Recombinant Lactobacillus plantarum Demethylmenaquinone methyltransferase (ubiE)

What is Demethylmenaquinone methyltransferase (ubiE) and what is its functional significance in L. plantarum?

Demethylmenaquinone methyltransferase (ubiE) is a critical enzyme involved in the biosynthesis of menaquinones (vitamin K2), which function as electron carriers in bacterial respiratory chains. In L. plantarum, the menaquinone biosynthesis pathway is notably incomplete, with the organism naturally possessing only two of the eight required genes (menA and menG) . This limited biosynthetic capacity restricts L. plantarum's ability to produce endogenous menaquinones and consequently impacts its respiratory metabolism.

The expression of recombinant ubiE in L. plantarum represents an attempt to reconstitute or enhance the menaquinone biosynthesis pathway, potentially enabling respiratory growth when supplemented with heme. Research has demonstrated that recombinant strains with enhanced menaquinone production show improved biomass formation, reduced acidification, increased resistance to oxygen, and better long-term storage stability .

Methodologically, researchers can assess ubiE functionality through:

• Quantification of menaquinone production using HPLC analysis

• Growth comparative assays under aerobic conditions with heme supplementation

• Measurement of NADH/NAD+ ratios as indicators of respiratory activity

• Analysis of acid production rates and final pH in culture media

What are the most effective expression systems for recombinant ubiE in L. plantarum?

Several expression systems have demonstrated efficacy for recombinant protein production in L. plantarum. Based on current research, the following methodological approaches have proven successful:

• pSIP Expression System: This inducible expression system, based on the sakacin promoter, has been successfully used for heterologous gene expression in L. plantarum. The system utilizes a peptide pheromone (IP-673) as inducer and provides tight regulation of gene expression . For ubiE expression, the pSIP409 vector has been modified to eliminate internal BsaI sites through site-directed mutagenesis, facilitating subsequent cloning steps .

• Direct Cloning Method: In vitro assembly and PCR amplification can generate sufficient quantities of recombinant DNA for transformation into L. plantarum WCFS1 without requiring an intermediate host like E. coli . This approach is particularly valuable when expressing genes potentially incompatible with E. coli.

• Synthetic Operon Construction: For complete pathway reconstitution, researchers have successfully employed synthetic operons with multiple inducible promoters. For example, the menaquinone biosynthesis pathway in L. plantarum was reconstituted by expressing six genes from L. lactis using a synthetic operon with two inducible promoters .

Transformation efficiency considerations:

• Electroporation typically requires 1-5 μg of plasmid DNA for successful transformation

• Cell wall weakening agents may improve transformation efficiency

• Expression should be verified via Western blotting, enzyme activity assays, and functional complementation studies

What experimental approaches can verify successful expression and functionality of recombinant ubiE?

Multiple experimental strategies can be employed to confirm both the expression and functional activity of recombinant ubiE in L. plantarum:

• Protein Expression Verification:

  • Western blotting using specific antibodies against ubiE or epitope tags

  • Flow cytometry for surface-displayed proteins

  • Mass spectrometry-based proteomic analysis

• Functional Complementation Assays:

  • Transformation of ubiE-deficient strains (e.g., L. lactis ΔmenG mutants) with the recombinant L. plantarum ubiE gene and assessment of menaquinone production restoration

  • Growth experiments under respiratory conditions (aerobic with heme supplementation)

• Menaquinone Production Analysis:

  • HPLC quantification of menaquinone forms (MK-1, MK-3, MK-8, MK-9) in cellular extracts

  • Comparison of menaquinone profiles between wild-type and recombinant strains under different growth conditions (anaerobic, aerobic, and respiration-permissive)

• Metabolic Impact Assessment:

  • Measurement of NADH/NAD+ ratios

  • Analysis of organic acid production patterns

  • Determination of biomass yields under different cultivation conditions

Research has shown that functional complementation of L. lactis menG mutants with L. plantarum ubiE genes (lpmenG1, lpmenG2) successfully restored menaquinone production and respiratory growth when supplemented with heme .

How does recombinant ubiE expression affect the respiratory capacity and growth characteristics of L. plantarum?

Expression of functional ubiE in L. plantarum significantly impacts respiratory metabolism and growth characteristics through several mechanisms:

• Enhanced Biomass Production:

  • Reconstitution of the menaquinone biosynthesis pathway in L. plantarum through expression of six genes from L. lactis (including ubiE) resulted in higher biomass formation, with optical density (OD600) increases from 3.0 to 5.0 upon induction .

  • Respiratory metabolism enables more efficient energy conservation compared to fermentative growth.

• Altered Central Metabolism:

  • Respiration reroutes pyruvate away from lactate accumulation, resulting in reduced acidification of the growth medium .

