物丙酸的转化率、产物3-羟基丙酸的积累性能及丙酸的耐受性均优于出发菌株。     

ILV2基因缺失对啤酒酵母生长与双乙酰代谢的影响

【目的】旨在应用分子生物学方法降低啤酒发酵液中双乙酰含量,改善啤酒感官质量。【方法】以酿酒酵母S2(Saccharomyces cerevisiae)为出发菌株,通过同源重组敲除四倍体啤酒酵母α-乙酰乳酸合成酶部分基因(ILV2),构建缺失一个和两个ILV2等位基因的突变株QI2-1和QI2-2,并进行啤酒发酵实验。【结果】ILV2基因的缺失,会导致菌株初始生长速率的降低。其中QI2-2较为明显,12 h时,突变株与出发菌株的生长速率达到一致。啤酒发酵结果表明,与出发菌株相比,突变株QI2-1双乙酰峰值与双乙酰最终含量分别降低17.50%和17.83%,而QI2-2分别降低51.67%和45.65%。其他啤酒指标如酒精度、发酵度、残糖和风味物质等略有变化,但都在优质啤酒指标范围内,符合啤酒发酵的质量要求。【结论】通过同源重组敲除部分ILV2基因和选育低产双乙酰菌株是降低啤酒双乙酰含量、提高啤酒质量的有效方法,具有一定的实际应用价值。     

海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化*

目的:对海洋来源的具有产纤溶酶能力的枯草芽孢杆菌(Bacillus subtilis)LC6-1进行紫外诱变,得到高产且稳定的突变株PW6-3,对该突变株发酵产酶的条件进行优化。方法:采用单因素和正交试验进行发酵培养基组分和培养条件的优化。结果:突变株PW6-3的酶活力为(6 960.21 ± 85.51)U/mL,较原始菌株提高了30.48%。以PW6-3为出发菌株,采用单因素及正交试验的方法对菌株进行发酵培养基组分与培养条件优化,最终得到的最佳培养基组分是:玉米淀粉30 g/L,玉米浆干粉40 g/L,CaCl₂ 3 g/L;最佳发酵培养条件是:32℃,转速200 r/min,接种量3%,pH 6.5,种龄18 h,发酵培养时间66 h,最终菌株的酶活力稳定在(9 203.63 ± 67.85)U/mL。结论:发酵工艺优化后,菌株PW6-3纤溶酶产量较诱变之前的菌株LC6-1提高72.53%,且发酵工艺成本较低,具有较好的经济效益。     

lysC定点突变及lysC、asdA串联表达对谷氨酸棒杆菌L-苏氨酸积累的影响

lysC、asdA基因分别编码的天冬氨酸激酶(Aspartate kinase,AK)和天冬氨酸半醛脱氢酶(Aspartate semi-aldehyde dehydrogenase,ASD)是L-苏氨酸合成途径中两个关键限速酶基因,其中AK受到代谢产物赖氨酸与苏氨酸的协同抑制。以选育获得的一株谷氨酸棒状杆菌T11(Corynebacterium glutamicum T11)为出发菌株,通过构建lysC-asdA串联表达盒,并对其关键限速酶基因lysC进行定点突变,突变位点为Ala279Thr,获得抗反馈抑制突变型编码基因lysCr-asdA,将其插入含强启动子tac的穿梭表达载体pZ8-1中成功构建串联表达质粒pZ8-1-lysCr-asdA转化出发菌株,筛选获得工程菌株T11/pZ8-1-lysCr-asdA。摇瓶发酵其L-苏氨酸产量达到7.18 g/L,较出发菌株提高27.8%。进一步的30 L发酵罐补料分批发酵结果显示,发酵60 h L-苏氨酸产量达65.5 g/L,糖酸转化率达到39.5%,较出发菌株分别提高29.5%和33.9%,为后续的进一步构建高产L-苏氨酸的谷氨酸棒杆菌工程菌株提供强有力的基础。     

米根霉交替呼吸途径高活性突变株的选育及其对富马酸合成的影响

米根霉交替呼吸强度与富马酸合成之间存在重要关联。以米根霉F-14为出发菌株,通过常压室温等离子体(ARTP)离子诱变技术,筛选出1株交替呼吸强度增强的突变菌株S-1。该菌株的交替呼吸初始强度是出发菌株F-14的3.5倍,然而,该菌株富马酸积累量(28 g/L)却远低于出发菌株(42 g/L)。进一步考察突变株S-1和出发株F-14菌株在发酵过程中交替呼吸及富马酸生产强度,总呼吸变化和还原力NADH/NAD+的变化。结果表明:突变株在富马酸发酵初始阶段,过高的交替呼吸强度反而降低了富马酸的生产速率,并导致了发酵后期菌体的早衰现象。交替呼吸强度与发酵进程存在适配性,只有当适配性达到最佳状态时,才有利于产物的高效积累。     

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。     

Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans

Abstract A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.     

