Genetic Improvement Of Industrial Dairy Lactic Acid Bacteria

Modern genetics research is founded upon the power to establish cellular and molecular functions through DNA manipulation, and LAB played an important role in the origin of this technology. In their landmark research on the ''transforming principle'' of S. pneumoniae, Avery and coworkers (1944) not only proved that DNA was the molecule of heredity, they also recognized the distinction between genetic material (DNA) and products of its expression (in this instance a capsular exopolysaccharide). In his discussion, Avery wrote:

Thus, it is evident that the inducing substance and the substance produced in turn are chemically distinct and biologically specific in their action . . .''; that these induced changes ''are predictable, type-specific, and heritable.'';

and therefore ''If . . . desoxyribonucleic acid actually proves to be the transforming principle. . . ., then nucleic acids of this type must be regarded not merely as structurally important but as functionally active in determining the biochemical activities and specific characteristics of pneumococcal cells.

Today, our ability to manipulate animals, plants, and microorganisms genetically to manufacture, modify, or improve products or processes has blossomed into a multibillion dollar enterprise that has revolutionized pharmaceutical, chemical, and agricultural industries. Many of the most exciting and successful industrial applications of biotechnology involve microbial products or whole microorganisms. In the agricultural sector, for example, microbial biotechnology has become an integral component of modern plant and animal production, agricultural waste management, and food processing operations. Although many of these applications rely on naturally occurring cells or cell products, use of recombinant DNA-derived microbial products in agricultural and food systems is now commonplace. However, a similar statement does not apply to live, genetically modified microorganisms (GMMs), whose applications in food and agriculture has essentially been drowned in a whirlpool of scientific, political, and social controversies. The undercurrents that created this vortex are complex and beyond the scope of this chapter; suffice it to say that in addition to scientific and regulatory hurdles, the sociopolitical climate regarding use of recombinant DNA technology in food systems ranges from outright opposition (e.g., Western Europe, Australia, and New Zealand) to cautiously acquiescent (e.g., North America and parts of Asia). A variety of genetically modified agricultural plants are now in commercial production in the latter countries, but general opposition to genetic engineering in agriculture will probably continue to resonate through the sociopolitical agendas of most other states for years to come. Change will come, but it will come faster if academicians, industry scientists, and governmental representatives work to facilitate open and reasoned public discussion on risks and benefits of biotechnology in agriculture, and to promulgate sound scientific guidelines and policies.

As we consider commercial applications for genetically modified starter LAB, it is important to recognize a few basic principles: (1) dairy starter technology can be traced to the late 19th century, and the long history of safe application of LAB in human food means dairy starter bacteria have GRAS status (generally regarded as safe for use in food by governmental regulatory agencies such as the U.S. Food and Drug Administration); (2) our knowledge of LAB genetics and physiology has already identified very clear strategies to improve the industrial performance of dairy LAB; and (3) many of these improvements can be effected by mutation or natural gene transfer (e.g., conjugation). From this perspective, one can envision several simple, yet industrially valuable, genetic alterations to dairy LAB that do not undermine the GRAS status of these bacteria or influence the nutritional composition of fermented dairy foods. Two examples of genetic improvements that meet these criteria involve intraspecific transfer of native plas-mids and by directed metabolic engineering through natural mutation.

A. Enhanced Phage Resistance by Intraspecific Transfer of Native Plasmids

As noted in Table 1 and Section III.C, bacteriophage resistance is one of several industrially important traits that may be encoded by plasmid DNA in lactococci, and many lactococcal phage resistance plasmids can be transferred by conjugation (Klaenhammer and Fitzgerald, 1994). Since conjugation is a natural form of gene transfer, dairy LAB that are genetically improved by this process do not command the regulatory and sociopolitical attention that is directed toward recombinant DNA technology. Sanders and coworkers (1986) were the first to capitalize on this fortuitous situation when they introduced pTRK2030, a conjugative lactococcal plasmid that encodes restriction/modification and abortive infection phage defense mechanisms, into commercial Cheddar cheese starter bacteria. This general strategy has since been emulated by other researchers (Klaenhammer and Fitzgerald, 1994), and conjugation-derived, bacteriophage-insensitive dairy starter cultures have been commercially available for many years.

Conjugation has also been used to obtain strains that contain two or more plasmids encoding complementary phage defense systems (Klaenhammer and Fitzgerald, 1994). This capability led Sing and Klaenhammer (1993) to propose an ingenious phage resistance strategy that is based upon rotation of different restriction/modification and abortive phage defense mechanisms within a singlestrain Lc. lactis starter background. Those investigators showed that rotation of isogenic phage-resistant derivatives—which differ in the types and specificities of phage defense mechanisms they encode—not only thwarts bacteriophage proliferation, it actually removes contaminating phages from the culture medium (because of the combined action of multiple abortive phage defense systems). By restricting the starter system to a single strain, this strategy also acts to reduce the potential for emergence of new phages in the dairy processing environment.

Although intraspecific conjugation of native phage resistance plasmids has been of great benefit to the dairy industry, the flexibility of this strategy is clearly limited to plasmids that are self-transmissible or mobilizable (see Sec. III.C). In some countries, this limitation has been overcome by electroporation with native phage resistance plasmids, and starter lactococci that have been improved by this process are now in widespread commercial use.

B. Metabolic Engineering for Diacetyl Production

Diacetyl is an industrially important ''buttery'' flavor and aromatic compound that is derived from citrate metabolism by LAB. Recent advances in our under standing of the genetics of citrate metabolism and mechanisms for diacetyl production have yielded several useful strategies to metabolically engineer Lc. lactis strains for enhanced diacetyl production (De Vos, 1996). One of the most promising avenues toward this goal involves inactivation of the gene encoding a-aceto-lactate decarboxylase (aldB), the enzyme that converts a-acetolactate to acetoin (see Fig. 10 in Chap. 7). This approach results in accumulation of a-acetolactate, the immediate precursor to diacetyl, which in turn leads to an increased concentration of diacetyl in the growth medium.

Inactivation of aldB can, of course, be directly achieved by replacement recombination (Swindell et al., 1996), but naturally occurring aldB mutants can also be isolated by growth selection in a medium that contains leucine but not valine. The latter approach is possible because a-acetolactate also serves as an intermediate compound in biosynthesis of leucine and valine, and leucine is an allosteric activator of a-acetolactate decarboxylase (Goupil-Feuillerat et al., 1997). Thus, wild-type lactococci cannot grow in such a medium, because leucine stimulates conversion of a-acetolactate to acetoin, leaving none to support valine biosynthesis. Any aldB mutants in the population, however, are able to synthesize valine in the presence of leucine and so will continue to grow. Regrettably, the industrial utility of this strategy is rather limited, because most commercial Lc. lactis strains are auxotrophic for branched-chain amino acids. To overcome this limitation, Curic et al. (1999) developed an inventive strategy wherein industrial strains are first transformed with recombinant plasmid-encoding enzymes for branched-chain amino acid biosynthesis. Selection for naturally occurring aldB mutants in the transformants can then be done as outlined above, and food-grade variants of that population obtained by subsequent plasmid curing. Since the final product of this work is a completely natural mutant that lacks any foreign DNA, strains that are improved by this approach are likely to see commercial application in the very near future.

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