2 Genetic Improvement of Microbial Starter Cultures
Susan K. Harlander
Fermentation has been used for preserving food for hundreds of years and virtually every culture has, as part of its diet, a variety of fermented milk, meat, vegetable, fruit, or cereal products. Microorganisms, including bacteria, yeasts, and mold, produce a wide range of metabolic end products that function as preservatives, texturizers, stabilizers, and flavoring and coloring agents. Several traditional and nontraditional methods have been used to improve metabolic properties of food fermentation microorganisms. These include mutation and selection techniques; the use of natural gene transfer methods such as transduction, conjugation and transformation; and, more recently, genetic engineering. These techniques will be briefly reviewed with emphasis on the advantages and disadvantages of each method for genetic improvement of microorganisms used in food fermentations.
TRADITIONAL GENETIC IMPROVEMENT STRATEGIES
Mutation and Selection
In nature, mutations (changes in the chromosome of an organism) occur spontaneously at very low rates (one mutational event in every 10e6 to 10e7 cells per generation. These mutations occur at random throughout the chromosome, and a spontaneous mutation in a metabolic pathway of interest for food fermentations would be an extremely rare event. The mutation rate can be dramatically increased by exposure of microorganisms to mutagenic agents, such as ultraviolet light or various chemicals, which induce changes in the deoxyribonucleic acid (DNA) of host cells. Mutation rates can be increased to one mutational event in every 10e1 or 10e2 cells per generation for auxotrophic mutants, and one in 10e3 to 10e5 for the isolation of improved secondary metabolite producers. A method of selection is critical for effective screening of mutants as several thousand individual isolates may need to be evaluated to find one strain with improved activity in the property of interest.
Mutation and selection techniques have been used to improve the metabolic properties of microbial starter cultures used for food fermentations; however, there are severe limitations with this method. Mutagenic agents cause random mutations, thus specificity and precision are not possible. Potentially deleterious undetected mutations can occur, since selection systems may be geared for only the mutation of interest. Additionally, traditional mutation procedures are extremely costly and time-consuming and there is no opportunity to expand the gene pool. In spite of these limitations, mutation and selection techniques have been used extensively to improve industrially important microorganisms and, in some cases, yields of greater than 100-times the normal production level of bacterial secondary metabolites have been achieved.
Natural Gene Transfer Methods
The discovery of natural gene transfer systems in bacteria has greatly facilitated the understanding of the genetics of microbial starter cultures and in some cases has been used for strain improvement. Genetic exchange in bacteria can occur naturally by three different mechanisms: transduction, conjugation, and transformation.
Transduction involves genetic exchange mediated by a bacterial virus (bacteriophage). The bacteriophage acquires a portion of the chromosome or plasmid from the host strains and transfers it to a recipient during subsequent viral infection. Although transduction has been exploited for the development of a highly efficient gene transfer system in the gram-negative organism Escherichia coli, it has not been used extensively for improving microorganisms used in food fermentations. In general, transduction efficiencies are low and gene transfer is not always possible between unrelated strains, limiting the usefulness of the technique for strain improvement. In addition, bacteriophage have not been isolated and are not well characterized for most strains.
Conjugation, or bacterial mating, is a natural gene transfer system that requires close physical contact between donors and recipients and is responsible for the dissemination of plasmids in nature. Numerous genera of bacteria harbor plasmid DNA. In most cases, these plasmids are cryptic (the functions encoded are not known), but in some cases important metabolic traits are encoded by plasmid DNA. If these plasmids are also self-transmissible or mobilizable, they can be transferred to recipient strains. Once introduced into a new strain, the properties encoded by the plasmid can be expressed in the recipient. The lactic acid bacteria naturally contain from one to more than ten distinct plasmids, and metabolically important traits, including lactose-fermenting ability, bacteriophage resistance, and bacteriocin production, have been linked to plasmid DNA. Conjugation has been used to transfer these plasmids into recipient strains for the construction of genetically improved commercial dairy starter cultures.
