Recombinant Ashbya gossypii tRNA (guanine-N (7)-)-methyltransferase subunit TRM82 (TRM82)

What is the function of TRM82 in Ashbya gossypii?

TRM82 in Ashbya gossypii functions as a vital subunit of the tRNA m7G methyltransferase complex, partnering with TRM8. Based on homology with Saccharomyces cerevisiae, TRM82 plays a crucial dual role in the complex: it maintains cellular levels of TRM8 protein and stabilizes TRM8 in an active conformation. The TRM8/TRM82 complex is responsible for adding a methyl group to the N7 position of guanosine at position 46 (m7G46) in specific tRNAs, a modification essential for proper tRNA function and stability during protein translation . This modification mechanism is evolutionarily conserved, though interestingly bacterial species appear to accomplish the same function with a single protein rather than a two-protein complex .

How does the TRM8/TRM82 complex contribute to tRNA modification in A. gossypii?

The TRM8/TRM82 complex in A. gossypii catalyzes the formation of 7-methylguanosine (m7G) at position 46 in specific tRNAs, similar to its role in S. cerevisiae. The functional divisions within this complex are noteworthy: TRM8 contains both the S-adenosylmethionine (SAM) binding domain (required for methyl group donation) and the tRNA binding capacity . TRM82, by contrast, does not directly bind to tRNA or contain a SAM-binding domain, as shown through cross-linking studies . Instead, it appears to function primarily to stabilize TRM8 in an active conformation and regulate its cellular levels. Without TRM82, TRM8 protein levels drop dramatically (to <6% of normal levels) and even overexpressed TRM8 remains enzymatically inactive (<1% of normal activity) .

What characteristics distinguish A. gossypii as a model organism for TRM82 research?

Ashbya gossypii offers several distinct advantages as a model organism for studying TRM82 and related genes:

• A. gossypii is a filamentous fungus with multinucleated hyphae that has been extensively used for industrial production of riboflavin and other valuable metabolites .

• Despite its filamentous growth pattern, A. gossypii possesses a genome with extensive synteny to S. cerevisiae, making it valuable for comparative genomics studies .

• A. gossypii can effectively utilize various waste streams as carbon sources, including xylose-rich feedstocks, making it industrially relevant .

• The fungus has been successfully engineered for production of diverse compounds, demonstrating its genetic tractability .

• Recent development of new promoters and molecular tools has expanded the A. gossypii genetic engineering toolkit, facilitating studies of genes like TRM82 .

These characteristics make A. gossypii an excellent system for investigating the roles of TRM82 in metabolism, stress response, and translation regulation in a filamentous fungal context.

What are the optimal methods for purifying recombinant A. gossypii TRM82?

For purifying recombinant A. gossypii TRM82, researchers should consider the following optimized protocol based on approaches used for other A. gossypii proteins:

• Expression system selection: Express His-tagged TRM82 in E. coli, similar to the approach used for other A. gossypii proteins . For optimal activity, consider co-expressing with TRM8 since TRM82 stabilizes TRM8.

• Purification workflow:

  • Lyse cells in a Tris/PBS-based buffer with protease inhibitors

  • Perform Ni-NTA affinity chromatography for initial purification

  • Apply size exclusion chromatography for further purification

  • Consider ion exchange chromatography if additional purity is required

• Buffer optimization:

  • Maintain protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0 for stability

  • Add glycerol (5-50% final concentration) for long-term storage

  • Store as aliquots at -20°C/-80°C to avoid freeze-thaw cycles

• Special considerations:

  • Centrifuge vials briefly before opening to bring contents to the bottom

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Analyze purity by SDS-PAGE (aim for >90% purity)

  • For active enzyme studies, verify that TRM82 co-purifies with TRM8

The presence of TRM82 is critical for obtaining active methyltransferase complex, as TRM8 produced without TRM82 is essentially inactive (<1% normal activity) .

How can one assess the functional activity of recombinant TRM82?

