Recombinant Synechocystis sp. Uncharacterized AIPM/Hcit synthase family transferase sll1564 (sll1564), partial

What is sll1564 in Synechocystis sp. PCC 6803 and what is its significance in research?

sll1564 is an uncharacterized alpha-isopropylmalate/homocitrate (AIPM/Hcit) synthase family transferase in the cyanobacterium Synechocystis sp. PCC 6803. According to recombinant protein databases, it is alternatively annotated as "(R)-citramalate synthase" . The gene has gained research significance as it appears to be involved in metabolic pathways relevant to biofuel production, particularly in engineered strains producing isobutanol (IB) and 3-methyl-1-butanol (3M1B) . Researchers studying cyanobacterial metabolism often use this gene as a genetic engineering target or integration site.

What experimental considerations should be taken when working with recombinant sll1564?

When expressing recombinant sll1564, researchers should consider multiple expression systems including E. coli, yeast, baculovirus, or mammalian cells, as all have been successfully used to produce this protein with ≥85% purity . For research applications, the choice of expression system should align with experimental goals - prokaryotic systems (E. coli) for basic characterization and eukaryotic systems when post-translational modifications might be relevant. Purification typically employs affinity chromatography, and buffer conditions should be optimized to maintain enzyme stability during storage and functional assays.

How is sll1564 expression regulated in Synechocystis sp. PCC 6803?

Based on transcriptomic analyses, sll1564 expression appears to be influenced by metabolic engineering interventions. In recombinant Synechocystis strains actively synthesizing polyhydroxyalkanoates (PHA), sll1564 shows significant upregulation compared to control strains . This suggests that sll1564 expression may be linked to carbon metabolism regulation pathways and responds to changes in cellular metabolic flux. Further research is needed to elucidate specific transcription factors and regulatory elements controlling sll1564 expression under various environmental conditions.

What CRISPR-based methods are effective for studying sll1564 function?

Recent advances in CRISPR technology for Synechocystis sp. PCC 6803 provide multiple approaches for investigating sll1564:

• CRISPR Activation (CRISPRa): A rhamnose-inducible CRISPRa system using dCas12-SoxS protein fusion can upregulate sll1564. This approach has been demonstrated in studies targeting metabolic pathways in Synechocystis .

• CRISPR Interference (CRISPRi): dCas12a-mediated gene repression can be used to assess phenotypic consequences of sll1564 downregulation .

• CRISPR-Cas9 Editing: For complete knockout or targeted mutations, CRISPR-Cas9 can be delivered via conjugation using triparental mating with helper strain HB101 containing pRL443-AmpR plasmid .

When designing gRNAs for sll1564 targeting, researchers should consider:

• Position relative to transcriptional start site (TSS) affects activation efficacy

• The intrinsic strength of the target promoter influences results

• Multiple gRNAs may be necessary for robust phenotypic effects

How can protein-protein interactions involving sll1564 be investigated?

To elucidate the functional role of sll1564 through its interaction network, researchers should employ:

• Co-immunoprecipitation (Co-IP): Using epitope-tagged sll1564 to pull down interacting proteins, followed by mass spectrometry identification.

• Bacterial two-hybrid screening: Particularly suitable for identifying direct protein interactions in prokaryotic systems.

• Crosslinking mass spectrometry: To capture transient interactions within the native cellular environment.

• Proximity labeling approaches: Such as BioID or APEX2 fusion proteins to identify proximal proteins in the cellular context.

These methods can help establish whether sll1564 functions in isolation or as part of a larger metabolic complex in various cellular conditions.

What is the role of sll1564 in biofuel production pathways?

Several lines of evidence suggest sll1564 is involved in biofuel production pathways:

• sll1564 has been used as an integration site for kivDS286T, a key gene in the biosynthesis of isobutanol (IB) and 3-methyl-1-butanol (3M1B) .

• In engineered Synechocystis strains, the chromosomal context around sll1564 influences biofuel production efficiency, suggesting possible regulatory or metabolic interactions with the inserted pathway genes .

• Data from strain HX51 (which has three kivDS286T copies inserted at ddh, NS1, and sll1564 sites) shows:

This indicates that the genomic context of sll1564 affects biofuel production dynamics and that multiplexed targeting approaches yield better results .

How does sll1564 function compare to other AIPM/Hcit synthase family transferases?

While sll1564 remains largely uncharacterized, comparative analysis with other AIPM/Hcit synthase family proteins provides functional insights:

• Homology relationships: sll1564 shares structural features with characterized citramalate synthases from other organisms, particularly those from archaeal species like Methanocaldococcus jannaschii (CimA) .

• Substrate specificity: Based on related enzymes, sll1564 likely catalyzes the condensation of acetyl-CoA with pyruvate or similar α-keto acids to form citramalate-like compounds, contributing to alternative amino acid biosynthesis pathways.

• Functional divergence: Unlike well-characterized homologs, sll1564's specific metabolic role in Synechocystis appears to intersect with biofuel-relevant pathways, suggesting possible evolutionary adaptation of this enzyme class for specialized metabolism in cyanobacteria.

Future biochemical characterization should focus on determining substrate specificity and kinetic parameters compared to characterized family members from other organisms.

How does environmental stress affect sll1564 expression and function?

