Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei

Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.     

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly. Received: 4 May 1998 / Accepted: 23 April 1999     

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly.     

里氏木霉不同糖苷水解酶基因转录水平比较分析

以前期里氏木霉RNA-seq中发现的7个糖苷水解酶基因为对象,分析其不同条件下的表达特性,以期为寻找新的纤维素降解功能酶提供证据。运用生物信息学方法,分析了7个基因可能的编码产物和结构特征。以不同的产纤维素酶菌株（QM 9414、RUT C30）为材料,采用实时荧光定量PCR,对7个糖苷水解酶基因（编号4–10）在各种碳源条件下转录情况与主要的3个纤维素酶基因cbh1,cbh2,egl1（编号1–3）进行了比较分析。信息学分析表明,7个基因编码蛋白分属于GH47（4号、5号）,GH92（6–8号）,GH16（9号）,GH31（10号）糖苷水解酶家族,具有典型的信号肽序列。cbh1,cbh2,egl1基因在纤维素酶诱导条件下,转录水平均表现显著的增加,上调倍数以QM 9414菌株表现的最高。QM 9414菌株中,cbh1,cbh2,egl1基因在纤维素条件下的上调倍数显著高于乳糖,3个基因在RUT C30菌株中的转录水平则显示乳糖条件下上调幅度更大。7个糖苷水解酶基因也存在类似的情况,而且编码α-甘露糖苷酶和内切β-葡聚糖酶的8号、9号基因上调倍数在纤维素酶诱导条件下仅次于纤维素酶基因,而以甘油为碳源条件下,8号、9号基因上调倍数高于纤维素酶基因。4号基因在上述碳源条件下,转录水平变化不大。结果表明：4号基因可能是组成型表达。基因5、6、7、8、9、10的表达呈现明显的菌株和碳源依赖性,且在纤维素酶诱导条件下基本上是和3个纤维素酶基因共转录的。     

甘蔗花叶病毒E株系外壳蛋白基因原核表达载体的构建与表达

目的:用原核表达的方法获取大量带6个His标记的甘蔗花叶病毒E株系(ScMV-E)外壳蛋白(CP)。方法:用带有BamHⅠ和SalⅠ酶切位点的特异引物,以带有多个基因的重组质粒pNUSCP为模板,扩增出片段长度为942bp的ScMV-E外壳蛋白基因,亚克隆到pMD18-T载体上,转化E.coliDH5α,经双酶切检测获得阳性克隆。BamHⅠ和SalⅠ双酶切阳性克隆质粒,回收目的片段ScMV-E的CP基因。把目的片段插入表达载体pET29a( ),转化E.coliBL21(DE3),测序。结果:阳性质粒pET29a-CP在E.coliBL21(DE3)中得到大量特异表达。SDS-PAGE分析表明,该蛋白的相对分子质量约36000,与预测一致。结论:以上方法可以得到带6个His标记的目的蛋白,有利于纯化并获取高纯度的ScMV-E的外壳蛋白。     

Functional differences in yeast protein disulfide isomerases

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems

Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.     

酵母PMT1基因参与内质网应激的调控机制

【目的】内质网应激(Endoplasmic reticulum stress,ERS)可激活细胞保护性信号级联反应——未折叠蛋白质反应(Unfolded protein response,UPR)。研究表明,酵母细胞中的UPR信号通路由转录因子Hac1p和ERS感应因子Ire1p共同介导。前期研究发现:蛋白质-O-甘露糖转移酶1(Protein-O-mannosyltransferase 1,PMT1)基因缺失能延长酵母细胞的复制性寿命,其机制与上调UPR通路活性相关。本文进一步探讨PMT1基因缺失在酵母ERS反应中的作用。【方法】观察PMT1基因与IRE1或HAC1基因双缺失酵母菌株(pmt1?hac1?和pmt1?ire1?)在ERS反应条件下的克隆形成能力;通过比色法检测各菌株的细胞增殖活性;RT-PCR检测各菌株UPR通路下游部分靶基因的转录水平。【结果】与对照菌株比较,PMT1基因缺失菌株(pmt1?)在ERS反应条件下生长较慢,而HAC1和IRE1单基因缺失菌株(hac1?和ire1?)在ERS反应条件下无法存活;在hac1?或ire1?菌株的基础上进一步缺失PMT1基因,可以改善hac1?菌株在ERS反应条件下的生长状态;但缺失PMT1基因没有上调hac1?菌株UPR通路靶基因的转录水平。【结论】缺失PMT1基因可增强hac1?菌株对ERS诱导剂衣霉素的抗性,机制与已知的UPR通路不相关,提示可能存在其它途径参与ERS反应的调控。     

Effects of inactivation and constitutive expression of the unfolded- protein response pathway on protein production in the yeast Saccharomyces cerevisiae

One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens alpha-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both alpha-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in alpha-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of alpha-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation.