Partial strains of lactic acid bacteria and yeasts are defined as probiotics, microorganisms with health-promoting effects on humans and animals. These bacteria and fungi are Generally Recognized as Safe (GRAS) microorganisms among prokaryotes (Duche et al. 2023) and eukaryotes (Liu and Cheng 2023), respectively, and they have been defined by the Food and Agriculture Organization/World Health Organization as probiotics (Iqbal et al. 2014). Therefore, they are widely used in the food industry. Lactic acid bacteria is a collective term representing gram-positive bacteria that ferment carbohydrates to produce lactic acid (Dong and Li 2012). They can provide proteases, amino acids, vitamins, and other growth substances to the animal body, promoting the individual’s growth and development (Scholz-Ahrens et al. 2007). Additionally, lactic acid bacteria can activate the immune mechanisms of bodily fluids and cells in individuals, contributing to immune responses such as apoptosis in tumor cells (Yuan et al. 2011). Yeast is a fungus with a strong fermentation ability, rich nutritional value, and stable characteristics, including its gene expression regulation mechanism and protein modification systems. It can hydrolyze polysaccharides or proteins into monosaccharides or amino acids, facilitating animal digestion and nutrient absorption (Marklinder et al. 1995). Due to the various nutrients present in yeast, it is widely used in the processing and production of condiments, flour products, spices, and other foods (Zhang et al. 2023).
Currently, the genetic engineering of lactic acid bacteria and yeast is developing rapidly. Various enzymes, vitamins, and secondary products related to food production can be expressed and transformed in large quantities through foreign gene fragment insertion. However, establishing these genetically engineered microorganisms must be based on a premise that ensures human health and environmental protection. Therefore, the required reaction substrates, inducers, and screeners need to be food-grade. Selection markers are essential components for constructing genetically engineered microorganisms. Selection markers for resistance to antibiotics or heavy metals have been used in most previous genetic engineering programs. However, neither of these two markers are food-grade selection markers. Antibiotic resistance selection markers easily spread with the changing seasons and endanger human health and the whole biosphere (Trombert 2015). Heavy metal resistance selection markers can cause the host cells to introduce toxic heavy metal ions in the culture process, which can affect the health of individuals via heavy metal toxicity in the gastrointestinal tract (Plavec and Berlec 2020).
Establishing safe and stable selection markers in food-grade expression vectors is a hot topic in current research (Huang and Li 2007; He et al. 2012). Presently, only dominant and complementary selection markers can be used. Dominant and antibiotic resistance selection markers share the same principle, and bacteriocins are representative of these screening agents (Xiang et al. 2007), but there are not many types of these selection markers. Complementary selection markers affect the host microorganism through the natural environment or artificial means and change their traits, lowering their survival in the corresponding basic or specific medium. Then, if the strain can grow normally by converting some specific selection marker genes related to the metabolic pathway, the transformants are obtained. D-alanine, threonine, purines, pyrimidines, and other substances that are harmless to humans have been used as screeners to establish food-grade expression vectors (He et al. 2012). Sugar utilization markers are complementary selection markers, and their screeners include lactose, melibiose, sucrose, D-xylose, glucosamine, and N-acetylglucosamine. Typically, lactic acid bacteria and yeast use carbohydrates as the carbon source for their growth during cultivation. Using sugar metabolism-associated key genes as selection markers and the corresponding carbon sources as screeners has the following two advantages (Tian et al. 2017): first, the transformant can obtain sufficient energy on the screening plate, rather than being in a hungry state, and second, in the case of non-purification, carbohydrates are edible, whereas in the case of purification, the purified protein exhibits no toxicity or sensitization. Therefore, when the inducer is of food-grade, genetically engineered microorganisms with safe and stable food-grade microorganisms as hosts, expression vectors containing sugar utilization marker elements, and no non-food-grade functional DNA can not only meet the primary conditions of a GRAS microbial expression system (Huang and Li 2007), but also ensure the safety of the produced food.
This review aims to summarize the experimental results of food-grade expression vector construction with various sugar utilization markers as screening elements using lactic acid bacteria and yeast as host microorganisms and compare and analyze them.
Among all the sugar utilization markers, lactose has been widely used as the screener for constructing food-grade expression vectors (Table I). Because of the presence of the lactose metabolism-associated coding gene on the expression vector, after successful transformation, the recombinant microorganisms grew yellow or blue colonies on the screening plate containing acid-base indicators (Platteeuw et al. 1996), such as bromocresol purple or 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside.
