Recombinant Ashbya gossypii Mitochondrial inner membrane magnesium transporter LPE10 (LPE10)

What is the known biological function of LPE10 in Ashbya gossypii?

LPE10 is characterized as a mitochondrial inner membrane magnesium transporter in Ashbya gossypii, responsible for maintaining magnesium homeostasis within mitochondria . As a key component of the mitochondrial transport machinery, it likely plays a critical role in energy metabolism and other magnesium-dependent mitochondrial processes. Methodologically, researchers can investigate its function through gene knockout studies comparing wild-type and LPE10-deficient strains, examining parameters such as growth rates, stress tolerance, and magnesium-dependent enzymatic activities. Complementation studies with homologous transporters from other organisms can further validate functional conservation.

How is the LPE10 protein structurally characterized and what functional domains have been identified?

The Ashbya gossypii LPE10 protein consists of 330 amino acids with the expression region spanning positions 14-330 . Based on its sequence and predicted structure, LPE10 likely contains multiple transmembrane domains characteristic of membrane transporters. Key structural approaches include:

• X-ray crystallography or cryo-electron microscopy for detailed structural determination

• Hydropathy plot analysis to identify transmembrane domains

• Site-directed mutagenesis of conserved residues to identify functional domains

• Protein modeling based on homologous transporters with known structures

The amino acid sequence suggests specific functional domains that may be involved in magnesium binding and transport across the inner mitochondrial membrane. Researchers investigating structure-function relationships should focus on conserved residues likely involved in ion recognition and passage.

How can I establish a reliable expression system for studying recombinant LPE10?

When establishing an expression system for recombinant LPE10, consider the following methodological approach:

• Selection of expression host: While E. coli is commonly used, eukaryotic hosts such as yeast (Saccharomyces cerevisiae) may be more suitable for proper folding of this eukaryotic membrane protein.

• Vector design: Include the LPE10 gene sequence (ADR049W) with appropriate fusion tags for detection and purification (His-tag, GFP, etc.) .

• Optimization strategy:

  • Codon optimization for the chosen expression host

  • Use of strong inducible promoters

  • Temperature and induction optimization

  • Addition of molecular chaperones to assist proper folding

• Verification methods:

  • Western blotting with specific antibodies

  • Functional assays to confirm transporter activity

  • Subcellular localization using fluorescence microscopy (for tagged constructs)

A methodological challenge to anticipate is the potential toxicity of overexpressed membrane proteins to the host system, which may require careful regulation of expression levels.

What purification strategies are most effective for recombinant LPE10 protein?

Purifying membrane transporters like LPE10 presents specific challenges requiring a tailored approach:

• Solubilization phase:

  • Use mild detergents (DDM, LMNG) to extract LPE10 from membranes without denaturation

  • Consider nanodiscs or amphipols as alternative solubilization methods for maintaining native conformation

• Chromatography strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged LPE10

  • Size exclusion chromatography for further purification and assessment of oligomeric state

  • Ion exchange chromatography as a polishing step

• Quality assessment:

  • SDS-PAGE with Coomassie staining for purity evaluation

  • Western blotting for protein identification

  • Circular dichroism to verify secondary structure integrity

  • Dynamic light scattering to assess homogeneity and aggregation state

• Activity verification:

  • Liposome reconstitution assays to confirm magnesium transport function

  • Patch-clamp electrophysiology for direct measurement of transport activity

Researchers should monitor protein stability throughout the purification process and adjust buffer conditions (pH, ionic strength, glycerol content) to maintain LPE10 in its native conformation.

What functional assays can be used to measure LPE10 magnesium transport activity?

