lldD E. coli
Description
Gene and Protein Structure
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Gene: The lldD gene (b3605, JW3580) is part of the lldPRD operon, which includes lldP (permease), lldD (dehydrogenase), and lldR (regulatory protein) .
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Protein: The recombinant lldD enzyme is a 45.3 kDa polypeptide with a 24-amino acid His-tag for purification. It contains 420 amino acids (1–396) and is non-glycosylated .
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Active Site: Utilizes FMN (flavin mononucleotide) as a cofactor, enabling redox reactions between pyruvate and L-lactate .
| Property | Value | Source |
|---|---|---|
| Molecular Weight | 45.3 kDa | |
| Amino Acid Sequence | MGSSHHHHHH... (full sequence in ) | |
| Optimal pH | 8.0 | |
| Expression System | E. coli (recombinant) |
L-Lactate Production
Engineered E. coli strains overexpressing lldD from Bacillus coagulans (e.g., strain 090B3) achieve high L-lactate yields:
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Production Yield: 142.2 g/L L-lactate under temperature-shifting fermentation (37°C growth, 42°C production) .
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By-Products: Minimal acetate, pyruvate, or succinate (<1.2 g/L) due to optimized metabolic flux .
Catabolic Utilization
Under anaerobic conditions, lldD enables E. coli to oxidize L-lactate to pyruvate, feeding into energy-generating pathways (e.g., TCA cycle) .
LldR Transcription Factor
LldR regulates the lldPRD operon via a dual role:
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Repressor: Binds to operator sites O1 and O2, inhibiting transcription in the absence of L-lactate .
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Activator: L-Lactate induces a conformational change in LldR, disrupting DNA looping and enabling transcription .
Additional Regulatory Inputs
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ArcAB Two-Component System: Activates lldPRD under anaerobic conditions .
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PdhR: Pyruvate-sensing transcription factor indirectly modulates lactate metabolism .
Strain Engineering for Bioproduction
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Thermodynamic Optimization: Engineered lldD variants (e.g., from Lactobacillus or Streptobacillus) improve L-lactate production by shifting equilibrium toward lactate synthesis .
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Acid Resistance: LldR regulates genes for glutamate-dependent acid resistance (e.g., gadW, gadY) and membrane lipid remodeling (e.g., lpxP) .
Lactate-Inducible Systems
LldD-containing operons are conserved across E. coli, Cupriavidus, and Pseudomonas spp., enabling lactate-responsive genetic circuits for synthetic biology .
Enzyme Properties
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Catalytic Activity: Oxidizes hydroxybutyrate (alternative substrate) and reduces pyruvate to L-lactate .
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Purification: Recombinant lldD is purified via affinity chromatography (His-tag) and stored in Tris-HCl buffer with glycerol/DTT .
Industrial Production
Product Specs
Q&A
lldD in E. coli: Research-Focused FAQs
What regulatory mechanisms control lldD expression, and how do researchers validate these interactions?
The lldPRD operon (including lldD) is regulated by:
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LldR: A lactate-responsive transcription factor that activates lldPRD in the presence of L-/D-lactate .
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ArcAB: A two-component system that represses lldPRD under anaerobic conditions .
Methodological approaches:
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Perform electrophoretic mobility shift assays (EMSAs) with purified LldR protein and the lldP promoter region .
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Use reporter plasmids (e.g., lacZ fusions) to quantify promoter activity in ΔlldR or ΔarcB mutants .
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Conduct chromatin immunoprecipitation (ChIP) to confirm in vivo binding of LldR to the lldPRD operon .
How do conflicting reports about constitutive vs. inducible lldD expression in E. coli arise, and how can they be resolved?
Discrepancies may stem from:
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Strain-specific variations: Wild-type vs. engineered strains (e.g., lactate-overproducing mutants) .
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Carbon source availability: lldD is constitutively expressed at low levels but strongly induced by lactate .
Resolution strategies:
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Compare transcript levels (via RNA-seq) in E. coli K-12 vs. metabolic engineering strains like B0013-070 .
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Test induction kinetics using lactate analogs (e.g., glycolate) to rule out cross-regulation .
What experimental challenges arise when engineering E. coli for enhanced L-lactate production via lldD manipulation?
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Thermodynamic limitations: l-iLDH activity is temperature-sensitive. Strains expressing thermophilic L-LDH variants show improved lactate yields at 42°C .
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By-product accumulation: Competing pathways (e.g., acetate synthesis) require knockout of pta-ackA or poxB .
Optimization workflow:
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Introduce a temperature-inducible promoter (e.g., λpL/pR) to decouple growth (37°C) and production (42°C) phases .
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Use flux balance analysis (FBA) to predict optimal gene knockout targets .
How does lldD interact with acid resistance systems in E. coli during lactate exposure?
LldR (regulator of lldPRD) co-activates glutamate-dependent acid resistance genes (e.g., gadE), enabling survival under lactic acid stress .
Experimental validation:
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Perform lactic acid tolerance assays comparing wild-type and ΔlldR strains .
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Measure intracellular pH using pH-sensitive fluorescent dyes (e.g., BCECF-AM) in lactate-challenged cells .
Key finding:
| Strain | Viability (%) at 120 mM L-lactate |
|---|---|
| Wild-type | 78 ± 5 |
| ΔlldR | 32 ± 4 |
What advanced techniques are used to map the global regulatory network of lldD in E. coli?
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Genomic SELEX (gSELEX): Identifies all LldR-binding sites genome-wide, revealing novel targets like membrane lipid biosynthesis genes .
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CRISPRi-based repression: Silences lldD to study its metabolic interactions with central carbon metabolism .
Integration with multi-omics:
Product Science Overview
To enable E. coli to produce L-lactate, scientists have employed genetic engineering techniques. This involves cloning and expressing L-lactate dehydrogenase genes from different bacteria into E. coli. The process typically includes the following steps :
- Gene Cloning: L-LDH genes from various bacteria are cloned into plasmids.
- Gene Expression: These plasmids are then introduced into E. coli strains, enabling the expression of L-LDH.
- Metabolic Engineering: Specific genes in E. coli are deleted or modified to enhance L-lactate production. For example, deleting the ldhA gene to block D-lactate formation and deleting the lldD gene to prevent the conversion of L-lactate to pyruvate .
Recombinant L-LDH produced in E. coli is a single, non-glycosylated polypeptide chain. It typically contains 420 amino acids and has a molecular mass of approximately 45.3 kDa. The recombinant enzyme is often fused with a His-tag at the N-terminus to facilitate purification using chromatographic techniques .
The production of L-lactate using engineered E. coli has significant biotechnological applications. L-lactic acid is an important chiral molecule used in various industries, including food, pharmaceuticals, and biodegradable plastics. The ability to produce L-lactate efficiently through microbial fermentation offers a sustainable and cost-effective alternative to traditional chemical synthesis methods .
One of the main challenges in producing L-lactate using E. coli is balancing the competition between cell growth and lactate synthesis. The enzymatic properties, especially the thermodynamics of L-LDH, play a crucial role in regulating metabolic pathways and optimizing lactate production. Future research aims to further enhance the efficiency of L-lactate production by exploring new genetic modifications and optimizing fermentation processes .
In conclusion, the recombinant L-Lactate Dehydrogenase from E. coli represents a significant advancement in metabolic engineering, offering promising solutions for sustainable production of valuable biochemicals.