There are some limitations in the application of conjugation for strain improvement. To exploit the use of conjugative improvement requires an understanding of plasmid biology and, in many cases, few conjugative plasmids encoding genes of interest have been identified or sufficiently characterized. Conjugation efficiencies vary widely and not all strains are able to serve as recipients for conjugation. Moreover, there is no opportunity to expand the gene pool beyond those plasmids already present in the species.
Certain microorganisms are able to take up naked DNA present in the surrounding medium. This process is called transformation and this gene transfer process is limited to strains that are naturally competent. Competence-dependent transformation is limited to a few, primarily pathogenic, genera, and has not been used extensively for genetic improvement of microbial starter cultures. For many species of bacteria, the thick peptidoglycan layer present in gram-positive cell walls is considered a potential barrier to DNA uptake. Methods have been developed for enzymatic removal of the cell wall to create protoplasts. In the presence of polyethylene glycol, DNA uptake by protoplasts is facilitated. If maintained under osmotically stabilized conditions, transformed protoplasts regenerate cell walls and express the transformed DNA. Protoplast transformation procedures have been developed for some of the lactic acid bacteria; however, the procedures are tedious and time-consuming, and frequently parameters must be optimized for each strain. Transformation efficiencies are often low and highly variable, limiting the application of the technique for strain improvement.
The above mentioned gene transfer systems have become less popular since the advent of electroporation, a technique involving the application of high-voltage electric pulses of short duration to induce the formation of transient pores in cell walls and membranes. Under appropriate conditions, DNA present in the surrounding medium may enter through the pores. Electroporation is the method of choice for strains that are recalcitrant to other gene transfer techniques; although optimization of several parameters (e.g., cell preparation conditions, voltage and duration of the pulse, regeneration conditions, etc.) is still required.
Genetic engineering provides an alternative method for improving microbial starter cultures. This rapidly expanding area of technology provides methods for the isolation and transfer of single genes in a precise, controllable, and expedient manner. Genes that code for specific desirable traits can be derived from virtually any living organism (plant, animal, microbe, or virus). Genetic engineering is revolutionizing the science of strain improvement and is destined to have a major impact on the food fermentation industry.
Although much of the microbial genetic engineering research since the advent of recombinant DNA technology in the early 1970s has focused on the gram-negative bacterium Escherichia coli, significant progress has been made with the lactic acid bacteria and yeast. Appropriate hosts have been identified, multifunctional cloning vectors have been constructed, and reliable, high-efficiency gene transfer procedures have been developed. Further, the structural and functional properties, as well as the expression in host strains, of several important genes have been reported. Engineered bacteria, yeast, and molds could also be used for the production of other products, including food additives and ingredients, processing aids such as enzymes, and pharmaceuticals.
Metabolism and Biochemistry of the Host
A necessary prerequisite for the application of genetic engineering to any microorganism is a fundamental understanding of the metabolism and biochemistry of the strain of interest. Although for hundreds of years the metabolic potential of microbial starter cultures has been exploited, in many cases little is known about specific metabolic pathways, the regulation of metabolism, or structural and functional relationships of critical genes involved in metabolism. This information is essential for the design of genetic improvement strategies, as it provides the rationale for selection of desirable gene(s) and assures that once inserted into a new host, the gene(s) will be appropriately expressed and regulated as predicted.
Plasmid-free, genetically characterized and highly transformable hosts, coupled with multifunctional expression vectors, provide the necessary tools for transfer, maintenance, and optimal expression of cloned DNA in microbial starter cultures. Many microbial starter cultures harbor plasmid DNA, and although most plasmids remain cryptic, resident plasmids interfere with identification of plasmid-containing transformants. Use of plasmid-free hosts also eliminates plasmid incompatibility problems and the possibility of cointegrate formation between transforming and endogenous plasmids. It is important to note that plasmid-free strains are used for the development of model systems; however, ultimately it will be necessary to engineer commercial strains.
A vector can be defined as a vehicle for transferring DNA from one strain to another. Plasmids are frequently used for this purpose because they are small autonomously replicating circular DNA forms that are stable and relatively easy to isolate, characterize, and manipulate in the laboratory. Native plasmids do not naturally possess all of the desirable features of a vector (e.g., multiple cloning sites, selectable marker(s), ability to replicate in several hosts, and so forth). Therefore, genetic engineering is frequently used to construct multifunctional cloning vectors. Although antibiotic resistance markers greatly facilitate genetic engineering in microbial systems, vectors derived solely from food-grade organisms may be critical in obtaining regulatory approval for use of the organisms, as antibiotic resistance determinants may not be acceptable in food systems.
