Recombinant Bacillus amyloliquefaciens Riboflavin biosynthesis protein RibD (ribD)

What is the function of the RibD protein in riboflavin biosynthesis?

RibD functions as a bifunctional enzyme in the riboflavin biosynthetic pathway (RBP), serving as both a deaminase and reductase (EC 3.5.4.26). In the riboflavin biosynthesis pathway, RibD catalyzes two consecutive reactions in the conversion pathway from GTP to riboflavin. The enzyme specifically performs the deamination and reduction steps in the middle of the pathway, converting the intermediate compounds in the process of synthesizing the vitamin B2 precursor .

Unlike some other riboflavin biosynthesis genes that may have regulatory elements such as the FMN riboswitch (as seen with ribB in E. coli), the ribD gene seems to be regulated differently. In E. coli, rib genes including ribD are scattered across the genome, which differs from the operon structure seen in organisms like Bacillus subtilis where the genes form a cluster (ribD-ribE-ribBA-ribH) .

How does the organization of riboflavin biosynthesis genes in B. amyloliquefaciens compare to other bacterial species?

Based on comparative genomics, Bacillus species tend to organize their riboflavin biosynthesis genes differently than E. coli. While E. coli has its rib genes scattered across the genome, Bacillus subtilis (closely related to B. amyloliquefaciens) has these genes organized in an operon structure (ribD-ribE-ribBA-ribH) .

This different genetic organization has implications for gene regulation and expression strategies when attempting to enhance riboflavin production. The clustered operon structure in Bacillus species may facilitate coordinated expression of the riboflavin biosynthesis genes, potentially offering advantages for metabolic engineering approaches compared to species with scattered gene arrangements .

What expression systems are most effective for recombinant production of B. amyloliquefaciens RibD?

For recombinant production of riboflavin biosynthesis proteins like RibD, several expression systems have proven effective in related research. The pET expression system has been successfully used for riboflavin biosynthesis genes, as demonstrated in studies with E. coli BL21. For example, researchers have used the pET vector system to overexpress riboflavin biosynthesis genes, achieving significant production increases .

When working specifically with B. amyloliquefaciens proteins, consider:

• Expression host: E. coli BL21(DE3) remains a popular choice due to its well-characterized nature and high protein expression capabilities

• Vector selection: pET series vectors provide strong, inducible expression under the T7 promoter

• Induction parameters: IPTG concentration optimization is crucial, as studies have shown that fermentation conditions including IPTG concentration significantly affect riboflavin production

• Temperature: Lower induction temperatures (20-25°C) may improve soluble protein expression for enzymes like RibD

What growth conditions are optimal for B. amyloliquefaciens cultivation in laboratory settings?

B. amyloliquefaciens strains generally grow well under standard laboratory conditions. Based on research with B. amyloliquefaciens BLB369, the following conditions have been found effective:

• Temperature: Typically 25-37°C, with 30°C being common for routine cultivation

• Media options:

  • LB (Luria-Bertani) medium is suitable for routine growth

  • For specialized purposes, MSY medium has shown superior results compared to both M9Y and LB media in certain strains

• pH: Around 7.0-7.5

• Aeration: Good aeration through shaking (150-200 rpm) improves growth

• Growth phase: Culture for 48 hours typically yields sufficient cell density for most experimental purposes

Environmental control is particularly important as B. amyloliquefaciens forms endospores that allow it to withstand extreme conditions including acid and high temperatures .

How can CRISPR/Cas9 be employed to enhance riboflavin production through RibD manipulation in B. amyloliquefaciens?

CRISPR/Cas9 technology offers precise genetic manipulation opportunities for enhancing riboflavin production in B. amyloliquefaciens. Based on successful approaches in related systems, a comprehensive strategy would include:

• Target identification: While ribD is important, consider the entire pathway regulation. In E. coli studies, researchers identified the FMN riboswitch as a key regulatory element affecting ribB expression that could be deleted using CRISPR/Cas9 .