Lactose utilization markers.
Recombinant vector | Marker gene | Host | Strain number | Reference |
---|---|---|---|---|
ADH | Li et al. 1996 | |||
SBT2195 | Hashiba et al. 1992 | |||
NZ3900 | Cao et al. 2020 | |||
NZ3900 | Platteeuw et al. 1996 | |||
NZ3000 | Tagliavia et al. 2019 | |||
ATCC® 4356™ | Deng et al. 2011 | |||
LP01 | Wang et al. 2023 | |||
CECT 5276 | Gosalbes et al. 2000 |
All the lactic acid bacteria can metabolize lactose (Fan et al. 2006). Therefore, the β-galactosidase coding gene of lactic acid bacteria must be knocked out via gene editing techniques so the recipient bacterium can mutate into Lac-phenotype. Lac+ phenotype can be recovered when the recombinant plasmid containing the β-galactosidase coding gene is imported to such defective strain. β-Galactosidase is a key excision enzyme that catalyzes lactose hydrolysis to generate glucose and galactose. Some β-galactosidase also exhibit transglycosylation activity and can transfer galactosyl groups to various substrates to generate transglycosylation products with various prebiotic functions (Posno et al. 1991b; Delgado-Fernandez et al. 2020). β
Using inexpensive lactose as a selection marker can save the process cost and facilitate the co-production of milk-related products because lactose is also present in milk. After successful construction, the recombinant microorganisms and their products can be combined with some commonly used milk-based media to produce various healthy, nutritious, or better-tasting foods. Using the lactose promoter in plasmid vectors with lactose-selective markers and inducing with lactose can help achieve good expression (Wang et al. 2023); this construction can exhibit dual effects of the screening of exogenous lactose and lactose-selective marker expression. Selecting the target host bacteria would be a better and more straightforward method than using lactic acid bacteria with only β-galactosidase (lacF) activity, knocking out its lacF gene and constructing it into a plasmid vector (MacCormick et al. 1995; Platteeuw et al. 1996). There levant research analysis results show that the selection marker of β
Similar to β-galactosidase, α-galactosidases are also a type of excision enzyme that can be divided into two categories: one that targets galactomannan and hydrolyzes low-Mr substrates to varying degrees and the other that resolves melibiose into galactose and glucose (Shibuya et al. 1998). Present studies have shown that the α-galactosidase selection marker exhibits the latter’s activity, which has multiple coding genes from different species. There are mainly four types of markers that are used for genetic engineering construction (Table II):
Melibiose utilization markers.
Recombinant vector | Marker gene | Host | Strain number | Reference |
---|---|---|---|---|
MG1363, SMQ-741, SMQ-561, IL1404 | Boucher et al. 2002 | |||
ML23 | Gu et al. 2014 | |||
RD733 | Labrie et al. 2005 | |||
NZ3900 | Xu et al. 2007 | |||
MS-1 | Guo et al. 2010 | |||
NZ9800 | Jeong et al. 2006 | |||
JLS400 | Sridhar et al. 2006 |
The production of α-galactosidase has a particular physiological importance, as many soy products contain melibiose, which humans and monogastric animals cannot digest and metabolize, and its excessive consumption may lead to stomach pain, nausea, and diarrhea (Jeong et al. 2006). Because both lactic acid bacteria and yeast are a part of the native intestinal microbiota, we can establish “α-galactosidase-producing, foodgrade, genetically engineered microorganisms as healthcare products to improve the digestion of soy products. “α-Galactosidase is also used as a starter culture for soybean milk fermentation (Sridhar et al. 2006); therefore, establishing such genetically engineered microorganisms is beneficial for producing better soybean products by the metabolizing undesirable byproducts during the production process.
Temperature dramatically affects “α-galactosidase activity in different species. A study showed that the recombinant plasmid
Using the “α-galactosidase-encoding gene as a foodgrade selection marker to construct genetically engineered strains may make these methods more convenient. However, it is important to note that in host microorganisms using melibiose as the carbon source, the intracellular acquisition of melibiose requires permease (
In contrast, the
The resident plasmid of PPE1.0, a subspecies of
Cloning the scrA and scrB genes on the plasmids can establish a food-grade selection marker with sucrose as the only carbon source (Table III). In a study, the two coding genes, namely
Sucrose utilization markers.