An alternative vector strategy involves the development of linear fragments of DNA that are capable of integrating into the host chromosome via homologous recombination. Although transformation frequencies are very low, the advantage of the integrative vector is that transformed genetic information is targeted to the chromosome where it will be more stably maintained. Insertion sequences (IS elements) naturally present in the chromosome that can transpose chromosomal DNA to plasmids could be used as an alternative strategy for developing integrative vectors for some strains of lactic acid bacteria.
Efficient Gene Transfer Systems
Once gene(s) have been identified and cloned into the appropriate vector in the test tube, they must be introduced into a viable host. Since the recombinant DNA is a naked DNA molecule, gene transfer systems based on protoplast transformation and electroporation are most applicable in genetic engineering experiments. High transformation efficiencies (greater than 104 to 105 transformants per kilogram of DNA) greatly facilitate screening and identification of appropriate transformants. Electroporation is the transformation procedure of choice for most microbial strains.
Transfer of structural genes to a new host using genetic engineering does not guarantee that the genes will be expressed. To optimize expression of cloned genes, efficient promoters, ribosome-binding sites, and terminators must be isolated, characterized, and cloned along with the gene(s) of interest. Identification of signal sequences essential for secretion of proteins outside the cell may be useful for situations where microbial starter cultures are used to produce high-value food ingredients and processing aids. Secretion into the medium greatly facilitates purification of such substances.
Properties of Interest
Several properties could be enhanced using genetic engineering. For example, bacteriocins are natural proteins produced by certain bacteria that inhibit the growth of other often closely related bacteria. In some cases, these antimicrobial agents are antagonistic to pathogens and spoilage organisms commonly found as contaminants in fermented foods. Transfer of bacteriocin production to microbial starter cultures could improve the safety of fermented products.
Acid production is one of the primary functions of lactobacilli during fermentation. Increasing the number of copies of the genes that code for the enzymes involved in acid production might increase the rate of acid production, ensuring that the starter will dominate the fermentation and rapidly destroy less-aciduric competitors.
Certain enzymes are critical for proper development of flavor and texture of fermented foods. For example, lactococcal proteases slowly released within the curd are responsible for the tart flavor and crumbly texture of aged cheddar cheese. Cloning of additional copies of specific proteases involved in ripening could greatly accelerate the process.
An engineered Saccharomyces cerevisiae (baker's yeast), which is more efficient in leavening of bread, has been approved for use in the United Kingdom and is the first strain to attain regulatory approval. This strain produces elevated levels of two enzymes, maltose permease and maltase, involved in starch degradation.
There are a number of issues that must be resolved before genetically engineered starter cultures could be used in food. Engineered strains will need to be approved for use by appropriate regulatory agencies. To date, no engineered organisms have been approved in the United States, and specific criteria for approval have not been established by the Food and Drug Administration.
The public must be assured that the products of biotechnology are safe for consumption. If consumers have the perception that the products are not safe, the technology will not be utilized. Although genetic engineering is probably safer and more precise than strain-improvement methods used in the past, most U.S. consumers are not aware of the role of bacteria in fermented foods and do not have a fundamental understanding of recombinant DNA technology, and they may be unwilling to accept the technology. This may be less of a problem in developing countries where improved microbial starter cultures could provide significantly safer and more nutritious foods with longer shelf life and higher quality.
Another limitation is that genetic improvement of microbial starter cultures requires sophisticated equipment and expensive biological materials that may not be available in developing countries. Where equipment and materials are available in industrialized countries, there may be little incentive for researchers to improve strains that would probably not be used in their own countries.
Genetic improvement of microbial starter cultures is most appropriate for those fermentations that rely solely or primarily on one microorganism. In many cases, our knowledge about the fermentation is limited, making selection of the target strain very difficult. Since many food fermentation processes are complex and involve several microorganisms, genetic improvement of just one of the organisms may not improve the overall product.
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