Kluyveromyces lactis is one of few yeast species that can utilize lactose or galactose as a sole source of carbon and energy. It can be isolated from dairy products and is used at industrial scale for preparation of the enzyme lactase (β-galactosidase) [22]. Lactose utilization is accomplished by inducing both lactose transport and an intracellular β-galactosidase (EC 3.2.1.23) activity more than 100- fold above a moderate basal level [29]. Furthermore, K. lactis is an organism that has been designated as GRAS (generally recognized as safe) by the American Food and Drug Administration. K. lactis enzymes of the Leloir pathway catalyze the conversion of galactose to a more metabolically useful, glucose‐6–phosphate. This pathway is required as galactose itself cannot be used for glycolysis directly [10]. Galactose is catabolized by inducing enzymes of the Leloir pathway: ATP-a-D-galactose-1-phosphotransferase ("galactokinase," EC 2.7.1.6), uridine diphosphoglucose-D-galactose-l-phosphate uridylyltransferase, ("transferase," EC 2.7.7.10), and uridine diphosphogalactose-4-epimerase ("epimerase," EC 5.1.3.2) [2]. The lactose–galactose (LAC/GAL) regulon in K. lactis is induced by lactose or galactose [52]. Utilization of lactose or galactose requires induction of transcription of LAC4 (β-galactosidase), LAC12 (lactose permease), GAL1 (galactokinase), GAL7 (galactose-1-phosphate uridylyltransferase) and GAL10 (uridine diphosphoglucose 4-epimerase), which are tightly linked and highly regulated [28]. Gal1p besides having galactokinase activity has a positive transcriptional regulatory function required for induction of the LAC/GAL regulon [31], [43]. However, several checkpoints can potentially reduce GOS production in the wild type K. lactis strain. Hence, knocking out certain genes after careful analysis of the regulon would be essential to obtain an economically feasible yield of GOS.

Chemical synthesis of GOS is also feasible, but it requires many reaction steps due to the necessary selective protection of the hydroxyl groups, which is not the case with enzymatic synthesis. Also, the environmental impact of toxic reagents would be far greater with chemical than with enzymatic synthesis [15]. Therefore, enzymatic processes are more feasible, more environmentally useful, and less expensive than the chemical processes. Enzymatic hydrolysis and trans-galactosylation of lactose has technological and environmental benefits, since it provides products free of disaccharides, which can be consumed by intolerants, improves the characteristics of final products, and reduces environmental impacts, as it allows the processing of whey, a dairy by-product that can be converted into galacto-oligosaccharides [13]. Enzyme concentration and initial lactose concentration are considered to be the crucial factors affecting GOS yield [51].

β-D-galactosidases (EC 3.2.1.23) are enzymes that catalyse the hydrolysis of terminal non-reducing β-D-galactose units from β-D-galactosides. As an enzyme, β-galactosidase has hydrolysis activity that cleaves the disaccharide lactose to produce galactose, which enters Leloir pathway, and glucose, which ultimately enters glycolysis [45]. β-galactosidases also have transgalactosylation activity that makes them very attractive for obtaining galactooligosaccharides [35]. β-Galactosidases are useful for treating lactose intolerance [6], for texture enhancement [26], and are frequently used in the food industry to increase the sweetening power of natural saccharides [45]. They are also useful in cheese, whey treatment and transformation [27]. The β-galactosidase from the yeast K. lactis is one of the most frequently used β-galactosidases in the biotechnological industry due to its biochemical properties: it has an optimal neutral pH and higher stability compared to β-galactosidases from other microbes, such as fungal galactosidases [7].

The increasing trend in health and environmentally conscious consumers has fueled the research on prebiotics and probiotics [40]. Gut microflora and probiotics are known to play a crucial role in determining digestive health and gut-induced immunity. However, the availability of an appropriate non-digestible dietary prebiotic is critical in deciding the composition and/or activity of gastrointestinal microbiota, making it an important factor for determining the gut health and overall wellbeing [17]. Galacto-oligosaccharides (GOS), a class of non-digestible short-chain (3-10 molecules) carbohydrate oligomer of galactose and glucose are demonstrated to provide health benefits and to improve the quality of many foods that is synthesized primarily from lactose. It is the main milk sugar, and is associated with dairy products. GOS are structurally related to human milk oligosaccharides (HMOs) and both families exhibit prebiotic properties. Although GOS concentration in milk is low, there is a great potential to produce them from by-products of the dairy industry [18], [54]. GOS can be produced by enzymatic trans-galactosylation of lactose using β-galactosidase [26].

In this study, mutants with gal7gene (galactotransferase activity), gal1 gene (galactokinase activity) disruptionand both were created by fusion overlap extension PCR-based generation of gene knockout [57]. Wild type, gal7k mutant (Δgal7kLAC++), gal1z mutants (Δgal1zLAC+) and double mutants with both gal1z &gal7kdisruption (Δgal1z Δgal7kLAC+++) strains were collectively analysed and compared for the constitutively improved β-galactosidase expression during early stationary phase and for their significant trans-galactosylation activity. The strains and its potential value in industrial applications needing high yield has been validated by measuring their trans-galactosylation activity in prebiotic galacto-oligosaccharides production.