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Lactococcus lactis
Scientific classification Edit this classification
Domain: Bacteria
Kingdom: Bacillati
Phylum: Bacillota
Class: Bacilli
Order: Lactobacillales
Family: Streptococcaceae
Genus: Lactococcus
Species:
L. lactis
Binomial name
Lactococcus lactis
(Lister 1873)
Schleifer et al. 1986
Subspecies

L. l. cremoris
L. l. hordniae
L. l. lactis
L. l. lactis bv. diacetylactis
L. l. tructae

Lactococcus lactis is a gram-positive bacterium used extensively in the production of buttermilk and cheese,[1] but has also become famous as the first genetically modified organism to be used alive for the treatment of human disease.[2] L. lactis cells are cocci that group in pairs and short chains, and, depending on growth conditions, appear ovoid with a typical length of 0.5 - 1.5 μm. L. lactis does not produce spores (nonsporulating) and are not motile (nonmotile). They have a homofermentative metabolism, meaning they produce lactic acid from sugars. They've also been reported to produce exclusive L-(+)-lactic acid.[3] However,[4] reported D-(−)-lactic acid can be produced when cultured at low pH. The capability to produce lactic acid is one of the reasons why L. lactis is one of the most important microorganisms in the dairy industry.[5] Based on its history in food fermentation, L. lactis has generally recognized as safe (GRAS) status,[6][7] with few case reports of it being an opportunistic pathogen.[8][9][10]

Lactococcus lactis is of crucial importance for manufacturing dairy products, such as buttermilk and cheeses. When L. lactis ssp. lactis is added to milk, the bacterium uses enzymes to produce energy molecules (ATP), from lactose. The byproduct of ATP energy production is lactic acid. The lactic acid produced by the bacterium curdles the milk, which then separates to form curds that are used to produce cheese.[11] Other uses that have been reported for this bacterium include the production of pickled vegetables, beer or wine, some breads, and other fermented foodstuffs like soymilk kefir, buttermilk, and others.[12] L. lactis is one of the best characterized low GC Gram positive bacteria with detailed knowledge on genetics, metabolism and biodiversity.[13][14]

L. lactis is mainly isolated from either the dairy environment, or plant material.[15][16][17] Dairy isolates are suggested to have evolved from plant isolates through a process in which genes without benefit in the rich milk were lost or downregulated.[14][18] This process, called genome erosion or reductive evolution, has been described in several other lactic acid bacteria.[19][20] The proposed transition from the plant to the dairy environment was reproduced in the laboratory through experimental evolution of a plant isolate that was cultivated in milk for a prolonged period. Consistent with the results from comparative genomics (see references above), this resulted in L. lactis losing or downregulating genes that are dispensable in milk and the upregulation of peptide transport.[21]

Hundreds of novel small RNAs were identified by Meulen et al. in the genome of L. lactis MG1363. One of them, LLnc147, was shown to be involved in carbon uptake and metabolism.[22]

Cheese production

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L. lactis subsp. lactis (formerly Streptococcus lactis)[23] is used in the early stages for the production of many cheeses, including brie, camembert, Cheddar, Colby, Gruyère, Parmesan, and Roquefort.[24] The use of L. lactis in dairy factories is not without issues. Bacteriophages specific to L. lactis cause significant economic losses each year by preventing the bacteria from fully metabolizing the milk substrate.[24] Several epidemiologic studies showed the phages mainly responsible for these losses are from the species 936, c2, and P335 (all from the family Siphoviridae).[25]

The state Assembly of Wisconsin, also the number one cheese-producing state in the United States, voted in 2010 to name this bacterium as the official state microbe; it would have been the first and only such designation by a state legislature in the nation,[26] however the legislation was not adopted by the Senate.[27] The legislation was introduced in November 2009 as Assembly Bill 556 by Representatives Hebl, Vruwink, Williams, Pasch, Danou, and Fields; it was cosponsored by Senator Taylor.[28] The bill passed the Assembly on May 15, 2010, and was dropped by the Senate on April 28.[28]

Therapeutic benefits

[edit]

The feasibility of using lactic acid bacteria (LAB) as functional protein delivery vectors has been widely investigated.[29] Lactococcus lactis has been demonstrated to be a promising candidate for the delivery of functional proteins because of its noninvasive and nonpathogenic characteristics.[30] Many different expression systems of L. lactis have been developed and used for heterologous protein expression.[31][32][33]

Lactose fermentation In one study that sought to prove that some fermentation produced by L. lactis can hinder motility in pathogenic bacteria, the motilities of Pseudomonas, Vibrio, and Leptospira strains were severely disrupted by lactose utilization on the part of L. lactis.[34] Using flagellar Salmonella as the experimental group, the research team found that a product of lactose fermentation is the cause of motility impairment in Salmonella. It is suggested that the L. lactis supernatant mainly affects Salmonella motility through disruption of flagellar rotation rather than through irreversible damage to morphology and physiology. Lactose fermentation by L. lactis produces acetate that reduces the intracellular pH of Salmonella, which in turn slows the rotation of their flagella.[35][36] These results highlight the potential use of L. lactis for preventing infections by multiple bacterial species.

