谷氨酸棒杆菌3-脱氧-α-阿拉伯庚酮糖酸-7磷酸合成酶基因的克隆与过量表达

本实验对谷氨酸棒杆菌(Corynebacterium glutamicum) AS10111及其亚硝基胍诱变获得的丙氨酸和酪氨酸双营养缺陷型、抗3- 甲基色氨酸突变菌株JLC1 中3- 脱氧- α- 阿拉伯庚酮糖酸-7 磷酸合成酶(DS)基因进行克隆和序列分析,将突变体来源DS 基因构建pZ8-1-DSM 重组质粒,在谷氨酸棒杆菌突变株JLC1 中进行表达,重组菌株JLC1(pZ8-1-DSM)DS 酶活力分别是宿主菌和野生型菌株DS 酶活力的5.9 和7.3 倍,色氨酸产量达到14.90g/L,较宿主菌提高了65%。这表明通过在谷氨酸棒杆菌中过量表达3- 脱氧- α- 阿拉伯庚酮糖酸-7 磷酸合成酶基因可以有效提高该酶活力和色氨酸的产量。     

二硫键稳定的抗氯霉素单链抗体的构建

为构建一种食品残留检测用二硫键稳定的抗氯霉素单链抗体(sdsFvCAP)。以抗氯霉素抗体可变区基因为模板,通过引物突变PCR 在轻链和重链的可变区分别引入一个半胱氨酸定点突变,以重叠延伸PCR 组装成sdsFvCAP。在大肠杆菌系统强启动子T7 驱动下,sdsFvCAP 基因不能单独表达,而通过同义突变降低其5＇端序列GC 含量则可实现高效表达。表达产物主要以包涵体形式存在,经β- 环糊精分子伴侣系统复性可获得亲和力与母本抗体相似的sdsFvCAP。所构建的sdsFvCAP 具有取代单克隆抗体用于氯霉素残留检测的潜力。     

β-Ketoacyl-acyl Carrier Protein Synthase I (KASI) Plays Crucial Roles in the Plant Growth and Fatty Acids Synthesis in Tobacco

Fatty acids serve many functions in plants, but the effects of some key genes involved in fatty acids biosynthesis on plants growth and development are not well understood yet. To understand the functions of 3-ketoacyl-acyl-carrier protein synthase I (KASI) in tobacco, we isolated two KASI homologs, which we have designated NtKASI-1 and NtKASI-2. Expression analysis showed that these two KASI genes were transcribed constitutively in all tissues examined. Over-expression of NtKASI-1 in tobacco changed the fatty acid content in leaves, whereas over-expressed lines of NtKASI-2 exhibited distinct phenotypic features such as slightly variegated leaves and reduction of the fatty acid content in leaves, similar to the silencing plants of NtKASI-1 gene. Interestingly, the silencing of NtKASI-2 gene had no discernibly altered phenotypes compared to wild type. The double silencing plants of these two genes enhanced the phenotypic changes during vegetative and reproductive growth compared to wild type. These results uncovered that these two KASI genes had the partially functional redundancy, and that the KASI genes played a key role in regulating fatty acids synthesis and in mediating plant growth and development in tobacco.     

代谢工程改造蛋氨酸代谢途径构建高产L-蛋氨酸大肠杆菌

以Escherichia coli BL21(DE3)为出发菌株,敲除其蛋氨酸合成途径中关键酶的阻遏基因metJ,使菌株积累蛋氨酸达22 mg/L. 在此基础上,利用紫外诱变筛选育出一株抗蛋氨酸结构类似物的蛋氨酸高产菌株YB12,使蛋氨酸产量提高到60 mg/L. 通过过量表达蛋氨酸合成途径中的metA和cysE基因及编码蛋氨酸转运蛋白的yeaS基因,YB12菌株积累蛋氨酸量提高到251 mg/L. 结果表明,蛋氨酸在胞外的积累受多个基因、多种代谢途径调控,单独敲除某个基因或改造某个途径不能使蛋氨酸大量合成和积累,对多个代谢途径共同改造是构建蛋氨酸工程菌的最有效方法.     

The SKS1 gene of Saccharomyces cerevisiae is required for long-term adaptation of snf3 null strains to low glucose

The SKS1 gene was originally identified as a multicopy suppressor of the growth defect of snf3 null mutations on low glucose concentrations. Snf3p is required for the rapid induction of HXT2 during growth on low substrate concentrations. Loss of Snf3p leads to a dramatic delay in expression of HXT2. Adaptation to low substrate concentrations does not occur in snf3 sks1 double null mutant strains, suggesting that SKS1 is required for the glucose-dependent expression of HXT2 in the absence of Snf3p activity. Over-expression of SKS1 leads to over-expression of Hxt2p, thus explaining the mechanism of suppression of the snf3 defect. SKS1 defines a novel, Snf3p-independent pathway for the expression of Hxt2p. Under certain growth conditions, over-expression of SKS1 itself leads to a growth defect which is diminished in snf3 hxt2 double mutants. This suggests that over-expression of Hxt2p at physiologically inappropriate times is detrimental to the cells. © 1998 John Wiley & Sons, Ltd.     

Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.