While direct data on sll1564's response to environmental stress is limited, insights can be drawn from studies on Synechocystis sp. PCC 6803 stress responses:

• Studies on related genes in Synechocystis (such as sll1558) show significant upregulation under acidic stress conditions (pH 6.0) as well as other stressors including high salt, high osmolality, high/low temperature, and ultraviolet-B stress .

• Different Synechocystis substrains (GT-L, GT-B, PCC-B) show varying stress sensitivities to light wavelength, temperature, and salt concentration , suggesting strain-specific regulation of stress response genes that may include sll1564.

Researchers investigating sll1564's role in stress response should consider:

• Monitoring expression changes under various stress conditions

• Phenotypic analysis of sll1564 mutants under stress

• Assessing metabolic flux changes involving sll1564 pathways during stress adaptation

What are the technical challenges in multiplex CRISPR targeting of sll1564?

Advanced research involving multiplex CRISPR targeting of sll1564 faces several technical challenges:

• Variable gRNA efficiency: When targeting sll1564 for CRISPRa/CRISPRi, the efficiency of different guide RNAs varies substantially, requiring empirical optimization .

• Strain-specific effects: The genetic background significantly impacts targeting outcomes - identical gRNAs targeting sll1564 produce different effects in different strain backgrounds (e.g., HX11 vs. HX51) .

• Complex regulation dynamics: Data from the HX51 strain shows that single targeting of sll1564 unexpectedly led to target gene downregulation, while multiplexed targeting with other sites produced activation .

• Transcript detection limitations: Transcript levels of genes integrated at sll1564 and NS1 sites could not be differentiated in qPCR readings, complicating quantitative analysis .

These challenges highlight the need for robust controls and multiple targeting strategies when using CRISPR systems to manipulate sll1564 expression.

How can contradictory data on sll1564 function be reconciled?

When encountering contradictory results regarding sll1564 function:

• Strain-specific effects: The phenotypic consequences of sll1564 manipulation vary between different Synechocystis strains. For example, dual targeting of NS1 and ddh loci resulted in opposite effects on isobutanol and 3-methyl-1-butanol production in HX11 and HX51 strains, despite identical constructs and gRNAs .

• Temporal dynamics: The impact of sll1564 manipulation changes over time. The highest CRISPRa-induced improvement was observed at day 4, while at later timepoints, differences between targeted and non-targeted strains diminished .

• Contextual dependencies: The complex and dynamic system-wide metabolic regulation influences how sll1564 manipulation affects cellular phenotypes. Researchers should consider factors beyond direct transcriptional effects, including:

  • Substrate availability

  • Feedback regulation

  • Metabolic burden of heterologous expression

  • Growth phase-dependent regulation

What computational approaches can predict sll1564 structure and function?

Advanced computational methods to elucidate sll1564 structure and function include:

• Homology modeling: Using solved structures of characterized AIPM/Hcit synthase family proteins as templates for predicting sll1564 structure.

• Molecular dynamics simulations: To predict substrate binding sites, conformational changes during catalysis, and potential allosteric regulation mechanisms.

• Metabolic flux analysis: Genome-scale metabolic models of Synechocystis can predict how sll1564 perturbation affects carbon flux through central metabolic pathways.

• Protein-protein interaction networks: Based on homology and co-expression data, computational approaches can predict the interaction network of sll1564.

• Ancestral sequence reconstruction: Evolutionary analysis of the AIPM/Hcit synthase family can provide insights into the functional divergence of sll1564 in cyanobacteria.

These computational approaches, when integrated with experimental validation, can significantly accelerate characterization of this uncharacterized enzyme.

How might sll1564 be engineered for enhanced biofuel production?

Based on current knowledge, several engineering strategies for sll1564 could enhance biofuel production:

• Promoter engineering: Replacing the native sll1564 promoter with stronger or inducible promoters to modify expression levels.

• Protein engineering: Directed evolution or rational design approaches targeting the catalytic domain to enhance specificity for biofuel-relevant substrates.

• Integration optimization: The genomic context of sll1564 affects biofuel pathway efficiency - optimizing the integration of heterologous genes near sll1564 could exploit potential regulatory advantages.

• Multiplexed regulation: Combined CRISPRa targeting of sll1564 alongside other metabolic genes showed synergistic effects on biofuel production , suggesting comprehensive pathway engineering approaches would be beneficial.

• Synthetic metabolic modules: Creating artificial metabolic channels by fusing sll1564 with other enzymes in the biofuel pathway could enhance metabolic flux.

What experimental systems best model the native function of sll1564?

To accurately characterize the native function of sll1564, researchers should consider:

• Growth condition optimization: Synechocystis cultures should be cultivated in controlled environments such as flat panel photobioreactors under defined light conditions (red, blue, white) of specific intensities (up to 790 μmol(photons) m-2 s-1), controlled temperatures (23°C–60°C), and precise CO2 concentrations (400-15,000 ppm) .

• Strain selection considerations: Different Synechocystis substrains (GT-L, GT-B, PCC-B) show phenotypic variations in growth rate, photosynthesis efficiency, dry weight, and cellular composition . Researchers should clearly document which substrain is used and be aware that results may vary between substrains.

• In vitro reconstitution: Purified recombinant sll1564 should be tested with potential substrates in biochemical assays to definitively establish enzymatic function.

• Metabolomic profiling: Comprehensive analysis of metabolite changes in wild-type versus sll1564 mutant strains under various conditions can reveal the metabolic pathways affected.

These experimental systems, when combined, can provide complementary insights into the native function of this uncharacterized enzyme.