大肠杆菌发酵生产重组人颗粒溶素

目的:在FUS-50L生物反应器中利用补料分批培养技术培养表达含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS株,生产重组颗粒溶素(GNLY)。方法:选取含pET28a( )-GNLY的BL21(DE3)pLysS菌株单个克隆分级培养,将制备的二级种子液接种于发酵罐中。在发酵过程中,控制溶氧为30%～50%,温度为37℃;在基础培养基内生长4h后,补加以甘油为碳源的补料,继续生长到11h;加入葡萄糖至终浓度为1%,30℃诱导表达6h;收集菌体,纯化制备目的蛋白。利用Western印迹检测重组蛋白的抗原性,用CFU方法检测其生物学活性。结果:发酵液中最终菌体密度达80g/L;纯化所得重组蛋白约占菌体总蛋白的5%,含量为60mg/L;经鉴定所获重组颗粒溶素有较好的免疫学活性和生物学活性。结论:用含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS表达系统,可得到具有生物活性的重组颗粒溶素,为大批量生产提供了条件。     

蛇毒锯鳞蝰素融合蛋白的发酵与纯化

研究大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合蛋白的发酵和纯化工艺。将Ecs基因插入表达载体pTXB1,转化E.coliBL21(DE3)构建工程菌。对工程菌进行补料分批培养并诱导表达,研究培养基、培养和诱导时间对工程菌生长和目的蛋白表达的影响,几丁质亲和层析纯化Ecs融合蛋白,经DTT裂解后,检测Ecs活性。发酵后菌体湿重可达75g/L,融合蛋白表达量约占总蛋白的35%,重组质粒在BL21宿主菌中传代稳定。亲和层析纯化后,得到Ecs单体,得率为28mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。优化了Ecs融合基因工程菌的发酵和纯化条件,为规模化生产奠定基础。     

高密度培养大肠杆菌YK537/pSBHL11生产重组人细胞白介素-3

在B.Braun ES-10型15L和NBS BioFlo 3000型5L发酵罐中,利用补料分批培养技术高密度表达培养含重组质粒pSBHL-11的大肠杆菌YK537,生产重组人白细胞介素-3(IL-3),发现在发酵过程中,限制性流加甘油,控制溶解氧在30%～40％左右、30℃生长11h,42℃诱导培养4h,能将发酵液中最终菌体密度从OD₁₆600提高到OD₅₃600(相当于每升发酵液含106克湿菌体),并且保持了白细胞介素-3的表达量,占菌体总蛋白的30%左右,含量超过3.3%g/L,使IL-3包涵体产量从湿重2.2g/L提高到8.5 g/L,纯化步骤比较简单,超声破菌后经两次洗涤纯度就达到70%以上。     

真养产碱菌利用高果糖浆积累聚β-羟基丁酸的研究

真养产碱菌(Alcaligenes eutrophus)H16能以果糖为碳源在无机合成培养基上积累聚p-羟基丁酸(PHB)。将该菌用富含果糖的高果糖浆(HFS)培养,PHB的积累可达到果糖发酵水平,但发现高浓度的果糖和葡萄糖对菌体生长及PHB积累有抑制作用。采用补料分批培养技术可降低果糖和葡萄糖的抑制。并可大幅度提高产量,菌体干重达16—20g/L,PHB产量7.0—7.6g/L,PHB的产率达0.24g/g果糖。     

幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺的研究

通过不同培养基、不同葡萄糖浓度、不同溶氧条件、补料与非补料对幽门螺杆菌尿素酶B亚单位(UreB)基因工程菌的菌体生长与外源蛋白表达量的影响的比较 ,建立了稳定、适宜的幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺。多批实验结果证明 ,菌体单产可达 86 g/L ,目的蛋白的表达率为 38.2 %。     

pta基因敲除对L-色氨酸发酵的影响

[目的]探究pta基因缺失对大肠杆菌发酵生产L-色氨酸的影响.[方法]运用Red重组技术敲除pta基因,构建pta缺失株E.coli TRTH△pta.利用30 L发酵罐进行分批补料发酵试验,考察重组菌E.coliTRTHApta发酵0生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、发酵液中NH4+浓度及变化....     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