Assessing functional activity of recombinant TRM82 requires monitoring its effects on the TRM8/TRM82 methyltransferase complex activity. The following methodological approach is recommended:

• In vitro methyltransferase assay:

  • Reconstitute the TRM8/TRM82 complex by combining purified proteins

  • Prepare substrate tRNA either by in vitro transcription or isolation from trm8Δ/trm82Δ strains

  • Set up reactions containing the complex, tRNA substrate, and S-adenosylmethionine (SAM)

  • Incubate at 30°C in appropriate buffer conditions

  • Detect m7G formation through:

    • Incorporation of radiolabeled methyl groups from [3H]-SAM

    • Mass spectrometry analysis of modified nucleosides

    • Antibody detection of m7G modification

• Control experiments:

  • TRM8 alone (should show minimal activity)

  • TRM82 alone (should show no activity)

  • TRM8 from trm82Δ strain (should be inactive)

  • TRM8/TRM82 complex (full activity)

• TRM82 functionality assessment:

  • Measure TRM8 protein levels in the presence/absence of TRM82 via western blot

  • Compare TRM8 enzyme activity with wild-type vs. mutant TRM82 variants

  • Assess complex formation using analytical size exclusion chromatography

  • Evaluate TRM8-tRNA crosslinking efficiency with or without TRM82

The research indicates that TRM82's primary function is maintaining TRM8 stability and conformation rather than direct catalytic activity, so assays measuring TRM8 levels and activity serve as proxies for TRM82 functionality .

What strategies should be employed for studying TRM82-TRM8 interactions in A. gossypii?

To effectively study TRM82-TRM8 interactions in A. gossypii, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Generate epitope-tagged versions of TRM82 and TRM8

  • Prepare lysates under conditions that preserve protein-protein interactions

  • Immunoprecipitate one protein and detect the presence of the partner

  • Include RNase treatment controls to determine if the interaction is RNA-dependent

• Biochemical characterization:

  • Perform size exclusion chromatography to analyze complex formation

  • Use surface plasmon resonance to measure binding kinetics

  • Apply analytical ultracentrifugation to determine stoichiometry

  • Employ thermal shift assays to assess complex stability

• Functional complementation:

  • Express bacterial TRM8 homologs (which function without TRM82) in A. gossypii trm8Δ/trm82Δ strains

  • Test whether bacterial enzymes can complement both single and double mutants

  • Create chimeric proteins between A. gossypii and bacterial methyltransferases

• Cross-linking studies:

  • Perform in vitro UV cross-linking with TRM8, TRM82, and tRNA substrates

  • Analyze which proteins directly contact the RNA (evidence suggests only TRM8 cross-links to tRNA)

  • Use chemical cross-linkers to map protein-protein interaction interfaces

Research reveals that TRM8 efficiently cross-links to pre-tRNA Phe in both the presence and absence of TRM82, indicating TRM8 contains the tRNA binding site . Additionally, though TRM8 retains some capacity to bind tRNA without TRM82, it requires TRM82 for full enzymatic activity and protein stability .

How does TRM82 overexpression affect metabolic pathways in A. gossypii?

Recent research demonstrates that overexpression of the TRM8/TRM82 complex significantly impacts cellular metabolism, particularly in terpenoid biosynthesis pathways:

• Enhanced squalene biosynthesis:

  • Overexpression of Trm8/Trm82 complex significantly increases squalene production in yeast

  • This enhancement is linked to broader metabolic regulation effects

• Upregulation of central carbon metabolism:

  • Transcriptome analysis revealed upregulation of glycolysis genes (HXK1, PGI1, PFK1, TDH1, TDH3, ENO1, ENO2, CDC19)

  • TCA cycle genes were similarly upregulated

  • These changes likely increase acetyl-CoA availability, a precursor for terpenoid biosynthesis

• Impact on amino acid biosynthesis:

  • Altered expression of genes involved in tryptophan biosynthesis (ARO3, ARO1, TRP2, TRP3, TRP4, TRP5)

  • Changes in histidine biosynthesis pathways

• Enhanced terpenoid pathway:

  • The overexpression of TRM8/TRM82 led to increased expression of MVA pathway genes

  • This resulted in elevated production of terpenoids, including squalene and potentially other valuable compounds

These findings suggest that TRM82, beyond its direct role in tRNA modification, indirectly influences central metabolism through effects on translation or other regulatory mechanisms, making it a potential target for metabolic engineering in A. gossypii .

What role does TRM82 play in stabilizing TRM8 protein levels in A. gossypii?

