葡糖酸醋杆菌内源复制片段的克隆

采用改进的碱裂解法提取Gluconacetobacter hansenii ATCC23769自发不产膜突变体的内源隐蔽质粒。用不同的限制性内切酶对混合质粒直接进行酶切,酶切后的片段混合物与pUC18载体连接构建重组载体。重组载体回转入G.hansenii ATCC23769获得隐蔽质粒上具有复制能力的片段,序列结果分析表明:该片段上没有某些其他质粒所具有的Rep蛋白。利用该片段,构建了能同时在大肠杆菌和葡糖酸醋杆菌中复制的质粒载体,体内的抗生素抗性实验证明该载体具有良好的稳定性。     

发酵工程

当木醋杆菌(通cetobaeter xylinum)ATCC 23769在每升含有209葡萄糖、59酵母膏、59蛋白陈、2.79硫酸钠和1.15g柠檬酸的非特定培养基上生长时,合成了     

CRISPR/dCas9干扰bcsD基因表达调控细菌纤维素结构

木葡糖酸醋杆菌(Gluconacetobacter xylinus)是细菌纤维素的主要生产菌株。在该菌中,BcsD是纤维素合酶的亚基之一,参与细菌纤维素的组装过程。利用CRISPR/dCas9系统调控bcsD基因的表达量,获得了一系列bcsD基因表达量不同的木葡糖酸醋杆菌。通过分析细菌纤维素的结构特征发现,细菌纤维素的结晶度和孔隙率随着木葡糖酸醋杆菌中bcsD表达量的变化而发生改变。其中孔隙率的变化范围在59.95%–84.05%之间,结晶度的变化范围在74.26%–93.75%之间,而细菌纤维素的产量并未因bcsD的表达量变化而发生显著下降。结果表明,bcsD的表达量低于55.34%后,细菌纤维素的孔隙率显著上升,并且细菌纤维素的结晶度与bcsD的表达量呈正相关。最终,通过干扰bcsD基因的表达,实现了一步发酵木葡糖酸醋杆菌获得了产量稳定且结构不同的细菌纤维素。     

PspA外源表达对枯草芽孢杆菌168蛋白质分泌的影响

PspA同源物广泛存在于细菌和高等生物的组织中.在本研究中克隆了来源于地衣芽孢杆菌的PspA基因,并将其克隆于用于大肠-芽孢穿梭诱导表达载体pDG-StuI中构建重组质粒pDG-PspA.将构建的诱导表达型的重组质粒转化到Bacillus subtilis 168中,研究PspA的外源表达对该菌的生长,总蛋白分泌,以及Sec分泌途径中α-淀粉酶分泌的影响,结果表明,PspA基因的外源表达,在发酵过程后期能在一定程度上提高总蛋白的分泌量,在发酵过程后期能在一定程度上提高分泌的α-淀粉酶浓度.     

短短小芽孢杆菌大肠杆菌穿梭分泌表达载体的构建

应用PCR技术从具有分泌蛋白能力强且没有胞外蛋白酶活性的短短小芽孢杆菌50中分离出细胞壁蛋白基因的多启动子和信号肽编码序列,利用它与质粒pUB110和pKF3-起构建成穿梭分泌表达载体pBKE50,将α0淀粉酶基因引入该载体转化短短小芽孢杆菌50后,发现α－淀粉酶可以活性形式分泌表达,此工作为下一步建立短短小芽孢杆菌高效分泌表达系统奠定了基础。     

短短小芽孢杆菌启动子筛选载体的构建

为了筛选短短小芽孢杆菌强启动子元件,应用PCR技术从枯草杆菌168中分离出α-淀粉酶基因,用其作为报告基因与质粒pUB110和pKF3一起构建了启动子筛选载体pKB/A.将短短小芽孢杆菌细胞壁蛋白基因启动子引入该载体构建成重组质粒pKB/PA,电穿孔法转化短短小芽孢杆菌50后发现α-淀粉酶以活性形式分泌表达.结果表明短短小芽孢杆菌启动子筛选载体构建成功.     