Secretion of Interleukin-10 Genetically engineered L. lactis can secrete the cytokine interleukin-10 (IL-10) for the treatment of inflammatory bowel diseases (IBD), since IL-10 has a central role in downregulating inflammatory cascades[37] and matrix metalloproteinases.[38] A study by Lothar Steidler and Wolfgang Hans[39] shows that in situ synthesis of IL-10 by genetically engineered L. lactis requires much lower doses than systemic treatments like antibodies to tumor necrosis factor (TNF) or recombinant IL-10.

The authors propose two possible routes by which IL-10 can reach its therapeutic target. Genetically engineered L. lactis may produce murine IL-10 in the lumen, and the protein may diffuse to responsive cells in the epithelium or the lamina propria. Another route involves L. lactis being taken up by M cells because of its bacterial size and shape, and the major part of the effect may be due to recombinant IL-10 production in situ in intestinal lymphoid tissue. Both routes may involve paracellular transport mechanisms that are enhanced in inflammation. After transport, IL-10 may directly downregulate inflammation. In principle, this method may be useful for intestinal delivery of other protein therapeutics that are unstable or difficult to produce in large quantities and an alternative to the systemic treatment of IBD.[citation needed]

Tumor-suppressor through Tumor metastasis-inhibiting peptide KISS1 Another study, led by Zhang B, created a L. lactis strain that maintains a plasmid containing a tumor metastasis-inhibiting peptide known as KISS1.[40] L. lactis NZ9000 was demonstrated to be a cell factory for the secretion of biologically active KiSS1 protein, exerting inhibition effects on human colorectal cancer HT-29 cells.

KiSS1 secreted from recombinant L. lactis strain effectively downregulated the expression of Matrix metalloproteinases (MMP-9), a crucial key in the invasion, metastasis, and regulation of the signaling pathways controlling tumor cell growth, survival, invasion, inflammation, and angiogenesis.[41][42][43] The reason for this is that KiSS1 expressed in L. lactis activates the MAPK pathway via GPR54 signaling, suppressing NFκB binding to the MMP-9 promoter and thus downregulating MMP-9 expression.[44] This, in turn, reduces the survival rate, inhibits metastasis, and induces dormancy of cancer cells.

In addition, it was demonstrated that tumor growth can be inhibited by the LAB strain itself,[45][46] due to the ability of LAB to produce exopolysaccharides.[47][48] This study shows that L. lactis NZ9000 can inhibit HT-29 proliferation and induce cell apoptosis by itself. The success of this strain's construction helped to inhibit migration and expansion of cancer cells, showing that the secretion properties of L. lactis of this particular peptide may serve as a new tool for cancer therapy in the future.[49]

References

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Lactococcus lactis

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Lactococcus lactis is a Gram-positive, spherical coccus-shaped bacterium that belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, and family Streptococcaceae, characterized by its non-motile, non-spore-forming, and facultative anaerobic nature.[1] It primarily ferments carbohydrates, such as lactose, into lactic acid through homolactic fermentation, enabling its essential role in dairy product production.[2] With a genome size of approximately 2.3–2.5 million base pairs and a low G+C content of about 35 mol%, it exhibits genetic features that support rapid growth at temperatures between 10–40°C (optimum around 30°C) and the production of bacteriocins like nisin for antimicrobial activity.[3] As a cornerstone of the dairy industry, L. lactis serves as a primary starter culture in the fermentation of cheeses (e.g., Cheddar), yogurts, and other fermented milks, where it drives acidification to lower pH, enhances texture through exopolysaccharide production, and develops complex flavors via volatile compounds and proteolysis.[4] Recognized as generally regarded as safe (GRAS) by regulatory bodies like the FDA, it has been used for centuries in food preservation and imparts nutritional enhancements, including vitamins such as folate, riboflavin, and vitamin K2.[2] The bacterium exists in subspecies like l. lactis subsp. lactis and cremoris, with dairy-adapted strains showing genome erosion and specialized genes for lactose metabolism and phage resistance, distinguishing them from plant-origin isolates.[1] Beyond food applications, L. lactis has emerged as a versatile microbial cell factory in biotechnology due to its well-characterized genetics, plasmid-based systems, and food-grade status, enabling the production of recombinant proteins, therapeutics (e.g., interleukin-10 for inflammatory bowel disease), and vaccines.[2] It also demonstrates probiotic potential, surviving gastrointestinal conditions (e.g., pH 2.5–3 and bile salts), modulating gut microbiota, reducing pathogens through bacteriocins, and offering health benefits like cholesterol reduction and immunostimulation when consumed at doses of 10⁶–10⁹ viable cells daily.[4] These attributes underscore its transition from a traditional fermentative organism to a tool in modern industrial and health sciences.[3]