• Guide RNA design:

  • Design guide RNAs targeting regulatory regions of ribD

  • Consider multiple guide RNAs to test different modification approaches

  • Ensure specificity by checking for off-target effects in the B. amyloliquefaciens genome

• Deletion vs. modification approach:

  • Complete deletion of regulatory elements (as done with the FMN riboswitch in E. coli)

  • Promoter replacement to increase expression

  • Point mutations to optimize enzyme activity

• Verification methods:

  • RT-qPCR to confirm increased transcript levels (as demonstrated in E. coli where ribB transcript levels improved 2.78 to 3.05-fold following FMN riboswitch deletion)

  • Enzyme activity assays

  • Riboflavin quantification

• Multi-gene approach:

  • Consider co-targeting other genes in the pathway (ribA, ribB, ribC, ribE)

  • The coordinated overexpression of multiple pathway genes has shown synergistic effects in E. coli, increasing riboflavin production from 182.65 mg/L (single manipulation) to 437.58 mg/L (multiple manipulations plus regulatory element deletion)

What metabolic bottlenecks exist in the riboflavin biosynthetic pathway involving RibD, and how might they be overcome?

Several metabolic bottlenecks can limit riboflavin production in the biosynthetic pathway:

• Precursor availability:

  • The riboflavin biosynthesis pathway requires guanosine 5′-triphosphate (GTP) and D-ribulose 5-phosphate (Ru5P) as precursors

  • Strategy: Overexpression of zwf (glucose-6-phosphate dehydrogenase) has been shown to increase NADPH and precursor availability, resulting in a 74.66% increase in riboflavin production in engineered E. coli strains

• Regulatory feedback inhibition:

  • The FMN riboswitch regulates expression of certain rib genes like ribB in response to flavin levels

  • Strategy: Deletion of the FMN riboswitch using CRISPR/Cas9 improved ribB transcript levels and facilitated riboflavin production, with a 37.17% increase compared to strains without this modification

• Conversion to downstream products:

  • Riboflavin is converted to FMN and FAD by RibF

  • Strategy: Reduced expression of ribF through RBS replacement or knockdown using synthetic regulatory small RNA has been effective in other organisms

• Metabolic flux imbalance:

  • Imbalance between different enzymes in the pathway

  • Strategy: Fine-tuning expression levels of all enzymes, potentially with different promoter strengths for different genes

A comprehensive approach that addresses multiple bottlenecks simultaneously is likely to be most effective, as demonstrated by the 1,574.60 mg/L riboflavin yield achieved in E. coli through combined strategies .

How can multi-omics approaches be applied to study the impact of RibD overexpression on cellular metabolism in B. amyloliquefaciens?

Multi-omics approaches offer powerful tools to comprehensively analyze the effects of RibD overexpression:

• Transcriptomics analysis:

  • RNA-seq to profile global gene expression changes

  • RT-qPCR for targeted validation of key pathway genes

  • Analysis of potential regulatory responses, similar to the analysis performed in mice treated with B. amyloliquefaciens

• Proteomics approach:

  • Quantitative proteomics (LC-MS/MS) to identify protein-level changes

  • Investigate not only RibD levels but also other proteins in the riboflavin pathway and related metabolic networks

  • Post-translational modifications that might affect enzyme activity

• Metabolomics integration:

  • Targeted metabolomics focusing on riboflavin pathway intermediates

  • Untargeted metabolomics to discover unexpected metabolic shifts

  • Stable isotope labeling to track carbon flux through the pathway

• Integrated analysis workflow:

  • Correlation analysis between transcripts, proteins, and metabolites

  • Pathway enrichment analysis to identify affected cellular processes

  • Flux balance analysis to predict metabolic rerouting

• Experimental design considerations:

  • Include time-course sampling to capture dynamic responses

  • Compare wild-type, RibD overexpression alone, and RibD overexpression with additional pathway modifications

  • Control for growth phase effects by normalizing samples appropriately

Similar multi-omics approaches have been successfully applied to study B. amyloliquefaciens in other contexts, such as analyzing its effects on intestinal microbiome and host metabolism .

What are the most accurate methods for quantifying RibD enzyme activity in B. amyloliquefaciens extracts?

Accurate quantification of RibD enzyme activity requires specialized approaches addressing its bifunctional nature:

• Separate assays for deaminase and reductase activities:

  Deaminase activity:

  • Substrate: 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate

  • Detection: Monitor decrease in substrate or increase in product (5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate)

  • Spectrophotometric method: Measure absorbance changes at specific wavelengths

  Reductase activity:

  • Substrate: 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate

  • Detection: Monitor NADPH consumption at 340 nm

  • Coupled assay options for increased sensitivity

• Continuous vs. endpoint measurements:

  • Continuous measurements provide kinetic parameters (Km, Vmax)

  • Endpoint measurements may be more practical for high-throughput screening

• Extract preparation considerations:

  • Buffer optimization: Test multiple buffer systems (phosphate, Tris) with different pH values

  • Enzyme stabilization: Include glycerol (10-20%) and reducing agents (DTT or β-mercaptoethanol)

  • Cell disruption: Sonication or mechanical disruption under controlled temperature

• Activity verification approaches:

  • Recombinant purified enzyme as positive control

  • Specific inhibitors to confirm assay specificity

  • Substrate analogs to verify enzyme specificity

• Advanced analytical methods:

  • HPLC-based assays for direct product quantification

  • LC-MS methods for highly sensitive detection of pathway intermediates

  • Radiometric assays using labeled substrates for highest sensitivity

How does temperature and pH affect the stability and activity of recombinant RibD protein?

