Recombinant Ashbya gossypii Long chronological lifespan protein 2 (LCL2)

What is Ashbya gossypii LCL2 and how does it relate to other fungal orthologs?

LCL2 (Long Chronological Lifespan Protein 2) in A. gossypii is orthologous to the S. cerevisiae LCL2 protein with a high bitscore of 133, indicating significant sequence conservation . According to ortholog data, A. gossypii LCL2 (Q74ZY6) and S. cerevisiae LCL2 (Q08045) have an inparalog score of 1.0, suggesting they are functionally equivalent proteins . This conservation extends to other fungi, with varying degrees of similarity:

To investigate conservation experimentally, researchers should employ:

• Multiple sequence alignment of LCL2 sequences

• Phylogenetic analysis to determine evolutionary relationships

• Domain analysis to identify conserved functional regions

• Complementation assays testing functional equivalence between orthologs

What role does LCL2 play in Ashbya gossypii biology?

While the search results do not provide specific information about LCL2's function in A. gossypii, its name suggests involvement in chronological lifespan regulation (survival time of non-dividing cells). To determine its function, researchers should:

• Generate LCL2 knockout strains using CRISPR/Cas9 systems adapted for A. gossypii

• Compare phenotypes between wild-type and knockout strains, particularly focusing on:

  • Growth characteristics during trophic and productive phases

  • Stress responses, especially to environmental and secretion stresses

  • Chronological and replicative lifespan measurements

• Investigate genetic interactions with known lifespan regulators

• Examine expression patterns during different growth phases

A. gossypii transitions between trophic and productive phases, with significant physiological and transcriptional changes , making it important to study LCL2's role throughout these developmental stages.

What expression systems are most effective for recombinant A. gossypii LCL2 production?

Several expression systems have been developed for A. gossypii recombinant protein production with varying efficiencies:

• Native promoter systems: A. gossypii promoters AgTEF and AgGPD have shown up to 8-fold improvement over heterologous promoters like ScPGK1 .

• Vector designs: One-vector strategies containing all required modules have been optimized for A. gossypii, typically including:

  • Strong native promoters (AgTEF or AgGPD)

  • Appropriate terminator sequences

  • Selection markers (G418 resistance is common)

  • Secretion signal peptides when applicable

• Genome engineering tools:

  • CRISPR/Cas9 system adapted for A. gossypii enables marker-free engineering

  • Cre-loxP system allows removal and reuse of selection markers for multiple modifications

For optimal LCL2 expression, employ native A. gossypii promoters and consider the culture medium composition carefully. Glycerol as a carbon source has shown a 1.5-fold improvement over glucose for recombinant protein production .

What purification strategies are recommended for recombinant LCL2 from A. gossypii?

While specific LCL2 purification protocols aren't detailed in the search results, the following methodological approach is recommended based on general A. gossypii protein purification principles:

• Expression optimization:

  • Include an affinity tag (His-tag, FLAG) for easier purification

  • Consider secretion signals if extracellular production is desired

  • Use strong native promoters like AgTEF or AgGPD

• Extraction methods:

  • For intracellular proteins: Cell disruption methods appropriate for filamentous fungi (mechanical disruption with glass beads, enzymatic digestion of cell wall)

  • For secreted proteins: Direct recovery from culture supernatant

• Purification workflow:

  • Initial capture: Affinity chromatography based on fusion tags

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Quality control: SDS-PAGE (reference ranges 7-209 kDa, pI 4-6)

• Special considerations for A. gossypii:

  • High riboflavin content may interfere with some detection methods

  • The filamentous nature affects extraction efficiency

  • Culture harvesting by vacuum filtration using sterile paper filters

How can CRISPR/Cas9 be optimized for LCL2 manipulation in A. gossypii?

A specialized CRISPR/Cas9 system has been adapted for A. gossypii that allows precise genetic modifications . For LCL2 manipulation, implement the following protocol:

• System components:

  • Cas9 expression module using the TEF1 promoter and CYC1 terminator

  • sgRNA expression module under control of A. gossypii SNR52 promoter

  • Donor DNA for homologous recombination

• Design considerations for LCL2 targeting:

  • Select 20bp target sequences with NGG PAM sites

  • Design donor DNA with 40-50bp homology arms flanking the intended modification

  • Include selection markers or screening strategies

• Transformation protocol optimization:

  • Transform A. gossypii spores in the "germling" state for optimal efficiency

  • Incubate spores at 28°C for 10-12h to reach optimal germination stage

  • Collect spores by vacuum filtration using sterile paper filters

  • Approximately 10^8 spores should be used for transformation

• Verification strategies:

  • PCR confirmation of intended modifications

  • Sequencing to verify precise edits

  • Phenotypic analysis of mutants

This system enables various modifications including gene deletions, insertions, and nucleotide substitutions without permanent marker integration .

