Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Construction of thermotolerant yeast expressing thermostable cellulase genes

Kluyveromyces marxianus NBRC1777 was identified as a thermotolerant yeast and was developed as a host for the expression of thermostable cellulase genes. The previously isolated genes for thermostable endo-beta-1,4-glucanase, cellobiohydrolase, and beta-glucosidase were introduced into the chromosome of K. marxianus and successfully expressed under the control of high-expression promoters. The recombinant K. marxianus expressing cellulase genes became able to grow in synthetic medium containing cellobiose or carboxymethyl-cellulose as the single carbon source. Moreover, the recombinant strain produced 43.4 g/L ethanol from 10% cellobiose. These results suggest that K. marxianus may afford a useful host system, which may be applicable to the simultaneous saccharification and fermentation and the foundation of cellulose consolidated bioprocessing.     

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermesindicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichiacoli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonasmobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.     

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353. © 2008 Wiley Periodicals, Inc.     

黄瓜膨胀素的重组表达及活性分析

目的:提高纤维素的酶水解效率和开发高效的纤维素酶水解过程。方法:采用RT-PCR方法从黄瓜胚轴细胞中分离了膨胀素S1的cDNA,并使之与毕赤酵母表达质粒pPICZ(A连接,形成重组质粒pPICZ(A-exs1。通过电转化方法,用质粒pPICZ(A-exs1转化巴氏毕赤酵母GS115,得到重组菌株P.pastoris-exs1。在该重组菌株中,膨胀素的基因通过同源重组整合在毕赤酵母的染色体上,并处于毕赤酵母甲醇氧化酶启动子的下游。重组菌株P.pastoris-exs1在甲醇诱导下可合成并分泌膨胀素。结果:培养上清液没有纤维素酶活性,但具有破坏滤纸纤维素结晶结构的能力。培养上清液与里氏木霉纤维素酶等量混合后,可使纤维素酶的滤纸酶活力提高50%。结论:采用巴氏毕赤酵母GS115重组成功表达了黄瓜膨胀素,其表达产物可以促进纤维素酶对滤纸的水解。     

产外切葡聚糖酶真菌的筛选鉴定及毕赤酵母中的表达

【目的】外切葡聚糖酶是纤维素酶组分中一类对结晶纤维素有降解作用的酶类,如何提高外切葡聚糖酶活力是研究的关键问题。【方法】从筛选鉴定得到的一株产外切葡聚糖酶酶活较高的黑曲霉Asp-524菌株出发,通过PCR技术克隆得到外切葡聚糖酶基因序列,生物学信息分析后,构建了毕赤酵母诱导型表达载体,实现了该基因在毕赤酵母中的成功表达。【结果】抗性筛选得到的阳性转化子,用终浓度为1%甲醇诱导5 d后,酶活达到4.74 U/mL。酶学性质分析显示重组外切葡聚糖酶最适pH为5.0,pH稳定性分析显示在pH为4.0-6.0范围内相对稳定,酶活能保持在最高酶活力的80%以上,最适反应温度为50°C,经60°C保温1 h后,酶活仍能保持80%以上。【结论】结果说明该外切葡聚糖酶具有较好的热稳定性和pH稳定性,这一研究为纤维素酶的实际应用奠定了一定基础。     

Heterologous expression of Neurospora crassa cbh1 gene in Pichia pastoris resulted in production of a neutral cellobiohydrolase I

The high production cost of cellulase is one of the limitations that hinder the commercialization of lignocellulose-based biorefineries. As one of the important cellulases, Neurospora crassa cellulase is not so intensively investigated as T. reesei cellulase. In this study, the cbh1gene (NCU07340) cloned from N. crassa was expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promotor. Six transformants with the highest resistance to G418 were selected by two rounds of transformant screening, among which the most robust producer of the recombinant cellobiohydrolase I (CBHI) has an Avicelase activity of 0.22 U/mL. After fermentation optimization, it was improved to 0.30 U/mL. Interestingly, the optimal temperature and pH of the recombinant CBHI were 60°C and 7.2, respectively, and it was quite stable within the wide ranges of temperature and pH. This work is a good example for the future improvement and optimization of N. crassa cellulase.     

Characterization of Trichoderma reesei endoglucanase ii expressed heterologously in Pichia pastoris for better biofinishing and biostoning

The endoglucanase II of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase II is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut(+) transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase II was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.     

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.     