芽孢杆菌分泌型表达载体的构建

以地衣芽孢杆菌(B．Lichenformis)α－淀粉酶信号肽编码区为基础构建了具有Pst I、NheⅠ、SmaⅠ、BamHⅠ、SalⅠ多酶切点的芽孢杆菌分泌型载体pAMY403,其分泌效率与质粒pAMY 413相同,而表达水平提高了50％,并能使外源β－内酰胺酶基因正常表达和分泌。粒pAMY403多酶切点可以满足外源基因插入产生非融合蛋白质,也可满足以三种不同读码框架插入外源基因产生融合蛋白质,因而能适应构建不同分泌型工程菌株的要求。     

大麦α-淀粉酶和黑曲霉糖化酶在酿酒酵母中的表达和分泌

将大麦α 淀粉酶和黑曲霉糖化酶cDNA重组进同一大肠杆菌 酵母穿梭质粒构建含双基因的表达分泌载体 pMAG1 5 .用原生质体转化法将 pMAG1 5引入酿酒酵母 (S .cerevisiae  GRF1 8) ,在酵母PGK基因的启动子和转录终止信号及本身的信号序列的调控下 ,实现大麦α 淀粉酶和糖化酶的高效表达 ,99%以上的酶活力分泌至培养基中 .构建的酿酒酵母菌株GRF1 8( pMAG1 5 )在含 1 5 %可溶性淀粉的培养基中 ,培养 47h能水解 99%的淀粉 ,并能发酵产生酒精     

葡糖淀粉酶基因的分子生物学研究

从酶活、底物、产物、应用及空间结构等方面论述了葡糖淀粉酶的一般生物学特性及应用,分析了其不同来源的基因同源度。着重从分子生物学角度阐述了葡糖淀粉酶基因在多种表达系统中的分泌表达情况,并对其基因在曲霉及酵母中的高效表达及分泌引导功能进行了初步阐述和探讨。     

美洲拟鲽抗菌肽Pleurocidin在大肠杆菌中的高效分泌表达及优化

为在大肠杆菌中分泌表达Pleurocidin,并提高融合蛋白的分泌效率,将Pleurocidin基因和Cherry DNA序列通过平末端连接融合,再将融合基因整合到p ET22b(+)载体中,转化大肠杆菌BL21(DE3);乳糖诱导表达。成功构建含p ET22b(+)-CP重组质粒的基因工程菌,用乳糖诱导获得高效表达。在诱导16 h时加入甘氨酸可以显著提高融合蛋白Cherry-Pleurocidin的分泌效率。用稀盐酸水解融合蛋白的酸敏感位点,再进一步分离纯化即得到r-Pleurocidin,其对大肠杆菌DH5α和枯草芽孢杆菌BS168具有明显的抑菌活性。结果表明成功构建了高效表达Pleurocidin的大肠杆菌基因工程菌,获得有活性的r-Pleurocidin。     

AcsA–AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA–AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA–AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA–AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.     

Taxonomic heterogeneity of strains comprising Gluconacetobacter hansenii

The taxonomic standing of Gluconacetobacter hansenii was clarified through phenotypic characteristics, quinones, DNA base composition, DNA relatedness, and the production of gluconic and ketogluconic acids from glucose. All strains that Gosselé et al. (Syst. Appl. Microbiol., 4, 338-368, 1983) employed in the establishment of Acetobacter hansenii (=G. hansenii) were used in this study. Phenotypic differences were shown among the strains of G. hansenii, suggesting heterogeneity within the species. The major ubiquinone was Q-10 for all strains of G. hansenii, except for strain IFO 3296, which was characterized by Q-9. This excluded IFO 3296 from the species G. hansenii and placed it in the genus Acetobacter. DNA relatedness revealed four distinct homology groups (I, II, III, and IV) among strains of the species. Group I was distinguished from the other genomic groups by a lower G1C range from 58.9 to 59.2 mol%. Groups II, III, and IV showed higher G+C contents of 60.4 to 62.2, 60.8, and 61.7 mol%, respectively. Groups I and IV produced both 2- and 5-ketogluconic acids from glucose, and Group III produced only 2-ketogluconic acid. Group II included strains that produced both 2- and 5-ketogluconic acids and strains that produced only 2-ketogluconic acid. It is clear that G. hansenii consists of genotypically heterogeneous strains comprising four homology groups (I, II, III, and IV). Since group I contains the type strain (IFO 14820(T)=LMG 1527(T)) of the species, this group is designated as the species G. hansenii.     