What advantages does the Cre-loxP system offer for complex genetic engineering of A. gossypii LCL2?

The Cre-loxP recombination system has been successfully adapted for A. gossypii, providing several methodological advantages for complex genetic manipulations :

• Marker recycling: The system allows removal and reuse of selection markers, enabling multiple sequential genetic modifications using limited marker options.

• Implementation strategy:

  • Design disruption cassettes with selection markers flanked by loxP sites

  • After selection and verification, induce Cre recombinase expression

  • Recombination between loxP sites removes the marker

  • Verify marker excision and proceed with subsequent modifications

• Applications for LCL2 research:

  • Generate clean deletions without marker interference

  • Create multiple modifications in regulatory regions

  • Introduce domain mutations while maintaining native regulation

  • Combine with CRISPR/Cas9 for precise editing

• Versatility:

  • Suitable for both laboratory and industrial strains

  • Does not require predetermined genetic backgrounds

  • Compatible with various selection systems including URA3 and ADE1 auxotrophic markers

This technique is particularly valuable when studying complex protein functions requiring multiple precise genetic modifications.

How can researchers assess the impact of LCL2 on A. gossypii chronological lifespan?

To investigate LCL2's role in chronological lifespan regulation, implement the following methodological approaches:

• Genetic manipulation:

  • Generate LCL2 deletion mutants using CRISPR/Cas9

  • Create complemented strains expressing wild-type or modified LCL2

  • Develop strains with tagged LCL2 for localization studies

• Chronological lifespan assays:

  • Culture cells to stationary phase in defined media

  • Monitor viability over time using standard plate count methods

  • Calculate survival curves and mean/maximum lifespan

  • Compare wild-type, deletion, and complemented strains

• Stress response analysis:

  • Challenge with various stressors (oxidative, heat, nutritional)

  • Monitor survival rates under stress conditions

  • Examine transcriptional responses using RT-qPCR or RNA-seq

  • Compare with known stress response patterns in A. gossypii

• Phase-specific analysis:

  • Examine LCL2 function during trophic versus productive phases

  • Monitor transcriptional changes during phase transitions

  • Investigate potential roles in riboflavin production regulation

A. gossypii undergoes significant transcriptional reprogramming during phase transitions and stress responses , making temporal analysis critical for understanding LCL2 function.

What approaches can determine LCL2's subcellular localization and protein interactions?

Understanding LCL2's subcellular localization and interactome is essential for functional characterization:

• Localization techniques:

  • Fluorescent protein tagging: Create LCL2-GFP or LCL2-mCherry fusions

  • Microscopy analysis: A. gossypii visualization requires specialized techniques due to its filamentous growth

  • Comparative analysis: SWAT-GFP and mCherry fusion proteins have been successfully used in A. gossypii to localize proteins to specific compartments

• Protein interaction studies:

  • Affinity purification-mass spectrometry (AP-MS)

  • Yeast two-hybrid screening

  • Proximity-dependent biotin identification (BioID)

  • Co-immunoprecipitation with tagged LCL2

• Dynamics analysis:

  • Live-cell imaging to track LCL2 movement

  • Nuclear-cytoplasmic shuttling (especially relevant given A. gossypii's multinucleated nature)

  • Association with stress granules or specific organelles

• Methodological considerations for A. gossypii:

  • A. gossypii grows as multinucleated hyphae with unique nuclear dynamics

  • Nuclei are constantly in motion within hyphae, potentially affecting protein localization patterns

  • Cytoplasmic microtubule dynamics differ significantly from S. cerevisiae

Studies with other A. gossypii proteins have revealed both expected and unexpected localizations compared to S. cerevisiae orthologs, highlighting the importance of direct experimental verification .

How does secretion stress affect LCL2 expression and function in A. gossypii?

