Construction of a promoter collection for genes co-expression in filamentous fungus Trichoderma reesei

Trichoderma reesei is the preferred organism for producing industrial cellulases. However, cellulases derived from T. reesei have their highest activity at acidic pH. When the pH value increased above 7, the enzyme activities almost disappeared, thereby limiting the application of fungal cellulases under neutral or alkaline conditions. A lot of heterologous alkaline cellulases have been successfully expressed in T. reesei to improve its cellulolytic profile. To our knowledge, there are few reports describing the co-expression of two or more heterologous cellulases in T. reesei. We designed and constructed a promoter collection for gene expression and co-expression in T. reesei. Taking alkaline cellulase as a reporter gene, we assessed our promoters with strengths ranging from 4 to 106 % as compared to the pWEF31 expression vector (Lv D, Wang W, Wei D (2012) Construction of two vectors for gene expression in Trichoderma reesei. Plasmid 67(1):67–71). The promoter collection was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. We observed higher activities of both cellulose degradation and biostoning by the co-expression of an endoglucanase and a cellobiohydrolase than the activities obtained by the expression of only endoglucanase or cellobiohydrolase. This study makes the process of engineering expression of multiple genes easier in T. reesei.     

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

Regulation of gene expression in industrial fungi: <Emphasis Type="Italic">Trichoderma</Emphasis>

The genus Trichoderma comprises a group of filamentous ascomycetes that are now widely used in industrial applications because of their ability to produce extracellular hydrolases in large amounts. In addition, strong inducible promoters together with high secretory capacity have made Trichoderma an attractive host for heterologous protein production. Several promoters of genes encoding hydrolytic enzymes have been investigated in detail regarding their cis-acting elements and trans-acting factors. Potent inducer molecules, for both xylanolytic and cellulolytic enzyme systems, have been identified and characterized. Furthermore, models for the recognition of the insoluble substrates cellulose and xylan have been developed based on a large set of experiments. This mini-review summarises the considerable amount of data accumulated over the past three decades.     

Efficient production of recombinant DNA proteins in Saccharomyces cerevisiae by controlled high-cell-density fermentation

High levels of expression of heterologous proteins (from 5 to 15% of total cell proteins) in the budding yeast Saccharomyces cerevisiae have been obtained previously by the use of the inducible strong hybrid promoter UASGAL/CYC1, in batch as well in continuous cultures. However, in order to maximize the yield of heterologous proteins, a computer controlled fed-batch fermentation is essential. For this reason we have developed a fed-batch system based on a semiconductor gas detector that measures ethanol in the outflow gases. The optimal conditions are described for very high production (up to 1550 mg/liter), with both high productivity (up to 100-120 mg/liter/h) and high yield (up to 15 mg of protein/g of dry biomass), of heterologous protein driven by the UASGAL/CYC1 promoter in a completely computer controlled fed-batch fermentation of budding yeast. However, high production was dependent upon the addition of a large amount of galactose. The process was improved by developing a new, more easily inducible, vector system obtained by subcloning the GAL4 gene.     

Determination of the cellulolytic activity ofMicromycetes by an express method

An express method of testing the cellulolytic activity ofTrichoderma viride in media with cellulose-containing substrates by the rate of glucose production has been elaborated. The optimum temperature selections and duration of sample incubation (1 h, 50°C) have been described, and the temperature coefficient for the scale-down of the enzyme complex from 50 to 30°C (0.77) has been determined.     

An Escherichia coli expression vector for high-level production of heterologous proteins in fusion with CMP-KDO synthetase

The construction of a vector which overproduces the enzyme, CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase or CKS) and its use as an expression vector for producing heterologous proteins in E. coli is described. The vector, which includes a modified lac promoter and synthetic ribosome binding site upstream of the native kdsB gene (encoding CKS), produces CKS at levels as high as 70% of the total cellular proteins. Several heterologous gene sequences have been fused to the 3'-end of the kdsB gene with resulting protein fusions produced at a level of up to 40% of the total cellular proteins.     

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.     

Systems for applied gene control in Saccharomyces cerevisiae

Saccharomyces cerevisiae is frequently used in biotechnology, including fermentative processes in food production, heterologous protein production and high throughput developments for biomedicine. Accurate expression of selected genes is essential for all these areas. Systems that can be regulated are particularly useful because they allow controlling the timing and levels of gene expression. We examine here new expression systems that have been described, including improvements of classical ones and new strategies of artificial gene control that have been applied in functional genomics.     

