Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Biodegradation of Toxic Compounds and Biomass Utilization
Cellulose Utilization

The structural framework of almost all terrestrial plants consists of polymers: Lignin, hemicellulose, and Cellulose. Combining in various proportions, they form lignocellulosic material (Table 13.8), which accounts for the bulk of biomass remaining in vast quantities as waste from agriculture, the woodworking industry, and other human economic activities. This waste must be processed or utilized as industrial feedstock. From this perspective, lignocellulosic Materials can be divided into three classes.

✵ Plants themselves, specifically cultivated to obtain cellulose, building materials, or livestock feed (cotton, wood, hay).

✵ Plant residues remaining after crop harvesting and Processing, and after wood processing (straw, rice husks, sugarcane bagasse, wood chips, sawdust, etc.).

✵ Household waste (used paper, cardboard, etc.).

Class="center">Table 13.8. Composition of various lignocellulosic materials¹⁾

¹⁾ From Brown, Philos. Trans. R. Soc. Lond. B. 300: 305—322, 1983.

Lignocellulose Components

Lignin is a globular, irregular, insoluble polymer (mol. mass >10 000) consisting of phenylpropane units (Fig. 13.13). During lignin formation, the molecules of this aromatic substance link together randomly via various chemical bonds that resist Enzymatic Hydrolysis or chemical Cleavage. In plants, lignin forms a complex with hemicellulose, which encloses the vascular bundles. Lignin provides plant rigidity as well as resistance to mechanical damage and microbial action.

Fig. 13.12. Insertion of the α-amylase Gene into the L. plantarum chromosome. The α-amylase gene is inserted into the cbh gene of the E. coli–L. plantarum shuttle plasmid, which is then used to transform L. plantarum Cells. As a result of Crossing Over between the cbh loci of the plasmid and chromosomal DNA, L. plantarum clones resistant to erythromycin and possessing α-amylase activity are formed. When transformed cells are grown for a sufficiently long period (at least 30 generations) under non-selective conditions, an intrachromosomal double crossover can occur, leading to the elimination of the erythromycin resistance gene Erm^r, the chromosomal cbh gene, and the plasmid DNA.

Hemicelluloses are short-chain heterogeneous polymers consisting of hexose (six-carbon sugars, such as glucose, mannose, and galactose) and pentose (five-carbon sugars, such as xylose and arabinose) units. All hemicelluloses can be divided into three main types: xylans, whose backbone consists of poly-β-1,4-Xylan molecules with attached arabinose, glucuronic, and arabinoglucuronic acids; Mannans, consisting of glucomannans and galactomannans; and arabinogalactans. The type of hemicellulose typically depends on its origin; thus, xylans are usually found in hardwoods, while glucomannans are found in softwoods.

Cellulose, the simplest component of lignocellulose, is the most abundant natural polymer. Its long chains consist of D-glucose residues linked by β-1,4-bonds (Fig. 13.14). Upon hydrolysis, cellulose, like starch, yields glucose, but these starting materials themselves have different structures. Starch is an energy-storing molecule in which glucose residues are linked in such a way that the polymer chains cannot arrange themselves in an orderly fashion, forming a network Structure that is easily permeable to Water; therefore, it dissolves in water and is easily hydrolyzed by amylases and glucoamylases. In cellulose, the polymer chains are packed to form a crystal-like structure impermeable to water; therefore, cellulose is insoluble in water and resistant to hydrolysis. Cellulose is a highly valuable material from which many products (e.g., ethanol) can be obtained. However, it must first be released from its complex with lignin and hemicellulose. To achieve this, the lignocellulosic material can be treated with a strong acid or strong alkali, or subjected to high Temperature and pressure. In any case, the energy costs required for this will significantly increase the cost of the final product.

Fig. 13.13. Structure of lignin. Only some of the possible ways of linking phenylpropane residues (a six-carbon aromatic compound containing an alkyl group) are shown.

The annual production of lignocellulose is enormous, which is why there is an ongoing search for more efficient Methods of enzymatic degradation of cellulose (and, to a lesser extent, hemicellulose). In addition, Methods for the selective chemical and Enzymatic cleavage of lignin are being developed.

Isolation of Prokaryotic Cellulase Genes

Many Bacteria and Fungi are capable of degrading cellulose through the synergistic action of several Enzymes called cellulases.

Fig. 13.14. Segment of a cellulose polymer chain. Glucose residues are linked HEAD-to-tail by β-1,4-bonds.

