Introduction
Metabolic engineering uses genetic manipulation methods to improve the productivity and yield of native products that are novel to biological systems synthesized by microorganisms and improve cell adaptations to adverse conditions (Leonard, Lim, Saw, Koffas, & Microbiology, 2007; Tomar & De, 2013). The genetic alteration of the cell is a common factor in almost all types of metabolic engineering.
Recombination involving plasmid preparations was one of the first techniques used, and it is still used today. As a result, metabolic engineering can be viewed as a practical manifestation and natural progression of methods first developed in the biological field. It moves away from the idea of individual enzyme and toward integrating entire systems to accomplish the objectives set out at the start of any project. Recombinant DNA technology can now be used to design and execute more rational metabolic alterations (Lixin, 2000).This allows for more logical metabolic modifications to be designed and implemented using recombinant DNA technology (Wenzel & Müller, 2005).
Metabolic engineering places a premium on the modification pathway and analysis. This is a field of metabolic engineering that has overgrown in the last few years, and computing power continues to increase exponentially. The analytical part comprises the experiment and modelling techniques, e.g., labelling diagrams and metabolic flux analysis (MFA). MFA enables the formal investigation of cellular responses to genetic and environmental perturbations in terms of metabolic fluxes (Antoniewicz, 2020).
Due to the essential fermentation role in food processing and preparation, microorganisms are continually encountered in these processes. Bacteria play an essential role in these microorganisms: they are used as a bio-catalyst in forming organic acids, amino acids, vitamins, and carbohydrates, either as bacteria or as products synthesized by bacteria. Bacterial food additives may be used in dietary supplements, texturizers, acidulates, preservatives, emulsifiers, surfactants, thickeners, or functional food ingredients (Partridge et al., 2019). Food that has the potential to enhance one's health is known as a functional food. Amino acids are used as a feed of animals, pharmaceuticals, and food industries (Mayra-Makinen, Bigret, & Dekker-, 2004). They can also be used as sweeteners and flavor enhancers.
Improvement in central metabolic pathways
In these pathways, rerouting of carbon is required to produce the desired product. This can be done by raising the accessibility of precursor metabolites. There are various valuable strategies for redirecting the flow of carbon towards the synthesis of the desired product. Central metabolic pathways are modified to produce a higher amount of histidine and other amino acids with the help of planned modification of cellular metabolism to produce desired products (Bar-Even, Flamholz, Noor, & Milo, 2012). By increasing, the accessibility of ribose-5- phosphate by transketolase deficient strain. By raising the amount of erythrose-4 phosphate, we can raise amino acid production. This can be achieved by two main factors; over expression of transketolases and inactivation of phosphoenolpyruvate system dependent on phosphotransferase system. This can inflate the phosphoenol synthase.
Modification of biosynthetic Shikimate pathways
Over-expression of chorismate mutase enzyme as important biochemical intermediate plants and microorganisms is required in this pathway to increase phenylalanine expression (Russ et al., 2020). it is also termed the Shikimate pathway because it has one substrate (Chorismate) and one product (prephenate) (Tzin & Galili, 2010) . To increase the tyrosine and phenylalanine, simultaneous inflation of chorismate mutase and prephenate dehydrates is done. The phenomenon of Chorismate in the synthesis of aromatic amino acids and secondary metabolites has shown in Figure 1.
Figure 1
Chorismate, a central branch point metabolite insynthesising aromatic amino acids and secondary metabolites (Tzin et al., 2010)
Modification of transport system
The intracellular concentration of amino acids and feedback inhibition can be decreased or avoided by altering the amino acids transport system (Maser, Peebo, Vilu, & Nahku, 2020). Two examples can understand the significance of this strategy. When C-glutamicum produces the L- tryptophan, which results in the accumulation of L-tryptophan in the extracellular medium, there is a backflow of it into cells. It results in the production of severe feedback inhibition during the biosynthesis of tryptophan. Ikeda and Katsumata solve this problem by lowering the level of tryptophan uptake due to creating the mutants, which result in 10 to 20% accumulation of tryptophan more than in their parent. There can be an increase in cysteine's fermentation yield, which is another excellent example of transport engineering (Antoniewicz, 2020). This can be obtained by the overexpression of multidrug efflux genes in the strain of E.coli, which produce cysteine.
