What modification was done?
Lactic acid bacteria (LAB) have a long history in application of fermented food products. Progress in gene technology allows their modification by introducing new genes or by modifying their metabolic functions. These modifications are important as may lead to improvements in food technology. LAB are widely used as starter cultures for fermentation in the dairy, meat and other food industries. Their properties have been used to manufacture products like cheese, yoghurts, fermented milk products, beverages, sausages, and olives. Genetic alteration of LAB can be divided into two that is uncontrolled (natural) and controlled (directed).
Natural events such as insertion sequence elements (Visser et al. 2004), radiation, erroneous DNA replication or transcription, and other factors lead to spontaneous mutation in LAB. Spontaneous mutations in single genes can lead to altered lactose metabolism, citrate uptake, and increased proteolytic activity. In Lactobacillus bulgaricus a spontaneous insertion sequence (IS) element-mediated deletion of the lacZ gene resulting in limited fermentation capacity by altering the lactose metabolism. Therefore, yoghurt, made with these altered L. bulgaricus, is not suffering from post-fermentation acidification during the shelf life period (Mollet and Delley, 1990).
In the fermentation of Roquefort cheeses, L. lactis strains were randomly mutated, and selection was based on carbon dioxide production that improves the quality of the cheese. After random mutagenesis with the mutagen NNG, production levels of the butter flavour compound diacetyl that was increasing were selected. The selection of a spontaneously mutated L. lactis strain that overproduced diacetyl, responsible for the butter flavour in many fresh dairy products, is described by Monnet et al. 2000.
The nutritional value of vitamins in fermenting bacteria can also be increased. To increase vitamin B2 (riboflavin) production levels in the dairy starter bacterium L. lactis, bacterial cells were exposed to increasing concentrations of roseoflavin. Roseoflavin-resistant strain will be selected with deregulated riboflavin biosynthesis and increased production levels of riboflavin, up to 1 mg/L. Modified L. lactis will have a daily recommended intake (DRI) of 1.3 mg for riboflavin.
Removal of undesirable compounds from raw food materials can also be done by genetic engineering of LAB. Undesirable sugar lactose is degraded only partially such as galactose in traditional yoghurt fermentation. Galactose that is degraded will cause galactosemia and also cataract. Therefore, a selection of spontaneous galactose-fermenting mutants of Streptococcus thermophilus, that contain up-mutations in the gal operon is important to remove undesirable galactose.
Besides, directed mutagenesis is widely applied in research to improve fermented food products. For example, cheese ripening can be enhanced by over expression of peptidase genes in LAB via self-replicating plasmids. Moreover, the expression of gdh from Peptoniphilus asaccharolyticus, encoding glutamate dehydrogenase, into L. lactis increases the production of α-ketoglutarate (Rijnen et al. 2000). This enhances the degradation of amino acids, which also benefits the cheese ripening process. During processes of cheese ripening, lantibiotics in dairy starter strains could prevent spoilage from Gram positive bacteria. This can be done by production of lantibioticsto. Lantibioticsto prevent growth of Listeria monocytogenes and Lactobacillus fermentum.
In S. thermophilus the phosphoglucomutase gene was inactivated resulting in improved exopolysaccharide production enhancing the viscosity of the fermented food product (Levander et al. 2002). Engineering of exopolysaccharide production in L. lactis was also achieved by using a self-cloning strategy. Other than that, l-alanine is found in the high added value products. By introduction of a Bacillus subtilis (formerly B. natto) alanine dehydrogenase gene into a L. lactis strain deficient in lactate production, pyruvate conversion was pushed in the direction of alanine. The subsequent inactivation of the host gene encoding alanine racemase leads to the production of the stereo specific and thus highly valuable l-alanine (Hols et al. 1999).
To remove the undesirable sugars from the gastro-intestinal tract, the use of recombinant DNA technology in the development of probiotic strains is important. α-Galactosides such as raffinose and stachyose in raw agromaterial like soy will cause digestion problems in a lot of people. As humans do not have ability to produce intestinal α-galactosidases, these sugars pass into the lower GI-tract where they are fermented by gas-producing bacteria, rendering intestinal pain and flatulence. The expression of genes encoding enzymes that are able to degrade raffinose, stachyose, etc, such as α-galactosidases (Boucher et al. 2002; Silvestroni et al. 2002). Therefore, when these sugars pass through the GI tract, they will be degraded by the probiotic LAB and not by the resident gas-forming bacteria.
Why was it done?
Food demand increases as the population keeps growing rapidly. Lactic acid bacteria (LAB) play an important role in food manufacturing process. It is responsible to produce lactic acid during fermentation. Lactic acid produced from the fermentation will be used to produce fermented dairy products such as yogurt and cheese. Therefore, large quantities of LAB needed to meet the requirements for industry production.
Genetically modified LAB are designed as they have a better quality than natural LAB. Genetically modified LAB are able to produce desired compounds as cell factories in closed systems. Cell factory system is a technique for large scale production of cells. This technique reduces the possibility of the undesirable microorganisms production and thus the risk of contamination will be lowered (Plavec & Berlec, 2020). Genetically modified LAB are suitable for high production food factories because of the large amount of bacterias produced in a short time.