Several complementary approaches can be employed to assess LPE10 transport activity:

• Liposome-based transport assays:

  • Reconstitute purified LPE10 into liposomes

  • Load liposomes with fluorescent magnesium indicators (Mag-Fura-2, Mag-Fluo-4)

  • Monitor fluorescence changes upon addition of external magnesium

  • Quantify transport kinetics (Km, Vmax) under varying conditions

• Cellular magnesium measurement:

  • Express LPE10 in model cell systems lacking endogenous magnesium transporters

  • Use magnesium-sensitive fluorescent dyes to monitor intracellular magnesium levels

  • Apply selective inhibitors to confirm specificity

• Electrophysiological approaches:

  • Patch-clamp recordings of membranes containing LPE10

  • Measurement of current changes in response to magnesium concentration gradients

• Isotope flux assays:

  • Use radioactive 28Mg to directly measure transport rates

  • Compare uptake in vesicles with and without functional LPE10

Each approach offers different advantages, and combining multiple methods provides more robust characterization of LPE10 transport properties.

How can CRISPR-Cas9 be implemented for LPE10 functional studies in A. gossypii?

CRISPR-Cas9 offers powerful approaches for studying LPE10 function in A. gossypii:

• Gene knockout strategy:

  • Design guide RNAs targeting the LPE10 (ADR049W) locus

  • Include appropriate homology arms for repair template

  • Screen transformants using PCR and sequencing verification

  • Validate knockout by RT-PCR and Western blotting

• Domain modification approach:

  • Create precise mutations in functional domains using HDR templates

  • Design mutations based on conserved residues identified through sequence alignment

  • Generate a series of systematic mutations to map critical residues

• Transcriptional regulation:

  • Implement CRISPRa/CRISPRi for modulation of LPE10 expression

  • Create conditional expression systems to study dosage effects

• Tagged variant generation:

  • Insert fluorescent or affinity tags for localization and interaction studies

  • Ensure tags don't interfere with protein folding or function

The filamentous nature of A. gossypii requires optimization of transformation protocols, with special attention to mycelial growth stages for optimal CRISPR-Cas9 efficiency. Initial validation in simpler model systems before moving to A. gossypii may increase success rates.

How might LPE10 function impact riboflavin production in engineered A. gossypii strains?

The potential relationship between LPE10 function and riboflavin production can be explored through several investigative approaches:

• Expression correlation analysis:

  • Compare LPE10 expression levels across high and low riboflavin-producing strains

  • Analyze transcriptomics data to identify correlations between LPE10 and riboflavin biosynthetic genes

• Magnesium dependency evaluation:

  • Assess whether riboflavin biosynthetic enzymes require magnesium as a cofactor

  • Investigate if mitochondrial magnesium levels regulated by LPE10 affect riboflavin precursor availability

• Metabolic flux analysis:

  • Trace carbon flow in LPE10-modified strains using 13C-labeled substrates

  • Determine if altered magnesium homeostasis affects flux through pathways feeding into riboflavin biosynthesis

• Engineering approach:

  • Create LPE10 overexpression and knockdown variants in production strains

  • Analyze resulting changes in riboflavin yield and production kinetics

A. gossypii is widely utilized for industrial riboflavin production , and understanding how mitochondrial magnesium transport interfaces with this process could lead to novel optimization strategies for vitamin production.

What role might LPE10 play in A. gossypii's ability to utilize different carbon sources?

Investigating LPE10's role in carbon source utilization requires a systematic approach:

• Growth phenotyping:

  • Compare growth of wild-type and LPE10-modified strains on various carbon sources (glucose, xylose, waste streams)

  • Measure consumption rates of different sugars in continuous culture

• Enzyme activity analysis:

  • Assess magnesium-dependent enzymes involved in alternative carbon metabolism pathways

  • Determine if LPE10 disruption affects their function

• Metabolic adaptation study:

  • Analyze transcriptional and proteomic changes in LPE10 mutants grown on different carbon sources

  • Identify compensatory mechanisms that emerge when magnesium transport is compromised

A. gossypii can effectively use various waste streams, including xylose-rich feedstocks , and mitochondrial function is critical for energy metabolism. The magnesium transport mediated by LPE10 may influence the activity of key metabolic enzymes involved in alternative carbon source utilization.

How does magnesium transport via LPE10 potentially affect monoterpene production in engineered A. gossypii?