Use of analytical tools in a toolbox
The MFA has been used to modify the production of many amino acids, including the two essential amino acids (Chotani et al., 2000; Ivanov, Stoimenova, Obreshkova, Saso, & Equipment, 2013). Both amino acids have the most significant production volume in the world. For example, L-glutamic acid has 800,000 tons/year, and L-lysine has 600,000 tons/year.
Glutamate
Several processes and pathways have been contributed to the improvement of the glutamate producing by C-glutamicum, such as modification and analysis of biosynthetic and central metabolic pathways. The MFA shows the estimation of fluxes by the label experiments (Antoniewicz, 2020). It explains the relative contribution of different pathways. During the metabolic branch point of 2- oxoglutarate glutamate, there is a relationship established by MFA between the decline of the oxoglutarate dehydrogenase complex and the flux distribution. It suggests that they could be limiting the production of glutamate by metabolic flux through anaplerotic pathways.
L-lysine
One other example of labelling experiments based on MFA is the L-Lysine (Ooyen, Noack, Bott, Reth, & B, 2012) . The comprehensive approaches have been used to assess all the significant effluxes in the C-glutamicum metabolism. It reveals the pattern which is used for designing ME strategies. For this PPP the high capacity for NADPH reoxidation, and the futile cycle between the C4 compound of the TCA cycle and the C3 compound of glycolysis. This metabolic fluxislow (Holms, 1987). The NADPH is required in high amount byheterologous NADPH dependent glutamate dehydrogenase mutant, and it results in the increasing flux of TCA cycle and here the isocitrate step of TCA cycle results in the HencedecreasesPPP flux because of the NADPH requirements, which are lower. For the homologous NADPH dependent glutamate-dependent glutamate dehydrogenase mutant, the inverse is true. So, the NADPH requirements are high, so the PPP flux is also higher, and there is a lower TCA cycle.
Use of lactic acid bacteria in the production of amino acid
The different types of amino acids can also be produced using the LAB (Toe et al., 2019) . The metabolic engineering of Lactococcus lactis produces L- alanine which is the product of fermentation. In the lactate dehydrogenase deficient strain, the Bacillus sphaericus. Alanine dehydrogenase results in the rerouting of the carbon flux towards the alanine. Finally, by disrupting the gene encoding the alanine racemase, the stereospecific production of L- alanine was achieved (Ferreira & Guido, 2018).
Multifuncional organic acids
Organic acids are organic compounds having acidic properties. Carboxylic acids are the most common organic acids having a carboxyl group (-COOH). Some are strong organic acids, and some are weak organic acids, but both are helpful in food production. Examples of organic acids are lactic acid, acetic acid, formic acid, etc (Clark, 1989). In many food industries, the production of organic acids through different processes and techniques such as fermentation proved very beneficial because of their widespread applications in beverage industries, food preservation, and sweeteners and flavor enhancers. For this purpose, many types of bacteria, such as E.coli, go through different mixed acid fermentation of many sugars, for example, glucose, and produce good products like formate, acetate, ethanol, and succinate et (Clark, 1989). Anaerobic fermentative pathways have the following functions: It produces energy in this anaerobic environment through phosphorylation at a substrate level. It provides a source to regenerate NAD+.
Metabolic pathways
Following are some examples of metabolic pathways of E.coli for the production of organic acids include succinate, lactate, and acetic acid (Yamauchi, Hirasawa, Nishii, Furusawa, & T, 2014).
