高产γ-氨基丁酸酵母菌株的亚硝基胍诱变选育

目的:探讨亚硝基胍诱变选育高产γ-氨基丁酸酵母菌株的方法。方法:使用亚硝基胍对酵母菌株进行诱变;采用含溴甲酚绿的YEPD培养基筛选突变菌,采用薄层层析法和比色法鉴定变异菌株发酵液中的γ-氨基丁酸及其含量;对突变菌株连续继代培养4代,测定各代发酵液中γ-氨基丁酸的含量,鉴定诱变菌株的遗传稳定性。结果:亚硝基胍诱变酵母的最佳浓度为1.0g.L-1,最佳诱变时间为15min;获得了5株突变菌株,菌落呈绿色;薄层层析法鉴定突变菌株都能产γ-氨基丁酸;诱变菌发酵液中的γ-氨基丁酸含量各异,但高于对照,且增长幅度很大;对突变菌株后代遗传稳定性进行了鉴定,结果表明突变菌株4遗传性较稳定。结论:采用1.0g.L-1的亚硝基胍溶液处理酵母菌15min,经筛选鉴定,获得了一株遗传稳定的高产γ-氨基丁酸的酵母菌株。     

虾青素高产菌株的选育

采用激光（Laser）、紫外线（UV）、亚硝基胍（NTG）及其复合诱变法夫酵母菌,再用高浓度葡萄糖培养基选育经复合诱变的法夫酵母菌。结果表明：与原始出发菌相比,菌株经诱变后其虾青素产量有显著提高,其中复合诱变的是青素产量可达7.26μm/ml。诱变高产菌株遗传稳定性好。     

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。     

黄原胶生产菌无色素黄单胞菌的选育和发酵条件的研究

从实验室保存菌株黄原胶生产菌野油菜黄单胞菌XG30-1出发,经亚硝基胍诱变,筛选到一株不产生黄色素的突变菌株XG30-1-SW,发酵产物的红外吸收图谱鉴定与出发菌株一致。该菌株以葡萄糖或蔗糖为碳源、大豆粉或大豆分离蛋白为氮源、pH7．0为最适发酵条件,产量最高可达33g/L,连续多次传代后突变性状能稳定遗传。     

酵母超氧物歧化酶高产菌的选育

采用常规筛选方法从300多株不同种属的酵母菌中筛选到两株细胞生物量和超氧物歧化酶(SOD)含量都较高的菌株(酿酒酵母,编号为Y-8和Y一111)作为实验出发菌.经单倍体分离、N-甲基-N-亚硝基-N’-硝基胍(MNNG)诱变和群体杂交等手段,从中选育出一株细胞生物量略高于实验出发菌、超氧物歧化酶高达1350U/g湿菌体的SOD高产菌株(编号为ZDF-48),它的SOD产量分别为实验出发菌株Y-8和Y-111的2.2 倍和2.4倍.经分离纯化后蛋白含量及超氧物歧化酶活性测定等研究,证明我们选育出的ZDF-48是一株生产超氧物歧化酶的优良品系.     

海藻糖产生菌的诱变育种

目的：从土壤中筛选得到高产海藻糖的酵母菌,并且进行物理诱变进一步提高海藻糖的产量。方法：从土壤中分离纯化获得10株酵母菌株,经初步菌种鉴定属瓶形酵母属;并从中筛选得到1株高产海藻糖的酵母菌株Y5,然后对其进行了紫外线诱变。结果：选育出性能优良菌株Y59,其生产性能比出发菌株提高了2．26倍,发酵液中海藻样含量达到2908．5μg/ml。结论：紫外线诱变可以有效地提高海藻糖的产量。     

L-异亮氨酸产生菌的选育

以黄色短杆菌BF420为出发菌株,经紫外线和亚硝基胍复合诱变处理后,单菌落分离筛选到一株营养缺陷型突变菌株BF35(Lys-).进一步采用氨基酸结构类似物S-2-氨基乙基-L-半胱氨酸(AEC)、α-氨基丁酸(α-AB)进行抗性筛选,获得一株带有遗传标记的L-异亮氨酸高产突变株BF3510(Lys-+AECr +a-ABr).该菌株在培养基未优化的条件下摇瓶产酸量为6.4g·L-1,比出发菌株增加了83％.     

