Production of R- and S-1,2-propanediol in engineered Lactococcus lactis.
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ABSTRACT:
SUBMITTER: Sato R
PROVIDER: S-EPMC8368392 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Microbial production of commodity chemicals has gained increasing attention and most of the focus has been on reducing the production cost. Selecting a suitable microorganism, which can grow rapidly on cheap feedstocks, is of key importance when developing an economically feasible bioprocess. We chose Lactococcus lactis, a well-characterized lactic acid bacterium, as our microbial host to produce pyruvate, which is a commodity chemical with various important applications. Here we report the engineering of Lactococcus lactis into becoming an efficient microbial platform for producing pyruvate. The strain obtained, FS1076 (MG1363 Δ3 ldh Δpta ΔadhE Δals), was able to produce pyruvate as the sole product. Since all the competitive pathways had been knocked out, we achieved growth-coupled production of pyruvate with high yield. More than 80 percent of the carbon flux was directed toward pyruvate, and a final titer of 54.6 g/L was obtained using a fed-batch fermentation setup. By introducing lactose catabolism into FS1076, we obtained the strain FS1080, which was able to generate pyruvate from lactose. We then demonstrated the potential of FS1080 for valorizing lactose contained in dairy side-streams, by achieving a high titer (40.1 g/L) and high yield (78.6%) of pyruvate using residual whey permeate (RWP) as substrate. The results obtained, show that the L. lactis platform is well-suited for transforming lactose in dairy waste into food-grade pyruvate, and the yields obtained are the highest reported in the literature. These results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) to valuable products.
| S-EPMC7815928 | biostudies-literature
Project description:The potential advantages of hyaluronic acid (HA) production by metabolically-engineered Lactococcus lactis is constrained by the lower molecular weight and yield of HA obtained in these strains, compared to natural producers. Earlier studies have correlated lower HA yield with excessive lactate production in L. lactis cultures (Chauhan et al., 2014). In the present study, a three-fold increase was observed in the amount as well as molecular weight of HA produced by recombinant ldh-mutant L. lactis strains. The diversion from lactate production in the ldh-mutant strains resulted in excess ethanol and acetoin production and higher NAD+/NADH ratio in these cultures. The initial NAD+/NADH ratio showed a positive correlation with HA molecular weight as well as with the HA-precursor ratio (UDP-GlcUA/UDP-GlcNAc). The influence of NAD+/NADH ratio on regulation of the concerned metabolic pathways was assessed by transcriptional analysis of key genes having putative binding sites of the NADH-binding transcriptional factor, Rex.
| S-EPMC5779726 | biostudies-literature
Project description:Lectins have been increasingly utilized as carriers for targeted drug delivery based on their specific binding to glycans located on mammalian cells. This study employed two lectins, B subunit of bacterial Shiga holotoxin (Stx1B) and fungal Clitocybe nebularis lectin (CNL), for surface display on the lactic acid bacterium Lactococcus lactis. The specific adhesion of these engineered, lectin-displaying L. lactis to cancer cells was evaluated. The expression and surface display of both lectins on L. lactis were demonstrated by western blotting and flow cytometry, respectively. MTS assays revealed that recombinant Stx1B had no effect on Caco-2 cell viability at concentrations of ≤25 µg/mL, whereas CNL was non-toxic even at relatively high concentrations of ≤250 µg/mL. Stx1B bound to Caco-2, HT-29 and HeLa cells after 1 h of incubation. CNL bound to Caco-2 cells and recognized several glycoproteins in HT-29 and Caco-2 cell homogenates of which a 70 kDa protein predominated. Confocal microscopy revealed adhesion of Stx1B-displaying L. lactis to HeLa, Caco-2, and, to a lesser extent, HT-29 cells; CNL-displaying L. lactis showed a relatively similar level of adherence to HT-29 and Caco-2 cells. Thus, lectin-displaying L. lactis might serve as a carrier in targeted drug delivery when coupled to a therapeutic moiety.
| S-EPMC7911926 | biostudies-literature
Project description:Cheese produced with Lactococcus lactis is the main source of vitamin K2 in the Western diet. Subclinical vitamin K2 deficiency is common, calling for foods with enhanced vitamin K2 content. In this study we describe analyses of vitamin K2 (menaquinone) production in the lactic acid bacterium L. lactis ssp. cremoris strain MG1363. By cloning and expression from strong promoters we have identified genes and bottlenecks in the biosynthetic pathways leading to the long-chained menaquinones, MK-8 and MK-9. Key genes of the biosynthetic menaquinone pathway were overexpressed, singly or combined, to examine how vitamin K2 production can be enhanced. We observed that the production of the long menaquinone polyprenyl side chain, rather than production of the napthoate ring (1,4-dihydroxy-2-naphtoic acid), limits total menaquinone synthesis. Overexpression of genes causing increased ring formation (menF and menA) led to overproduction of short chained MK-3, while overexpression of other key genes (mvk and llmg_0196) resulted in enhanced full-length MK-9 production. Of two putatively annotated prenyl diphosphate synthases we pinpoint llmg_0196 (preA) to be important for menaquinone production in L. lactis. The genes mvk, preA, menF, and menA were found to be important contributors to menaquinone levels as single overexpression of these genes double and more than triple the total menaquinone content in culture. Combined overexpression of mvk, preA, and menA increased menaquinone levels to a higher level than obtained individually. When the overproducing strains were applied for milk fermentations vitamin K2 content was effectively increased 3-fold compared to the wild type. The results provide a foundation for development of strains to ferment foods with increased functional value i.e., higher vitamin K2 content.
