Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris.

Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS-PAGE gels. Carbohydrate content determination and SDS-PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild-type enzyme. More seriously, the yeast-expressed enzyme showed non-wild-type secondary structure by circular dichroism. We conclude that P. pastoris may not serve as an adequate host for the site-directed mutagenesis of T. reesei CBH I.     

里氏木霉纤维二糖水解酶Ⅱ在毕赤酵母中的高效表达

本工作采用巴氏毕赤酵母Pichiapastoris表达系统进行了里氏木霉Trichodermareesei纤维二糖水解酶Ⅱ(CellobiohydrolaseII)的表达。用RT-PCR的方法从经稻草粉诱导的里氏木霉培养物中分离出纤维二糖水解酶Ⅱ的基因,将其插入到巴氏毕赤酵母的表达载体pPICZαA中,并使之处于α-因子信号肽序列的下游,得到重组质粒pPICZαA-cbh2。通过电穿孔的方法用线性化的pPICZαA-cbh2转化巴氏毕赤酵母GS115菌株,经过大量筛选后得到可以高效表达纤维二糖水解酶的毕赤酵母菌株P.pastorisCBHⅡ1。在甲醇诱导的条件下培养P.pastorisCBHⅡ1,培养液中的CMC活性可达到3.82U/mL,SDS-PAGE分析结果表明纤维二糖水解酶在P.pastorisCBHⅡ1中的表达量远远高于里氏木霉。对表达产物进行了LC-MS分析,结果表明所表达的蛋白为里氏木霉的纤维二糖水解酶。     

Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.     

Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters

Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K(m) values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.     

敲除组蛋白去乙酰化酶基因hdac对里氏木霉纤维素酶表达的调控作用

[背景]里氏木霉(Trichoderma reesei)是木霉属中产纤维素酶最具代表性的真菌之一,表观遗传调控是不涉及DNA序列变化的可遗传变化,组蛋白去乙酰化是其中一种。组蛋白去乙酰化酶(histone deacetylase,HDAC)负责脱乙酰化,敲除去乙酰化酶基因可引起菌株孢子、菌丝及纤维素酶活性等的一系列改变。[目的]通过敲除里氏木霉组蛋白去乙酰化酶基因(histone deacetylase,hdac)建立了里氏木霉hdac缺失突变株(T.reesei△hdac),以研究对纤维素酶基因表达的调控作用。[方法]利用Split-Maker技术构建了组蛋白去乙酰化酶基因敲除表达盒,并转化了里氏木霉T.reesei QM9414。经PCR及Southern blotting验证正确后,对突变体T.reesei△hdac连续7 d检测滤纸酶活(filter paper activity,AFP)、羧甲基纤维素钠酶活(carboxymethyl cellulase activity,CMCA),利用RT-qPCR检测纤维素酶及其相关基因cbh1、egl1和xyr1的表达。[结果]突变体T.reesei△hdac两种酶活力均显著高于出发菌株,分别高出8.00、30.00 IU/mL。突变体T.reesei△hdac纤维素酶及其相关基因cbh1、egl1和xyr1的转录水平分别为出发菌株T.reesei QM9414的6.50、6.01和4.51倍。[结论]里氏木霉中纤维素酶的基因表达明显受到组蛋白去乙酰化酶基因(hdac)的调控,这为研究里氏木霉表观遗传调控对纤维素酶的影响提供了新的证据。     

用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因

目的:为提高β-葡萄糖苷酶的产量,用毕赤酵母取代理氏木霉用于生产,以弥补理氏木霉在大规模生产中的缺陷。方法:用套叠PCR法从理氏木霉基因组中扩增β-葡萄糖苷酶基因(bglⅠ)。用T4DNA连接酶和限制性DNA内酶将bglⅠ重组于P.pastoris表达载体pPIC9K的多克隆位点,获得含bglⅠ的重组表达载体pPIC9K-bglⅠ。通过电转法将其pPIC9K-bglⅠ载体转化于P.pastoris基因组,筛选高G418抗性以及高表达bglⅠ酶的重组子作为工程菌。结果:用BMGY-BMMY培养基体系,在摇瓶中发酵48 h,表达BglⅠ30 mg/L,在P.pastoris中表达的BglⅠ能水解对硝基苯-β-D-葡萄糖苷具有β-葡萄糖苷酶活性。其酶活力为56 U/L发酵液。结论:通过这种方法,可以成功地用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