  • Changes in central metabolism can be quantified through:

    • Measurement of organic acid profiles by HPLC

    • Analysis of gene expression using qRT-PCR

    • Determination of enzymatic activities (lactate dehydrogenase, pyruvate oxidase, NADH oxidase)

• Increased Oxidative Stress Tolerance:

  • Respiratory cultures show improved survival under oxidative stress conditions compared to fermentative cultures .

  • Tolerance can be assessed by challenging cultures with hydrogen peroxide, menadione, or other ROS-generating compounds.

• Electron Transport Chain Functionality:

  • Function of the electron transport chain can be evaluated by measuring oxygen consumption rates using oxygen electrodes.

  • Cytochrome bd oxidase activity (encoded by cydABCD) can be assessed spectrophotometrically.

How does ubiE expression influence the extracellular electron transfer capabilities of L. plantarum?

Recent research has identified that L. plantarum can perform extracellular electron transfer (EET), a process partially enabled by menaquinones as electron carriers . The expression of recombinant ubiE and enhancement of menaquinone biosynthesis impacts this capability through several mechanisms:

• Electron Shuttle Functionality:

  • L. plantarum can convert DHNA (1,4-dihydroxy-2-naphthoic acid) to MK-6 and MK-7, but still relies on exogenous electron shuttles for EET .

  • Recombinant ubiE expression may enhance the conversion efficiency of precursors to functional menaquinones.

• EET Pathway Gene Expression:

  • Growth of L. plantarum in media containing DHNA and ferric ammonium citrate (FeAC) induces FLEET (flavin-mediated extracellular electron transfer) pathway genes including ndh2 and pplA .

  • Measurement of gene expression using qRT-PCR reveals upregulation of these key EET components.

• Environmental Acidification Effects:

  • Quinone-cross feeding from other lactic acid bacteria to L. plantarum results in accelerated environmental acidification during the early exponential phase of growth .

  • Co-cultures of L. plantarum with quinone-producing bacteria (e.g., L. lactis TIL46) showed greater acidification (pH 5.26 ± 0.03) compared to co-cultures with non-producing strains (pH 6.16 ± 0.03) within 6 hours of incubation .

Experimental approaches to assess EET capability include:

• Ferrihydrite reduction assays using spectrophotometric methods

• Chronoamperometry measurements using polarized electrodes

• Analysis of redox indicator dyes (e.g., DCPIP) reduction rates

• Gene expression analysis of EET pathway components

What are the implications of recombinant L. plantarum expressing ubiE for immunomodulation and gut microbiota interactions?

Recombinant L. plantarum strains have demonstrated significant potential as vehicles for mucosal vaccines and immunomodulatory applications. The expression of ubiE and resulting changes in respiratory metabolism may influence these properties:

• Impact on Gut Microbiota:

  • Recombinant L. plantarum can modulate gut microbial diversity, as demonstrated by increased Shannon-Wiener index in studies with recombinant strains .

  • Beta diversity analysis shows altered microbial structure mediated by recombinant L. plantarum .

• Immune Response Modulation:

  • Recombinant L. plantarum expressing fusion proteins (e.g., P14.5 of African swine fever virus and IL-33) enhances gut bacterial functions in metabolism and immune regulation .

  • Studies report increased levels of IgG and IgG1 in serum and secretory IgA (sIgA) in feces, along with enrichment of CD4+ T cells and IgA+ B cells .

• Mucosal Immunity Enhancement:

  • Recombinant L. plantarum can activate dendritic cells in Peyer's patches, increase CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes, and affect CD4+ and CD8+ cell proliferation .

  • Higher B220+IgA+ cell numbers in Peyer's patches and increased IgA levels in the lungs and intestinal segments have been observed .

• Methodology for Assessing Immunomodulatory Effects:

  • Flow cytometry analysis of immune cell populations

  • ELISA measurement of specific antibodies

  • Immunofluorescence staining for tissue-specific IgA expression

  • qRT-PCR for cytokine expression profiling

What are the key challenges in achieving stable and consistent expression of functional ubiE in L. plantarum?

Researchers face several significant challenges when working with recombinant L. plantarum expressing ubiE:

• Plasmid Stability Issues:

  • Recombinant plasmids may show instability during prolonged cultivation without selection pressure.

  • Assessment of plasmid stability requires:

    • Serial subculturing without antibiotics

    • Periodic plating on selective and non-selective media

    • PCR verification of plasmid maintenance

• Expression Level Variability:

  • Expression levels can vary significantly based on growth phase, media composition, and induction conditions.

  • For the pSIP system, induction with peptide pheromone IP-673 at a final concentration of 25 ng/mL when cultures reach OD600 of 0.3 has shown good results .

  • Monitoring expression through:

    • Western blotting at different time points

    • Enzyme activity assays throughout growth

    • qRT-PCR for transcript level quantification

• Cofactor Availability:

  • Functional activity of ubiE requires S-adenosylmethionine (SAM) as methyl donor.

  • Availability of this cofactor may limit enzyme activity even with high expression levels.

