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

Molecular Biotechnology of Microbial Systems
Biodegradation of Toxic Compounds and Biomass Utilization
Utilization of Starch and Sugars

Starch, the primary storage polysaccharide of plants, is a mixture of D-glucose homopolymers—both linear (amylose) and branched (amylopectin). The amylose molecule consists of 1∙10²—4∙10⁵ D-glucose residues linked by α-1,4-bonds (Fig. 13.9, A), while amylopectin consists of short (17—23 D-glucose residues linked by α-1,4-bonds) linear chains connected by 1,6- and 1,3-bonds, forming a highly branched Structure containing 1∙10⁴—4∙10⁷ glucose residues (Fig. 13.9, B). The degree of branching and The ratio of amylose to amylopectin vary depending on the species and age of the plant from which the starch was obtained.

Class="center">Table 13.3. Growth of parental and recombinant Pseudomonas strains on various Aromatic Compounds¹⁾

¹⁾ Modified from Suyama et al., J. Bacteriol. 178: 4039—4046, 1996.

²⁾ Symbols: +++ good growth; ++ moderate growth; + poor growth; — very poor growth or no growth.

³⁾ The P. putida strain KF715-D5 was obtained by replacing the bphAl Gene in strain KF715 with the todCl gene from strain F1

Fig. 13.9. A. Enzymatic Hydrolysis of amylose. B. Enzymatic hydrolysis of amylopectin. Blue circles represent D-glucose residues.

Industrial production of fructose and ethanol

Starch is widely used in the food and brewing industries; it is first hydrolyzed into low-molecular-weight components and then converted into Other Compounds, primarily fructose and ethanol. The Key Enzymes required for starch hydrolysis and subsequent conversions are α-amylase, glucoamylase, and glucose isomerase. Their cost accounts for approximately 30% of the total cost of all enzymes currently used in the industry.

Fig. 13.10. Industrial production of fructose and ethanol from starch.

Industrial production of fructose and ethanol from starch is a multi-step process involving the following enzymatic and non-enzymatic stages (Fig. 13.10).

1. Gelatinization of milled grain (usually corn, which has a starch content of about 40%). To achieve this, the grain is treated with pressurized steam, which disrupts the starch granules and makes the starch accessible for subsequent enzymatic hydrolysis. The resulting product has a gelatinous consistency.

2. Liquefaction. The gelatinized starch is cooled to 50—60 °C, and α-amylase is added. This hydrolyzes the accessible α-1,4-bonds, forming low-molecular-weight Polysaccharides. The high Temperature enhances the enzyme's penetration into the gelatinized starch and increases The rate of hydrolysis.

3. Saccharification (complete hydrolysis) of low-molecular-weight polysaccharides (both linear and branched) to glucose molecules. This is carried out by glucoamylase.

The final product of this Treatment is glucose, which can then be converted into ethanol (via Yeast Fermentation) or fructose (using glucose isomerase). Due to the high efficiency of the latter process, cheaper fructose is used instead of sucrose in food preparation and brewing in North America. In the industrial production of fructose, starch is typically obtained from corn, so the final product is called high-fructose corn syrup, or simply high-fructose syrup, although it consists of roughly equal proportions of fructose and glucose.

The enzyme α-amylase randomly hydrolyzes α-1,4-bonds in amylose and amylopectin molecules, producing a mixture of glucose, maltose (two glucose residues linked by an α-1,4-bond), maltotriose (three glucose residues linked by an α-1,4-bond), and a range of α-dextrins, which are branched fragments of amylopectin chains (Fig. 13.9). Although α-amylase can be isolated from many microorganisms, for industrial purposes it is typically obtained from Bacillus amyloliquefaciens.

Sometimes, to break down starch, β-amylase is used instead of or alongside α-amylase. β-Amylase hydrolyzes every second α-1,4-bond starting from the ends of the amylose and amylopectin chains, yielding maltose residues and various β-dextrins. The enzyme glucoamylase hydrolyzes α-1,3-, α-1,4-, and α-1,6-bonds. However, it hydrolyzes α-1,4-bonds less efficiently than α-amylase and is therefore typically used in combination with it. The primary function of glucoamylase is to cleave the branch points in dextrin molecules, converting them into glucose. This and Other Enzymes are used to reduce the carbohydrate (dextrin) content in regular beers to produce so-called light and dry beers. Glucoamylase treatment is usually performed prior to fermentation, though these two processes can be combined. Glucoamylase is synthesized by many microorganisms, but it is typically obtained from the fungus Aspergillus niger.