Based on detailed studies in S. cerevisiae, TRM82 plays critical roles in maintaining both TRM8 protein stability and enzymatic activity:

• Regulation of TRM8 protein levels:

  • In trm82Δ strains, TRM8 protein levels decrease to <6% of wild-type amounts

  • This reduction occurs without changes in TRM8 mRNA levels, indicating post-transcriptional regulation

  • TRM8 levels are fully restored upon introduction of a plasmid expressing TRM82

• Activation of TRM8 enzymatic function:

  • TRM8 overexpressed in trm82Δ strains is essentially inactive (<1% normal activity)

  • This indicates TRM82 is required not just for TRM8 stability but also for its catalytic function

  • TRM82 likely stabilizes TRM8 in an active conformation

• Mechanistic implications:

  • TRM82 may function as a chaperone for TRM8, preventing its misfolding or aggregation

  • It likely shields TRM8 from proteolytic degradation systems

  • The interaction may induce conformational changes in TRM8 required for substrate binding or catalysis

This dual role in both protein stability and enzymatic activation distinguishes TRM82 from subunits in other tRNA modification complexes, where functions are more clearly partitioned between subunits (e.g., in Gcd10p/Gcd14p m1A58 methyltransferase complex, one subunit binds SAM and the other binds tRNA) .

How can TRM82 be exploited for metabolic engineering in A. gossypii?

The recent discovery that TRM8/TRM82 overexpression enhances terpenoid biosynthesis opens new avenues for metabolic engineering applications in A. gossypii:

• Enhanced production of high-value compounds:

  • A. gossypii is already used for riboflavin production and has shown potential for producing monoterpenes like limonene and sabinene at impressive yields (383 mg/L and 684.5 mg/L respectively)

  • TRM82 overexpression could further enhance these production capabilities by boosting central carbon metabolism

• Optimization strategies:

  • Co-overexpress TRM82 with key pathway enzymes like HMG1 and ERG12 to synergistically enhance production

  • Combine TRM82 overexpression with modifications to the native ERG20 (F95W mutation) to redirect flux toward desired products

  • Implement the NPP synthase orthogonal pathway alongside TRM82 overexpression

• Valorization of waste feedstocks:

  • A. gossypii can effectively utilize xylose-rich feedstocks and various waste streams

  • TRM82 overexpression could enhance the efficiency of converting these waste resources to high-value products

  • Mixed formulations of corn-cob lignocellulosic hydrolysates with sugarcane or beet molasses have shown promise

• Metabolic engineering targets:

  • Focus on enhancing glycolysis and acetyl-CoA production, which are upregulated by TRM82 overexpression

  • Consider co-optimization of tRNA pool alongside TRM82 expression for maximum translational efficiency

  • Explore combinatorial approaches involving both transcriptional and translational engineering

This approach represents an innovative strategy that leverages translational modification machinery to enhance metabolic output, potentially opening new paradigms in metabolic engineering .

What promoters are most effective for TRM82 expression in A. gossypii?

Recent advances in A. gossypii promoter development provide several optimized options for TRM82 expression:

• Strong constitutive promoters:

  • P_CCW12: Recently identified as a novel strong promoter in A. gossypii

  • Traditional options: P_TEF and P_GPD remain reliable choices for strong expression

• Regulatable promoters:

  • Carbon source-regulatable promoters: Allow controlled expression based on available carbon sources

  • These are particularly valuable for metabolic engineering applications where conditional expression is desired

• Promoter selection considerations:

  • Expression level requirements: Match promoter strength to the desired TRM82 expression level

  • Temporal control: Consider whether constitutive or inducible expression is more appropriate

  • Metabolic burden: Very strong promoters may impose growth penalties

  • Compatibility with target pathways: Choose promoters not affected by the same regulatory elements as target pathways

• Evaluation methodology:

  • The Dual Luciferase Reporter (DLR) Assay has been adapted for promoter analysis in A. gossypii using integrative cassettes

  • This system provides quantitative assessment of promoter performance under various conditions

For TRM82 overexpression aimed at enhancing terpenoid biosynthesis, a strong constitutive promoter like P_CCW12 would be appropriate to ensure consistent high-level expression. For functional studies requiring more physiological expression, the native TRM82 promoter or a moderate-strength constitutive promoter would be more suitable .