Succinic acid
Succinic acid can be produced from different strains of bacteria like obligate anaerobe Anaerobiospirillum succiniciproducens, Enterococcus sp. and Actinobacillus, which have been investigated (Stols, Donnelly, & Microbiology, 1997). However, the metabolic engineering of Escherichia coli is focused on producing succinate in the form of the primary fermentation product. Wild type strains of E.coli produce succinate as a minor product in fermentation, but after some genetic manipulations, one can increase succinate production and reduce its by-product generation. For example, in the first step of succinate production, increasing the flux overexpressing PEP carboxylase can increase succinate production from 12% to 45% (Millard, Chao, Liao, Donnelly, & Microbiology, 1996). Another technique is done when pyruvate to succinate form by transforming a wild type strain of E.coli with plasmid pTrc99A-PYC, which shows rhizobium pyruvate carboxylase. This step also increases the yield of succinate production by 17% (Gokarn, Eiteman, & Altman, 1998). For its production, glucose is converted into 2 molecules of PEP by gene ptsG (a gene that encodes the enzyme IICBGlc). Then, PEP is converted into OAA (Oxaloacetic acid) by amplification in its gene ppc (gene which encodes PEP carboxylase). OAA by reaction with 2 hydrogens is converted into malate by the mdh gene (which encodes malate dehydrogenase). Malate is converted into fumarate and then into succinate (Millard et al., 1996).
Lactic acid
Many organisms generate D-lactic acid, and some lactic acid bacteria, like Lactobacillus bulgaricus, produce a large amount of pure D-Lactic acid (Benthin, Villadsen, & Biotechnology, 1995). L-Lactic acid can also be produced from lactic acid bacteria such as Lactobacillus amylophilus, Lactobacillus helveticus and Lactobacillus delbrueckii (Vickroy et al., 1985) . Nevertheless, lactic acid bacteria are not considered beneficial to produce lactic acid because they have complex nutritional needs, have less growth rate, and do not utilize pentose or its incomplete utilization (Hoet & Stainer, 1970). So, other bacterial strains suppose E.coli (Chang, Jung, Rhee, & J, 1999) , is then considered efficient to produce pure lactic acid by metabolic engineering. For example, the strain of E.coli RR1 metabolized glucose into D-lactic acid and produced less succinate as by product under anaerobic conditions. For L-lactic acid production, a gene named L-lactate dehydrogenase from the Lactobacillus casei bacteria was introduced into a strain ldhA. This recombinant strain metabolises glucose into L-lactic acid, a primary product of fermentation in this strain (Wang, Yu, & Chou, 2002). Glucose is converted into 2PEP and then into pyruvate by ppc gene, which encodes PEP carboxylase, then it is converted into lactic acid by modification in gene ldhA ( gene which encodes lactate dehydrogenase).
Acetic acid
Acetic acid is produced as the main product of fermentation used in food markets like meat preservation, vinegar, etc. Microbes that are used for its production are Saccharomyces cerevisiae, Clostridium spp., and Acetobacter aceti. The strain of E.coli W3110 was metabolically engineered for the production of acetic acid from glucose (Causey, Zhou, Shanmugam, & J, 2003). Firstly, glucose is converted into 2PEP, and then by the involvement of the ppc gene, it is converted into two molecules of pyruvate. Pyruvate is then converted into an intermediate called Acetyl Co-A by pflB (gene which encodes pyruvate-formate lyase) converted into 2Acetyl-P by pta gene involvement encodes the enzyme phosphotransacetylase. Acetyl-P has done the dephosphorylation by removing ATP and converted into acetate, which is our product of interest (Wolfe, 2005) .
Vitamin
Vitamins are nutritious or practical foods (Ottaway, 2012). They function as cofactors of various enzymes concerned in numerous biochemical reactions, and they are essential additives inside the diet of humans. A few examples of the use of metabolic engineering to supply three vitamins (riboflavin, folate, and ascorbic acid) in microorganism strains (Leblanc, Taranto, Molina, & J, 2010) .
