核黄素产生菌阿舒假囊酵母(Eremothecium ashbyii)抗反馈抑制突变株的选育

在调查阿舒假囊酵母(Eremothecium ashbyii)营养要求的基础上,设计了适合该菌生长的合成培养基,在合成培养基上诱变筛选得到了数株抗嘌呤拮抗物8-AG的突变株。选择其中三株U_(95-1)、U_(95-2)和U_(95-3)进行传代和摇瓶发酵试验,U_(95-3)的核黄素发酵单位低于出发菌,未经传代的U_(95-1)、U_(95-2)比出发菌株的发酵水平分别提高15.5％和9.8％,经5～10代传接,其产核黄素的遗传性状稳定。该研究表明,从代谢控制角度通过减轻核黄素合成途径中重要调节酶的反馈抑制对提高E. ashbyii的核黄素产量和稳定性是可行的,为该菌的菌种改良提供了一条遗传育种途径。     

N-甲基-N’-硝基-N-亚硝基胍对短小芽孢杆菌AS 1.271菌株的诱变作用

1. MNNG在 pH6时较为稳定,在碱性条件下分解极为迅速,较易受光照作用而分解。 2．MNNG诱发短小芽孢杆菌(Bacillus pumilus) AS 1.271 菌株获得最高突变率的适宜条件是：菌龄为对数生长期的早期,采用0．05M pH6的TM缓冲液,所用剂量为1000微克／ 毫升,于30℃处理45分钟,在这样的处理条件下,营养缺陷型突变菌株可达20％左右。 3．在所获得的1775株突变菌株中,有947株经过营养缺陷型的鉴定,其中有核酸碱基、氨基酸、维生素的单一和双重营养缺陷型突变体,核酸碱基缺陷型中,腺嘌呤缺陷型较多,氢基酸缺陷型中以组氨酸、谷氨酸、蛋氨酸、精氨酸等缺陷型所占的比例较大。     

核黄素产生菌阿舒假囊酵母(Eremothecium ashbyii)抗反馈抑制突变株的选育

在调查阿舒假囊酵母(Eremothecium ashbyii)营养要求的基础上,设计了适合该菌生长的合成培养基,在合成培养基上诱变筛选得到了数株抗嘌呤拮抗物8-AG的突变株。选择其中三株U_(95-1)、U_(95-2)和U_(95-3)进行传代和摇瓶发酵试验,U_(95-3)的核黄素发酵单位低于出发菌,未经传代的U_(95-1)、U_(95-2)比出发菌株的发酵水平分别提高15.5％和9.8％,经5～10代传接,其产核黄素的遗传性状稳定。该研究表明,从代谢控制角度通过减轻核黄素合成途径中重要调节酶的反馈抑制对提高E. ashbyii的核黄素产量和稳定性是可行的,为该菌的菌种改良提供了一条遗传育种途径。     

复合诱变选育高产GSH的菌株

目的:选育出GSH的高产菌株.方法:以产GSH的产朊假丝酵母(Candida utilis)为出发菌株,利用紫外-超声波复合诱变.结果:筛选得到ZnCl2抗性突变株UU3,GSH菌体含量为53.50mg·g-1,比出发菌株提高137.03%.结论:突变株UU3遗传性能稳定,可做进一步研究.     

黑曲霉原生质体诱变选育β-葡萄糖苷酶高产菌株

本研究报道了以原生质体诱变技术选育高产β-葡萄糖苷酶的黑曲霉菌株,并研究了其发酵特性。以黑曲霉CGMCC3.316为出发菌株,通过紫外诱变得到突变株3-3M。然后以3-3M为供试菌株,研究了其原生质体制备与再生的条件。最后通过原生质体诱变,选育得到一株β-葡萄糖苷酶活力较高的突变株60B-3D。该菌株具有良好的遗传稳定性,酶活力平均达到23IU/mL,与出发菌株CGMCC3.316相比提高39%。此外,该菌株的木聚糖酶活力也有所增加。同时考察了黑曲霉60B-3D的发酵特性,并与3-3M和出发菌株进行比较,结果表明该菌株有较高的蛋白分泌能力。本研究为发酵生产β-葡萄糖苷酶提供了一株良好的供试菌株。     

肌苷产生菌枯草杆菌7171-6-1菌株的选育

本文报告由枯草杆菌(Bacillus subtilis) No．101野生型为出发菌株,经过硫酸二乙酯(Dicthyl Sulfate,DES)、8-氮杂鸟嘌呤(8-Azagnaninc,8-AG)处理获得了一株腺嘌呤、硫胺素双重营养缺陷型突变株7171-6-1,在以葡萄糖为碳源的发酵培养基中,经60—72小时、30—32℃发酵,肌苷产量达11．65克／升。     

应用营养缺陷型回复突变的方法筛选枯草杆菌(Bacillus subtilis)α-淀粉酶的高产菌株

用甲基磺酸乙脂(EMS)处理枯草杆菌(Bacillus Subtilis)BF-7658的孢子,从中分离到氨基酸、维生素和嘌呤、嘧啶的单项和多重营养缺陷型共47株。营养缺陷菌株和原养型的出发菌株比较,除MA-46突变株的α-淀粉酶活力稍高于出发菌株外,其余均有不同程度的下降。α-淀粉酶活力降至原水平1/6—1/9左右的维生素或嘌呤嘧啶缺陷型VPP-36,VPP-37,酶活力完全丧失的脂肪族氨基酸缺陷型NA-27等,经EMS诱发回复突变后,从回复体中分离到α-淀粉酶高产菌株十余株,最高增产幅度比原始菌株达15％以上。     

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％.     