Taxonomy and Classification

Species Description

Lactococcus lactis is a bacterial species classified within the genus Lactococcus in the family Streptococcaceae, order Lactobacillales, class Bacilli, and phylum Bacillota (formerly known as Firmicutes).[5] Originally described as "Bacterium lactis" by Joseph Lister in 1873 based on its role in milk souring, it was reclassified as Streptococcus lactis in the early 20th century.[6] In 1985, Karl-Heinz Schleifer and colleagues proposed the establishment of the genus Lactococcus to accommodate S. lactis and related taxa, distinguishing them from other streptococci through 16S rRNA sequencing, DNA-DNA hybridization, and phenotypic analyses that revealed significant genetic and physiological divergences, such as differences in peptidoglycan composition and fermentation patterns.[7] The species is defined by key morphological and physiological traits that align with its lactic acid bacterial lineage. Lactococcus lactis consists of Gram-positive cocci that are spherical or ovoid in shape, typically arranged in pairs or chains, and measure approximately 0.5 to 1.2 μm in diameter.[8] These bacteria are non-motile and non-spore-forming, exhibiting a facultative anaerobic metabolism that allows growth under both aerobic and anaerobic conditions, with optimal temperatures around 30°C.[9] They are catalase-negative and homofermentative, primarily producing L-lactic acid from carbohydrate fermentation, which underscores their adaptation to nutrient-rich environments like plant material and dairy sources.[10] The type strain for Lactococcus lactis is designated as Lactococcus lactis subsp. lactis NCDO 604, originally isolated from milk and now maintained as ATCC 19435 in culture collections worldwide; this strain serves as the reference for species identification and genomic studies.[11]

Subspecies and Strains

As of 2021, Lactococcus lactis is classified into two subspecies: L. lactis subsp. lactis and L. lactis subsp. hordniae.[6] The subspecies L. lactis subsp. lactis is mesophilic with optimal growth around 30°C and is widely employed as a starter culture in cheese production due to its rapid acidification activity through lactose fermentation to lactic acid. Within subsp. lactis, citrate-positive variants known as biovar diacetylactis are distinguished by their ability to ferment citrate, leading to the production of diacetyl, a key aroma compound responsible for buttery flavors in fermented dairy products.[12][13] In contrast, the former L. lactis subsp. cremoris—important for flavor enhancement in cheeses like Cheddar through production of compounds such as diacetyl and acetoin—has been elevated to the species level as Lactococcus cremoris sp. nov.[14][15] The subspecies L. lactis subsp. hordniae, isolated from the plant-hopper Agallia constricta, is less commonly associated with industrial applications.[16] Notable strains include IL1403 and NZ9000, which serve as models in research. The strain IL1403, a plasmid-cured derivative of the industrial isolate IL594, was the first L. lactis genome to be fully sequenced in 2001, providing a foundational reference for genetic studies with its 2.37 Mb chromosome containing genes for carbohydrate metabolism and stress responses.[17] NZ9000, derived from the plasmid-free laboratory strain MG1363 (originally classified as subsp. cremoris, now L. cremoris), is engineered with the nisin-controlled expression (NICE) system and functions as a primary host for heterologous protein production in biotechnological applications.[18][19] Strains of L. lactis exhibit significant genetic and phenotypic variations, particularly in plasmid content, which influences traits like phage resistance and lactose metabolism. Plasmids in dairy-adapted strains, such as those in subsp. lactis, often encode lactose fermentation operons (e.g., lac operon for β-galactosidase activity) and citrate utilization pathways, enabling efficient milk sugar breakdown.[20][21] Phage resistance mechanisms, including abortive infection (Abi) systems like AbiB and AbiD1, are predominantly plasmid-borne and differ among strains; for instance, industrial isolates may carry multiple such plasmids, conferring broad insensitivity to lytic phages that threaten fermentation.[22][23] In industrial applications, strain selection prioritizes rapid acid production rates to ensure timely coagulation in dairy fermentations, alongside bacteriophage insensitivity to maintain process reliability. High-performing strains, such as those with optimized acidification (e.g., reaching pH 5.2 in 6 hours in milk), are chosen for their balance of these traits, often derived from natural variants with enhanced plasmid-encoded defenses against common phages like those in the 936 or c2 families.[4][24]

Biology and Physiology

Morphology and Cellular Structure

Lactococcus lactis cells are Gram-positive cocci with a spherical to ovoid shape, typically measuring 0.5 to 1.5 μm in diameter, and they commonly occur as single cells, pairs, or short chains of 2 to 20 cells depending on strain and growth conditions.[3][4] The arrangement in chains arises from incomplete separation during cell division, a feature observed across various strains.[25] The cellular envelope features a thick peptidoglycan layer characteristic of Gram-positive bacteria, which provides mechanical strength and shape maintenance, supplemented by teichoic acids that anchor to the peptidoglycan and promote adhesion to surfaces and other cells.[26][27] These teichoic acids, including lipoteichoic acids linked to the cytoplasmic membrane, constitute a minor phosphorus component (<1%) but play key roles in cell wall organization and interactions.[26] L. lactis lacks spores for survival under stress, flagella for motility, and true capsules, though a protective polysaccharide pellicle covers the surface in wild-type strains, masking the underlying peptidoglycan.[28] Surface proteins, including pili-like structures, are present and enable biofilm formation by facilitating cell aggregation and attachment to substrates.[28] Electron microscopy reveals a smooth, featureless surface morphology in wild-type L. lactis cells, with the peptidoglycan organized into 25-nm-wide oblique bands beneath the polysaccharide layer, as visualized by atomic force microscopy on live cells.[26] In mutants lacking surface polysaccharides, the exposed peptidoglycan shows a more irregular nanoscale texture, highlighting the protective role of the outer layer.[26] Transmission electron microscopy of dividing cells occasionally shows invaginations of the cytoplasmic membrane, interpreted as mesosome-like structures involved in septation, while the lactate dehydrogenase enzyme is primarily localized in the cytoplasm adjacent to the membrane for efficient pyruvate reduction.[29][30]