Optimizing temperature and pH conditions is crucial for maintaining RibD stability and activity:

• Temperature effects:

  • Enzyme activity typically shows a bell-shaped curve with temperature

  • Optimal activity temperature range: Likely 25-37°C based on B. amyloliquefaciens growth preferences

  • Thermal stability considerations:

    • Measure half-life at different temperatures

    • Pre-incubation studies to determine inactivation kinetics

    • Consider stabilizing agents (glycerol, trehalose) for long-term storage

• pH dependence:

  • Dual functionality of RibD may result in different pH optima for deaminase vs. reductase activities

  • Recommended pH screening range: 6.0-9.0 with 0.5 pH unit intervals

  • Buffer systems to test:

    • Phosphate buffer (pH 6.0-8.0)

    • Tris-HCl buffer (pH 7.0-9.0)

    • MOPS buffer (pH 6.5-7.9)

• Combined temperature-pH interaction:

  • Create activity profiles at different temperature-pH combinations using a matrix design

  • Identify conditions where both activities are reasonably maintained

  • The bifunctional nature may require compromise conditions that maintain both activities

• Long-term stability parameters:

  • Storage stability at different temperatures (4°C, -20°C, -80°C)

  • Freeze-thaw stability assessment

  • Effect of stabilizing agents (glycerol, BSA, metal ions)

• Application-specific considerations:

  • For in vitro assays: Optimize for maximum activity

  • For structural studies: Focus on conditions promoting stability

  • For in vivo expression: Consider physiological conditions of B. amyloliquefaciens

What is the optimal fermentation strategy for enhancing riboflavin production in recombinant B. amyloliquefaciens strains overexpressing RibD?

Based on successful approaches with related riboflavin-producing strains, an optimized fermentation strategy would include:

• Medium optimization:

  • Base medium selection: MSY medium has shown better results than M9Y and LB for certain B. amyloliquefaciens strains

  • Carbon source: Glucose is effective, with 40 g/L being used successfully in fed-batch fermentation for riboflavin production

  • Nitrogen sources: Yeast extract and peptone combinations

  • Trace elements: Metal elements can significantly impact production

• Process parameters:

  • Temperature: 30-37°C range with potential for temperature shifts during production phase

  • pH control: Maintain at optimal range for B. amyloliquefaciens (typically pH 7.0-7.5)

  • Dissolved oxygen: Maintain high aeration rates as riboflavin biosynthesis is aerobic

  • Induction timing: For IPTG-inducible systems, optimize concentration and induction point based on growth curve

• Feeding strategies:

  • Fed-batch fermentation with glucose feeding has achieved riboflavin titers of 1,574.60 mg/L in engineered E. coli

  • Exponential feeding based on growth rate

  • Feedback-controlled feeding based on glucose concentration

• Process monitoring:

  • Online monitoring of pH, temperature, dissolved oxygen

  • Offline sampling for cell density (OD600), glucose concentration, and riboflavin production

  • Real-time analytics for process control decisions

• Scale-up considerations:

  • Maintain similar oxygen transfer rates across scales

  • Adjust mixing parameters to prevent shear damage

  • Consider pH and temperature gradients in larger vessels

The combination of genetic engineering (ribD overexpression) with optimized fermentation conditions has the potential to significantly increase riboflavin production, as demonstrated by the 8.6-fold increase (from 182.65 to 1,574.60 mg/L) achieved in E. coli through similar approaches .

How can protein engineering approaches be applied to improve the catalytic efficiency of RibD?

Protein engineering of RibD offers opportunities to enhance its catalytic properties through several strategic approaches:

Successful protein engineering could potentially address pathway bottlenecks more effectively than simple overexpression approaches, especially if combined with pathway-level optimizations.

What analytical methods provide the most accurate quantification of riboflavin in B. amyloliquefaciens fermentation samples?