The connection between LPE10-mediated magnesium transport and monoterpene production can be investigated through:

• Cofactor requirement analysis:

  • Determine magnesium dependency of enzymes in the monoterpene biosynthetic pathway

  • Assess if mitochondrial magnesium levels affect MEP/MVA pathway enzyme activities

• Metabolic engineering strategy:

  • Create combinatorial strains with modified LPE10 expression and overexpressed terpene synthases

  • Test monoterpene production in these strains under varying magnesium concentrations

• Compartmentalization study:

  • Investigate if mitochondrial-cytosolic magnesium exchange affects precursor availability

  • Analyze subcellular localization of rate-limiting steps in monoterpene biosynthesis

Engineered A. gossypii strains have demonstrated capability to produce various monoterpenes, including limonene and sabinene (with yields reaching ~700 mg/L) , establishing this organism as a promising platform for terpene production. Understanding the role of magnesium homeostasis in these processes could further enhance production efficiency.

What are the potential interaction partners of LPE10 in the mitochondrial membrane?

Identifying LPE10 interaction partners represents an advanced research direction that can be approached through:

• Proximity-dependent biotin labeling:

  • Express LPE10 fused to BioID or APEX2 in A. gossypii

  • Identify biotinylated proteins in the vicinity of LPE10 using mass spectrometry

  • Validate potential interactions through reciprocal tagging experiments

• Co-immunoprecipitation strategy:

  • Generate antibodies against LPE10 or use epitope-tagged variants

  • Perform pull-downs under conditions that preserve membrane protein interactions

  • Identify co-precipitated proteins by mass spectrometry

• Genetic interaction screening:

  • Conduct synthetic lethality screens with LPE10 partial loss-of-function mutants

  • Identify genes whose disruption exacerbates LPE10 phenotypes

  • Create an interaction network based on genetic dependencies

• Split-reporter assays:

  • Test candidate interactions using split-GFP or split-luciferase complementation

  • Visualize interaction dynamics in living cells

Understanding the LPE10 interactome would provide insights into how magnesium transport is regulated and coordinated with other mitochondrial processes in A. gossypii.

How does mitochondrial magnesium transport via LPE10 influence metabolic flux in A. gossypii?

This sophisticated research question requires advanced methodological approaches:

• Comprehensive metabolomics:

  • Compare metabolite profiles between wild-type and LPE10-modified strains

  • Use untargeted mass spectrometry to identify unexpected metabolic changes

  • Perform time-course analyses during different growth phases

• 13C metabolic flux analysis:

  • Feed LPE10 mutant and control strains with 13C-labeled carbon sources

  • Trace isotope incorporation through central carbon metabolism

  • Develop computational models to quantify flux differences

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Identify regulatory points affected by altered magnesium homeostasis

  • Construct predictive models of metabolic adaptation

• In vivo metabolite sensing:

  • Deploy genetically encoded sensors for real-time monitoring of key metabolites

  • Correlate metabolite fluctuations with magnesium availability

This research direction could reveal how mitochondrial magnesium levels serve as a regulatory mechanism for metabolic adaptation in A. gossypii, potentially informing new engineering strategies.

What is the relationship between LPE10 function and production of other high-value compounds in A. gossypii?

Beyond established applications in riboflavin and monoterpene production, A. gossypii shows potential for producing various valuable compounds. The relationship between LPE10 function and these processes can be explored through:

• Biolipid production analysis:

  • Compare lipid profiles in wild-type and LPE10-modified strains

  • Analyze fatty acid composition and accumulation under various conditions

  • Test if magnesium transport affects lipid droplet formation and composition

• Folate biosynthesis investigation:

  • Examine if folate production is influenced by LPE10-mediated magnesium transport

  • Test if supplementation with magnesium can enhance folate yields in engineered strains

• Protein expression system development:

  • Evaluate if LPE10 function affects recombinant protein yields and quality

  • Determine if magnesium homeostasis impacts protein folding and secretion

A. gossypii has been developed for applications beyond riboflavin production, including recombinant proteins, single cell oils (SCOs), and flavor compounds . Understanding how fundamental processes like magnesium transport interface with these applications could expand the biotechnological potential of this organism.