Recombinant Oligotropha carboxidovorans Serine hydroxymethyltransferase (glyA)

Basic Research Questions

• What is Oligotropha carboxidovorans and why is it significant for glyA studies?

Oligotropha carboxidovorans OM5 is an aerobic carboxidotrophic bacterium capable of chemolithoautotrophic growth using CO2, CO, and H2 as carbon and energy sources. It possesses remarkable metabolic versatility, being able to grow using syngas (a mixture of CO and H2 generated by organic waste gasification) as well as heterotrophically in standard bacteriological media . This metabolic flexibility makes it an excellent model organism for studying the role of core metabolic enzymes such as GlyA under different growth conditions.

The organism contains a megaplasmid (pHCG3) that encodes genes essential for autotrophic growth, including the Calvin-Benson-Bassham cycle components, CO dehydrogenase, and hydrogenase . Recent advances in transformation protocols and genetic manipulation techniques for O. carboxidovorans have established it as an emerging platform for investigating the function of key metabolic enzymes like GlyA under varying growth conditions .

• What is the glyA gene and what function does serine hydroxymethyltransferase serve in bacterial metabolism?

The glyA gene encodes serine hydroxymethyltransferase (SHMT, EC 2.1.2.1), an enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. Based on comparative genomics, bacterial glyA genes show significant conservation, with BLAST searches revealing approximately 55% amino acid identity between E. coli glyA and related bacterial orthologs .

SHMT serves several critical functions in bacterial metabolism:

• Provides one-carbon units for the synthesis of purines, thymidylate, and methionine

• Facilitates the interconversion of serine and glycine, linking multiple amino acid biosynthetic pathways

• Connects to threonine metabolism through interaction with threonine aldolase pathways

• Contributes to cellular redox balance through folate metabolism

• Plays a role in C1 assimilation, particularly important for organisms utilizing C1 compounds

The enzyme belongs to the PLP-dependent (pyridoxal 5'-phosphate) family of enzymes and typically functions as a tetramer in most bacteria.

• What genetic manipulation techniques have been established for O. carboxidovorans glyA studies?

Recent advances have established several genetic manipulation techniques for O. carboxidovorans, which can be applied to glyA studies:

• Electroporation-mediated transformation has been successfully demonstrated for O. carboxidovorans, enabling the introduction of foreign DNA

• Gene deletion and gene exchange protocols via two-step recombination have been developed, facilitating the construction of defined mutants

• Inducible expression systems have been established, allowing controlled expression of native or heterologous genes

• Plasmid-based expression systems enable stable maintenance of recombinant constructs

For specific glyA manipulations, researchers can utilize techniques analogous to those used for other bacterial glyA genes, such as:

• Amplification of the entire glyA gene using PCR with appropriate primers

• Cloning into standard vectors (like pUC18) for genetic manipulation

• Construction of promoterless glyA with added restriction sites for controlled expression

• Implementation of inducible promoters (like the IPTG-inducible tac promoter) for conditional expression

These techniques provide a comprehensive toolkit for investigating glyA function through recombinant approaches in O. carboxidovorans.

• What experimental methods can be used to assess O. carboxidovorans GlyA activity?

Several experimental approaches can be employed to assess the activity of recombinant O. carboxidovorans GlyA:

Direct activity assays:

• HPLC-based quantification of glycine formation, as demonstrated in similar enzymatic systems

• Spectrophotometric assays coupling the SHMT reaction with NADH or NADPH-dependent enzymes

• Colorimetric assays that detect formaldehyde formation through reaction with chromogenic reagents

Indirect assays:

• Threonine aldolase activity assays that monitor the spontaneous decarboxylation of amino-ketobutyrate to aminoacetone, which can be quantified with Ehrlich's reagent

• Complementation assays in glyA-deficient strains to assess functional activity

Typical reaction conditions include:

• Buffer: Tris-HCl or potassium phosphate (200-250 mM, pH 8.4-8.6)

• Cofactors: Pyridoxal 5'-phosphate (2 mM)

• Substrates: L-serine or L-threonine (10-200 mM)

• Additional components: NAD+ for coupled assays

Samples are typically incubated at 30°C for 30-120 minutes, followed by protein precipitation with trichloroacetic acid, neutralization, and analysis using HPLC or other detection methods .

• How does O. carboxidovorans GlyA relate to orthologs in other bacterial species?