表达重组葡激酶-水蛭素融合蛋白的大肠杆菌工程菌的高密度培养

采用溶氧反馈的分批培养流加补料的方法高密度培养重组大肠杆菌BL21(DE3)生产重组葡激酶-水蛭素融合蛋白。通过摇瓶培养对菌种和培养条件的初步筛选,采用溶氧反馈的流加补料策略,进行了5L发酵罐的合成培养基和复合培养基的发酵工艺的研究。通过对培养条件的不断优化,重组葡激酶-水蛭素融合蛋白在大肠杆菌BL21(DE3)里得到了高效表达,菌体密度最终达到115g/L(WCW)以上,可溶性重组融合蛋白占菌体总蛋白的30%以上,含量约为1.1~1.2g/L。5L发酵罐的发酵工艺参数在40L发酵罐中进行了放大培养,结果表明该工艺能有效的放大,可适用于工业生产。     

破菌时可自动降解宿主核酸的大肠杆菌BL21(DE3)的lpxM突变株构建

研究利用Red同源重组技术对常用大肠杆菌表达宿主菌BL21(DE3)进行改良, 构建破菌时可自动降解宿主核酸的大肠杆菌表达宿主菌, 该菌株可望有助于解决因破菌时宿主菌染色体核酸释放给后续纯化重组蛋白工作带来的困难。将N端连有OmpA的信号肽的S. aureus nucleaseB(nucB)表达框整合至E. coli BL21(DE3)的lpxM位点, 改造后菌株(称为BLN)经诱导能表达nucB、并分泌至周质空间, 这样可使宿主核酸免受该酶“毒性”影响, 菌体裂解后, nucB释放,能自动降解宿主核酸。BLN菌体生长状态以及表达外源重组蛋白的能力与出发菌基本一致。     

乙酸对重组大肠杆菌BL21产酶的影响及作用机理研究

旨在研究发酵过程中产生的乙酸对重组大肠杆菌BL21(DE3)/p ET15b-Tre S的生长以及重组海藻糖合酶基因表达的影响,并对其作用机理进行探讨。通过外源添加乙酸(钠)的方法,研究了乙酸钠对重组菌BL21(DE3)/p ET15b-Tre S生长曲线的影响;利用环境扫描电子显微镜,观察了重组菌形态的变化情况;通过测定菌液的电导率值变化、菌液上清中OD260的变化,研究了乙酸(钠)对菌体细胞膜渗透性、完整性的影响;通过测定菌液上清中β-半乳糖苷酶的活性检测了乙酸钠对菌体细胞内膜的影响;利用荧光分析法检测了乙酸对菌体膜蛋白构象的影响;采用SDS-PAGE电泳研究了乙酸钠对菌体重组海藻糖合酶表达量的影响。结果表明,乙酸(钠)会对重组菌的生长产生一定的抑制作用,可以导致菌体细胞的表面出现凹陷、皱缩;并会影响细胞膜的渗透性、完整性,使得一些细胞内容物发生泄漏;影响细胞膜上的膜蛋白构象,对膜的结构造成一定程度的破坏;对重组海藻糖合酶的表达产生影响。乙酸(钠)对重组菌菌体的生长及重组海藻糖合酶基因的表达有影响,并且菌体细胞膜是其作用的一个靶点。     

产L-苏氨酸重组大肠杆菌的构建和发酵性能

【背景】大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌。【目的】敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株。【方法】利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysC~(fbr)、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中。【结果】以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶Ⅲ (AKⅢ)的基因lysC、磷酸果糖激酶Ⅱ基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35g/L,比出发菌株增加9.9%。随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysC~(fbr))、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株。该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20%,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L。【结论】单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法。     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Novel electrochemical-enzymatic model which quantifies the effect of the solution Eh on the kinetics of ferrous iron oxidation with Acidithiobacillus ferrooxidans

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.     