复合诱变在青霉高产菊粉酶菌株选育中的应用

以Penicillium sp．B01为出发菌株,经吖黄素或DES（硫酸二乙酯）分别与^60Co-γ射线对其孢子悬液进行复合诱变。经过初筛和复筛,在30μg／mL吖黄素诱变时间2h,剂量率为4．11Gy／min的^60Co-γ射线辐射使累计剂量为20．55Gy复合诱变的条件下,筛选出一株菊粉酶活比出发菌株高32％的突变菌株B01-A13-Co31。经同工酶电泳验证,变异株与出发菌株相比酶带有所变化。将此菌种连续传代6次进行产酶性能的稳定性测定,表明此菌株具有良好的遗传稳定性。     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet

Aims:  The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ).
Methods and Results:  Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro . Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2·3-fold as that of the original strain.
Conclusions:  A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture.
Significance and Impact of Study:  This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.     

基因组重排与传统育种相结合选育大观霉素高产菌株

以壮观链霉菌(Streptomyces spectabilis)为研究对象,采用基因组重排技术与传统诱变育种相结合的方法选育大观霉素的高产菌株.通过原生质体紫外诱变获得壮观链霉菌突变体群体,高产突变菌株间进行两轮的基因组重排,筛选的高产菌株用NTG诱变得新霉素和链霉素的抗性突变菌株,抗性突变菌株间进行两轮基因组重排,从...     

Endo-1,4-[beta]-Glucanase,Xyloglucanase, and Xyloglucan Endo-Transglycosylase Activities Versus Potential Substrates in Ripening Tomatoes

In ripening fruits of tomato (Lycopersicon esculentum L. var 83-G-38), the amounts of cellulose and xyloglucan (XG) remained constant during tissue softening, but the relative molecular weight (Mr) of XG decreased markedly and the Mr of cellulose declined slightly. These changes could have been due to activities of non-specific endo-1,4-[beta]-glucanases and/or buffer-soluble XG endo-transglycosylase, both of which increased when tissue firmness declined most rapidly. Tomato extracts also reduced the viscosity of XG solutions, especially in the presence of added XG oligosac-charides. This depolymerizing (XGase) capacity differed from [beta]-glucanase and XG transglycosylase activity (a) by being almost entirely buffer insoluble, and (b) by declining precipitously during fruit softening. Although it disappeared from ripe fruit, XGase may have functioned in promoting wall loosening at earlier stages of fruit development when its activity was highest. By contrast, during aging of fruit in the ripening-inhibited mutant rin there was no change in Mr of XG or cellulose, and activities of [beta]-glucanases and XG transglycosylase were lower than in wild-type tomato. Nevertheless, some softening of the fruit did take place over time and XG amounts declined, possibly because high XGase activity was maintained in the mutant, unlike in wild-type fruit.     

Biochemical study of the relationship of extracellular glucan to adherence and cariogenicity in Streptococcus mutans and an extracellular polysaccharide mutant.

A mutant of Streptococcus mutans, GS-5, which differed in extracellular polysaccharide (EPS) produced from sucrose, was used to study the role of EPS in the production of dental caries. The mutant proved to be identical to the parent strain in sugar fermentation, growth rate, and serotype. Strain GS-5 synthesized an EPS, which in electron micrographs appeared to be of fibrillar structure, whereas the mutant produced no fibrillar material but only a globular EPS. Analysis of the EPS revealed that about 30% of the glucose units in the GS-5 polymer carried (1-3)-like bonds either as branch points or as part of the linear backbone and that the mutant material contained only about 3% of these linkages. When grown in sucrose broth, the proportion of the mutant culture adherent to the glass vessel was dramatically less than that of the parent strain. Caries scores produced in conventional rats by the mutant were significantly lower than those obtained with the parent strain. Since the only difference discovered between strain GS-5 and the mutant was the inability of the mutant to synthesize either a fibrillar EPS or an EPS with more than about 3% (1-3)-like linkages, it was concluded that the fibrillar EPS of strain GS-5 contained about 30% (1-3)-like linkages and was necessary for adherence of the bacteria to surfaces and for production of dental caries in test animals.