In some microorganisms, they are part of the cellulosome, a protein complex located on The Cell surface. These enzymes are as follows:

✵ endoglucanase, which hydrolyzes β-1,4-bonds between adjacent glucose residues in loosely packed regions of cellulose, creating breaks in the middle of the chain (Fig. 13.15)

✵ exoglucanase, which cleaves broken cellulose chains from the non-reducing ends to yield glucose, cellobiose (two glucose residues), and cellotriose (three glucose residues)

✵ cellobiohydrolase, which is often present in cellulolytic fungi and is a type of exoglucanase that cleaves fragments of 10 or more glucose residues from the non-reducing ends of cellulose molecules

✵ β-glucosidase, or cellobiase, which catalyzes The conversion of cellobiose and cellotriose to glucose (Fig. 13.15).

Fig. 13.15. Cellulose biodegradation. Chain hydrolysis begins with the cleavage of ß-1,4-bonds in loosely packed regions by endoglucanase. Then, exoglucanase(s) and cellobiohydrolase(s) cleave Oligosaccharides from the non-reducing end of partially hydrolyzed chains. Subsequently, ß-glucosidase catalyzes the conversion of cellobiose and cellotriose into glucose.

The degradation of cellulose by cellulolytic microorganisms is slow and often incomplete. Therefore, attempts have been made to genetically engineer microorganisms with higher cellulase activity. To achieve this, prokaryotic and eukaryotic genes encoding individual Enzymes of the cellulase complex were isolated.

Prokaryotic endoglucanase genes were cloned and identified using the following simple yet effective approach.

1. A DNA clone library of a cellulolytic prokaryotic Organism was constructed by cloning in E. coli, and the recombinant cells were grown for 12 h on a solid medium containing a selective antibiotic.

2. The resulting colonies were overlaid with Agar containing carboxymethylcellulose (CMC), a soluble cellulose derivative, and incubated at 37 °C for several more hours. During this time, partial degradation of CMC molecules occurred near the colonies that synthesize and secrete endoglucanase. Transformed cells that synthesize but do not secrete endoglucanase are unable to degrade this substrate, as its large molecules cannot enter the cell.

3. Regions where CMC hydrolysis had occurred were detected using Congo red, a dye non-toxic to bacteria, and a sodium chloride solution. Congo red selectively binds to cellulose, staining it red, and binds weakly to low-molecular-weight saccharides, leaving them yellowish. Treatment with sodium chloride stabilizes the color. Colonies producing secreted endoglucanase were surrounded by a yellow halo, while the Background of undegraded CMC remained red.

Using this approach, endoglucanase genes were isolated from Streptomyces, Clostridium, Thermoanaerobacter, Thermomonospora, Erwinia, Pseudomonas, Cellvibrio, Ruminococcus, Cellulomonas, Fibrobacter, and Bacillus.

To detect recombinant clones synthesizing exoglucanase, immunoscreening was used, which allows identification of the target protein using specific Antibodies; protein secretion is not required for this. Recombinant cells were lysed in situ (with chloroform vapor), cytoplasmic Proteins were transferred to a nylon or nitrocellulose membrane, and an immunological assay was performed. The replica plating method used in this process allowed the preservation of viable cells for further studies.

Prokaryotic ß-glucosidase genes were isolated by transforming E. coli with a DNA clone library obtained from a microorganism producing this enzyme, and selecting transformants capable of growing on minimal medium with cellobiose as the sole carbon source. Clones exhibiting ß-glucosidase activity could also be detected using a medium containing a chromogenic substrate (e.g., 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside) or MacConkey cellobiose agar; under these conditions, the colonies turn red.

Isolation of eukaryotic cellulase genes

Screening of cDNA or Genomic Libraries by Hybridization with a heterologous probe is not very effective for identifying cellulase genes because their nucleotide sequences vary considerably among different organisms. Therefore, new approaches are needed to isolate mRNAs of cellulolytic enzymes from fungi or plants. Unfortunately, these mRNAs constitute only a small fraction of total mRNA; thus, libraries must be enriched with these mRNAs or cDNAs, and cDNA clones lacking the target sequence must be eliminated. To isolate some eukaryotic cellulase genes, a "differential hybridization" method was used, The Essence of which is as follows (Fig. 13.16).

Fig. 13.16. Identification of cDNA clones encoding eukaryotic cellulases by differential hybridization.

1. mRNA is isolated from cells grown in a medium without cellulose (uninduced cells) and from cells grown in the presence of cellulose or its derivatives to increase cellulase production (induced cells).

2. Each mRNA population is fractionated on a sucrose density gradient. The mRNA of each fraction is translated in a cell-free system based on rabbit reticulocytes or wheat germ. The Molecular Weight of the corresponding proteins is determined, and an immunological assay is performed to identify the fraction (or fractions) containing cellulase mRNA. Translation products are separated by Polyacrylamide gel Electrophoresis, and bands derived from induced cells that are absent in uninduced cells are identified; the material in these bands represents proteins whose synthesis was induced by cellulose.

