Biocatalyst Design for Stability and Specificity - American Chemical

Chapter 19

Recombinant β-Glucosidase of Trichoderma reesei Tim Fowler Genencor International, Inc., 180 Kimball Way,

Downloaded by MONASH UNIV on September 14, 2013 | http://pubs.acs.org Publication Date: December 11, 1993 | doi: 10.1021/bk-1993-0516.ch019

South San Francisco, CA 94080

Recent advances in the genetic manipulation of T. reesei provide a molecular tool with which the cellulase enzyme composition can be altered to provide new and useful mixtures. In addition, mutagenesis experiments can be designed to answer direct questions regarding cellulase gene regulation and mechanism of enzyme action. ß-glucosidase is one member of a family of cellulase enzymes that act synergistically to hydrolyze cellulose to glucose. Studies in our laboratory have focused on the role of extracellular ß-glucosidase in the regulation of the other enzymes of the cellulase complex of T. reesei and its function in the production of glucose from cellulose. To this end we have cloned the gene encoding the extracellular ß-glucosidase (bgll) from Trichoderma reesei. (1). Re-introduction of the bgll gene back into the host in extra copies gives increased expression of ß-glucosidase and results in a cellulase complex that has an increased rate of glucose production from cellulosic substrates. Results of bgll gene disruption experiments and proposed site directed mutagenesis of the enzyme are described.

In this chapter I will describe the cellulase complex of Trichoderma reesei and in particular the genetic manipulation of this complex to produce novel strains in which the cellulase complex profile and activity has been altered. I will focus on the extracellular β-glucosidase enzyme encoded by the bgll gene. Historically, filamentous fungi have been used to produce many enzymes, antibiotics and other biochemical products. However, methods for DNA-mediated transformation of industrially important fungi only became generally available in the mid 80s. This achievement provided a viable alternative to classical genetics for rapid improvement of existing fungal strains which produce salable commodities. The industrially important deuteromycete, Trichoderma reesei, is an example of such a fungal species. T. reesei secretes large quantities of a hydrolytic mixture of enzymes that act in concert to degrade crystalline cellulose to glucose. These cellulases are used in a variety of applications; examples include the extraction offruitand vegetable juices, animal feed and silage processing, and malting and brewing. In addition, the 0097-6156/93/0516-0233$06.00/0 © 1993 American Chemical Society

In Biocatalyst Design for Stability and Specificity; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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BIOCATALYST DESIGN FOR STABILITY AND SPECIFICITY

introduction of genetic engineering techniques to Trichoderma is leading to rapid advances in our understanding and manipulation of the cellulase complex. The cellulase enzyme complex of T. reesei consists of two known cellobiohydrolases, CBHI and CBHII (EC 3.2.1.91), at least two endoglucanases, EGI and EGII (EC 3.2.1.4), and at least one β-glucosidase (EC 3.2.1.21). The endoglucanases and cellobiohydrolases are believed to act synergistically to hydrolyze crystalline cellulose to small cello-oligosaccharides (mainly cellobiose). The small oligo-dextrins are subsequently hydrolyzed to glucose by β-glucosidase. The exact biochemistry, regulation and synergy between the different cellulases of T. reesei are the subject of a great deal of investigation. Wild type strains of T. reesei produce extracellular cellulase at levels of a few grams per liter and in molar ratios of about CBHI (60) : CBHII (20) : EGI (10) : βglucosidase (1) (5,6). In the biotechnology industry, selection of T. reesei production strains by mutagenesis and screening have resulted in strains capable of secreting in excess of 40 g/L (38,39). The cellulase products from these strains can be further modified by blending with other cellulase preparations (sometimes enriched for specific components). In addition, variation in fermentation conditions enables a measure of control over the ratios of secreted enzymes. However, these methods tend to be expensive and/or labor intensive. Methods for genetic manipulation of T. reesei became possible with the introduction of both dominant and auxotrophic markers (2,3,4) and techniques such as electroporation and protoplast fusion for introducing the foreign D N A into the fungal cells. These techniques are now being employed to manipulate the cellulase complex at the genetic level. Most recently, genetic engineering of T. reesei strains enables the complete removal or overproduction of either individual or multiple components of the cellulase complex. This means novel strains with cellulase profiles specifically tailored to defined applications can now be made (See Table I from ref. 7). The role of the β-glucosidase component in cellulose hydrolysis and in the regulation of the cellulase complex is currently under investigation at Genencor International. Presumably one function of β-glucosidase is the breakdown of the cellobiose produced by the cellobiohydrolases and endoglucanases to provide glucose as a carbon source. Another function of β-glucosidase may be to use glucose (via a transglycosylation reaction) to produce oligosaccharides that have been shown to act as potent inducers of the cellulase complex (5,41,42). Cell fractionation, immunoflourescence and electron microscopic localization indicate that the majority of β-glucosidase is associated with the outer integuments of T. reesei (37,43,44,45,46). It therefore makes teleological sense that for the organism to make more efficient use of available cellobiose, the major portion of the detectable βglucosidase activity remains associated with the cell wall (8,9,10). It is believed that the association of β-glucosidase with the cell wall is a significant factor in the reduced ability of commercial preparations of cellulase to produce glucose. Improvement of the cellulase preparations by the addition of purified β-glucosidase (11,12,13) or isolation of mutant strains of T. reesei that have increased levels of β-glucosidase are possible solutions to this problem (40). We have begun to investigate the role of the β-glucosidase enzyme in cellulase system at the molecular level by cloning and sequencing of the extracellular βglucosidase gene, bgll, from T. reesei (1). We demonstrate that transformation of the bgll gene into the T. reesei genome in multiple copies can be used to generate strains with significant increase in extracellular β-glucosidase activity. Finally site directed mutants of the bgll gene have been created to locate residues central to catalytic activity. The long term goal of these experiments is to identify mutant forms of the Églucosidase enzyme that possess novel and useful enzymatic properties. The deletion