Cloning of cellulose synthesis related genes from Acetobacter xylinum ATCC23769 and ATCC53582: comparison of cellulose synthetic ability between strains.

About 14.5 kb of DNA fragments from Acetobacter xylinum ATCC23769 and ATCC53582 were cloned, and their nucleotide sequences were determined. The sequenced DNA regions contained endo-beta-1,4-glucanase, cellulose complementing protein, cellulose synthase subunit AB, C, D and beta-glucosidase genes. The results from a homology search of deduced amino acid sequences between A. xylinum ATCC23769 and ATCC53582 showed that they were highly similar. However, the amount of cellulose production by ATCC53582 was 5 times larger than that of ATCC23769 during a 7-day incubation. In A. xylinum ATCC53582, synthesis of cellulose continued after glucose was consumed, suggesting that a metabolite of glucose, or a component of the medium other than glucose, may be a substrate of cellulose. On the other hand, cell growth of ATCC23769 was twice that of ATCC53582. Glucose is the energy source in A. xylinum as well as the substrate of cellulose synthesis, and the metabolic pathway of glucose in both strains may be different. These results suggest that the synthesis of cellulose and the growth of bacterial cells are contradictory.     

Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D. occidentalis AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.     

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).     

Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining δ-integration and polyploidization through cell fusion. Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase/α-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The α-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

Abstract The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10–20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase ( gla A), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any gla A coding sequence. The portion of gla A used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.     

Ethanol production and fermentation characteristics of recombinant saccharomyces cerevisiae strains grown on starch

The production of ethanol from starch has been investigated in three genetically modified Saccharomyces cerevisiae strains (YPG/AB, YPG/MM, and YPB-G). Two of the three strains produce the Aspergillus awamori glucoamylase together with either the Bacillus subtilis (YPG/AB) or the mouse (YPG/MM) α-amylase as separately secreted polypeptides. YPB-G, on the other hand, secretes a bifunctional fusion protein that contains both the B. subtilis α-amylase and the A. awamori glucoamylase activities. Substrate utilization, biomass growth, and ethanol production were all studied in both starch- and glucose-containing media. Much higher growth rates were found when any of the three strains were grown on glucose. YPG/AB showed the most efficient utilization of starch for ethanol production with the lowest levels of reducing sugars accumulating in the medium. The superior performance of YPG/AB as compared to YPB-G was found to correlate with its higher level of α-amylase activity. The ethanol production levels of YPG/AB in starch- and glucose-containing media were found to be comparable. YPB-G, which secretes the bifunctional fusion protein, could produce ethanol in media with starch concentrations above 100 g l⁻¹ while YPG/MM did not produce ethanol from starch because of its negligible secretion of glucoamylase.     

Statistical analysis of optimal culture conditions for Gluconacetobacter hansenii cellulose production

AIM: The purpose of this study was to analyse the effects of different culture parameters on Gluconacetobacter hansenii (ATCC 10821) to determine which conditions provided optimum cellulose growth. METHODS AND RESULTS: Five culture factors were investigated: carbon source, addition of ethanol, inoculation ratio, pH and temperature. jmp Software (SAS, Cary, NC, USA) was used to design this experiment using a fractional factorial design. After 22 days of static culture, the cellulose produced by the bacteria was harvested, purified and dried to compare the cellulose yields. The results were analysed by fitting the data to a first-order model with two-factor interactions. CONCLUSIONS: The study confirmed that carbon source, addition of ethanol, and temperature were significant factors in the production of cellulose of this G. hansenii strain. While pH alone does not significantly affect average cellulose production, cellulose yields are affected by pH interaction with the carbon source. Culturing the bacteria on glucose at pH 6.5 produces more cellulose than at pH 5.5, while using mannitol at pH 5.5 produces more cellulose than at pH 6.5. The bacteria produced the most cellulose when cultured on mannitol, at pH 5.5, without ethanol, at 20 degrees C. Inoculation ratio was not found to be a significant factor or involved in any significant two-factor interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings give insight into the conditions necessary to maximize cellulose production from this G. hansenii strain. In addition, this work demonstrates how the fractional factorial design can be used to test a large number of factors using an abbreviated set of experiments. Fitting a statistical model determined the significant factors as well as the significant two-factor interactions.