Accurate riboflavin quantification requires robust analytical methods suitable for complex fermentation samples:

• HPLC-based methods:

  • Reversed-phase HPLC with fluorescence detection (ex: 450 nm, em: 530 nm)

  • Column recommendation: C18 columns (150 × 4.6 mm, 5 μm particle size)

  • Mobile phase: Typically methanol/water or acetonitrile/water mixtures with buffering agents

  • Sample preparation: Dilution, filtration, and potential deproteinization

  • Quantification range: 0.01-100 mg/L with appropriate dilutions

• Spectrophotometric/spectrofluorometric methods:

  • Direct absorbance measurement at 444 nm

  • Fluorescence measurement (ex: 450 nm, em: 530 nm)

  • Advantages: Rapid, minimal sample preparation

  • Limitations: Potential interference from other compounds in complex media

  • Standard curve preparation: Use pure riboflavin standards in matching matrix

• LC-MS/MS methods:

  • Highest specificity and sensitivity

  • Multiple reaction monitoring (MRM) for riboflavin quantification

  • Internal standard recommendation: Isotopically labeled riboflavin

  • Particularly valuable for detecting pathway intermediates alongside end product

• Sample preparation considerations:

  • Centrifugation to remove cells (10,000 rpm for 15 min)

  • Filtration through 0.22 μm filters

  • Potential extraction methods for intracellular riboflavin

  • Stability concerns: Protect samples from light

• Method validation requirements:

  • Linearity: R² > 0.99 over the working range

  • Precision: RSD < 5% for replicate measurements

  • Accuracy: Recovery of 95-105% from spiked samples

  • LOD/LOQ determination: Signal-to-noise ratios of 3:1 and 10:1, respectively

The method selection should balance required sensitivity, sample throughput, and available instrumentation, with HPLC-fluorescence detection offering a good compromise for routine analysis.

How can researchers address poor expression or insolubility issues when working with recombinant RibD protein?

Addressing expression and solubility challenges with recombinant RibD requires a systematic approach:

• Expression optimization strategies:

  • Codon optimization for the expression host

  • Testing multiple expression vectors with different promoter strengths

  • Evaluating different E. coli strains (BL21, Rosetta, Origami)

  • Optimizing induction parameters:

    • IPTG concentration (0.1-1.0 mM)

    • Induction temperature (15-37°C)

    • Induction time (3-24 hours)

  • Addition of rare tRNA-encoding plasmids if rare codons are present

• Solubility enhancement approaches:

  • Fusion tags screening:

    • Solubility enhancers: MBP, SUMO, Trx, GST

    • Affinity tags: His6, FLAG, Strep-tag II

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Expression at reduced temperatures (15-25°C)

  • Addition of solubility enhancers to growth media:

    • Osmolytes (sorbitol, glycine betaine)

    • Low concentrations of non-denaturing detergents

• Protein refolding strategies if inclusion bodies form:

  • Denaturation with 6-8 M urea or guanidinium chloride

  • Stepwise dialysis for gradual refolding

  • On-column refolding with immobilized metal affinity chromatography

  • Addition of folding enhancers (arginine, low concentrations of detergents)

• Construct design considerations:

  • Domain analysis to ensure complete domains are included

  • Flexible linkers between domains if working with the bifunctional enzyme

  • Testing truncated constructs focusing on individual domains

  • Site-directed mutagenesis of aggregation-prone regions

• Analytical methods to monitor progress:

  • SDS-PAGE for expression level assessment

  • Western blotting for specific detection

  • Solubility fractionation to quantify soluble vs. insoluble expression

  • Activity assays to confirm functional folding

What are common pitfalls in riboflavin pathway engineering and how can they be overcome?

Engineering the riboflavin biosynthetic pathway presents several challenges that researchers should anticipate:

• Metabolic burden effects:

  • Challenge: Overexpression of multiple pathway genes can place significant burden on cellular resources

  • Solution: Use balanced expression strategies with different promoter strengths and plasmid copy numbers

  • Evidence: In engineered E. coli strains, coordinated expression of pathway genes resulted in optimal riboflavin production

• Redox balance disruption:

  • Challenge: Riboflavin biosynthesis affects cellular redox state (NADPH consumption)

  • Solution: Co-overexpress zwf (glucose-6-phosphate dehydrogenase) to increase NADPH availability

  • Evidence: zwf overexpression increased riboflavin production by 74.66% in engineered E. coli

• Feedback inhibition:

  • Challenge: FMN and FAD can inhibit pathway enzymes or repress gene expression

  • Solution: Delete or modify regulatory elements like the FMN riboswitch

  • Evidence: FMN riboswitch deletion increased ribB transcript levels 3.05-fold and riboflavin production by 37.17%