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of the bgll gene from T. reesei and the use of this strain as a host for the site directed mutants of β-glucosidase are also described


Isolation and Overexpression of the bgll Gene in T. reesei. We have previously described the cloning and sequencing of the gene encoding the extracellular β-glucosidase gene (bgll) from T. reesei (1). The primary structure of the deduced extracellular β-glucosidase protein has several properties of note. The predicted molecular weight of the encoded β-glucosidase protein is 74,341 daltons. The size and composition is in good agreement with the protein purified by Chirico and Brown (14). A 31 amino acid secretion signal peptide precedes the mature amino terminus of β-glucosidase as deduced from the amino terminal peptide sequence and homologies to the other cellulase genes (75,16,17,18,19). The primary amino acid sequence of β-glucosidase shows 7 potential N-linked glycosylation sites (20). The bgll coding region is interrupted by two putative introns of 70 and 64 base pairs that show homology to the consensus splice signals emerging for T. reesei and other filamentous fungi (27). The following sections describe experiments in which the cellulolytic capacity of T. reesei strains was altered by transformation with extra copies of the bgll gene, resulting in the overexpression of extracellular β-glucosidase. Alternatively, transformation with a vector designed to disrupt the bgll gene resulted in a strain with no apparent extracellular β-glucosidase activity. The ability to increase expression of interesting and/or commercially valuable proteins by genetic engineering has recently become possible in filamentous fungi. As a general rule increasing the copy number of a fungal gene through transformation usually leads to increased expression of that gene. Recent examples of this phenomenon include glucose oxidase (goxA, ref. 31), glucoamylase (glaA, ref. 32), and prepro-polygalacturonidase II (pgall, ref. 33) expressed in A. niger. We have undertaken a similar approach in obtaining enhanced expression of extracellular βglucosidase. Extra copies of a genomic clone of bgll were introduced into the genome of T. reesei using the transformation vector called pSASB-glu (Figure 1). Positive selection of T. reesei transformants containing extra copies of bgll was made using the A. nidulans amdS gene as a selectable marker (2). Trichoderma does not contain a functional equivalent of the amdS gene and is therefore unable to utilize acetamide as a sole nitrogen source unless the amdS gene is stably inherited during transformation. Several stable transformants were seen to contain multiple copies of the bgll gene (T. Fowler, unpublished Southern blot results). The cellulase products from these multicopy bgll gene transformants showed an increase in the rate of glucose release from Avicel. One transformant was chosen for a more detailed analysis and was shown to contain 5-10 additional copies of the bgll gene integrated into the genome, which gave rise to a 4.2 fold increase in bgll mRNA levels (See figure 5 from ref. 7). In addition, data is given for the action of the cellulase preparation (named C5X) from the transformant on cellobiose, Avicel and phosphoric acid swollen cellulose (PSC). A n increase in the rate of production of glucose from the cellulosic substrates was observed for each substrate (See Figure 5 from ref. 7). These data suggest that integration of additional copies of bgll in the genome of T. reesei leads to an increase in specific message levels and corresponding extracellular protein levels. Furthermore the cellulolytic activity of T. reesei strains is specifically improved by transformation with the bgll gene. Increased saccharification observed from relatively pure substrates were an indicator that similar results upon treatment of less well defined cellulosic materials may be obtained. The resulting increase in the rates of hydrolysis may have potential applications for biomass conversion and ethanol production.