产纤溶酶少根根霉菌株的诱变筛选

目的:通过对自南方小酒药中筛选得到的1株产纤溶酶的少根根霉Or株的诱变筛选,提高原有菌株的产酶能力。方法:以Or为出发菌株,进行亚硝基胍、紫外线、Co60诱变,以血纤维蛋白平板法为检测方法,筛选高产酶突变株。结果:经诱变传代后得到高产突变株8B,其产酶活性稳定为291.05U/mL,为原菌株产酶活力的6.34倍。该菌在血琼脂平板上不产生溶圈,诱变后孢子成熟提前8h。结论:物理诱变和化学诱变交替应用,能显著提高少根根霉Or株单位体积发酵液的产酶量,并能缩短孢子的成熟周期。该菌不具有溶血性,与已有报道的产纤溶酶的菌株不同。     

利用特比萘芬抗性筛选赤霉素高产菌株及相关发酵特性的研究

【目的】诱变、筛选赤霉素高产菌株,并明确该菌遗传稳定性及部分发酵特性。【方法】利用亚硝基胍、60Co-γ射线以及复合诱变的方法,结合抑真菌剂-特比萘芬抗性筛选,定向选育赤霉素高产突变株;通过琼脂斜面传代实验确定遗传稳定性;发酵罐培养了解其部分发酵性状。【结果】藤仓赤霉菌单细胞悬液先后经过终浓度为300 mg/L亚硝基胍30 min和60Co-γ射线300戈瑞下复合诱变,得到抗120μg/L特比萘芬的赤霉菌突变株。摇瓶复筛,确定赤霉素突变菌株的赤霉素合成能力,其中ZNL13-3菌产赤霉素效价为2 215±35 mg/L,较之诱变前发酵效价平均提高了11.87%。ZNL13-3菌株经连续15次试管斜面传代考察,菌株赤霉素合成的稳定性较好,能维持原发菌株93.2%,具有良好的遗传稳定性和生产应用前景。5 L发酵罐培养,比较菌丝浓度和产物浓度随发酵时间变化趋势,ZNL13-3菌比生产速率优于原发菌株。【结论】赤霉菌抗特比萘芬的变异特性与赤霉素高产之间存在某种对应关系,为进一步定向优化筛选优良菌株提供参考。     

耐酸性α-淀粉酶高产菌株的选育

从酒曲中筛选得到1株高产耐酸性α-淀粉酶的野生菌株Tx-78,经鉴定为曲霉属的黑曲霉(Aspergil-lus niger)。以Tx-78作为出发菌株,通过紫外线(UV)诱变和亚硝基胍(NG)诱变,获得了遗传稳定的高产菌株UN78358,其产酶量可达到1 120 U/mL。     

酵母产γ-氨基丁酸发酵条件的研究

目的：研究酵母产γ-氨基丁酸的发酵条件,提高其产γ-氨基丁酸的能力。方法：以高产γ-氨基丁酸的酵母突变株为材料,通过单因素实验研究培养温度、摇床转速、接种量、种龄和培养时间等条件对菌株发酵生产γ-氨基丁酸的影响。结果：最适发酵条件为：培养温度30℃,摇床转速220 rpm,接种量4%,种龄为2d的种子菌,培养时间4d。在此发酵条件下,变异菌发酵液中γ-氨基丁酸含量高达2.588 g.L-1,较优化前提高了53%。结论：发酵条件的优化,提高了菌株产γ-氨基丁酸的能力。     

高产γ-氨基丁酸低产桔霉素红曲霉(Monascus ruber)突变子1047筛选与发酵特性研究

以红曲米中筛选到的红色红曲霉菌株G为原始菌株,通过农杆菌介导T-DNA插入突变技术,成功构建了含有1 483株红曲霉突变子的T-DNA插入突变库。用HPLC等方法从突变库中筛选出10株γ-氨基丁酸(GABA)产量稳定高于原始菌株G的突变子,薄层层析结合HPLC技术分析了10株突变子发酵液中桔霉素的含量;其中突变子1047的GABA产量较高,为1.169g/L,是原始菌株G(GABA产量0.472g/L)的247.67%,桔霉素含量稳定低于原始菌株G。以红曲霉菌株G和突变子1047为实验材料,通过5 L发酵罐发酵并定时取样,HPLC等方法精确分析发酵液各种活性物质的含量;结果显示,突变子1047生长速度稍快于原始菌株G;GABA、莫纳可林K(Monacolin K)、红曲色素色价分别为2.201 g/L、83.892 mg/L、21.984 U/mL,是原始菌株G的279.67%、108.01%和182.35%;而桔霉素产量为1.976 mg/L,是原始菌株G的41.71%。因此,利用TDNA插入的方法对红曲霉进行育种,能产生稳定的遗传变化,在红曲霉资源的保护和利用上有一定潜力。     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance. This is disadvantageous for several biotechnological applications, e.g. the preparation of freeze doughs. We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance ('fil' mutants) using a heat shock selection protocol. We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase. It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity. The fil1 mutant displays a reduction in the glucose-induced cAMP increase, trehalase activation and loss of heat resistance. Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway. Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain. These results show that high stress resistance and high fermentation activity are compatible biological properties. Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.     