Growth Conditions and Metabolism

Lactococcus lactis is a mesophilic bacterium with an optimal growth temperature of approximately 30°C, though it can grow over a range of 10–40°C depending on the strain.[31] It thrives at a pH between 6.0 and 6.9, with initial media pH often adjusted to around 7.0 to support robust proliferation before acidification occurs.[32] As a facultative anaerobe, L. lactis prefers microaerophilic conditions for growth but can shift to fermentative metabolism under strict anaerobiosis or respire when heme is available in the medium.[33] The metabolism of L. lactis is predominantly homofermentative, converting carbohydrates such as lactose or glucose into lactic acid through the Embden-Meyerhof-Parnas (glycolysis) pathway.[34] This process yields approximately 90% L(+)-lactic acid as the primary end product, with minor byproducts like acetate or ethanol formed under aerobic or stressed conditions.[35] The high optical purity of L(+)-lactic acid distinguishes L. lactis from heterofermentative lactic acid bacteria. Key enzymes facilitate lactose catabolism: lactose is transported into the cell via the lactose-specific phosphotransferase system (Lac-PTS), followed by hydrolysis of the phosphorylated form by phospho-β-galactosidase (LacG) to yield glucose-6-phosphate and galactose-6-phosphate, which enters the tagatose-6-phosphate pathway.[36] Lactate dehydrogenase (Ldh) then reduces pyruvate to L(+)-lactic acid, regenerating NAD⁺ essential for continued glycolysis.[37] L. lactis requires specific nutrients for growth, including carbohydrates like lactose or glucose as carbon and energy sources, along with peptides and amino acids for protein synthesis.[38] It is auxotrophic for several vitamins, notably riboflavin and pantothenate, which must be supplied externally due to incomplete biosynthetic pathways.[38] Additionally, L. lactis cannot synthesize heme and relies on exogenous sources to enable cytochrome-mediated respiration under oxygen exposure.[39] To cope with acidification during growth, L. lactis employs stress responses including acid tolerance via the F₀F₁-ATPase, which pumps protons out of the cell using ATP hydrolysis to maintain intracellular pH homeostasis. Strain variations may influence metabolic efficiency, such as altered lactic acid yields in adapted isolates.[40]

Industrial Applications in Food

Role in Dairy Fermentation

Lactococcus lactis serves as a primary starter culture in the production of various dairy products, particularly cheeses such as Cheddar and Gouda, where it drives rapid acidification of milk through homolactic fermentation of lactose into lactic acid.[37] This process typically lowers the pH from approximately 6.7 to 5.2–5.5 within 4–6 hours at 30°C, promoting the coagulation of milk proteins like casein to form the curd essential for cheese structure.[41] Strains of L. lactis subsp. lactis and subsp. cremoris are commonly employed in mesophilic starters for these semi-hard cheeses, ensuring consistent acid development during the initial manufacturing stages.[42] Beyond acidification, L. lactis contributes to flavor and texture development via proteolytic activity and production of aroma compounds. The cell-envelope proteinase PrtP hydrolyzes caseins into oligopeptides, which are transported intracellularly and further broken down by peptidases, releasing free amino acids that serve as precursors for volatile flavor molecules and enhance cheese ripening.[43] In L. lactis subsp. lactis biovar diacetylactis (also known as subsp. lactis var. diacetylactis), citrate metabolism yields diacetyl and acetaldehyde, key contributors to the buttery and creamy aromas in cheeses and cultured butters.[44] These metabolic pathways, building on the bacterium's basic lactic acid fermentation, support the sensory profile without dominating the overall process.[37] Historically, L. lactis has been integral to dairy fermentation since ancient times, with formalized use in 19th-century European cheesemaking through back-slopping of whey or fermented milk to propagate natural starters.[42] However, bacteriophage infections have long challenged production by lysing starter cells and causing slow or failed acidification, leading to economic losses; mitigation strategies include rotating phage-resistant strains within multi-strain blends.[42] In modern practices, defined-strain cultures—comprising specific, characterized isolates—are preferred over traditional undefined whey-based starters for their predictability, reduced phage risk, and scalability in direct-vat inoculation systems.[42]