Heterologous gene expression in bacterial systems under reduced oxygen tensions. Small-scale optimization precedes industrial fermentation

The expression of heterologous fusion proteins from the anaerobically inducible Escherichia coli nitrite reductase nirB promoter has been described using a number of different industrial regimes, but which have proved impractical for scaling down to suit primary research purposes. This paper describes the novel application of microbiological gas sachets generating anaerobic and microaerophilic environments to evaluate the inducible expression under the influence of nirB of heterologous proteins by attenuated vaccine strains of Salmonella typhimurium. The conditions of reduced oxygen tension model those found in lymphoid organs colonized by Salmonella in vivo and so can be used to optimize the vaccine dose prior to administration. Modeling in vivo promoter inducibility to monitor the stability of a plasmid within attenuated vaccine strains of bacteria offers an attractive alternative to antibiotic resistance, which is not permitted for clinical use in humans. This technological advance may be utilized to optimize heterologous gene expression in any microaerophilic bacterial system as a pilot, prior to production-scale applications.     

Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization

Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction.     

Recent advances on the GAP promoter derived expression system of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review. Supported by the National High-tech R&D Program (863 program) (No.2007AA021307).     

Screening of optimal cellulases from symbiotic protists of termites through expression in the secretory pathway of Saccharomyces cerevisiae

For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.     

米曲霉外源表达系统研究进展

丝状真菌米曲霉是发酵工业的重要菌种,具有强大的蛋白分泌能力和较高的食品安全性,可作为表达外源蛋白的细胞工厂。近年来,米曲霉全基因组序列的测序完成和基于表达序列标签的基因组学研究,为深入研究米曲霉外源表达系统提供了条件。从基因组学进展、遗传转化体系等方面综述了米曲霉作为外源蛋白表达宿主的研究进展。针对米曲霉在外源蛋白表达中存在的瓶颈,提出构建蛋白酶缺陷株、使用强启动子、融合表达等策略,以提高外源蛋白的表达和产量。最后介绍了米曲霉表达系统的应用,利用米曲霉代谢工程菌生产工业用酶和次级代谢产品具有良好的前景。     

Engineering new metabolic capabilities in bacteria: lessons from recombinant cellulolytic strategies

Cellulose waste biomass is the most attractive substrate for 'biorefinery strategies' producing high-value products (e.g. fuels or plastics) by fermentation. However, traditional biomass bioconversions are economically inefficient multistep processes. Thus far, no microorganisms able to perform single-step fermentation into products (consolidated bioprocessing; CBP) have been isolated. Metabolic engineering is currently employed to develop recombinant microorganisms suitable for CBP. The heterologous expression of extracellular proteins (e.g. cellulases or hemicellulases) is the key feature of recombinant cellulolytic strategies, conferring cellulolytic ability to microorganisms exhibiting high product yields and titers. Although more molecular tools are becoming available, efficient heterologous expression of secreted proteins is still a challenge. The present review summarizes both bottlenecks and solutions of organism engineering for biomass biorefinery strategies.     

Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal, proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed. Received: 5 July 1996 / Accepted: 6 September 1996     

Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene （cbh1） promoter optimization

To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene （cbhl） promoters was obtained. The region from -677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from -620 to -820 of the modified cbhl promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbhl promoter, obtaining promoters with copy numbers 2, 4, and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase （gus） reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.     

A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting a high seed oleic acid phenotype

As opposed to first-generation biotechnology products, such as pest-resistant crops and herbicide-resistant crops, second-generation products often utilize plant-derived, homologous or heterologous genes and/or promoters. In this study, we evaluated the ability of a promoter from a gene encoding a major storage protein in cottonseed to drive an antisense sequence of the cotton FAD2 gene to down-regulate the activity of Delta-12 desaturase enzyme in cottonseeds. The oleic acid level in the transgenic cottonseeds approximately doubled from the wild-type level of 15%, with a concomitant decrease in the level of linoleic acid. A more extensive study of one line revealed a higher degree of seed-to-seed variability in the transgenic phenotype. A thorough investigation was conducted to determine the impact of the use of a homologous promoter to drive a transgene on the activity of the endogenous promoter. The results showed that the use of the homologous alpha-globulin B promoter for transgenic purposes did not adversely affect the expression of alpha-globulin B storage protein in cottonseed. The results obtained in this investigation on the use of a homologous promoter and antisense technology will be useful in the design of strategies to alter biosynthetic pathways for nutritional quality improvements and for the production of heterologous proteins of commercial value in seeds.