| S-EPMC7093718 | biostudies-literature
Project description:BackgroundDysregulated production of interleukin (IL)-6 is implicated in the pathology of inflammatory bowel disease (IBD). Neutralization of IL-6 in the gut by safe probiotic bacteria may help alleviate intestinal inflammation. Here, we developed Lactococcus lactis with potent and selective IL-6 binding activity by displaying IL-6-specific affibody on its surface.ResultsAnti-IL-6 affibody (designated as ZIL) was expressed in fusion with lactococcal secretion peptide Usp45 and anchoring protein AcmA. A high amount of ZIL fusion protein was detected on bacterial surface, and its functionality was validated by confocal microscopy and flow cytometry. Removal of IL-6 from the surrounding medium by the engineered L. lactis was evaluated using enzyme-linked immunosorbent assay. ZIL-displaying L. lactis sequestered recombinant human IL-6 from the solution in a concentration-dependent manner by up to 99% and showed no binding to other pro-inflammatory cytokines, thus proving to be highly specific for IL-6. The removal was equally efficient across different IL-6 concentrations (150-1200 pg/mL) that were found to be clinically relevant in IBD patients. The ability of engineered bacteria to capture IL-6 from cell culture supernatant was assessed using immunostimulated human monocytic cell lines (THP-1 and U-937) differentiated into macrophage-like cells. ZIL-displaying L. lactis reduced the content of IL-6 in the supernatants of both cell lines in a concentration-dependent manner by up to 94%. Dose response analysis showed that bacterial cell concentrations of 107 and 109 CFU/mL (colony forming units per mL) were required for half-maximal removal of recombinant and macrophage-derived IL-6, respectively.ConclusionThe ability of ZIL-displaying L. lactis to bind pathological concentrations of IL-6 at common bacterial doses suggests physiological significance.
| S-EPMC9287920 | biostudies-literature
Project description:Lactococcus lactis subsp. lactis is a food bacterium that has been utilized for decades in food fermentation and the development of high-value industrial goods. Among these, nisin, which is produced by several strains of L. lactis subsp. lactis, plays a crucial role as a food bio-preservative. The gene expression for nisin synthesis was evaluated using qPCR analysis. Additionally, a series of re-transformations of the strain introducing multiple copies of the nisA and nisRK genes related to nisin production were developed. The simultaneous expression of nisA and nisZ genes was used to potentiate the effective inhibition of foodborne pathogens. Furthermore, qPCR analysis indicated that the nisA and nisRK genes were expressed at low levels in wild-type L. lactis subsp. lactis. After several re-transformations of the strain with the nisA and nisRK genes, a high expression of these genes was obtained, contributing to improved nisin production. Also, co-expression of the nisA and nisZ genes resulted in extremely effective antibacterial action. Hence, this study would provide an approach to enhancing nisin production during industrial processes and antimicrobial activity.
| S-EPMC10085027 | biostudies-literature
Project description:BACKGROUND: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. METHODOLOGY AND FINDINGS: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. CONCLUSIONS: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.
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Project description:Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.
| S-EPMC2708441 | biostudies-literature
Project description:α-Phosphoglucomutase (α-PGM) is an enzyme that is essential for the growth of Lactococcus lactis. The enzyme links bacterial anabolism with sugar utilization through glycolysis by catalyzing the reversible interconversion of glucose 6-phosphate and α-glucose 1-phosphate. The gene encoding α-PGM was cloned and overexpressed in L. lactis. The purified protein was functionally active and was crystallized with ammonium sulfate as a precipitant using vapour-diffusion and seeding techniques. Optimized crystals diffracted to 1.5 Å resolution at a synchrotron source.
| S-EPMC3433211 | biostudies-literature
Project description:Nisin produced by certain Lactococcus lactis strains is commercially used in meat and dairy industries because of its effective antibacterial activity and food safety characteristics. It has been proved that the antibacterial activity could be enhanced when combined with other antimicrobial agents. In this study, we demonstrated that nisin and 3-phenyllactic acid (PLA) in combination displayed excellent combinational antibacterial activity against foodborne pathogens including S. xylosus and M. luteus. The potential application in food preservation was further verified via microbial analysis during the storage of meat and milk, and determination of strawberry rot rate. Scanning electron microscopy observation indicated a distinct mode of PLA with nisin, which may target at the dividing cell, contributing to their combinational antibacterial effect of nisin and PLA. Considering the positive results, a nisin-PLA co-producing strain was constructed based on the food-grade strain L. lactis F44, a nisin Z producer. By the knockout of two L-lactate dehydrogenase (LDH) and overexpression of D-LDH Y25A, the yield of PLA was significantly increased 1.77-fold in comparison with the wild type. Anti-bacterial assays demonstrated that the fermentation product of the recombinant strain performed highly effective antibacterial activity. These results provided a promising prospect for the nisin-PLA co-expressing L. lactis in food preservation on account of its considerable antibacterial activity and cost-effective performance.
| S-EPMC7901885 | biostudies-literature