STARTER CULTURES AND FERMENTED MILK PRODUCTS

Lesson 13. Lesson 13 SCOPE AND NEED, CHARACTERISTICS WHICH CAN BE MANIPULATED, GENETIC TOOLS – MUTATION, CONJUGATION, TRANSFORMATION, TRANSDUCTION, ELECTROPORTION, PROTOPLAST FUSION AND GENETIC ENGINEERING

Module 9. Genetic improvement of starters

Lesson 13
SCOPE AND NEED, CHARACTERISTICS WHICH CAN BE MANIPULATED, GENETIC TOOLS – MUTATION, CONJUGATION, TRANSFORMATION, TRANSDUCTION, ELECTROPORTION, PROTOPLAST FUSION AND GENETIC ENGINEERING

13.1 Introduction

Modern approaches towards starter and protective culture improvement rely on advances in molecular biology. For most microorganisms used for food production, genetechnological methods have been well developed. By recombinant DNA technology, ‘tailor-made’ starter and protective cultures may be constructed so as to combine technically desirable features. A single strain which normally would fail to accomplish a given ‘task’ may now be improved so as to meet a set of requirements necessary for a specific production or preservation process (e.g. wholesomeness, no off-flavour production, overproduction of bacteriocins or particular enzymes).In addition, undesirable properties (e.g. mycotoxin or antibiotic production by cheese moulds) may be eliminated by techniques such as ‘gene disruption’.

Most strains of lactic acid bacteria employed as starter cultures contain plasmids as well as chromosomalgenes. Plasmid-borne genes are of particular importance both because of their accessibility for molecular genetic analysis and because they code for many properties essential for successful starter activity. There is considerable interesting applying knowledge of the genetics of lactic acid bacteria to improve existing or produce new strains. Both conventional techniques such as conjugation and recombinant DNA technology are used and directed integration of genetic material into the bacterial chromosomes, is also being investigated with the intention of improving the stability of genetically manipulated cultures.

13.2 Characteristics Which Can be Manipulated

Natural isolates usually produce commercially important products in very low concentrations and therefore every attempt is made to increase the productivity of the chosen organism. Increased yields maybe achieved by optimizing the culture medium and growth conditions, but this approach will be limited by the organism’s maximum ability to synthesize the product. The potential productivity of the organism is controlled by its genome and, therefore, the genome must be modified to increase the potential yield.

Research will continue on the plasmid biology, gene transfer system characterization, cloning, gene expression, and regulation of metabolic properties vital for producing fermented food products

Dairy field deals with two variables.

1. The bacteria: used generally as starter cultures for the production of lactic acid,flavours, proteolysis and also in the by-product utilization.

2.Enzymes: they are used for various metabolic changes in dairy foods for bringing desirable changes.

Starter failure due to antibiotic residues in milk could be eliminated by the isolation of drug resistant starter strains.

13.2.1 Properties of interest

• 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 flavour and texture of fermented foods. For example,lactococcal proteases slowly released within the curd are responsible for the tart flavour and crumbly texture of aged cheddar cheese. Cloning of additional copies of specific proteases involved in ripening could greatly accelerate the process.

• Diacetyl is an industrially important compound used to impart buttery flavour and aroma to many foods. Diacetyl is derived from citrate metabolism by LAB, and knowledge of the enzymology and genetics of citrate metabolism has identified several strategies to metabolically engineer L. lactiss trains for enhanced diacetyl production.One effective avenue toward this end involves inactivation of the gene encoding alpha-acetolactate decarboxylase (aldB), the enzyme that converts alpha-acetolactate to acetoin. This mutation promotes an accumulation of alpha-acetolactate, the immediate precursor to diacetyl, which in turn leads to an increased level of diacetyl in the growth medium.

• Bacteriocins are naturalproteins 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 bacterioc in production to microbial starter cultures could improve the safety of fermented products.

• An engineered Saccharomycescervisiae (baker's yeast), which is more efficient in leavening of bread,has been approved for use in the United Kingdom and is the firststrain to attain regulatory approval. This strain produces elevated levels of two enzymes, maltose permease and maltase, involved in starch degradation.

• Bacteriophage resistance
• Rennet substitutes
• Lactose intolerance
• Microbial polysaccharides

13.2.2 Genetic tools – mutation, conjugation, transformation,transduction, electroporation, protoplast fusion and genetic engineering

The potential productivity of the organism is controlled by its genome and, therefore, the genome must be modified to bring improvement in desired characteristics to increase the potential yield. The process of strain improvement involves the continual genetic modification of the culture, followed by reappraisals of its cultural requirements.