Improving the efficiency of fructose and ethanol production

The cost of producing ethanol or fructose from milled grain is primarily determined by the cost of the enzymes, which are typically used only once. Therefore, developing new approaches for the low-cost, large-scale production of these enzymes could significantly reduce the cost of the final products. This can be achieved in several ways.

✵ Use fast-growing recombinant microorganisms utilizing an inexpensive substrate for enzyme overproduction. This is more cost-effective than obtaining enzymes from wild-type microorganisms.

✵ Use α-amylase variants (either naturally occurring or engineered) that exhibit higher activity and allow liquefaction to be carried out at 80—90 °C. This will accelerate the hydrolysis of gelatinized starch and save energy otherwise spent on cooling it to the temperature at which hydrolysis is typically performed.

✵ Modify the α-amylase and glucoamylase genes so that the encoded enzymes share the same temperature and pH optima. This will allow the liquefaction and saccharification steps to be combined.

✵ To find or engineer an enzyme that will efficiently degrade raw starch, thereby eliminating the gelatinization step and saving a significant amount of energy.

✵ To engineer a fermenting microorganism that synthesizes and secretes glucoamylase, eliminating the need to add it during the fermentation process.

Research is currently underway to determine whether such approaches are feasible.

Genes encoding α-amylase have been isolated from many microorganisms, including B. amyloliquefaciens and the thermophilic bacterium B. stearothermophilus. To do this, their chromosomal DNA was extracted, partially digested with the restriction endonuclease Sau3AI, and inserted into the BamHI-digested plasmid pUB110, which contains a unique BamHI site and carries a kanamycin resistance gene. The resulting clone library was used to transform B. subtilis Cells lacking α-amylase activity; transformants were selected for kanamycin resistance and screened for their ability to synthesize and secrete α-amylase using a starch-iodine assay. For this, plates with colonies formed by transformants at 65 °C on starch-containing solid medium were exposed to iodine vapor. Colonies producing α-amylase were surrounded by a clearly visible halo, indicating starch hydrolysis in their vicinity. A positive starch-iodine test indicates METABOLISM/31.html">Transcription of the α-amylase gene under the control of its own promoter (the vector contains no promoter) and the presence of a signal required for secretion (as substrate molecules are too large to enter The Cell). The ability to obtain α-amylase genes from various sources has allowed researchers to introduce modifications necessary for utilizing these genes in specific Industrial processes.

The feasibility of eliminating the saccharification step in ethanol production from starch was demonstrated as follows. Full-length glucoamylase cDNA isolated from the fungus Aspergillus awamori was cloned into a Saccharomyces cerevisiae plasmid under the control of the promoter and transcription termination regulatory sequences of the yeast enolase gene (ENO1). A "laboratory" strain of S. cerevisiae transformed with this plasmid acquired glucoamylase activity and was able to convert soluble starch to ethanol.

Unfortunately, certain properties of this strain (sensitivity to high ethanol concentrations, inefficient expression of glucoamylase cDNA, and plasmid maintenance only under specific selective pressure) make it unsuitable for industrial use. However, these drawbacks were successfully overcome. First, glucoamylase production was increased approximately fivefold by deleting a 175-bp negative regulatory region of the ENO1 promoter from the plasmid. Second, the yeast origin of Replication was removed from the plasmid, and a DNA segment homologous to a region of the yeast chromosome was inserted, thereby converting it into an integrating vector that integrates into the yeast chromosome and is stably maintained in the cell. Third, another strain of S. cerevisiae (brewer's yeast), which is resistant to high ethanol concentrations, was used as the host cell for the plasmid modified in this manner.

As a result, two new yeast strains were obtained that hydrolyze and ferment soluble starch more efficiently than S. diastaticus, a natural amylolytic (starch-hydrolyzing) yeast closely related to S. cerevisiae (Table 13.4). The "brewer's" strain of S. cerevisiae with the integrated glucoamylase gene performed more efficiently than the "laboratory" strain carrying the same gene on a multicopy plasmid, which likely indicates plasmid instability and the loss of the introduced glucoamylase gene. Prior to the Introduction of the glucoamylase gene, both the "laboratory" and "brewer's" S. cerevisiae strains were unable to utilize soluble starch. The plasmid-borne and integrated A. awamori glucoamylase cDNA was under the control of the regulatory sequences of the ENO1 gene, from which the 175-bp negative regulatory region had been deleted. Specific selective pressure was applied to maintain the plasmid. To increase glucoamylase production, several copies of its gene were integrated into the chromosomal DNA of the fungus A. niger. It turned out that glucoamylase activity did not correlate with the gene copy number but was highly dependent on the chromosomal site of integration. Thus, simply increasing the gene copy number is insufficient to enhance The production of the active enzyme.