Recombinant vector | Marker gene | Host | Strain number | Reference |
---|---|---|---|---|
LL108, LL302 | Leenhouts et al. 1998a | |||
MG1363 | Leenhouts et al. 1998b |
Although sucrose is a considerably inexpensive sugar in the fermentation industry, studies have shown that this selection system requires high copy numbers of sucrose metabolism-associated genes to obtain sufficient expression for the stable growth of the host bacteria (Huang and Li 2007). Moreover, the recombinant microorganisms grow very slowly in a sucrose-containing culture medium, which may not be suitable for large-scale fermentations (Leenhouts et al. 1998b). However, further studies are needed to improve and optimize this selection system.
In many bacteria utilizing D-xylose as a carbon source, which have four genes involved in D-xylose catabolism, namely
Owing to the lack of one or multiple D-xylose metabolism-associated genes, most lactic acid bacteria species (Lokman et al. 1991) and yeast cannot utilize D-xylose for fermentation (Jetti et al. 2019). This feature can be exploited to construct food-grade selection marker vectors using D-xylose as the sole carbon source through genome sequence analysis of relevant coding genes lacking in the targeted host (Table IV). The
D-xylose utilization markers.
Recombinant vector | Marker gene | Host | Strain number | Reference |
---|---|---|---|---|
ATCC® 393™, NCDO 1193 | Posno et al. 1991 | |||
Ethanol Red, CLIB382, CEN.PK113-7D | Stovicek et al. 2015 |
D-xylose is widely used in food processing and starch industries and is entirely safe (Tian et al. 2017). However, its limitations include the large gene fragments that may hinder the normal replication of plasmids in some host cells. Moreover, similar to melibiose, D-xylose, which is highly pure, is considerably expensive (Huang and Li 2007). For the use of D-xylose as a sugar utilization marker to construct a genetically engineered strain, the plasmid load and the value of the fermented product need to be evaluated.
D-fructose-6-phosphate aminotransferase is the key catalytic enzyme in the hexosamine metabolic pathway, and its encoding genes (the
After deleting the glmS/gfat gene, the deficient microorganism needs exogenous glucosamine or N-acetylglucosamine in the culture medium to grow. When both are introduced into the cells in the absence of fructose-6-phosphate aminotransferase, the former is converted to GlcN6P, and the latter is converted to N-acetylglucosamine-6-phosphate (GlcNAc6P), which can also be obtained using GlcN6P as a substrate. Otherwise, such defective strains cannot grow properly (Bulik et al. 2003; Wu et al. 2011; Chen et al. 2012). In contrast to the sugars mentioned above as utilization markers, when the deficient strains were introduced with the expression vector containing the
Notably, yeast extract and tryptone should be removed from the culture plate because both contain trace amounts of glucosamine, which can affect the post-transformation screening of defective or recombinant strains (Chen et al. 2012). Some lactic acid bacteria, such as
Glucosamine and N-acetylglucosamine utilization markers.
Recombinant vector | Marker gene | Host | Strain number | Reference |
---|---|---|---|---|
glmS | NZ5332 | Chen et al. 2018 | ||
gfat | YHL6381 | Wu et al. 2011 |
With the advancements in biotechnology, the biological safety regarding genetic transformation has garnered much attention. As edible microorganisms, lactic acid bacteria and yeast have bright application prospects. Presently, many food-grade, genetically engineered microorganisms have been successfully constructed, and they have been applied in health care, fermentation, food processing, and other fields (Abrha et al. 2023; González-Orozco et al. 2023; Van de Voorde et al. 2023). The selection markers on vectors are an important part of the selection system for recombinant microorganisms. However, suppose the engineered microorganisms are cultured with a single carbohydrate as the carbon source for energy. In that case, plant growth may not be better than that when cultured with glucose, which is the most basic carbon source. Lactose and sucrose require a vector with a selection marker with a high copy number to obtain a good screening effect, and melibiose and xylose are expensive. They can increase production costs (Xiang et al. 2007).
The glucosamine-6-phosphate synthase-encoding gene is essential for the growth of various eukaryotic and prokaryotic microorganisms and is a newly established food-grade selective marker. It can compensate for the shortcomings of other sugar utilization markers, and engineered microorganisms can use multiple carbon sources to provide nutrition in large-scale industrial fermentation production without selection pressure. Therefore, the selection system with the glucosamine-6-phosphate synthase gene has great research value, and future studies may focus on exploiting its potential in various industries.