Recombinant Ashbya gossypii Mitochondrial group I intron splicing factor CCM1 (CCM1), partial

What is Ashbya gossypii and why is it used as a model organism?

Ashbya gossypii is a filamentous ascomycete fungus belonging to the yeast family Saccharomycetaceae. It serves as an excellent model organism for several reasons. First, it is closely related to unicellular yeasts such as Saccharomyces cerevisiae, sharing significant genomic similarity while exhibiting filamentous growth. This makes it ideal for studying the regulatory networks governing morphological differences between yeast and filamentous growth forms . Second, A. gossypii is known for naturally overproducing riboflavin (vitamin B2), making it industrially relevant. Third, the complete genome sequence of A. gossypii has been determined, facilitating comparative genomic analyses with other fungi. Finally, the fungus possesses a highly efficient homologous recombination system that allows for relatively straightforward genetic manipulation .

What are the key genomic features of A. gossypii?

A. gossypii possesses a compact genome that has been fully sequenced. Comparative genomic studies have revealed that A. gossypii and A. aceri have undergone chromosome number reduction from eight to seven chromosomes, while Eremothecium coryli has six chromosomes. In contrast, E. cymbalariae maintains the ancestral number of eight chromosomes . The A. gossypii genome sequence provided crucial evidence supporting the Whole Genome Duplication theory in the Saccharomyces lineage . Despite its filamentous growth pattern, A. gossypii's genetic makeup is remarkably similar to that of S. cerevisiae, making it an invaluable comparative model for understanding the genetic basis of morphological development.

How does recombinant expression affect the structure and function of CCM1?

Recombinant expression of CCM1 may influence its structure and function in several ways. When expressed recombinantly, CCM1 is typically produced as a partial protein rather than the full-length native form, which may affect its folding, stability, and activity. The recombinant expression system itself can introduce modifications not present in the native protein. For instance, A. gossypii has been shown to glycosylate many of its secreted proteins, with approximately 67% of its predicted secretome containing at least one N-glycosylation site . If recombinant CCM1 undergoes differential post-translational modifications compared to the native protein, this could significantly impact its splicing activity and interactions with other proteins or RNA molecules.

What are the established methodologies for genetic manipulation of A. gossypii?

A. gossypii benefits from a versatile genetic toolkit that has expanded significantly in recent years. The traditional approach leverages the fungus's highly efficient homologous recombination system for gene targeting . More recent advances have introduced CRISPR/Cas9 and CRISPR-Cpf1 systems adapted specifically for A. gossypii, enabling efficient multiplex genome editing . The available toolbox includes:

• Different marker genes for selection

• Regulatable promoters for controlled expression

• Cre-lox based marker removal systems

• CRISPR/Cas9 for precise genome editing

• CRISPR-Cpf1 for multiplex genome editing using T-rich PAM sequences (5'-TTTN-3')

For CCM1 specifically, researchers can employ these techniques to create knockout strains, introduce point mutations, or express tagged versions of the protein for localization studies.

How can CRISPR-Cpf1 be utilized for manipulating CCM1 in A. gossypii?

The CRISPR-Cpf1 system provides several advantages for manipulating the CCM1 gene in A. gossypii. Unlike Cas9, which requires a 5'-NGG-3' PAM sequence, Cpf1 from Lachnospiraceae bacterium recognizes T-rich PAM sequences (5'-TTTN-3'), expanding the range of potential target sites within the CCM1 locus . The system has been validated for introducing large deletions in multiple auxotrophic marker genes (HIS3, ADE2, TRP1, LEU2, and URA3) in A. gossypii .

To manipulate CCM1 using CRISPR-Cpf1:

• Design crRNAs targeting the CCM1 locus, considering that target sequence selection significantly affects editing efficiency

• Construct a CRISPR-Cpf1 plasmid containing the crRNA and, if desired, donor DNA for homology-directed repair

• Transform A. gossypii with the plasmid

• Select transformants using appropriate markers

• Confirm editing through PCR, sequencing, or functional assays

The multiplex capabilities of the Cpf1 system also allow for simultaneous targeting of CCM1 and other genes of interest, enabling comprehensive studies of genetic interactions in a single transformation event .