乳酸菌素Gassericin T基因的人工合成及其组成型表达载体的构建

目的:为了研究乳酸菌素Gassericin T的作用机制及应用价值,人工合成Gassericin T基因并构建能高效表达外源蛋白的毕赤酵母组成型表达载体。方法:应用PCR方法从毕赤酵母染色体中扩增GAP启动子,经测序正确后与已线性化的不含pAOX1启动子的毕赤酵母诱导型表达载体pPIC9K连接,转化大肠杆菌DH5α。根据Gassericin T的基因序列,把Gassericin T的结构基因gatA的密码子转换成毕赤酵母偏爱的形式,设计了6条59nt的寡聚核苷酸引物,通过3次连续PCR反应,人工合成gatA片段(简称gat基因),经测序正确后插入pGAP9K质粒的多克隆位点。结果:用GAP启动子(pGAP)取代了pPIC9K上的pAOX1,构建了毕赤酵母组成型表达载体pGAP9K;PCR拼接获得250bp的目的基因序列,将目的基因克隆于pGAP9K,获得组成型表达载体pGAP9K-gat。结论:为下一步在毕赤酵母中组成型表达外源蛋白,研究其作用机理和遗传机制奠定了基础。     

Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.     

Cloning and expression of a Melanocarpus albomyces steryl esterase gene in Pichia pastoris and Trichoderma reesei

The ste1 gene encoding a steryl esterase was isolated from the thermophilic fungus Melanocarpus albomyces. The gene has one intron, and it encodes a protein consisting of 576 amino acids. The deduced amino acid sequence of the steryl esterase was shown to be related to lipases and other esterases such as carboxylesterases. Formation of mature protein requires post-translational removal of a putative 18-amino-acid signal sequence and a 13-residue propeptide at the N-terminus. The intronless version of the Melanocarpus albomyces ste1 gene was expressed in Pichia pastoris under the inducible AOX1 promoter. The production level was low, and a large proportion of the total activity yield was found to be present intracellularly. However, the fact that steryl esterase activity was produced by P. pastoris cells carrying the expression cassette confirmed that the correct gene had been cloned. The ste1 gene was subsequently expressed in T. reesei under the inducible cbh1 promoter, and a clearly higher production level was obtained. About 60% of the total activity was bound to the fungal mycelium or to solid components of the culture medium, or existed as aggregates. Triton X-100 was successfully used to recover this activity. The heterologous production system in T. reesei provides a means of producing M. albomyces steryl esterase STE1 reliably in large scale for future studies.     

Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75

Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5 degrees C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.     

Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis

Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G+C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K-zbtb7a-his(6) was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS-PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.     

Cloning,sequence analysis and yeast expression of the egl1 gene from Trichoderma longibrachiatum

A gene (egl1) encoding an endoglucanase (EGL1) from Trichoderma longibrachiatum has been cloned and sequenced. This gene, homologous to the T. reesei egl1 gene, differs from it in the length of the introns (particularly the first one) and encoded protein. A cDNA fragment obtained by the rapid amplification of cDNA ends method, which takes advantage of the polymerase chain reaction, has been expressed in yeast under control of the cyc-gal inducible promoter and yeast clones able to secrete active enzyme have been obtained. Correspondence to: J. A. Pérez-González     

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).     

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.     

Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization

Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve its three dimensional structure. Pichia pastoris is a suitable expression system for the production of recombinant enzymes for NMR studies and therefore we expressed TYR2 in this host. As a result of extensive optimization, the production yield of active histidine tagged tyrosinase purified from P. pastoris shake flask cultures was increased from 2.5 to 24 mg/L. Correct copper concentration in the growth medium was critical for the expression of this copper containing enzyme. Our analysis showed that TYR2 expressed in P. pastoris is post-translationally modified; the C-terminal domain of 153 amino acids of the protein is proteolytically cleaved off from the catalytic domain and the only potential N-glycosylation site is glycosylated. The activities of TYR2 expressed in P. pastoris and T. reesei on diphenolic L-dopa and monophenolic L-tyrosine were rather similar. The TYR2 expressed in P. pastoris showed the same physicochemical properties in CD and unfolding assays as the native TYR2 enzyme. Uniform isotopic (15)N-labeling of TYR2 was carried out with (15)NH(4)SO(4) in minimal medium to assess the suitability of the expression system for investigation by NMR spectroscopy.     

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.