A. gossypii exhibits complex responses to secretion stress that likely impact LCL2:

• Secretion stress effects on A. gossypii:

  • DTT treatment causes substantial reduction in growth rate

  • Transcriptional down-regulation of genes involved in filamentous growth, glycosylation, and cell wall biosynthesis within 30 minutes of stress induction

  • Ribosomal protein-encoding genes down-regulation correlates with reduced growth rate

• Experimental approaches to study LCL2 under stress:

  • Monitor LCL2 expression levels during DTT-induced stress using RT-qPCR

  • Compare with expression patterns of known stress-responsive genes

  • Examine stress survival in wild-type versus lcl2Δ strains

  • Investigate LCL2 localization changes during stress response

• Potential connections to biotechnological applications:

  • If LCL2 affects stress tolerance, it could impact recombinant protein production

  • Understanding LCL2's role in stress response could inform strain engineering strategies

  • Connections between chronological lifespan and stress resistance may be leveraged for industrial strain improvement

The research would provide insights into both fundamental biology and potential biotechnological applications of A. gossypii.

How can A. gossypii LCL2 research contribute to industrial biotechnology applications?

A. gossypii is an important industrial organism with several biotechnological applications that LCL2 research could enhance:

• Riboflavin production optimization:

  • A. gossypii naturally overproduces riboflavin (vitamin B2) and is used industrially for its production

  • If LCL2 affects cellular lifespan or stress resistance, it could be targeted to improve production strains

  • LCL2 manipulation might extend productive phase duration or increase cell viability

• Recombinant protein production:

  • A. gossypii has been developed as a host for recombinant protein expression

  • Understanding LCL2's impact on secretory pathways could improve heterologous protein yields

  • If LCL2 affects aging or stress responses, its modification might enhance production strain robustness

• Metabolic engineering applications:

  • A. gossypii has been engineered for production of various compounds including lipids , inosine, and flavor compounds

  • Lifespan extension through LCL2 modification could improve production metrics

  • Integration with existing genetic tools (CRISPR/Cas9 , Cre-loxP ) could facilitate complex pathway engineering

• Methodological advantages of A. gossypii:

  • Utilization of low-cost culture media

  • Inexpensive downstream processing

  • Wide range of molecular tools for genetic manipulation

  • Potential for marker-free engineering via CRISPR/Cas9

The insights from LCL2 research could be applied to improve these established applications and potentially develop new biotechnological processes.

What novel phenotypic assays can detect subtle LCL2-dependent effects in A. gossypii?

Given the potential involvement of LCL2 in chronological lifespan and stress responses, specialized assays are needed to detect subtle phenotypes:

• High-resolution growth analysis:

  • Monitor hyphal tip elongation speeds (wild-type colonies show measurable radial growth rates)

  • Assess branch initiation frequency and elongation success rates

  • Examine hyphal morphology for defects (wavy hyphae, bulbous tips)

  • Quantify actin patch polarization at hyphal tips

• Stress-specific phenotypic assays:

  • Heat, osmotic, and cell wall stress sensitivity tests

  • Quantitative survival measurements under oxidative stress

  • Recovery kinetics after acute stress exposure

  • Nuclear dynamics assessment under stress conditions

• Phase transition analysis:

  • Monitor trophic-productive phase transition timing

  • Quantify riboflavin production during phase transitions

  • Examine transcriptional profiles during phase shifts

  • Assess metabolic shifts between growth phases

• Developmental phenotypes:

  • Spore germination efficiency and timing

  • Sporulation competence under various conditions

  • Hyphal network architecture analysis

  • Nuclear distribution and movement patterns

These specialized assays can reveal subtle phenotypes missed by conventional growth measurements, particularly important for proteins like LCL2 that may have regulatory rather than essential functions.

How can researchers address poor expression or instability of recombinant LCL2?

If encountering difficulties with LCL2 expression or stability, consider the following methodological approaches:

• Expression optimization:

  • Switch to native A. gossypii promoters (AgTEF, AgGPD) which show up to 8-fold improvement over heterologous promoters

  • Try glycerol instead of glucose as carbon source (1.5-fold improvement for other recombinant proteins)

  • Optimize codon usage if expressing non-native versions of LCL2

  • Remove problematic terminator sequences that might have autonomous replicating activity

• Stability enhancement:

  • Include stabilizing fusion partners (MBP, SUMO, etc.)

  • Optimize cultivation temperature (standard is 28°C)

  • Screen for protease-deficient production strains

  • Include appropriate protease inhibitors during extraction

• Solubility improvement:

  • Test different signal peptides for secreted expression

  • Analyze potential glycosylation sites that may affect folding

  • Express as truncated domains if full-length protein is problematic

  • Screen different buffer conditions for optimal stability

• A. gossypii-specific considerations:

  • Account for potential interference from high riboflavin content

  • Consider protein extraction methods optimized for filamentous fungi

  • Manage the complex morphology of A. gossypii cultures for consistent harvesting

Systematic optimization of these parameters should resolve most expression and stability issues with recombinant LCL2.