营养条件和流加发酵对放射型根瘤菌(Rhizobium radiobacter)产辅酶Q10的影响

利用放射型根瘤菌WSH2 6 0 1(RhizobiumradiobacterWSH2 6 0 1)重点考察了葡萄糖、蔗糖、玉米浆和蛋白胨、添加物以及流加发酵对细胞生长和产辅酶Q1 0 的影响 ,结果表明 ,葡萄糖和蔗糖适合于生产辅酶Q1 0 的最佳浓度分别为 30g L和 40g L ;辅酶Q1 0 发酵时玉米浆和蛋白胨的最适浓度分别为 11g L和 16g L ;添加蕃茄汁、玉米浆能提高发酵液的生物量 ,玉米浆、异戊醇、L 甲硫氨基酸等能促进辅酶Q1 0 的积累 ;与分批发酵相比 ,在 7L罐上流加蔗糖其细胞生物量 (DCW)和辅酶Q1 0 积累量增加 ,若在流加蔗糖的同时流加适当浓度的玉米浆能显著提高辅酶Q1 0 的产量 ,最大产量达到 5 2 .4mg L ;最大生物量 (DCW)和胞内辅酶Q1 0 含量 (C B值 )分别达到 2 6 .4g L和 2 .38mg g DCW ,比不流加的分批发酵分别提高 5 3 %和 33% ,比只流加蔗糖分别提高 2 4%和 2 6 %。     

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.     

Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides

For the commercial production of CoQ₁₀, batch-type fermentations were attempted in a 150-l fermenter using a mutant strain of R. sphaeroides. Optimum temperature and initial aeration rate were found to be 30°C and 2 vvm, respectively. Under optimum fermentation conditions, the maximum value of specific CoQ₁₀ content was achieved reproducibly as 6.34 mg/g DCW after 24 h, with 3.02 g/l of DCW. During the fermentation, aeration shift (from the adequate aeration at the early growth phase to the limited aeration in active cellular metabolism) was a key factor in CoQ₁₀ production for scale-up. A higher value of the specific CoQ₁₀ content (8.12 mg/g DCW) was achieved in fed-batch fermentation and comparable to those produced by the pilot-scale fed-batch fermentations of A. tumefaciens, which indicated that the mutant strain of R. sphaeroides used in this study was a potential high CoQ₁₀ producer. This is the first detailed study to demonstrate a pilot-scale production of CoQ₁₀ using a mutant strain of R. sphaeroides.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

重组大肠杆菌生产谷胱甘肽发酵条件的研究

研究了重组Ｅ．Ｃｏｌｉ产ＧＳＨ的发酵条件,重点考察了添加酵母膏、前体氨基酸和ＡＴＰ的影响。结果发现,前体氨基酸和ＡＴＰ均能促进胞内ＧＳＨ的积累,若在发酵０ｈ和１２ｈ分别加入２０ｇ／ＬＡＴＰ和９ｍｍｏｌ／Ｌ前体氨基酸,则细胞干重和胞内ＧＳＨ含量可分别比对照提高２４％和１４倍。应用正交试验得出的针对细胞干重和ＧＳＨ总量的最佳组合,最大细胞干重和ＧＳＨ总量比原试验中的最好结果分别提高了１０％和２６％。在分析了该菌对葡萄糖利用情况的基础上,对该菌进行了指数流加培养,２５ｈ细胞干重与发酵液内ＧＳＨ总量分别达到８０ｇ／Ｌ和８８０ｍｇ／Ｌ,比摇瓶最好结果分别提高了８３和４６倍。     

Lactate increases coenzyme Q10 production by Agrobacterium tumefaciens

By the optimization of nitrogen source for coenzyme Q₁₀ (ubiquinone, CoQ₁₀) production in Agrobacterium tumefaciens KCCM 10413 culture, the highest CoQ₁₀ production was achieved in medium containing corn steep powder (CSP). Components for a stimulatory effect on the production of CoQ₁₀ in CSP were screened, and lactate was found to increase dry cell weight (DCW) and the specific CoQ₁₀ content. In a fed-batch culture of A. tumefaciens, supplementation with 1.5 g of lactate l⁻¹ further improved DCW, the specific CoQ₁₀ content, and CoQ₁₀ production by 16.0, 5.8, and 22.8%, respectively. It has been reported that lactate stimulates cell growth and acts as an accelerator driving the tricarboxylic acid (TCA) cycle (Roberto et al. 2002, Biotechnol Let 24:427–431; Matsuoka et al. 1996, Biosci Biotechnol Biochem 60:575–579). In this study, lactate supplementation increased DCW and the specific CoQ₁₀ content in A. tumefaciens culture, probably by accelerating TCA cycle and energy production as reported previously, leading to the increase of CoQ₁₀ production.