Table 13.4. Hydrolysis of soluble starch (25%, w/v) by different yeast strains¹⁾

¹⁾ Modified from Cole et al., Bio/Technology 6: 417—421, 1988.

The enzyme glucose isomerase should more properly be called xylose/glucose isomerase, since the primary reaction it catalyzes is The conversion of the five-carbon monosaccharide D-xylose to D-xylulose, whereas the isomerization of D-glucose to D-fructose is a side reaction. Xylose/glucose isomerase has a lower catalytic constant k_cat and a higher Michaelis constant K_M for glucose than for xylose; this means that xylose binds more tightly to the enzyme and is converted to xylulose more rapidly than glucose is converted to fructose.

Intracellular enzymes, such as xylose/glucose isomerase, generally do not yield products of the same purity and quantity as extracellular (secreted) enzymes. Most enzymes used in industrial processes are not highly purified, and an extracellular enzyme preparation typically contains far fewer protein impurities than an intracellular extract. Furthermore, obtaining an intracellular enzyme extract requires harvesting cells from the culture medium, mechanically disrupting them, and removing the resulting debris. All of this increases the cost of the final product, xylose/glucose isomerase. To address this issue, the enzyme can be immobilized on a solid support and reused multiple times.

The isomerization of glucose to fructose is a reversible reaction, and the final yield of fructose is directly dependent on temperature, which is approximately 60 °C in most industrial processes. Increasing the temperature optimum and thermostability of xylose/glucose isomerase can enhance the fructose yield.

The thermophilic bacterium Thermus thermophilus produces a xylose/glucose isomerase that retains its activity and remains stable at 95°C, making it highly promising for industrial use. Unfortunately, the wild-type strain of T. thermophilus produces it in small quantities. To overcome this limitation, the xylose/glucose isomerase gene from T. thermophilus was isolated and expressed in E. coli and B. brevis using various promoters and ribosome-binding sites (Table 13.5). In the last system shown in Table 13.5, the enzyme was produced in an amount more than 1,000 times higher than the original level, making it suitable for industrial fructose production. Another possibility is to enhance the substrate Specificity of the enzyme. In one series of experiments, Site-Directed Mutagenesis was used to replace NUCLEOTIDES encoding one or Two Amino Acids of the xylose/glucose isomerase from the thermophilic bacterium Clostridium thermosulfurogenes. The Selection of sites for modification was based on data regarding the involvement of the corresponding amino acids in substrate binding. Replacing Tryptophan at position 139 with phenylalanine, or valine at position 186 with Threonine, resulted in a 1.7-fold and 2.6-fold increase in the catalytic efficiency (k_cat/K_M) of the enzyme toward glucose, respectively (Table 13.6), and a 2-fold and 7-fold decrease toward xylose. With the simultaneous replacement of both amino acids, the catalytic efficiency toward glucose increased 5.7-fold, while that toward xylose decreased 4.5-fold. Thus, the double substitution resulted in the enzyme, which was originally 17 times more active toward xylose, becoming 1.5 times more active toward glucose. The achieved modification of specificity and increase in thermostability of xylose/glucose isomerase will allow its use for the industrial conversion of glucose to fructose.

Table 13.5. Activity of T. thermophilus xylose/glucose isomerase in different Bacteria¹⁾

¹⁾ Adapted from Dekker et al., Appl. Microbiol. Biotechnol. 36: 727—732, 1992, with modifications.

²⁾ The data in the first row refer to the wild-type strain producing this enzyme. All other strains were obtained by transformation and contain the T. thermophilus xylose/glucose isomerase gene within a multicopy plasmid.

Table 13.6. Catalytic efficiency of wild-type and mutant forms of C. thermosulfurogenes xylose/glucose isomerase¹⁾

¹⁾ Adapted from Meng et al., Proc. Natl. Acad. Sci. USA 88: 4015—4019, 1991, with modifications.

Zymomonas mobilis

Substrate fermentation in industrial ethanol production is primarily carried out using S. cerevisiae, but the bacterium Zymomonas mobilis could be used as an alternative. This Gram-negative rod ferments glucose, fructose, and sucrose with a relatively high ethanol yield (Table 13.7). This is apparently due to reduced biomass accumulation during fermentation, which decreases The amount of substrate consumed for growth, leaving more for ethanol production. In breaking down 1 mole of glucose, yeast produces 2 moles of ATP, whereas Zymomonas, which utilizes a different metabolic pathway, produces only 1 mole. Historically, Zymomonas has been used for fermentation in the production of alcoholic beverages in tropical countries (for example, in Mexico to produce a beverage called pulque, which contains 3—5% alcohol and is made from agave sap).