Moreover, genetically modified LAB can withstand the environmental changes better than natural LAB. The function of bacteria will be affected in certain factors such as pH and temperature. A slight change in those factors can create a huge impact on the food production. The stability of genetically modified LAB enables the genetically modified food products, cheese and yogurt can be kept for a longer period. They can prolong the shelf life of food products.
The nutritional value of fermented foods can be improved by using genetically modified LAB. The complete riboflavin gene cluster will result in high riboflavin production. The high production level of riboflavin in food products shows that it exceeds the average servings of riboflavin daily (Sybesma et al. 2006). Nutritional value contained in the food will be increased as the quantity of riboflavin increases.
Texture of food products using genetically modified LAB can also be improved. Complete gene clusters will be transformed from one LAB strain to another one. The newly generated strains have the ability to alter the viscosity and texture of the food product (Germond et al. 2001). Viscosity of the food products increases when using genetically modified LAB. For example, yogurt becomes thicker when viscosity is high.
How was it done?
Lactic acid bacteria is a common microorganism which is mostly used in the food industry due to its fermentative and probiotic properties (Othman et al. 2017). Degradation of carbohydrate is the main metabolism of lactic acid bacteria which will produce lactic acid and also energy. Although lactic acid bacteria is widely used but it still contributes to some undesired spoilage and is easily affected by environmental factors (Florou-Paneri et al 2013). Hence, modification of lactic acid bacteria is done by using different methods.
One of the methods is genetic engineering of lactic acid bacteria (Plavec et al. 2020). There are three methods of genetic editing which are transformation, genome editing (DNA integration) and CRISPR-Cas-based genome editing (Börner et al. 2019). Transformation of DNA can occur naturally or artificially. Conjugative method is a natural method which achieves non- GMO lactic acid bacteria strains. In artificial methods, the cells are first washed with cell- envelope weakening solutions and external agents (Figure 1). This can help the cell for permeabilization and transformation process. Electroporation is the common method used because it is more simple, efficient and can be used in different genus of lactic acid bacteria. Electric pulse, chemical treatment or heat shock will be given to the cell to take up the plasmid DNA which contains the desired characteristics. Specific cell wall enzymes cleaves the cell wall of cells aiding the process of transformation. Protoplast fusion is also used to form hybrid cells.
Integration of DNA, also known as homologous recombination is the insertion or removal of gene of interest via integrative plasmid in a cell and recombined to form new characteristics. Since mostly bacteria cannot be transformed with the linear DNA, thus integrative plasmid is used (Heap et al. 2012). This method is time- consuming based on the curing and selecting of plasmid and integrants (Börner et al. 2019). Phage λ- or Rac prophage-derived exonuclease (Exo or RecE) and ssDNA binding protein (Beta or RecT) is required for the expression of double stranded DNA recombination and single stranded DNA respectively. A marker is needed to be inserted to the homologous region and undergo polymerase chain reaction (PCR) to verify whether it is wild type or mutant.
CRISPR is an adaptive immunity of prokaryotes against foreign invaders recognised by CRISPR RNAs (crRNAs). CRISPR-Cas-based system can be editing and silencing tools. Cas9 is an enzyme produced by CRISPR system and plays a role in binding to the desired DNA and cutting or silencing the gene while Cas 2 play a role in binding Cas1 to CRISPR locus for the adaption process. Cas9 counter- selection and repurposing endogenous systems can combine with the integrated gene. There are three types of CRISPR system. Based on Figure 1, the type II system is depicted for the repurposing endogenous systems. Plasmid based expression of the CRISPR array or synthetic single guide RNA on the native system can target the gene in the organism with the help of desired spacer under specific prerequisites (Börner et al. 2019). The process can occured when the system active under in vivo editing conditionwhile other component and protospacer adjacent moti (PAM) is showing recognition.
Gene silencing is a process inactivating enzyme in the system by mutating the active sites of Cas9 but does not cleave it. In this process no homologous recombinant is required which benefits the screening process with high potential of multiplication. Gene silencing LAB is still not famous in lactic acid bacteria but it is a tool to investigate the downregulation of essential genes (Luo et al. 2015).
Issues regarding the modification
Although genetically modified LAB have created great achievement in food processing, there are still many doubts regarding the technique. A lot of people are confused about the safety of consuming genetically modified LAB.
Genetically modified LAB have the probability to develop as reservoirs for antibiotic resistant (AR) genes (Plavec & Berlec, 2020). Antibiotic resistance is known as the ability of bacteria or microorganisms to resist the effects of an antibiotic to prevent them from being killed. Frequent consuming of genetically modified LAB will cause infectious bacteria resistant to the antibiotic. This creates a threat to public health because infections and diseases will spread widely in the population.
Some people can be sensitive to the genetically modified LAB especially the immunocompromised category. They have a weak immune system and are vulnerable to diseases. Genetically modified LAB products may have unexpected adverse reactions. Symptoms of weakened immune system included digestion problem, slow wound healing and constantly fighting recurrent infections.
Components inside the genetically modified LAB may cause allergic reaction for certain people. The nucleic acid techniques enable the introduction of DNA that can synthesise new substances in the genetically modified LAB that might trigger allergic reactions. The introduction of foreign DNA results in new substance formation and it may have allergic effects. Person with food allergy has symptoms such as an itchy red rash and even worse, inability to breath.