Several traditional and non traditional 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.

13.2.3 Mutation

The empirical approach to strain improvement involves subjecting a population of the microorganisms to a mutation treatment and then screening a proportion of the survivors of the treatment for improved productivity.

In nature, mutations(changes in the chromosome of an organism) occur spontaneously at very low rates (one mutational event in every 10⁶ to 10⁷ 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 mutationalevent in every 10¹ or 10² cells per generation for auxotrophic mutants, and one in 10³ to 10⁵ 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.

13.2.4 Transduction

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.

The value of transduction as a gene transfer process in starter cultures is limited, because only host strains which have the relevant bacteriophage receptors can be used and because the head capacity of the phage limits the amount of DNA that can be transferred. Transduction of lactose plasmid causes deletions to be generated because the whole material is too large to be accommodated in the phage head.Transduction sometimes causes lactose and proteinase genes to become integrated into the bacterial chromosome with two important consequences, the genes become stabilized by their new location and the levels of gene expression are reduced.

The transduction of streptomycin resistance in Lactococcus lactis ssp. lactis C2 and tryptophanin dependence in Lactococcus lactis ssp.lactis biovar diacetylactis 18-16 was reported. In Lactococcus lactis ssp. lactis C2 transduction of maltose and mannose markers and plasmid-encoded lactose genes were detected

13.2.5 Conjugation

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 harbour 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 bacterioc in 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.

Gene transfer by conjugation process has been widely reported in lactic streptococci.Conjugal transfer of plasmid-encoded lactose genes between marked strains of Lactococcus lactis ssp. lactis 712and from Lactococcus lactis ssp. lactisbiovar diacetylactis 18-16 into a plasmid-free derivative of Lactococcus lactis ssp. lactis C2. Although these transfer processes were initially detected as relatively low frequency events, high frequency donors have been isolated amongst the progeny from some mating experiments. These variant strains were first discovered in Lactococcus lactis ssp.lactis 712, exhibiting a novel cell aggregation phenomenon which causes a striking change both in colony morphology and in the appearance of broth cultures. Similar, aggregating progeny strains were found after lactose plasmid transfer from Lactococcus lactis ssp.lactis ML3, into a plasmid-free derivative of Lactococcus lactis C2. From an evolutionary standpoint it is of interest that cell aggregation is also an established feature of a sophisticated inducible conjugation system found to operate in some E.faecal is strains.

Various bacterioc in plasmids and the plasmid-like gene blocks for sucrose utilization and nisin production and for diplococcin production are able to effect their own transfer by conjugation.

Conjugation has considerable potential for the construction of new dairy starter strains. There is no theoretical limit to the amount of DNA that can be transferred and the promiscuous nature of pAMß1conjugation makes transfer between widely different strains a possibility.

13.2.6 Transformation

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 use dextensively for genetic improvement of microbial starter cultures. For many species of bacteria, the thick peptidogly can 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. Protoplast production and regeneration of lactic cultures involves cell wall digestion with lysozyme, mutanolysin or a combination of amylase and lysozyme.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. Although the initial transformation efficiencies were considerably improved, results are now being obtained with frequencies of about 10⁵ transformants per µg of DNA.

13.2.7 Electroporation

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.

13.2.8 Genetic engineering

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 has focused on the gram-negative bacterium E. 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.

Eventually the genetics of lactic acid bacteria will be applied to improve or produce new dairy starter cultures or strains. Two plasmid-encoded mechanisms of phage resistance, restriction modification and adsorption blocking, have been discovered. The genes for these properties will be introduced into a variety of starter strains either on their existing plasmids or after their isolation by gene cloning. A system of starter strains with much improved protection from bacteriophage attack could result. Stabilization of lactose and protein hydrolyzing enzymes by insertion into the chromosome has been achieved in L.lactis ssp lactis C2 and 712 and this approach may be extended to generally stabilize starters and eliminate slow variants.

The proteolytic enzymes and their roles in cheese maturation and off-flavour production will be better defined. Genes for different proteinases will be exchanged between strains, and control over the levels of gene expression will be possible. The most rewarding development may be the creation of strains capable of accelerating the ripening of Cheddar cheese either by manipulating existing starter genes or by the introduction of new genes from non-dairy sources.