How can CRISPR-Cas9 be implemented for studying TRM82 function in A. gossypii?

Implementing CRISPR-Cas9 for TRM82 manipulation in A. gossypii requires consideration of this organism's unique characteristics:

• Guide RNA design:

  • Select target sites in TRM82 with minimal off-target potential

  • Account for A. gossypii's GC content (~50%) when designing guides

  • For functional studies, target conserved domains identified through homology with S. cerevisiae TRM82

• Delivery system optimization:

  • Use appropriate promoters for Cas9 expression (such as the strong promoters identified in recent research)

  • Express guide RNAs using RNA polymerase III promoters

  • Consider transient vs. stable expression strategies

• Homology-directed repair templates:

  • Design with sufficient homology arms (700-1000 bp recommended)

  • Include selection markers appropriate for A. gossypii

  • For functional studies, create point mutations in key residues rather than complete knockouts

• Special considerations for A. gossypii:

  • Account for the multinucleated nature of A. gossypii hyphae

  • Multiple rounds of selection may be required to achieve homokaryotic mutants

  • Verify edits both genetically and phenotypically

• Experimental design options:

  • Gene deletions to study complete loss of function

  • Point mutations to study specific domain functions

  • Promoter replacements to modulate expression levels

  • C-terminal tagging for localization and interaction studies

This approach enables precise genetic manipulation of TRM82 to explore its roles in tRNA modification, protein stabilization, and metabolic regulation within the context of A. gossypii's unique cellular architecture .

What methods are most effective for analyzing TRM82's impact on tRNA modification profiles?

To comprehensively assess how TRM82 affects tRNA modification profiles in A. gossypii, researchers should employ these complementary methodologies:

• High-throughput sequencing approaches:

  • Hydro-tRNAseq: Allows detection of modified nucleosides through their effect on reverse transcriptase

  • NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry): Enables quantitative assessment of tRNA modifications

• Targeted analysis methods:

  • Primer extension assays: Detect m7G by reverse transcriptase stops

  • Northern blot with antibodies specific to m7G modifications

  • High-resolution mass spectrometry of isolated tRNAs

• Functional readouts:

  • Polysome profiling to assess translation efficiency changes

  • Ribosome profiling to identify specific mRNAs affected by loss of m7G modifications

  • tRNA charging assays to determine aminoacylation efficiency

• Comparative analysis:

  • Compare modification profiles between wild-type, trm82Δ, and TRM82-overexpression strains

  • Examine modification changes across different growth conditions and stress responses

  • Correlate tRNA modification changes with metabolic alterations observed in transcriptomic data

This multi-faceted approach provides a comprehensive view of how TRM82-dependent modifications affect the tRNA pool and subsequently influence translation and metabolism. The impact on tRNA modification is particularly relevant given the observed effects of TRM8/TRM82 overexpression on terpenoid biosynthesis and central metabolism .

How can researchers investigate the connection between TRM82 activity and terpenoid biosynthesis?

To investigate the mechanistic link between TRM82 activity and enhanced terpenoid biosynthesis, researchers should implement these methodological approaches:

• Genetic manipulation experiments:

  • Create a series of strains with varying TRM82 expression levels

  • Generate catalytically inactive TRM82 mutants to separate protein presence from activity

  • Construct strains with enhanced terpenoid pathways with and without TRM82 overexpression

• Metabolic analysis:

  • Measure terpenoid compound levels using GC-MS or LC-MS

  • Perform 13C metabolic flux analysis to track carbon flow through central metabolism

  • Quantify precursor metabolites including acetyl-CoA and mevalonate pathway intermediates

• Translational impact assessment:

  • Analyze translation efficiency of glycolysis and terpenoid pathway enzymes

  • Examine tRNA modification status of codons enriched in these pathways

  • Perform ribosome profiling to identify transcripts with altered translation in TRM82 mutants

• Integration with existing metabolic engineering platforms:

  • Test TRM82 overexpression in strains optimized for monoterpene production

  • Combine with other enhancement strategies like HMG1 and ERG12 overexpression

  • Test performance on various carbon sources, including waste feedstocks

By systematically investigating these connections, researchers can develop optimized A. gossypii strains that leverage TRM82's impact on translation and metabolism to enhance production of valuable terpenoid compounds .