热稳定高比活纤维素酶在Pichia pastoris中的表达研究

Bacillus amyoliquefaciens DL-3纤维素酶具有热稳定高比活多功能的酶学特性,本文根据该酶的氨基酸序列合成了其编码基因(cel),构建了pPIC9K-cel表达载体,并用P.pastoris进行了表达。工程菌株三角瓶发酵酶活性达0.50 U/mL,酶解滤纸的产物为低聚糖,表明人工合成的热稳定高比活纤维素酶基因在P.pastoris中可以正常表达、加工及分泌,重组酶的分子量由天然酶的53 kDa增加至68 kDa,糖基化严重。     

Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from Coptotermes formosanus and expressed in E. coli

An endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in Escherichia coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrates. The nCfEG was more active and stable than tCfEG even though the latter could be purified to near homogeneity with a simple procedure. The differential activities of nCfEG and tCfEG were also evidenced by hydrolytic products they produced on different substrates. On CMC, both acted as an endoglucanase, randomly hydrolyzing internal β-1,4-glycosidic bonds and resulting in a smear of polymers with different lengths, although cellobiose, cellotriose, and cellotetraose equivalents were noticeable. The hydrolytic products of tCfEG were one unit sugar less than those produced by nCfEG. Using filter paper as substrate, however, the major hydrolytic products of nCfEG were cellobiose, cellotriose and trace of glucose; those of tCfEG were cellobiose, cellotriose and trace of cellotetraose, indicating a property similar to that of cellobiohydrolase, an exoglucanase. The results presented in this report uncovered the biochemical properties of the recombinant cellulase derived from the intact gene of Formosan subterranean termites. The recombinant cellulase would be useful in designing cellulase-inhibiting termiticides and incorporating into a sugar-based biofuel production program.     

Isolation, characterization and manipulation of cellulase genes

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.     

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml⁻¹, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.     

极端耐热纤维素酶Cel74的表达、纯化与特性

从海栖热袍菌中克隆出编码热稳定性的纤维素酶基因,以热激载体pHsh为表达质粒,构建重组质粒phsh—Ceff4,并转化至大肠杆菌中进行表达。基因表达产物通过热处理和离子交换层析,重组酶纯度达电泳纯。对纯化的重组酶酶学性质研究表明,最适反应温度85℃,最适反应pH4．6,pH4．5—6．0之间酶的相对酶活在80％以上。Co^2＋对酶活性有促进作用,Ca^2＋、Mg^2＋、Zn^2＋不影响酶活性,而Cu^2＋、Ni^2＋、Mn^2＋对酶活性有抑制作用。     

A useful method integrating production and immobilization of recombinant cellulase

The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase (celA), cellobiohydrolase (celK), and β-glucosidase (gls) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli, each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose–hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives of Escherichia coli KO11

This study demonstrates a new approach to reduce the amount of fungal cellulase required for the conversion of cellulose into ethanol. Escherichia coli KO11, a biocatalyst developed for the fermentation of hemicellulose syrups, was used to produce recombinant endoglucanase as a co-product with ethanol. Seven different bacterial genes were expressed from plasmids in KO11. All produced cell-associated endoglucanase activity. KO11(pLOI1620) containing Erwinia chrysanthemi celZ (EGZ) produced the highest activity, 3,200 IU endoglucanase/L fermentation broth (assayed at pH 5.2 and 35 degrees C). Recombinant EGZ was solubilized from harvested cells by treatment with dilute sodium dodecyl sulfate (12.5 mg/ml, 10 min, 50 degrees C) and tested in fermentation experiments with commercial fungal cellulase (5 filter paper units/g cellulose) and purified cellulose (100 g/L). Using Klebsiella oxytoca P2 as the biocatalyst, fermentations supplemented with EGZ as a detergent-lysate of KO11(pLOI1620) produced 14%-24% more ethanol than control fermentations supplemented with a detergent-lysate of KO11(pUC18). These results demonstrate that recombinant bacterial endoglucanase can function with fungal cellulase to increase ethanol yield during the simultaneous saccharification and fermentation of cellulose. (c) 1997 Wiley & Sons, Inc. Biotechnol Bioeng 55: 547-555, 1997.     

曲霉与木霉纤维素酶系基因组的属间遗传表达相容性

选用三类典型重组子3a、3b、A7-1和双亲本菌株AspersillusnigerAMSH、TrichodermareeseiQM9414为材料,按照所设计的纤维素酶系基因的通用序列和同工酶分型方法,进行基因组DNA指纹和酶系同工酶多态性比较分析。旨在提供重组子中基因重组的分子证据,阐明远缘双亲本基因组间的遗传表达相容性,并讨论其杂种优势的分子基础。结果发现重组子中基因组DNA指纹的重组特征稳定遗传,并能够相容性增强表达重组后的羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶（βGlase）同工酶组分。纤维素酶系杂种优势的分子基础多样性包括：（1）3b中来自于双亲本部分编码βGlase的基因的杂合迭加和增强表达;（2）3a和A7-1中对应继承双亲本部分编码CMCase和βGlase的基因间协调性增强表达,并导致相应酶组分蛋白合成量的显著增加。由此综合提出了一个由βGlase介导的纤维素酶系活性调节和诱导合成调控的“双重协同增效”模型。此外还建立了考察重组子中杂种优势分子基础及其遗传稳定性的可行方法。