• Product conversion:

  • Challenge: Riboflavin is converted to FMN and FAD by RibF

  • Solution: Modulate ribF expression while maintaining essential cellular functions

  • Evidence: Approaches such as replacing native RBS of ribF with a weaker RBS or using synthetic regulatory small RNA have been effective

• Carbon flux limitations:

  • Challenge: Insufficient precursor availability (GTP and Ru5P)

  • Solution: Modulate central carbon metabolism to increase precursor supply

  • Evidence: Optimizing glucose concentration (40 g/L) in fed-batch fermentation significantly improved production

• Process scalability issues:

  • Challenge: Laboratory optimizations often don't translate to larger scales

  • Solution: Consider scale-up parameters during initial optimization

  • Evidence: Optimal fermentation conditions improved production from 437.58 mg/L to 611.22 mg/L, with further increases to 1,574.60 mg/L in fed-batch mode

How can researchers distinguish between the impacts of RibD overexpression versus other riboflavin pathway enzymes when analyzing production improvements?

Differentiating the specific contributions of RibD from other pathway enzymes requires targeted experimental design:

• Systematic single-gene studies:

  • Individually overexpress each pathway gene (ribA, ribB, ribC, ribD, ribE) in separate strains

  • Quantify riboflavin production and pathway intermediates for each strain

  • Compare transcript and protein levels to correlate expression with production

• Combinatorial expression analysis:

  • Create strains with different combinations of overexpressed genes

  • Design statistical models (e.g., Design of Experiments) to determine interaction effects

  • Reference example: The sequential construction of strains R1-R4 in E. coli, where combinations of gene overexpression and regulatory element deletion were evaluated

• Metabolite profiling:

  • Quantify all pathway intermediates using LC-MS/MS

  • Identify accumulation or depletion points to pinpoint bottlenecks

  • Compare profiles between RibD-only overexpression and multi-gene overexpression strains

• Enzyme activity assays:

  • Measure specific activities of all pathway enzymes in different strain backgrounds

  • Use enzyme kinetics data to model pathway flux

  • Correlate in vitro enzyme activities with in vivo production data

• Knockout/knockdown studies:

  • Use CRISPR interference or antisense RNA to create partial knockdowns of different pathway genes

  • Determine sensitivity of production to reduced activity of each enzyme

  • Complementation studies with varying expression levels

• Data analysis and modeling:

  • Apply metabolic control analysis to determine flux control coefficients

  • Use computational models to predict the impact of different enzyme level changes

  • Validate predictions with experimental data

The data table below illustrates how incremental pathway engineering can be used to differentiate contributions of various modifications:

What are the future directions for research on B. amyloliquefaciens RibD and riboflavin biosynthesis?

Future research on B. amyloliquefaciens RibD and riboflavin biosynthesis should focus on several promising directions:

• Comparative genomics and evolutionary studies:

  • Analyze RibD sequence and structure across various Bacillus species

  • Investigate the evolutionary relationships between mono- and bifunctional RibD enzymes

  • Explore the genomic context of riboflavin biosynthesis genes across bacterial species

• Advanced protein engineering:

  • Apply directed evolution specifically to B. amyloliquefaciens RibD

  • Explore structure-function relationships through crystallography and cryo-EM

  • Design chimeric enzymes combining advantageous properties from different species

• Systems biology approaches:

  • Develop genome-scale metabolic models of B. amyloliquefaciens

  • Apply multi-omics integration methods similar to those used in other B. amyloliquefaciens studies

  • Identify non-obvious metabolic interactions affecting riboflavin production

• Synthetic biology strategies:

  • Design synthetic operons optimizing gene arrangement and expression levels

  • Develop riboflavin-responsive genetic circuits for dynamic pathway regulation

  • Explore non-traditional hosts for heterologous expression of B. amyloliquefaciens RibD

• Application-focused research:

  • Investigate the potential probiotic applications of riboflavin-overproducing B. amyloliquefaciens strains

  • Explore B. amyloliquefaciens as a platform for in situ riboflavin delivery in agricultural or biomedical applications

  • Develop B. amyloliquefaciens strains capable of producing riboflavin from waste materials

• Novel analytical methods:

  • Develop biosensors for real-time monitoring of intracellular riboflavin levels

  • Apply single-cell technologies to understand population heterogeneity in riboflavin production

  • Implement machine learning approaches for predictive modeling of strain performance