Use in Other Fermented Foods

Lactococcus lactis plays a role in vegetable fermentations such as sauerkraut and kimchi, often as part of a mixed microbial community that includes Leuconostoc species, where it contributes to acidity development and preservation through lactic acid production and bacteriocin activity like nisin. In sauerkraut production, L. lactis subsp. lactis strains have been isolated from commercial fermentations and used as starters to enhance fermentation consistency and inhibit spoilage organisms. Similarly, in kimchi, L. lactis strains act as starters to extend shelf life by suppressing pathogens such as Lactobacillus plantarum and Pediococcus pentosaceus via antimicrobial compounds.[45][46][47] In fermented meat products like salami and sausages, L. lactis serves as a starter culture, contributing to microbial safety via bacteriocins and acidification, while also generating flavor compounds through lactate accumulation. Additionally, bacteriocin-producing L. lactis transconjugants have been incorporated into sausage fermentation to control Listeria monocytogenes growth in the presence of nitrite.[48][49][50] For beverage fermentations, L. lactis participates in kefir production through symbiotic interactions with yeasts and other lactic acid bacteria, contributing to the acidification and textural properties of the final product. In kefir grains, L. lactis subsp. lactis is among the dominant species that drive the initial stages of milk fermentation, producing lactic acid and exopolysaccharides that influence viscosity. In some traditional beers, such as chicha, L. lactis forms part of the natural bacterial community alongside Lactobacillus and Leuconostoc species, aiding in carbohydrate fermentation during the process.[51][52][53] Emerging applications of L. lactis extend to plant-based milk alternatives, where it is employed as a starter for fermenting oat, soy, and mung bean substrates to produce yogurt-like products with improved texture and nutritional profiles. These fermentations leverage L. lactis's ability to metabolize non-dairy carbohydrates, resulting in enhanced antioxidant capacities and sensory attributes comparable to dairy yogurts. Galactose-positive strains of L. lactis, often derived from nondairy environments, exhibit superior adaptation to these plant-derived sugars, enabling efficient acid production and flavor development in lactose-free media.[54][55][56][57]

Biotechnological and Therapeutic Uses

Probiotic Properties and Health Benefits

_Lactococcus lactis exhibits probiotic properties that promote gut health primarily through adhesion to the intestinal mucosa and modulation of the microbiota. Certain strains produce mucus-binding proteins (MUBs), such as MbpL in L. lactis BGKP1 and Muc in L. lactis TIL448, which facilitate specific binding to mucin glycans in the intestinal mucus layer, enhancing transient colonization and persistence in the gastrointestinal tract.[58] This adhesion mechanism supports competitive exclusion of pathogens and contributes to barrier function integrity. Additionally, L. lactis modulates the gut microbiota by producing bacteriocins like nisin, which inhibit the growth of pathogens such as Listeria monocytogenes through membrane disruption and pore formation, thereby reducing pathogen colonization without significantly altering commensal populations.[59] The bacterium also demonstrates immunomodulatory effects, particularly through its surface polysaccharides, including exopolysaccharides (EPS). For instance, EPS from L. lactis Z-2 increases the expression of anti-inflammatory cytokines such as IL-10 and TGF-β in intestinal tissues, promoting immune tolerance and reducing pro-inflammatory responses.[60] Specific strains, like L. lactis T-21 isolated from wild cranberries, exhibit anti-inflammatory properties; a 2024 randomized, double-blind, placebo-controlled study showed that daily intake of 25 mg T-21 improved skin conditions in individuals with atopic predisposition by reducing trans-epidermal water loss and enhancing skin brightness, indicating broader immunomodulatory benefits.[61] Another strain, L. lactis LB 1022, upregulates IL-10 production in atopic dermatitis models, suppressing Th2 cytokines (IL-4, IL-5, IL-13) and alleviating allergic symptoms through Treg-mediated immune suppression.[62] Therapeutic potential of L. lactis includes alleviation of allergies via natural IL-10 induction and cholesterol reduction through bile salt hydrolase (BSH) activity. In atopic dermatitis mouse models, L. lactis LB 1022 reduced IgE levels, mast cell infiltration, and histamine release, demonstrating anti-allergic effects independent of genetic modification.[62] For lipid management, strains like L. lactis subsp. lactis exhibit BSH activity that deconjugates bile salts (e.g., 2.47 U/ml with taurocholate), leading to cholesterol assimilation (up to 43.7% by growing cells) and precipitation, which lowers intestinal cholesterol absorption and supports hypocholesterolemic effects.[63] L. lactis holds a Generally Recognized as Safe (GRAS) status from the FDA, with multiple notices affirming its safety for use in food at levels up to 10^11 CFU per serving, and it lacks known virulence factors due to its non-pathogenic nature and long history in dairy fermentation.[64] Human trials confirm high tolerance; for example, a randomized, double-blind study administered 1×10^10 CFU/day of L. lactis GCWB1176 for 8 weeks without adverse effects, supporting its safety profile.[65] For probiotic delivery, L. lactis can be incorporated into fortified yogurts and cheeses, where immobilized cells maintain viability above 6 log CFU/g during refrigerated storage for up to 14 days, ensuring sustained health benefits.[66]

