Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

干酪乳杆菌L-乳酸脱氢酶在大肠杆菌中的表达、纯化及酶学性质

L-乳酸脱氢酶(L-lactate dehydrogenase,L-LDH)是发酵生产L-乳酸中催化丙酮酸转化成L-乳酸的关键酶。以干酪乳杆菌G-02(Lactobacillus casei G-02)基因组DNA为模板,克隆得到L-LDH基因(ldhL),经序列分析后将其连接到表达载体pET-28a(+)上,构建成重组质粒pET-ldhL转化到大肠杆菌BL21(DE3)中,实现ldhL基因的表达。30°C加入IPTG诱导表达后,经镍柱亲和层析纯化的重组蛋白样品通过SDS-PAGE分析,约在40 kD处出现显著的特异性条带。对表达的L-LDH生物学特异性研究显示:重组L-LDH的比酶活为1 722 U/mg,最适反应温度为40°C-45°C;果糖-1,6-二磷酸(FBP)为别构激活剂,使最适pH向中性方向偏移(pH为6.6-6.8),Mn2+可拓宽最适酶活pH范围;Mn2+、Ca2+和Mg2+对L-LDH有激活作用,而Zn2+对L-LDH有抑制作用。     

竹红菌素产生菌的筛选与鉴定

通过多点采集、反复筛选,从野生竹黄子实体中筛选出1株产北醌类光敏色素的真菌。通过对菌株的个体形态特征、菌落形态特征进行观察,结合其显微结构特征最终鉴定为竹黄菌（Shiraia bambuscola)。     

Seleno-lactobacillus

Lactic acid bacteria are nonpathogenic bacteria commonly used in food processing. An evaluation was made of the capacity to concentrate selenium in species of Lactobacillus. A selenium concentration of 1 μg/mL in the culture medium yielded in a bacterial content of 400 μg/g dry biomass. Dialysis and TCA precipitation experiments of a native intracellular extract proved that at least 80% of the total selenium is associated with organic molecules. Seleno-cysteine was identified as the only seleno-amino acid present in the intracellular selenoproteins. This study shows that species of the lactic acid bacteria are able to concentrate selenium intracellular as seleno-cysteine, which could be applied in supplementation studies.     

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose.     

交替假单胞菌(Pseudoalteromonas sp.)DY3菌株产内切葡聚糖酶的性质研究及基因克隆

从深海样品ESO109中分离到一株具有高内切葡聚糖酶活力的细菌DY3,16SrDNA序列分析表明该菌与交替假单胞菌属(Pseudoalteromonas sp．)的Pseudoalteromonas citrea和Pseudoalteromonas elyakovii的同源性为99％。PCR扩增DY3的内切葡聚糖酶基因celX全长1479bp,编码一个492AA的蛋白质。酶的氨基酸序列分析表明CelX与Rseudoalteromonas haloplanktis的内切葡聚糖酶CelG有95％的相似性,包括一个糖基水解酶家族5的催化结构域,一个连接序列和位于C端的的CBM5结构域。对酶性质的初步研究发现,CelX的最适温度为40℃,酶的最适pH在6～7之间。     

乳杆菌优良菌株的筛选及对小鼠阴道白假丝酵母菌过度增殖干预

目的探讨乳杆菌优良菌株的筛选及其对小鼠阴道白假丝酵母菌过度增殖干预作用。方法对德氏乳杆菌DM8909菌株进行诱变筛选,得到耐抗生素突变株208,以ICR小鼠为实验对象,用甲硝唑进行小鼠阴道菌群脱污染,将白假丝酵母菌接种到小鼠阴道内,构建小鼠阴道白假丝酵母菌过度增殖模型。采用阴道接种德氏乳杆菌DM8909突变株208菌液（2×108CFU/mL）处理,取其阴道冲洗液进行阴道菌群分析,并进行阴道上皮细胞的电镜检查。结果乳杆菌干预后显著清除阴道内的假丝酵母菌,肠杆菌恢复正常水平,与模型组比较,差异有统计学意义（P〈0.05）。结论德氏乳杆菌DM8909抗性菌株208能调整白假丝酵母菌在小鼠阴道内定植,对小鼠阴道上皮细胞有恢复作用。     

乙型脑炎病毒SA14—14—2株E蛋白主要抗原片段的高效表达

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZα-A,以电穿孔法转化酵母X-33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性.在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT-PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据.     

构建高产谷胱甘肽啤酒酵母基因工程菌提高啤酒抗老化能力的研究

根据同源重组的原理,将来源于啤酒酵母工业菌株G03的γ-谷氨酰半胱氨酸合成酶基因(GSH1)和筛选标记Kan取代质粒pRJ-5中18S rDNA内部约340bp的DNA片段,构建重组质粒pRKG。以pRKG为模版,PCR得到以18S rDNA为整合位点包含GSH1和Kan的基因片段18S rDNA::(Kan-GSH1)。用此片段转化啤酒酵母工业菌株G03,通过G418抗性筛选得到啤酒酵母工程菌。实验室小试表明,工程菌的谷胱甘肽含量比受体菌株提高16.6%,啤酒的抗老化能力得到了显著提高,而常规指标没有发生显著变化。连续传代5次后胞内GSH含量基本不变遗传稳定性良好。由于表达γ-谷氨酰半胱氨酸合成酶的基因来源于受体菌株自身,是通过自克隆技术改造工业啤酒酵母的一次有益的尝试。