  • Supplementation strategies may include:

    • Addition of methionine to culture media

    • Co-expression of SAM synthetase

• Growth Condition Optimization:

  • Optimal conditions for protein expression may differ from those for enzyme activity.

  • Systematic optimization of:

    • Temperature (typically 30-37°C)

    • pH (5.5-6.5)

    • Oxygen availability

    • Media composition

What methodological approaches have been successful in reconstituting the complete menaquinone biosynthesis pathway in L. plantarum?

Complete reconstitution of the menaquinone biosynthesis pathway in L. plantarum has been achieved using several strategic approaches:

• Synthetic Operon Construction:

  • Researchers have successfully reconstituted the incomplete menaquinone biosynthesis pathway in L. plantarum by expressing six genes from L. lactis homologous to the missing genes in a synthetic operon with two inducible promoters .

  • The synthetic operon approach requires:

    • PCR amplification of individual genes with appropriate restriction sites

    • Assembly into an operon structure with optimized ribosome binding sites

    • Introduction of multiple promoters for balanced expression

• Gene Verification Through Complementation:

  • Functionality of individual L. plantarum genes has been verified through complementation studies in L. lactis knockout strains.

  • Three L. plantarum biosynthesis genes (lpmenA1, lpmenG1, and lpmenG2) and two genes from L. buchneri (lbmenB and lbmenG) successfully reconstituted menaquinone production and respiratory growth in deficient L. lactis strains when supplemented with heme .

• Heterologous Expression System Selection:

  • For menaquinone pathway reconstitution in L. plantarum, the pSIP409 vector has been modified by eliminating internal BsaI sites using site-directed mutagenesis .

  • Vector backbone amplification with BsaI restriction sites and complementary overhangs facilitates the assembly of multiple genes.

• Verification of Pathway Functionality:

  • HPLC analysis to detect and quantify different menaquinone forms (MK-1, MK-3, MK-8, MK-9)

  • Growth experiments under respiratory conditions

  • Measurement of biomass formation under different cultivation conditions

  • Analysis of acid production rates and final pH

What are the emerging applications of recombinant L. plantarum expressing ubiE in biotechnology and medicine?

Recombinant L. plantarum expressing ubiE has potential applications in several cutting-edge areas:

• Enhanced Probiotic Formulations:

  • Respiratory-competent L. plantarum may show improved survival through the gastrointestinal tract.

  • Increased resistance to oxidative stress can enhance shelf-life and stability.

  • Altered metabolism may reduce undesirable acid production in situ.

• Vaccine Delivery Systems:

  • Recombinant L. plantarum has demonstrated efficacy as a vaccine delivery vehicle for mucosal immunity .

  • Expression of antigens like influenza virus HA1 with dendritic cell-targeting peptides has shown promising results in animal models .

  • Respiratory-competent strains may offer improved antigen delivery through enhanced survival and persistence.

• Microbiome Modulation:

  • Recombinant L. plantarum can significantly alter gut microbiota composition and function .

  • Enhanced respiratory metabolism may provide competitive advantages in the gut ecosystem.

  • Potential applications in addressing dysbiosis-related conditions.

• Biocatalysis Applications:

  • Respiratory-competent L. plantarum may serve as an improved host for whole-cell biocatalysis.

  • Enhanced biomass production and survival could increase process efficiency.

  • Altered redox balance may benefit certain biotransformation reactions.

• Biosensing and Bioelectronic Applications:

  • L. plantarum with enhanced EET capabilities through improved menaquinone production could be employed in microbial electrochemical systems .

  • Potential applications in biosensors and bioelectronic devices.

How does ubiE expression in L. plantarum influence cross-feeding interactions in microbial communities?

Menaquinone cross-feeding between lactic acid bacteria species significantly impacts community dynamics and metabolism:

• Quinone Cross-Feeding Effects:

  • Quinone cross-feeding from other lactic acid bacteria to L. plantarum accelerates environmental acidification during early growth phases .

  • Co-cultures of L. plantarum with quinone-producing L. lactis TIL46 showed greater acidification (pH 5.26 ± 0.03) compared to co-cultures with non-producing strains (pH 6.16 ± 0.03) within 6 hours .

• Growth Enhancement Through Co-cultivation:

  • L. plantarum cell numbers were 2-fold higher after 24h incubation with L. lactis, independent of menaquinone biosynthetic capacity .

  • Co-culture with L. mesenteroides increased L. plantarum cell numbers 4-fold after 8h incubation .

• Experimental Design for Co-Culture Studies:

  • Selection of appropriate partner strains with defined menaquinone production capabilities

  • Optimization of inoculation ratios and growth conditions

  • Species-specific enumeration methods (selective media, qPCR, flow cytometry)

  • Metabolite profiling to assess cross-feeding effects

• Methodological Considerations:

  • Use of defined media to control nutrient availability

  • Application of metabolic modeling to predict cross-feeding interactions

  • Employment of fluorescent labeling for strain-specific tracking

  • Implementation of transcriptomic analyses to identify responsive pathways