Engineering for Vaccine and Drug Delivery

_Lactococcus lactis has been genetically engineered as a live vector for mucosal delivery of vaccines and therapeutic proteins, leveraging its generally recognized as safe (GRAS) status and ability to colonize the gastrointestinal tract. A key approach involves the use of nisin-inducible expression systems, such as the pNZ8048 plasmid, which allows controlled production of heterologous proteins like antigens upon addition of the inducer nisin. This vector incorporates the strong P_nisA promoter and the Usp45 secretion signal peptide to facilitate extracellular secretion of the target protein, enabling high-level expression without the need for antibiotic selection markers, thus maintaining its food-grade properties.[67][68] Early demonstrations of L. lactis as a vaccine platform focused on mucosal immunization against tetanus, where recombinant strains expressing the tetanus toxin fragment C (TTFC) induced protective immunity in mice via nasal or oral routes. In a seminal 1997 study, nasal administration of L. lactis secreting TTFC elicited serum IgG and mucosal IgA responses, conferring survival against lethal toxin challenges in 80-100% of immunized mice. More recently, engineering efforts have targeted emerging pathogens, including SARS-CoV-2; preclinical studies from 2022-2024 have shown that oral or intranasal delivery of L. lactis expressing the COVID-19 spike protein or its receptor-binding domain triggers robust humoral and cellular immune responses in mice, including neutralizing antibodies and T-cell activation, positioning it as a needle-free vaccine candidate.[69][70][71][72] Beyond vaccines, L. lactis has been modified for in situ drug delivery in the gut, particularly for treating autoimmune and inflammatory conditions. Strains engineered to secrete proinsulin have demonstrated potential in reversing type 1 diabetes in mouse models by promoting regulatory T-cell responses and reducing insulitis upon oral administration. Similarly, L. lactis secreting interleukin-10 (IL-10) has shown therapeutic efficacy in murine colitis models, reducing inflammation by 50% through local cytokine modulation, with a Phase I clinical trial in Crohn's disease patients confirming safety and feasibility of oral delivery in 2006.[73][74][75] In a 2022 study, metabolic engineering of the lycopene biosynthesis pathway (via integration of crtEBI genes) enabled L. lactis to produce up to 1.5 mg/L of lycopene, an antioxidant that protects intestinal epithelial cells from reactive oxygen species stress, highlighting its role in targeted nutraceutical delivery.[76] Recent advancements include an early clinical evaluation in 2025, where engineered L. lactis delivering therapeutic agents combined with radiotherapy in three patients with recurrent solid tumors led to tumor reduction and enhanced systemic immunity, demonstrating feasibility in cancer therapy.[77] The advantages of L. lactis for these applications include its food-grade nature, absence of endotoxins (as a Gram-positive bacterium), and resilience in the gastrointestinal environment, with engineered strains achieving up to 70% survival through gastric and intestinal phases. However, challenges persist, such as rapid immune clearance by host defenses and limited persistence in the gut, which can reduce delivery efficiency and necessitate protective formulations like microencapsulation. Regulatory progress continues with ongoing preclinical evaluations for oral vaccines and advancing clinical applications in therapeutics as of 2025.[78][79][80]

Genomics and Genetic Research

Genome Structure and Sequencing

The genome of Lactococcus lactis consists of a single circular chromosome with a size ranging from approximately 2.25 to 2.59 Mb across strains, encoding roughly 2,000 to 2,500 protein-coding genes, and an average G+C content of about 35% https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-017-3650-5. Strains commonly harbor multiple plasmids, typically 1 to 8 in number and ranging from 2 to over 100 kb in size, with a lower G+C content of 30-40% compared to the chromosome; these plasmids often carry accessory genes such as the lactose operon (lacABC), which enables lactose metabolism in dairy environments https://academic.oup.com/femsre/article/30/2/243/2367769. The first complete genome sequence was reported for L. lactis subsp. lactis IL1403 in 2001, revealing a 2,365,589 bp chromosome with 2,311 predicted genes, 86% of which are protein-coding, along with 43 insertion sequence (IS) elements and integrated prophage regions https://www.ncbi.nlm.nih.gov/pmc/articles/PMC311110/. Key genomic features include CRISPR-Cas systems, which provide adaptive immunity against bacteriophages by incorporating spacer sequences from invading viral DNA, a critical defense mechanism in industrial fermentation settings where phage infections are common https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546414/. Mobile genetic elements such as IS elements and transposons are abundant, contributing to genomic plasticity and strain diversity through rearrangements and horizontal gene transfer; for instance, L. lactis subsp. cremoris genomes often exhibit more pseudogenes and IS elements, suggestive of ongoing genome decay and specialization https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-017-3650-5. Comparative genomics highlights subspecies-specific adaptations: L. lactis subsp. lactis genomes are generally larger and retain more genes associated with rapid growth and metabolic versatility on diverse substrates, supporting faster acidification in varied environments https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00004/full, whereas subsp. cremoris strains feature specialized clusters for flavor biosynthesis, including enhanced proteolysis and aminotransferase pathways that generate aroma compounds during cheese ripening https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-017-3650-5. These differences underscore the evolutionary divergence between plant-associated ancestors and dairy-adapted lineages, with plasmids playing a key role in acquiring traits like lactose utilization unique to subsp. cremoris https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365430/.