Comparative analysis indicates significant relatedness between O. carboxidovorans GlyA and orthologs in other bacterial species:

• BLAST searches reveal sequence similarities between bacterial SHMT enzymes, with approximately 55% amino acid identity observed between E. coli glyA and related bacterial orthologs

• Domain analysis through the Conserved Domain Database (CDD) identifies the same COG number (COG0112, GlyA) across bacterial SHMT enzymes, indicating functional conservation

• Protein family analysis using Pfam shows that these enzymes contain the same domain (PF00464, SHMT - Serine hydroxymethyltransferase)

Subcellular localization predictions using tools like TMH, SignalP, LipoP, and PSORT-B suggest that both E. coli GlyA and other bacterial SHMT orthologs are localized to the cytoplasm of the cell, lacking transmembrane helices or cleavage sites .

Functional conservation is further evidenced by:

• Shared enzyme commission number (E.C.2.1.2.1) across bacterial species

• Involvement in the same step of glycine biosynthesis and degradation pathways

• Conservation of key catalytic residues and active site architecture

Advanced Research Questions

• What factors influence the expression and activity of recombinant O. carboxidovorans GlyA in heterologous hosts?

Expression and activity of recombinant O. carboxidovorans GlyA in heterologous hosts is influenced by several critical factors:

For O. carboxidovorans proteins specifically, transformation protocols have been successfully established using electroporation, enabling the introduction of expression constructs . Inducible expression systems have been demonstrated to work effectively for O. carboxidovorans genes, with protocols for both chromosomal integration and plasmid-based expression .

When expressing O. carboxidovorans GlyA in E. coli, researchers should consider using vectors with the IPTG-inducible lac or tac promoter systems, which have been successfully employed for similar enzymes . Expression vectors containing appropriate affinity tags can facilitate purification while minimizing impact on enzyme activity.

• How does the proteome context affect GlyA function during adaptation to different growth conditions?

The function of GlyA in O. carboxidovorans is significantly influenced by the broader proteome context, particularly during adaptation between heterotrophic and autotrophic growth modes:

Proteomic analysis of O. carboxidovorans grown under different conditions reveals that adaptation to chemolithoautotrophic growth involves coordinated changes in multiple systems, including cell envelope components, oxidative stress response mechanisms, and metabolic pathways such as the glyoxylate shunt and amino acid/cofactor biosynthetic enzymes .

While GlyA-specific regulation is not explicitly detailed in the search results, SHMT function is likely integrated within this broader adaptive response:

• The metabolic shift from heterotrophy to autotrophy involves dramatic changes in carbon flux through central metabolism, affecting amino acid biosynthesis pathways where SHMT plays a key role

• RNA-Seq analysis comparing O. carboxidovorans grown heterotrophically with acetate versus autotrophically with CO2, CO, and H2 revealed numerous differentially expressed genes

• Autotrophic growth led to higher expression of genes related to CO2 fixation via the Calvin-Benson-Bassham cycle, CO metabolism, and H2 utilization

• What methodological approaches can be used to study O. carboxidovorans GlyA through isotopic labeling?

Isotopic labeling provides powerful approaches for investigating GlyA function in O. carboxidovorans:

Experimental design strategies:

• 13C-labeled substrate tracing to track carbon flux through the SHMT reaction

• Differential labeling patterns can distinguish between the forward and reverse directions of the reaction

• Combined with mass spectrometry analysis to determine incorporation patterns in downstream metabolites

This approach is analogous to the 13C-tracer analysis used to confirm the operation of the Calvin-Benson-Bassham cycle in engineered strains as mentioned in the literature .

Specific methodological considerations include:

For in vivo studies, cells can be grown with isotopically labeled substrates, followed by extraction and analysis of metabolites. For in vitro studies with purified recombinant GlyA, reaction kinetics with labeled substrates can provide mechanistic insights.

The resulting data requires sophisticated analysis pipelines to account for isotope dilution effects and to distinguish direct SHMT-catalyzed transformations from downstream metabolic events.

• How can genetic engineering approaches be used to manipulate O. carboxidovorans glyA for enhanced C1 assimilation?

Strategic manipulation of glyA in O. carboxidovorans could enhance its C1 assimilation capabilities through several approaches:

Expression optimization:

• Replacement of the native glyA promoter with stronger or inducible promoters to increase expression

• Implementation of the two-step recombination protocols established for O. carboxidovorans to enable precise genetic modifications

• Construction of defined glyA mutants using the recently established transformation and genetic manipulation techniques

Protein engineering strategies:

• Rational design of GlyA variants with altered kinetic properties, based on comparative analysis with other bacterial SHMT enzymes

• Modification of substrate binding sites to optimize the direction of the reversible SHMT reaction

• Engineering of cofactor binding to enhance catalytic efficiency

Integration with metabolic engineering:

• Coordinated manipulation of glyA alongside other genes involved in C1 metabolism

• Implementation of synthetic pathways that leverage SHMT activity for enhanced carbon fixation

• Creation of metabolic bottlenecks that channel carbon flux through SHMT-dependent pathways

Recent advances in O. carboxidovorans genetic manipulation make these approaches increasingly feasible:

• Electroporation-mediated transformation has been successfully established

• Gene deletion and exchange protocols via two-step recombination enable precise genetic modifications

• Inducible and stable expression systems for heterologous genes have been developed

These tools collectively provide a platform for sophisticated glyA manipulation strategies aimed at enhancing the organism's capacity for C1 assimilation and utilization.