5-氨基-4-氨甲酰咪唑核苷产生菌AIC-90菌种选育研究

已肌苷产生菌枯草杆菌GMI- 741(Ade - )为出发菌株 ,通过多因子诱变选育出具有非精确嘌呤缺陷型、丧失腺嘌呤脱氨酶的AICAR(5 -氨基 - 4-氨甲酰咪唑核苷 )产生菌AIC - 90 ,该菌株摇瓶发酵 72h产AICAR可达 2 0 .3g/L以上。     

结合缺陷型菌株显色法采用离子束诱变筛选高产L-精氨酸突变株

为快速高效筛选L-精氨酸高产突变株,建立一种缺陷菌株平板显色法并采用低能N+离子束对L-精氨酸生产用菌株钝齿棒杆菌SYPA5-5进行诱变处理,通过上述平板显色法筛选获得高产突变株.对突变株进行摇瓶发酵实验,最终选育出一株L-精氨酸产量较高且产酸性能比较稳定的突变菌株钝齿棒杆菌SYPA5-5-36.该菌株摇瓶发酵L-精氨酸产量可达35.85 g/L,比出发菌株提高了19.5％.因此,缺陷型菌株平板显色法可以用于快速、高效筛选高产L-精氨酸突变株.     

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria.     

Isolation and characterization of Candida membranifaciens subsp. flavinogenie W14-3, a novel riboflavin-producing marine yeast

We found that the marine yeast strain W14-3 isolated from seawater of China Eastern Sea could produce riboflavin. It is interesting to observe that the marine yeast strain produced a large amount of riboflavin in the medium containing xylose, sucrose, galactose and maltose under the conditions of vigorous shaking. The yeast strain was found to belong to Candida membranifaciens subsp. flavinogenie based on the results of routine and molecular identification. The protein sequences deduced from the partial genes encoding GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase in the yeast exhibited high identity with those of the corresponding enzymes for riboflavin biosynthesis in other yeasts. Fe³⁺ available in the medium repressed riboflavin production and expression of the genes responsible for riboflavin biosynthesis in the yeast. The results have evidenced that a riboflavin synthesis pathway indeed existed in the yeast. This is the first study to report that C. membranifaciens subsp. flavinogenie W14-3 from the marine environment could produce riboflavin.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

电视显微摄影技术在微生物教学及科研中的应用

通过对一株核黄素高产株阿舒假囊酵母Ｔ_３０的菌丝形态、孢子囊、孢子、发酵过程中出现的膨大菌体及发酵终点核黄素结晶产物的显微观察,介绍一种适用于微生物材料（如：细菌、霉菌、酵母菌等）、动植物材料（如：动物组织、植物花朵）、生物大分子物质（如：淀粉颗粒）、工业结晶（如：核黄素晶体）及金相实验、普通物理实验的显微观察、检测和记录的方法。这一技术可普遍地应用于电视教学及科研实践。     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.     

Eremothecium ashbyii mutants resistant to 8-azaguanine. II. Mutants with different degrees of resistance to 8-azaguanine]

Guanine, unlike adenine and hypoxanthine, can not eliminate the inhibitory effect of adenine analogues on the growth and flavinogenesis of Eremothecium ashbyii. Guanine does not restore riboflavin synthesis inhibited with 5-10(-3) M 8-azaguanine. Low adenine concentrations (10(-4)-3-10(-4) M), which do not influence the inhibitory effect of 5.-10(-3) M 8-azaguanine, restore the riboflavin synthesis in combination with guanine. On the basis of the data obtained as well as the data of biochemical analysis it is concluded that the riboflavin producer studied lacks guanosinemonophosphate reductase. The mutants resistant to various concentrations of 8-azaguanine have been obtained. In all mutants resistant to 8-azaguanine the efficiency of the incorporation of 14C-guanine and 14C-adenine into mycelium is decreased as compared with the susceptible strain. The mutant Azg-R 10 resistant to high (3-10(-3) M) concentrations of 8-azaguanine, 8-azaadenine and 2,6-diaminopurine secretes inosine-like compounds when grown in a synthetic medium. The stepwise increase of the mutant resistance to 8-azaguanine from 10(-4) M TO 3-10(-3) M did not result in further enhancement of riboflavin synthesis.     

Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]

Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production

AIMS: To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. METHODS AND RESULTS: Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680-685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103.5 or 57.4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g l(-1) at 62 h (yield on glucose of 0.651 g g(-1)) in a 7-l jar fermentor. CONCLUSIONS: The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. SIGNIFICANCE AND IMPACT OF THE STUDY: Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites.     

Isolation of Ashbya gossypii mutant for an improved riboflavin production targeting for biorefinery technology

AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. CONCLUSIONS: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.