Recent Genetic Modifications and Advances

Since the advent of CRISPR-Cas9 systems in the post-genomic era, targeted genome editing in Lactococcus lactis has advanced significantly, enabling precise knockouts and insertions to enhance industrial traits. From 2018 onward, researchers have developed one-plasmid-based CRISPR-Cas9 tools for efficient gene deletion in L. lactis, allowing for the investigation of stress-related pathways without relying on multiple vectors.[81] For instance, CRISPR-associated transposon systems have been adapted for guide RNA-directed insertions, facilitating stable genomic modifications in strains used for fermentation.[82] These tools have been applied to improve acid resistance, resulting in strains with significantly higher survival rates under low-pH conditions, as demonstrated in dairy simulation experiments.[83] In synthetic biology, metabolic engineering of L. lactis has leveraged modular assembly techniques to construct multi-gene pathways, expanding its role beyond traditional fermentation. Golden Gate assembly has been employed to reconstitute complex biosynthetic operons, allowing seamless integration of heterologous genes.[84] Recent efforts have focused on carotenoid biosynthesis via pathway optimization.[85] These advancements build on core genome features like modular plasmid backbones, which facilitate rapid prototyping of engineered strains.[21] Omics technologies have further illuminated genetic responses in L. lactis, with proteomics studies from 2024 revealing key regulators of stress adaptation. Quantitative proteomics of ccpA mutants under acid and oxidative stress identified over 200 differentially expressed proteins, including those in glycolysis and envelope remodeling, which enhance survival in fermented environments.[86] Complementing this, metagenomic surveys of fermented foods have mapped L. lactis interactions in microbial consortia, revealing strain-specific adaptations like prophage-encoded resistance genes that dominate in dairy matrices.[87] These insights from PMC-reviewed works underscore how omics data guide targeted modifications for robust starters.[88] Looking ahead, computational approaches are emerging for L. lactis strain design, integrating multi-omics data to predict resilient variants.[89] In therapeutic contexts, engineered L. lactis has been explored for delivering immunomodulators like IL-10 to address inflammatory conditions such as IBD.[90] These strains, modified with CRISPR for stable expression, continue to show promise in preclinical models.[91] Despite these advances, challenges persist in L. lactis genetic engineering, particularly plasmid instability, where curing rates can reach 50% during prolonged fermentation due to metabolic burden and replication conflicts.[21] Regulatory hurdles for food-grade genetically modified organisms (GMOs) include stringent GRAS assessments, as antibiotic markers and foreign DNA raise containment concerns, limiting commercial deployment.[92] Strategies like auxotrophic complementation and RM system bypassing are mitigating these issues, but integration into chromosomes remains essential for stability.[93]