• What role does GlyA play in redox homeostasis during O. carboxidovorans growth on different carbon sources?

GlyA likely plays a significant role in redox homeostasis during O. carboxidovorans growth on different carbon sources, though this must be inferred from general principles and the provided search results:

Proteome analysis has shown that adaptation to chemolithoautotrophic growth involves changes in oxidative homeostasis systems . The shift between heterotrophic growth on acetate and autotrophic growth on CO2/CO/H2 represents a dramatic change in cellular redox state.

GlyA's potential contributions to redox homeostasis include:

• The SHMT reaction connects to folate metabolism, which is intimately involved in cellular redox balance through NADPH-dependent reactions

• The interconversion of serine and glycine affects the availability of precursors for glutathione synthesis, a major cellular antioxidant

• One-carbon metabolism controlled by SHMT impacts the generation of reducing equivalents needed during autotrophic growth

During adaptation to chemolithoautotrophic growth, O. carboxidovorans shows regulation of proteins involved in oxidative homeostasis . The metabolism of CO and H2 as energy sources during autotrophic growth generates distinct redox challenges compared to heterotrophic growth on acetate.

GlyA activity may be regulated in response to these changing redox conditions, potentially through post-translational modifications or allosteric regulation. Understanding this relationship would require comparative analysis of GlyA activity under different growth conditions, coupled with measurements of cellular redox parameters.

• How can CRISPR-Cas systems be optimized for precise manipulation of glyA in O. carboxidovorans?

Optimizing CRISPR-Cas systems for precise manipulation of glyA in O. carboxidovorans requires addressing several technical considerations:

Vector system selection:

• Utilize the recently established transformation protocols for O. carboxidovorans, which have successfully employed electroporation for introducing foreign DNA

• Consider adapting the conditionally unstable replicon, Mex-CM4, which has shown potential for CRISPR-Cas applications requiring transient expression and fast extinction

• Newly identified extrachromosomal elements termed 'mini-chromosomes' based on repABC cassettes could provide stable maintenance of CRISPR components

Guide RNA design for glyA targeting:

• Analyze the O. carboxidovorans glyA sequence for optimal protospacer adjacent motif (PAM) sites

• Design guide RNAs with minimal off-target effects by performing whole-genome analysis

• Target conserved regions of glyA to ensure efficient editing

Delivery optimization:

• Leverage established two-step recombination protocols for O. carboxidovorans

• Incorporate inducible systems to control Cas9 expression, minimizing toxicity

• Consider temperature modulation during editing to optimize both transformation efficiency and CRISPR activity

Editing strategies:

• For gene knockout: Design guide RNAs targeting the beginning of the coding sequence

• For point mutations: Provide repair templates with the desired modifications flanked by homology arms

• For promoter replacement: Target the region upstream of the glyA coding sequence

Post-editing selection:

• Implement appropriate selection markers compatible with O. carboxidovorans

• Design screening strategies to identify successful edits without disrupting cellular function

• Consider counter-selection approaches to remove CRISPR components after editing

• What kinetic parameters characterize O. carboxidovorans GlyA and how do environmental conditions affect its catalytic properties?

While specific kinetic parameters for O. carboxidovorans GlyA are not provided in the search results, comparative analysis with other bacterial SHMT enzymes allows for predictions about its catalytic properties:

Predicted kinetic parameters for O. carboxidovorans GlyA:

Environmental factors likely influencing O. carboxidovorans GlyA activity:

• Carbon source (autotrophic vs. heterotrophic growth): Proteome analysis has shown that O. carboxidovorans adapts significantly when switching between acetate and syngas as carbon/energy sources

• Oxygen availability: As an aerobic organism, O. carboxidovorans GlyA has likely evolved to function optimally under aerobic conditions

• Temperature: The enzyme would be expected to show activity profiles aligned with the organism's growth temperature optima

Experimental methods to determine these parameters would utilize:

• Purified recombinant enzyme from expression systems that have been established for O. carboxidovorans

• Spectrophotometric or HPLC-based assays similar to those described for related enzymes

• Variable substrate concentrations to determine Michaelis-Menten parameters

• Testing under conditions mimicking different growth states to assess environmental effects

• How does glyA expression change in response to shifts between heterotrophic and autotrophic growth in O. carboxidovorans?