Riboflavin
Both Bacillus subtilis and Lactococcus lactis were designed to create riboflavin (Hao et al., 2013). In Bacillus subtilis, greater expression of the genes called rib (engaged with manufacturing of Vitamin B), prepared in a group, is finished by supplanting the two regulated promoters with constitutive ones were given from a phage. Although several duplicates of these four gene improvements had embedded specific websites inside the genome, an exclusive overexpression of ribs became crucial to attaining the most satisfactory productivity. The rib A gen. 83 encodes a bifunctional protein with dihydroxy-butanone phosphate synthetase pastime on the N-terminal and GTP cyclo hydrolase II motion on the C-terminal (Friedman, Shagina, Lussier, & Hripcsak, 2004). This confirmed that the beginning steps of riboflavin manufacturing restrained profitability within side the closing dispensed degree of pressure improvement. A metabolic exam of the wild-kind and riboflavin-turning in B. subtilis lines tracked down that the flux of the oxidative part of the pentose phosphate pathway turned into accelerated withinside the created pressure. Therefore, this department might also be in charge of prescribing, and new metabolic engineering structures are probably required for destiny pressure improvement. Lactococcus lactis is also being engineered to synthesis riboflavin through greater expressing the gene (rib A) encoding the catalyst (GTP cyclo hydrolase, catalyzing the number one reaction for its synthesis from GTP. These days, this catalyst spoke back to limitation of movement and overexpression of rib A delivered approximately a 3-fold greater in riboflavin synthesis.
Vitamin B11
Lactococcus lactis designed to synthesize vitamin B11 (Wang et al., 2021) . Techniques include the appearance of some genes engaged with the synthesis of vitamin B11 from GTP both independently and dependently and the appearance of heterologous y-glutamyl hydrolase (from people and mice). These methodologies have delivered approximately 3-6 fold expansions within the synthesis of folate and an amendment within the folate spatial dispersion (for instance, a pass from essentially intracellular to the extracellular gathering).
Vitamin C
Practically all natural cycles for generating vitamin C quit with manufacturing 2-keto-L-gulonic acid, which is later changed into ascorbic acid with the aid of using traditional chemical techniques innovation (i.e., esterification and lactonization). Consequently, the aim was targeted round designing strains organized to hand over 2-keto-L-gulonic acid from numerous sugars productively. Two strains of Gluconobacter oxydans had been designed to extrude over sorbitol into 2-keto-L-gulonic acid. Genes that encode the sorbitol dehydrogenase and sorbose dehydrogenase was cloned from glucose. Oxydans T-one hundred into glucose. Orydans G624, allowing the advent of 2- keto-L-gulonic acid in 3 stages. An Erwinia herbicola has been designed to supply 2-keto- L-gulonic acid from glucose in one fermentation step (Ikemi, 1994). E. coli typically creates 2,5-diketo- D-gluconic acid (2, five- DKG) through glucose oxidation but no longer have the chemical 2,5-diketo-D- gluconic acid reductase extra de over 2,5-diketo-D- gluconic acid into 2-keto-L- gulonic acid. Along with these modifications, E. coli became designed to communicate the gene encoding 2, 5-diketo-D-gluconic acid reductase from Corynebacterium spp. bringing approximately a recombinant strain that might change over glucose into 2-keto-L-gulonic acid in a one fermentation step. Moreover, this interplay has been streamlined, and 2-keto-L-gulonic acid concentrations > 120 g/L were accomplished (Levine, Rumsey, Daruwala, Park, & Wang, 1999).
Carbohydrates
A few non-digestible polysaccharides positively affect the improvement of gainful intestinal microflora, and along these lines are referred to by their capacity as prebiotics. Among them are galactooligosaccharides, glucooligosaccharides, transgalactooligosaccharides, and xylooligosaccharides. Lactic acid bacteria are a decent wellspring of non-digestible polysaccharides, basically, exopolysaccharides, which are additionally perceived by their commitment to the surface, taste insight, mouth feel and security of the last food item (Vilela, Cosme, & Inês, 2020). Given the accessible genetic data on qualities needed for exopolysaccharides biosynthesis in lactic acid bacteria, heterologous creation of exopolysaccharides has been accomplished in a lactic acid bacteria strain that could not deliver them. The comparative test has shown that exopolysaccharides with various pieces can be created from articulating a similar group of qualities due to one or the other selectivity in the fare and polymerization framework or restriction in the stock of forerunners. Exopolysaccharide creation in Lactococcus lactis can be expanded by overexpressing the quality of epsD, encoding a preparing glucosyltransferase (Looijesteijn, Trapet, Vries, Abee, & J, 2001). The two different instances of utilizing metabolic designing systems to improve exopolysaccharide creation and alter exopolysaccharide pieces in Lactococcus bacteria. Upregulation of fbp quality encoding fructose bisphosphatase brought about expanded Lactococcus bacteria development and exopolysaccharide combination utilizing fructose as a sugar source. Uridine diphosphate glucose pyrophosphorylase and uridine diphosphate galactose epimerase qualities of L. lactis to research their association in the biosynthesis of uridine diphosphate glucose and uridine diphosphate galactose, which are forerunners of glucose-and galactose- containing exopolysaccharide in Lactococcus lactis. Overexpression of the two qualities expanded the intracellular pools of Uridine diphosphate glucose and Uridine diphosphate galactose. These models delineate the part that metabolic design plays in the bacterial creation of polysaccharides (Mazmanian & Kasper, 2006).