References

  1. Comparative Genome Analysis of Lactococcus lactis Indicates Niche ...
  2. A review on Lactococcus lactis: from food to factory
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  4. Lactococcus lactis in Dairy Fermentation—Health-Promoting ... - MDPI
  5. https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=1358
  6. Species: Lactococcus lactis - LPSN
  7. Lactococcus - an overview | ScienceDirect Topics
  8. Lactic Acid Bacteria in Raw-Milk Cheeses: From Starter Cultures to ...
  9. Summary of Lactococcus lactis lactis IL1403, version 29.1 - BioCyc
  10. Lactococcus lactis subsp. lactis (Lister) Schleifer et al. - 19435 | ATCC
  11. Unit 2a. General functions, characteristics and classification of LAB ...
  12. Microbial interactions shape cheese flavour formation - Nature
  13. The microbes that give Cheddar cheese its distinct flavour
  14. Technological properties of Lactococcus lactis subsp. lactis bv ... - NIH
  15. Activation of the Diacetyl/Acetoin Pathway in Lactococcus lactis ...
  16. The complete genome sequence of the lactic acid bacterium ...
  17. Lactococcus lactis, an Attractive Cell Factory for the Expression of ...
  18. [PDF] NICE Expression System for Lactococcus lactis The ... - MoBiTec
  19. Complete Sequences of Four Plasmids of Lactococcus lactis subsp ...
  20. The Lactococcus lactis Pan-Plasmidome - Frontiers
  21. The Plasmid Complement of Lactococcus lactis UC509.9 Encodes ...
  22. Identification of Four Phage Resistance Plasmids from Lactococcus ...
  23. The art of strain improvement of industrial lactic acid bacteria without ...
  24. Lactococcus lactis vs. cremoris: Growth & Survival in Cheddar Cheese
  25. Imaging the nanoscale organization of peptidoglycan in living ...
  26. Peptidoglycan Structure Analysis of Lactococcus lactis Reveals the ...
  27. Genetics of Lactococci | Microbiology Spectrum - ASM Journals
  28. Formation of intracellular vesicles within the Gram+ Lactococcus ...
  29. lactate dehydrogenase, from Lactococcus lactis - PMC - NIH
  30. Adaptation of Lactococcus lactis to high growth temperature leads to ...
  31. https://pmc.ncbi.nlm.nih.gov/articles/PMC12592913/
  32. Respiration Capacity of the Fermenting Bacterium Lactococcus ...
  33. Lactococcus - Teuber - Major Reference Works - Wiley Online Library
  34. [PDF] Lactic acid production by a strain of Lactococcus lactis subs lactis ...
  35. [PDF] Metabolic shifts in Lactococcus lactis
  36. A review on Lactococcus lactis: from food to factory - PMC
  37. Nutritional requirements and media development for Lactococcus ...
  38. Respiratory Physiology of Lactococcus lactis in Chemostat Cultures ...
  39. Investigation of Flavor-Forming Starter Lactococcus lactis subsp ...
  40. Mesophilic and Thermophilic Cultures Used in Traditional ...
  41. Editing of the Proteolytic System of Lactococcus lactis Increases Its ...
  42. Fermentation Characteristics of Lactococcus lactis subsp. lactis ...
  43. DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut ... - NIH
  44. Characterization of Two Nisin-Producing Lactococcus lactis subsp.
  45. Extending the Shelf life of Kimchi with Lactococcus lactis Strain as a ...
  46. Nitrites in Cured Meats, Health Risk Issues, Alternatives to Nitrites
  47. [PDF] Role of Starter Cultures on the Safety of Fermented Meat Products
  48. The Curing Agent Sodium Nitrite, Used in the Production of ... - NIH
  49. The Microbiota and Health Promoting Characteristics of the ... - NIH
  50. Milk kefir: composition, microbial cultures, biological activities, and ...
  51. [PDF] Phylogenetic Diversity of the Bacterial Communities in Craft Beer
  52. Fermentation of plant‐based dairy alternatives by lactic acid bacteria
  53. Versatile Lactococcus lactis strains improve texture in both ... - NIH
  54. Nutritional value and sensory attributes of plant-based fermented milk
  55. Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates ...
  56. Surface Proteins of Lactococcus lactis: Bacterial Resources for Muco ...
  57. Bacteriocin-Producing Probiotic Lactic Acid Bacteria in Controlling ...
  58. Effects of an exopolysaccharide from Lactococcus lactis Z-2 on ...
  59. Effects of food containing Lactococcus lactis strain T21 on the ... - NIH
  60. Potential Anti-Allergy and Immunomodulatory Properties of ...
  61. In vitro assessment of hypocholesterolemic activity of Lactococcus ...
  62. [PDF] GRAS Notice (GRN) 1088 Agency Response Letter - FDA
  63. A Randomized Double-blind Placebo-controlled Clinical Trial of ...
  64. Indigenous Lactococcus lactis with Probiotic Properties - MDPI
  65. Development of Lactococcus lactis encoding fluorescent proteins ...
  66. Nisin inducible production of listeriolysin O in Lactococcus lactis ...
  67. Protection against tetanus toxin in mice nasally immunized with ...
  68. Protection against tetanus toxin in mice nasally immunized with ...
  69. Oral and intranasal immunization with food-grade recombinant ... - NIH
  70. Oral Immunization of Mice with Cell Extracts from Recombinant ...
  71. Oral Administration of Recombinant Lactococcus lactis Expressing ...
  72. Reversal of autoimmune diabetes by restoration of antigen-specific ...
  73. Treatment of murine colitis by Lactococcus lactis secreting ... - PubMed
  74. Engineered Probiotic Lactococcus lactis for Lycopene Production ...
  75. Enhanced gastrointestinal survivability of recombinant Lactococcus ...
  76. Therapeutic Drug Delivery by Genetically Modified Lactococcus lactis
  77. Oral Delivery of Lactococcus lactis Expressing Full-Length S Protein ...
  78. [PDF] One-plasmid based CRISPR-Cas9 editing for gene deletion in ...
  79. New Effective Method of Lactococcus Genome Editing Using Guide ...
  80. Improvement of γ-aminobutyrate biosynthesis by genetically ...
  81. Engineering Lactococcus lactis as a multi-stress tolerant ...
  82. Analysis and Reconstitution of the Menaquinone Biosynthesis ...
  83. Development of a Golden Gate Assembly-Based Genetic Toolbox ...
  84. Advances in biomanufacturing and recent biological applications of ...
  85. Quantitative proteomics and integrated stress response analysis of ...
  86. Integrated Transcriptomic and Proteomic Analyses Revealed the ...
  87. Fermented-Food Metagenomics Reveals Substrate-Associated ...
  88. MiFoDB, a workflow for microbial food metagenomic ... - ASM Journals
  89. Review Robustness: linking strain design to viable bioprocesses
  90. https://pmc.ncbi.nlm.nih.gov/articles/PMC12584474/
  91. Engineered bacteria and bacterial derivatives as advanced ...
  92. Safety Aspects of Genetically Modified Lactic Acid Bacteria - PMC
  93. Development of a Pre-Modification Strategy to Overcome Restriction ...
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