The regulation of glyA expression during shifts between heterotrophic and autotrophic growth in O. carboxidovorans likely involves complex transcriptional and post-transcriptional mechanisms:

RNA-Seq analysis comparing O. carboxidovorans grown heterotrophically with acetate versus autotrophically with CO2, CO, and H2 revealed numerous differentially expressed genes . While glyA was not specifically mentioned, the study found that genes encoding proteins required for autotrophic growth were much higher expressed during growth with synthesis gas .

Given SHMT's role in amino acid metabolism and one-carbon transfer, and considering that proteome analysis has shown that adaptation to chemolithoautotrophic growth involves changes in "amino acid/cofactor biosynthetic enzymes" , it's reasonable to hypothesize that glyA expression is regulated during this metabolic shift.

Potential regulatory mechanisms:

• Transcriptional regulation:

  • Carbon source-dependent transcription factors

  • Global regulators responding to changes in cellular energy status

  • Small RNAs controlling mRNA stability or translation

• Post-transcriptional regulation:

  • mRNA stability changes in response to metabolic shifts

  • Translational efficiency modulation

• Post-translational regulation:

  • Protein stability differences under different growth conditions

  • Activity modulation through allosteric regulation or PTMs

Experimental approaches to investigate this would include:

• qRT-PCR targeting glyA during growth transitions

• Western blot analysis comparing protein levels

• Enzyme activity assays under different growth conditions

• Reporter gene fusions to monitor promoter activity in real-time

• How can structural biology approaches inform the engineering of O. carboxidovorans GlyA for enhanced functionality?

Structural biology approaches provide crucial insights for engineering O. carboxidovorans GlyA with enhanced functionality:

While the specific crystal structure of O. carboxidovorans GlyA has not been reported in the provided search results, comparative analysis with other bacterial SHMTs can guide rational engineering strategies:

Domain architecture analysis:

• Bacterial SHMTs typically contain distinct N-terminal and C-terminal domains

• The N-terminal domain is involved in PLP binding while the C-terminal domain participates in substrate binding

• Based on comparative analysis between E. coli glyA and bacterial orthologs showing ~55% amino acid identity , key structural features are likely conserved in O. carboxidovorans GlyA

Engineering targets based on structural insights:

Implementation strategies:

• Site-directed mutagenesis based on homology models

• Directed evolution with appropriate selection schemes

• Domain swapping with other characterized SHMTs

• Computational design of novel active sites

These approaches can leverage the genetic manipulation techniques recently established for O. carboxidovorans, including electroporation-mediated transformation and gene exchange protocols via two-step recombination , to implement the designed modifications.

• What are the implications of GlyA function for metabolic engineering of O. carboxidovorans for C1 utilization platforms?

GlyA plays a pivotal role in the metabolic engineering of O. carboxidovorans for C1 utilization platforms:

Strategic importance for C1 metabolism:

• SHMT occupies a central position in one-carbon metabolism, connecting glycine/serine interconversion with folate-dependent C1 transfer reactions

• This position makes it a key enzyme for engineering enhanced C1 assimilation pathways

Specific engineering opportunities:

• Enhanced CO2 fixation:

  • O. carboxidovorans can grow autotrophically using CO2, CO, and H2

  • GlyA could be engineered to support more efficient carbon fixation through optimized one-carbon unit management

  • Integration with the Calvin-Benson-Bassham cycle already present in O. carboxidovorans

• Synthetic pathway construction:

  • Creation of novel pathways that utilize GlyA's reversible reaction for specialized product synthesis

  • Engineering of non-natural substrate utilization through GlyA modifications

• Growth optimization:

  • Fine-tuning GlyA expression levels to balance amino acid biosynthesis with one-carbon metabolism

  • Coordinated regulation with other key enzymes in C1 assimilation pathways

Implementation considerations:

• Genetic manipulation tools for O. carboxidovorans have been established, including transformation via electroporation and gene deletion/exchange protocols

• Inducible expression systems allow for controlled modulation of GlyA levels

• The organism's natural ability to grow on syngas provides a foundation for developing industrially relevant C1 utilization platforms

Balancing the forward and reverse SHMT reactions through protein engineering and expression regulation could be key to optimizing O. carboxidovorans for specific C1 utilization applications, potentially enabling more efficient conversion of waste gases into valuable products.