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Cytolase 123 is a Genencor cellulase preparation from a proprietary T. reesei strain. The same strain was used as the parent for transformation with extra copies of bgll resulting in the transformant that produce C5X. A comparison of the action of Cytolase 123 and C5X was made on a cellulosic waste floe from diaper manufacturing (figure 2). The cellulase preparation containing increased levels of β-glucosidase (C5X) released higher levels of glucose from the diaper waste over time than did an equivalent amount of Cytolase 123. Pretreatment of a variety of biological waste materials, such as agricultural residues, forestry waste, and municipal solid waste (MSW) gives rise to crude substrates that can subsequendy be used in separate hydrolysis fermentation (SHF) or simultaneous saccharification and fermentation reaction (SSF) to produce ethanol (34). We were interested in seeing whether the cellulase mixture, C5X, could improve the SSF process by providing more glucose as a substrate to ferment into ethanol. In the SSF process a single vessel is used to mix pretreated MSW, hydrolytic cellulase enzymes and an organism capable of fermenting the released glucose to ethanol (such as the yeast strain Saccharomyces cerevisiae). This simplification can be approximated in laboratory shake flask analysis using cellulase enzyme preparations, a cellulosic substrate and a yeast innoculum. Table I shows results from such an experiment on a paper fraction of municipal solid waste using Cytolase 123 and C5X as the cellulase enzyme mixtures. From this experiment, it appears that the enhanced β-glucosidase cellulase preparation increases the availability of glucose resulting in increased ethanol production from yeast fermentation. This result is especially pronounced at the lower dosages. One possible explanation for this observation is mat higher dosages of either cellulase mixture saturates the system with glucose and the resultant high alcohol levels become rate limiting. In conclusion, novel cellulase mixtures can be produced from genetic manipulation of a single T. reesei strain. In the friture these designer strains will result in simplified fermentation protocols and remove the need for supplementation of additional enzymes to existing enzyme preparation. Structural cellulose found in biomass materials is a mixture of crystalline cellulose, hemicellulose and lignin. Further improvement of strains of T. reesei for more rapid and complete conversion of cellulosic biomass may include the cloning and overexpression of the genes encoding hemicellulases and ligninolytic enzymes. Deletion of the bgll gene from T. reesei. We have described how the composition of the cellulase complex of T. reesei can be altered by introduction of extra copies of cellulase genes. Conversely, a targeted gene disruption can remove or interrupt cellulase gene coding sequences. Such mutations not only alter the overall composition and mode of action of the cellulase preparation but provide a host into which the disrupted gene can be reintroduced following site directed mutagenesis that is designed to modify the enzyme's activity. In addition, the manipulation of each individual component at die genetic level presents an opportunity to look in new ways at several questions central to the regulation of the cellulase complex and the hydrolysis of cellulose. For example, how is each gene is involved in the induction and regulation of the cellulase complex? What is the contribution of each component in the hydrolysis of cellulosic substrates? What is the exact composition of the Trichoderma cellulase complex and multiplicity of the enzyme species? Mutants of Trichoderma reesei lacking the coding sequence for the extracellular βglucosidase gene, bgll, were obtained by a targeted gene replacement event (Figure 3). A gene replacement vector was first constructed (illustrated in Figure 3). The vector



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Ssp/Sma Hind III

Figure 1. Trichoderma reesei bgll gene overexpression vector pSASB-glu. The genomic T. reesei bgll gene is contained on a 6.0 kb Hinc&U fragment. Figure source: from reference 1.

3η

Time (hrs)

Figure 2. Comparison of Cytolase 123 and C5X action on cellulosic diaper waste. The cellulase mixtures were prepared using the same fermentation conditions. Dosage of the cellulase was 0.4mg enzyme/lOOmg substrate.


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Table I. A Comparison of Cytolase 123 and C5X in the production of Ethanol from Cellulosic Biomass

Comparison of Cytolase 123 and C5X


Grams EtOH/Liter SSF RXN Cellulase Activity(FPU)/ Gram Cellulose (MSW)

Cytolase 123

C5X

6

1.3

3.8

12

3.3

4.5

18

4.3

5.5

24

5.0

5.8

30

5.3

5.8

36

5.3

5.8

a) The cellulase activity of a standard batch of Cytolase 123 was calculated and dosed as shown. C5X was then compared to Cytolase 123 by using an equivalent amount of protein. T. reesei Genome

2290bp

5 "2

c

_1_ \

/

2412bp

3C 5' o.

bgll 5' sequences

I

>

A . niger pyrG

73

Biocatalyst Design for Stability and Specificity - American Chemical

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