液泡蛋白酶B对酿酒酵母高温乙醇发酵效率的影响

【目的】提高酿酒酵母的高耐温性,从而提高菌株在高温下的乙醇发酵性能。【方法】利用染色体整合过表达酿酒酵母液泡蛋白酶B编码基因PRB1。【结果】在41 °C高温条件下进行乙醇发酵,过表达PRB1基因的重组酿酒酵母菌株可在31 h内消耗全部的葡萄糖,而对照菌株在相同时间内仅消耗不到一半的葡萄糖。【结论】利用蛋白酶B基因过表达可构建耐高温酿酒酵母菌株,提高在高温条件下乙醇的发酵效率。     

Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei

Two mutant strains of Amycolatopsis mediterranei VA17 and VA18 were isolated using physical (UV) and chemical (NTG) mutagens gave high rifamycin B than the parent type when grown in the same fermentation medium with a pH of 7.2, temperature 32v°C for a period of 12 days. The cultural conditions of both mutant strains are similar to the parent strain except temperature which was higher by 4v°C. By this mutation and selection study, rifamycin B production was improved from 1400 mg/l to 2450 mg/l.     

A strategy to prevent the occurrence of Lactobacillus strains using lactate-tolerant yeast Candida glabrata in bioethanol production

Contamination of Lactobacillus sp. in the fermentation broth of bioethanol production decreases ethanol production efficiency. Although the addition of lactate to the broth can effectively inhibit the growth of Lactobacillus sp., it also greatly reduces the fermentation ability of Saccharomyces cerevisiae. To overcome this conflict, lactate-tolerant yeast strains were screened. Candida glabrata strain NFRI 3164 was found to exhibit both higher levels of lactate tolerance and fermentation ability. Co-cultivation of C. glabrata was performed with Lactobacillus brevis and Lb. fermentum, which were reported as major contaminating bacteria during bioethanol production, in culture medium containing 2% lactate. Under these culture conditions, the growth of Lactobacillus strains was greatly inhibited, but the ethanol production of C. glabrata was not significantly affected. Our data show the possibility of designing an effective fuel ethanol production process that eliminates contamination by Lactobacillus strains through the combined use of lactate addition and C. glabrata.     

产γ -氨基丁酸乳酸菌的筛选及鉴定

从泡菜汁样品中初步筛选得到6株乳酸菌,采用纸层析和分光光度法对含1％谷氨酸的GYP发酵液中GABA含量分析,复筛得到一株产量较高的3#菌株,培养3d其GABA含量可达8.006 g/L.经形态学、生理生化特性分析及16S rDNA鉴定,所筛乳酸菌株为乳酸肠球菌.     

放线菌Streptomyces sp．FJ3的核糖体工程改良与活性产物的分离

[目的]利用核糖体工程抗性筛选技术,获得有抗菌活性突变株,并对突变株新产生活性物质进行研究.[方法]以三峡库区筛选出的无抗菌活性放线菌野生株为出发菌,通过单菌落挑选与平板划线培养,分离筛选具有链霉素和利福平抗性突变株;通过摇瓶发酵和对发酵液进行纸片法活性测定,获得抗金葡菌活性突变株;采用高效液相色谱法(HPLC)分析其发酵液组分,通过LC-MS对变化峰进行分析;进行16S rDNA及形态学鉴定.[结果]链霉素和利福平对放线菌菌株FJ3的MIC分别为0.5μg/mL和110μg/mL;在FJ3突变菌株中,共获得24株链霉素突变菌株和20株利福平突变菌株,抗菌活性筛选显示6株具有抗菌活性,其中2株链霉素突变菌株对金葡菌有强抑菌活性,采用Doskochilova溶剂系统纸层析结果表明,该活性物质为一种核酸类抗生素,HPLC和LC-MS显示该活性物质可能为硫藤黄菌素.[结论]利用核糖体工程技术可以改变放线菌的次级代谢,获得具有生物活性的突变株,拓展药源放线菌活性菌株新资源.