3. The cellulase-determining mRNA fractions from induced cells and their corresponding "counterparts" from the sucrose density gradient of uninduced cell mRNA are used individually as templates for cDNA synthesis.

4. cDNA from induced cells is cloned into a plasmid or phage vector, introduced into E. coli, plated, replica-plated, and screened using radiolabeled cDNA fractions from induced and uninduced cells as hybridization probes. Clones that hybridize with cDNA from induced cells but not with cDNA from uninduced cells may contain cellulase genes and are selected for further study.

5. To prove that the positive cDNA clones indeed encode cellulases, their DNA is hybridized with total mRNA from induced cells, the hybridized mRNA is translated in vitro in a cell-free system, and the resulting products are identified using antibodies specific to the enzymes of the cellulase complex.

6. The nucleotide sequence of each positive cDNA clone is determined to establish which clones encode identical and which encode different Proteins of the cellulase complex.

This scheme can be used to isolate any inducible eukaryotic genes.

Manipulation of cellulase genes. Cloned cellulase genes can be used for various purposes: to facilitate the purification of recombinant proteins using a cellulose-binding domain; and to produce commercial products (e.g., ethanol) from cellulose waste using microorganisms engineered with cellulase genes.

A cellulase molecule typically consists of three domains: a catalytic domain, a hinge region (often rich in Proline, Serine, and Threonine residues), and a cellulose-binding domain. The catalytic and binding domains function independently of each other. This division of Functions can be exploited by incorporating the nucleotide sequence of the cellulose-binding domain into a chimeric gene, the other part of which encodes a protein of commercial interest. To purify the resulting protein, its extract is passed through a Column packed with cellulose. Only the hybrid protein binds to the cellulose; it is then eluted, and the "cellulose" domain is removed by proteolysis. This system is similar to immunoaffinity Chromatography but is more cost-effective.

Most cellulase genes were originally cloned and expressed in E. coli, but they can be introduced into other microorganisms to generate new strains with useful properties. For instance, S. cerevisiae and Z. mobilis, which efficiently convert simple sugars (e.g., glucose) into ethanol, could convert cellulose directly into ethanol after the Introduction of cellulase genes. To test this hypothesis, several studies were conducted.

In one series of experiments, the endo- and exoglucanase genes from the bacterium Cellulomonas fimi, each under the control of an S. cerevisiae promoter and signal sequence, were subcloned into a plasmid vector and introduced into S. cerevisiae. Some transformants secreted both enzymes into the culture medium with an efficiency of approximately 70% and partially degraded the cellulose in filter paper and pretreated wood chips. The rate and extent of hydrolysis of these substrates increased upon adding ß-glucosidase, which cleaves cellobiose to glucose, to the mixture, but complete hydrolysis of cellulose did not occur. This is due to two feedback regulatory mechanisms: accumulating cellobiose inhibits cellulose hydrolysis, while glucose inhibits cellobiose cleavage. The ß-glucosidase gene was isolated from the fungus Trichoderma reesei, cloned into a multicopy plasmid, and reintroduced into T. reesei. The transformed strain produced a 5.5-fold excess of ß-glucosidase and degraded the cellulose derivative Avicel 33% faster than the untransformed strain. This Supports the evidence that ß-glucosidase facilitates the enzymatic degradation of cellulose and suggests that to construct more efficient cellulolytic microorganisms, ß-glucosidase genes should be integrated into existing strains.

Endoglucanase genes can be utilized not only for converting cellulosic waste into valuable products, but also for other Applications. Introducing an endoglucanase gene under the control of a constitutive Yeast Actin gene promoter into wine yeast can enhance the aroma of the resulting wine. This enhancement is associated with an increased concentration of at least 12 volatile compounds, including ethyl propionate, 2-butanol, isoamyl acetate, isoamyl alcohol, and isobutyric acid. Such genetic modification can help stabilize the Fermentation process and yield yeast strains tailored to produce wines with specific characteristics.

Researchers have also investigated the feasibility of using cellulases for the industrial bioconversion of paper waste into ethanol. In this process, the waste was partially hydrolyzed with cellulases at 45 °C, followed by the fermentation of the released glucose using S. cerevisiae at 37 °C without removing the enzymes. Based on the findings, it was estimated that this approach could yield 400 L of ethanol per 1 t of paper waste. If the 100 million t of paper waste generated annually in North America were entirely converted into ethanol and used as fuel, it could displace approximately 16% of gasoline consumption.