What methodologies are optimal for studying mitochondrial protein localization in A. gossypii?

For studying the localization of mitochondrial proteins like CCM1 in A. gossypii, researchers can employ several complementary approaches:

• Fluorescent protein tagging: Tagging CCM1 with fluorescent proteins such as GFP or mCherry can allow direct visualization of its localization within living cells. This approach has been successfully used in A. gossypii to study other proteins like AgSec3, AgSec5, and AgExo70 .

• Immunofluorescence microscopy: For proteins where direct tagging might interfere with function or localization, immunostaining with specific antibodies offers an alternative approach.

• Subcellular fractionation: Biochemical separation of mitochondria from other cellular components, followed by Western blotting, can confirm mitochondrial localization of CCM1.

• Growth-dependent analyses: As shown in studies of polarisome proteins, A. gossypii exhibits growth-speed-correlated localization patterns for certain proteins . For CCM1, comparing localization in slow-growing versus fast-growing hyphae might reveal functional insights.

When designing localization experiments, it's important to consider that A. gossypii's filamentous growth pattern creates distinct cellular compartments with potentially different protein distribution patterns.

How can researchers assess CCM1 splicing activity in vitro and in vivo?

Assessing CCM1 splicing activity requires specialized techniques targeting RNA processing:

In vitro assays:

• Reconstitution of splicing activity: Purified recombinant CCM1 can be tested for its ability to facilitate splicing of group I introns in a controlled environment with synthesized RNA substrates.

• RNA gel shift assays: To test binding of CCM1 to target introns, researchers can use electrophoretic mobility shift assays (EMSAs) with labeled RNA.

• Splicing kinetics measurements: Real-time monitoring of splicing reactions using fluorescent reporters can reveal the catalytic efficiency of CCM1.

In vivo approaches:

• RT-PCR analysis: Comparing the ratio of spliced to unspliced mitochondrial transcripts in wild-type versus CCM1-mutant strains.

• Northern blotting: Detecting accumulation of unspliced precursors or reduction in mature RNAs in CCM1-deficient strains.

• RNA-seq: Genome-wide assessment of splicing efficiency across all mitochondrial introns to identify specific targets of CCM1.

• Genetic complementation: Testing whether CCM1 variants can rescue defects in CCM1-deficient strains, providing insights into structure-function relationships.

What are the characteristics of protein secretion in A. gossypii?

A. gossypii possesses a well-characterized protein secretion system that has been exploited for recombinant protein production. Key features include:

• Secretome size and composition: The A. gossypii predicted secretome consists of 54 proteins (approximately 1% of its total proteome), all of which have homologs in closely related Saccharomycotina species . About 67% of these secreted proteins contain at least one N-glycosylation site, and 33% are predicted to have hydrolytic activity .

• Secretion capacity: A. gossypii secretes proteins at considerable levels, with reported values of 130 mg/L total protein in defined minimal medium (DMM) and 218 mg/L in rich medium (AFM) . This indicates its potential utility as a host for recombinant protein production.

• Secreted protein characteristics: Most secreted proteins from A. gossypii have isoelectric points between 4 and 6 and molecular weights above 25 kDa, though proteins secreted exclusively in rich medium may have slightly higher isoelectric points (6-8) .

• Secretion stress response: A. gossypii responds to secretion stress (e.g., DTT treatment) with transcriptional changes affecting filamentous growth, glycosylation, lipoprotein biosynthesis, and cell wall biosynthesis genes .

These characteristics would need to be considered when designing expression systems for recombinant CCM1 production.

How does secretion stress affect gene expression in A. gossypii, and what implications might this have for recombinant CCM1 production?