Low calories sugar
These are similar to sorbitol and trehalose, etc., add for weight reduction, due to which there is high shopping demand for them (Looijesteijn, Boels, Kleerebezem, Hugenholtz, & Microbiology, 1999). Tagatose is a low-calorie sugar that is ineffectively corrupted by the human body (Oh, 2007). It is viewed as both an antiplaque and prebiotic specialist. As of late, Lactoccus bacteria have been designed to create tagatose by upsetting the lacC or potentially lacD qualities, which brought about the creation of tagatose 6-phosphate or tagatose 1,6-diphosphate. Further hereditary adjustments brought about the creation of tagatose 1,6- diphosphate as the sole final result. Current endeavours are centered around the dephosphorylation of tagatose 1,6- diphosphate and discharge of the result, tagatose.
Aroma compounds
Diacetyl is a significant flavor compound in numerous dairy items like margarine, buttermilk, and cheeses (it gives a rich flavor) (Sales, Paulino, Pastore, & Bicas, 2018) . L. lactis has been designed to orchestrate diacetyl as the fundamental maturation item (Longo, Sanromán, & Biotechnology, 2006). Proficient diacetyl creation came about because of the inactivation of the aldB quality, which encodes acetolactate decarboxylase and overproduction of NADH-oxidase action. This methodology brought about the change of 80% of the carbon source into diacetyl. Another significant gathering of smell compounds is acquired from enzymatic corruption of amino acids, including isobutyrate, phenylacetaldehyde and indole, etc. Researchers have seen that overexpression of a heterologous glutamate dehydrogenase quality in L. lactis expanded the change of amino acids into smell compounds. In this manner, rather than adding ketoglutarate to cheddar, a GDH-delivering lactococcal strain could be utilized to upgrade amino corrosive corruption in fragrance compounds (Tananuwong, Lertsiri, & Agriculture, 2010).
Future trends and potential
Nature makes use of microorganisms to maintain our fermentation processes, and for lots of years, humankind has used yeasts, molds, and microorganisms to shape meals products like bread, beer, wine, vinegar, meats and cheese, furthermore as complex fish, meat and vegetables (Gonzalez, Andrews, Molitor, Asenjo, & Bioengineering, 2003). The key to the long run meals lies in microbiomes, the groups of microorganisms that colonize every viable circumstance in the world. By exploiting these ecosystems' ability, it is viable to offer better pleasant meals, growing the productivity, protection and belongings of meals provide chains. As the daily increase in population, we require more, quick and easy food that produces quickly and in massive amounts to get it according to their needs. Microorganisms have been a great help in getting such foods as many GMOs have been developed with the help of microorganisms for increased yield and fast growth (Hanlon & Sewalt, 2021).
Smart food
Smart food is highly nutrient food made with particular GMO crops that lead to quick growing and healthy nutritious and safe food that helps farmers for a daily wage. Producing, accessing, and consuming nutritionally rich and diverse foods is central to overcoming malnutrition and micronutrient deficiencies. A strong agricultural sector is vital to supporting rural livelihoods, and smart foods have a significant role to play in improving food security and protecting the environment (Maye, Culture, & Society, 2019) .
Protein sources
Protein is a very nutritional compound for the body and is mainly in the daily routine of people (Zhang et al., 2017). However, many people who cannot afford or do not have the resources to get meat cannot even consume it daily for that many proteins added things had been made using various microorganisms and similar techniques. One of the prominent examples is PROTEIN BARS; these are small bars containing a high level of proteins and other nutrients and can be obtained easily with low price than meat. Also, some more products, such as some cereals with high protein content, are now made for people who have any issue consuming meat. One of the common examples of cereals is Kellogs cereal that is protein-based (Tas, Shah, & Technology, 2021).