The primary difference between Zymomonas and S. cerevisiae as ethanol producers lies in the rate of ethanol formation, which is much higher in Zymomonas (Table 13.7). However, there are several biological and technical limitations that prevent The Use of Zymomonas for industrial ethanol production:

1) the limited range of carbon substrates it can utilize for ethanol synthesis; 2) difficulties in maintaining broad-host-range cloning vectors, and consequently foreign genes, within the bacterium; 3) natural resistance to the most common Antibiotics, which prevents the use of standard Antibiotic Resistance marker systems in cloning experiments.

Table 13.7. Comparison of Z. mobilis and S. cerevisiae as ethanol producers¹⁾

¹⁾ From Buchholz et al., Trends Biotechnol. 5: 199—204, 1987.

²⁾ The ethanol production rate was measured under standard fermentation conditions, and the volumetric rate was measured in continuous culture.

Despite all this, researchers have succeeded in introducing and expressing several foreign genes in Zymomonas. Most of these experiments aimed to expand the range of substrates it can utilize. For instance, genes encoding enzymes that hydrolyze lactose, starch, Cellulose, xylose, and cellobiose were introduced into Zymomonas. Although the transformed cells expressed all of these genes, in most cases they could not utilize these substrates as a sole carbon source. Imparting new catabolic properties to Zymomonas proved to be quite challenging, though in at least one instance, the microorganism was successfully used to produce ethanol from xylose-containing waste. At the very beginning of the efforts to engineer Z. mobilis strains capable of growing and producing ethanol on xylose, genes for glucose/xylose isomerase and xylulokinase—enzymes essential for xylose utilization—were introduced into the bacterium. However, the resulting transformants could not utilize the pentoses generated during xylose breakdown (xylulose-5-phosphate, ribulose-5-phosphate, and ribose-5-phosphate). Therefore, in the next step, a plasmid carrying two synthetic operons was introduced into Z. mobilis: one Operon encoded two xylose-assimilating enzymes, and the other encoded two pentose-metabolizing enzymes (transketolase and transaldolase) (Fig. 13.11). Transcription of the genes in the first operon was controlled by the strong constitutive promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene, while the second was controlled by the promoter of the Z. mobilis enolase gene. These two synthetic operons were cloned into an E. coli–Z. mobilis shuttle vector, which was then used to transform Z. mobilis. As expected, the transformed cells utilized xylose and converted pentoses to fructose-6-phosphate and glyceraldehyde-3-phosphate, which were subsequently converted to ethanol via the Entner–Doudoroff pathway. Furthermore, the transformants grew efficiently on glucose, xylose, and their mixture, converting xylose to ethanol with a high yield. This work demonstrated the feasibility of genetically engineering Z. mobilis to create an ethanol-producing microorganism that could utilize xylose, a byproduct of the wood and pulp-and-paper industries, as a carbon source.

Fig. 13.11. Zymomonas–E. coli shuttle vector carrying two operons, one containing the genes for enzymes required for xylose utilization (xylA and xylB), and the other containing the genes for enzymes involved in pentose metabolism (tktA and tal). Arrows indicate the direction of transcription from each promoter. p^eno — enolase gene promoter, p^gap — glyceraldehyde-3-phosphate dehydrogenase gene promoter, xylA — xylose isomerase gene, xylB — xylulokinase gene, tktA — transketolase gene, tal — transaldolase gene, Tet^r — tetracycline resistance gene, ori^E — E. coli origin of replication. Zymomonas DNA contains its own origin of replication.

Silage Production

Crops such as forage grasses, corn, and alfalfa are widely used as livestock feed, making it crucial to ensure proper storage conditions for many months. Traditionally, naturally occurring lactic acid bacteria are used for this purpose, utilizing the plant material as a substrate to synthesize lactic and acetic acids. These acids inhibit the growth of other microorganisms, thereby preserving the forage plant biomass (silage). If lactic acid bacteria are present on the fresh plant material in low numbers, a bacterial inoculant (usually Lactobacillus plantarum) must be added. Unfortunately, this measure is of little effect when the crop contains insufficient Water-soluble CARBOHYDRATES to support bacterial growth and lactic acid production.

To engineer a bacterium capable of efficient fermentation of plant material, the α-amylase gene from a non-silage strain of L. amylovorus was integrated into the chromosomal conjugated Bile acid hydrolase (cbh) gene of an L. plantarum strain (Fig. 13.12). This gene encodes an enzyme that is activated when the bacterium enters the animal's gut and, therefore, is not needed for silage formation. This work represents the first step toward developing L. plantarum strains that promote more efficient silage production from starch-rich crops, such as alfalfa.