Secretion stress significantly impacts gene expression in A. gossypii, with potential consequences for recombinant protein production including CCM1. Treatment with dithiothreitol (DTT), which induces ER stress by disrupting disulfide bond formation, causes wide-ranging transcriptional changes in A. gossypii . These changes include:

• Down-regulation of genes involved in:

  • Filamentous growth

  • Glycosylation and lipoprotein biosynthesis

  • Cell wall biosynthesis

  • Ribosomal proteins (particularly after 1 hour of DTT exposure)

• Up-regulation of genes involved in:

  • ER stress response

  • Protein folding

  • ER-associated degradation

These expression changes correlate with reduced growth rates in DTT-treated cultures. For recombinant CCM1 production, these findings suggest that secretion stress could significantly impact yield and quality. Strategies to mitigate secretion stress, such as co-expression of chaperones or optimization of culture conditions, might be necessary for efficient production of correctly folded and functional CCM1.

How can researchers investigate the relationship between CCM1 activity and riboflavin production in A. gossypii?

Investigating the relationship between CCM1 activity and riboflavin production requires integrated approaches connecting mitochondrial function to secondary metabolism:

This multi-faceted approach would help determine whether CCM1's role in mitochondrial RNA processing has downstream effects on the metabolic pathways leading to riboflavin biosynthesis.

What experimental designs are appropriate for studying the role of CCM1 in mitochondrial RNA processing networks?

Studying CCM1's role in mitochondrial RNA processing networks requires sophisticated experimental designs:

• Comprehensive mitochondrial transcriptome analysis:

  • RNA-seq focusing on mitochondrial transcripts

  • 3'-RACE and 5'-RACE to map precise ends of mitochondrial RNAs

  • Circular RT-PCR to detect and characterize lariat intermediates

• Protein-RNA interaction studies:

  • CLIP-seq (crosslinking immunoprecipitation followed by sequencing) to identify direct RNA targets of CCM1

  • RNA pull-down assays using biotinylated mitochondrial RNA sequences

  • Structural studies of CCM1-RNA complexes using X-ray crystallography or cryo-EM

• Genetic interaction networks:

  • Synthetic genetic array analysis using CCM1 mutants

  • Suppressor screens to identify genes that compensate for CCM1 deficiency

  • Double mutant analysis with other RNA processing factors

• Evolutionary comparative analysis:

  • Compare CCM1 function across different fungal species

  • Identify co-evolving mitochondrial introns and splicing factors

  • Transplant heterologous introns to test species-specific splicing dependencies

These approaches would provide a comprehensive understanding of CCM1's position within the broader network of mitochondrial RNA processing and gene expression.

How does A. gossypii CCM1 compare with homologous proteins in related fungi?

A comparative analysis of CCM1 across fungal species would reveal evolutionary conservation and specialization:

What insights from S. cerevisiae splicing factors can be applied to understanding CCM1 function in A. gossypii?

S. cerevisiae has been extensively studied as a model for mitochondrial RNA processing, providing valuable insights that may be applicable to A. gossypii CCM1:

• Conservation of core splicing machinery: Core components required for sporulation regulation in A. gossypii, such as IME1, IME2, IME4, and KAR4, are conserved with S. cerevisiae , suggesting similar conservation might exist for RNA processing factors.

• Differential requirements: Some factors essential in S. cerevisiae may be dispensable in A. gossypii, as demonstrated by SPO11, which decreases sporulation in S. cerevisiae when mutated but is apparently dispensable for sporulation in A. gossypii .

• Metabolic context: The high riboflavin production characteristic of A. gossypii suggests a distinct metabolic environment that may influence mitochondrial function and, consequently, requirements for RNA processing.

• Growth pattern implications: The filamentous growth of A. gossypii, as opposed to the unicellular nature of S. cerevisiae, may necessitate different spatial regulation of mitochondrial activity and thus RNA processing.

By combining knowledge from S. cerevisiae with A. gossypii-specific experiments, researchers can develop a more comprehensive understanding of CCM1 function in this filamentous fungus.