Lactic acid bacteria
Lactic acid bacteria have been beneficial in the making and preserving of food, and many more benefits regarding food have been discovered by using lactic acid bacteria (Agbor-Egbe & Mbome, 2006). Several daily use food products have been produced by lactic acid bacteria such as yoghurt, cheese and many more milk products that are used and a need in daily food also it is a cheap source too (Boels, Kranenburg, Hugenholtz, Kleerebezem, & Vos, 2001). This bacterium has been used to make products that help make whole food, such as yeast, which helps produce the bread of different flavors. For a long time, milk is being fermented by using these bacteria and further shortly, there are changes expected in milk as we know there is already lactose-free milk, but there are to be milk produced that will be high dose in calcium and any required component as milk would be manipulated in different ways through different processes. Probiotic and based farm products are likewise hired inside the closest destiny to deal with various fitness disorders. Current research recommends that carboxylic acid bacterium own anti-weight problems and anti-diabetic propensities on their hosts and consequently will play a crucial function in human fitness care (Annunziato et al., 2021) . Analysis inside the physics and chemical technological know-how homes of frozen dessert and its programs is also rising. These programs face certain hurdles collectively with technological (for much less advanced countries), consumer satisfactoriness of new realistic meals is likewise stimulated via means of culture, ethics, or faith. There would be love for added research at the genetic foundation for probiotic homes that can provide any knowledge regarding novel manipulation capabilities and pertinence in nutrients and fitness sectors (Alkay, Dertli, & Durak, 2021) considerable influence of microorganisms in them, but they would be beneficial and not harmful.
Plants and crops
The poor outcomes at the surroundings and human fitness resulting from the contemporary farming structures primarily based totally on the overuse of chemical fertilizers had been pronounced in lots of research. By contrast, bacterial inoculations produce superb outcomes on yields without inflicting this kind of harm (Muhammed, Memon, Baloch, Osman, & Ali, 2013). In recent years, the commercialization of bio-fertilizers has increased, and the variety of businesses and merchandise to be had is increasing international each year. In addition to the beautiful enhancement of crop manufacturing, much research has proven how the utility of microorganisms has good outcomes on meals exceptional including progressed vitamin, flavonoid and antioxidant content, amongst different benefits. This benefit is thrilling with admire to meals this is fed on raw, including result and plenty of vegetables, as those bioactive molecules are maintained up till the instant the meal is fed on. This overview specializes in gathering research that exhibits that microorganisms can act as plant probiotics of fruit and horticultural crops, vital forms of meals that shape a part of a healthful diet. It is consequently vital that agricultural productiveness be drastically improved with inside the following couple of decades. To this end, agricultural exercise is transferring closer to an extra sustainable and environmentally pleasant approach (Chowdhury & Yasmin, 2018). This consists of using transgenic vegetation and plant increase-selling microorganisms as part of mainstream agricultural exercise. It is anticipated that with inside the now no longer too remote future, plant increase selling microorganisms (PGPB) will start to update using chemical compounds in agriculture, horticulture, silviculture, and environmental clean-up strategies. While there won't be one easy method that may efficiently sell the increase of all vegetation beneath neath all conditions.
Conclusion
In this advanced era, microbial production of food products, nutrients, food supplements and food additives has been improved using gene manipulation techniques. In this challenge, genetic and metabolic engineering are playing an essential role in producing different types of genetically modified foods in the food industry. Human beings have greatly embraced the information of engineering of microorganisms to improve their lives. The products produced by genetically modified microorganisms are used for health benefits and the prevention of malnutrition problems. Furthermore, genetically modified foods such as food additives and some organic acids have been employed to develop a variety of foods, pharmaceuticals, chemicals, etc. This chapter focuses on the role of microbes in the food industry with particular reference to genetic and metabolically modified microorganisms. By overcoming the current challenges, we can increase the benefits.
