Recombinant Photorhabdus luminescens subsp. laumondii Acetyl-coenzyme A synthetase (acs), partial

What is Photorhabdus luminescens and what role does Acetyl-coenzyme A synthetase play in its metabolism?

Photorhabdus luminescens is a Gram-negative bioluminescent bacterium with a complex lifecycle that includes both pathogenic and mutualistic phases. It forms a symbiotic relationship with nematodes from the family Heterorhabditis while also being highly virulent to insect larvae .

Acetyl-coenzyme A synthetase (ACS) in P. luminescens plays a critical role in central metabolism by catalyzing the formation of acetyl-CoA from acetate, ATP, and coenzyme A. This reaction is fundamental as acetyl-CoA serves as a key metabolic intermediate connecting several pathways including:

• The tricarboxylic acid (TCA) cycle

• Amino acid metabolism

• Fatty acid metabolism

• Biosynthetic processes for polyketides and some terpenes

The enzyme operates through a two-step mechanism: first forming an acetyl-adenylate intermediate, followed by reaction with coenzyme A to form acetyl-CoA while releasing AMP .

How does the TCA cycle in P. luminescens relate to its dual lifestyle as both pathogen and mutualist?

The TCA cycle in P. luminescens appears to play a central role in the bacterium's ability to transition between pathogenic and mutualistic lifestyles. Research has demonstrated that interruptions in the TCA cycle can selectively impact these different aspects of P. luminescens biology:

• Mutations in TCA cycle enzymes, such as malate dehydrogenase (mdh), specifically impair secondary metabolism and mutualistic capabilities while leaving pathogenic functions intact

• The TCA cycle appears to act as a metabolic switch that regulates lifestyle decisions in P. luminescens

What expression systems are most effective for producing recombinant P. luminescens ACS?

While specific expression systems for P. luminescens ACS aren't detailed in the provided search results, effective expression of functionally related ACS enzymes provides guidance for researchers:

Expression optimization should include various parameters such as temperature, inducer concentration, and media composition. For functional studies, tags like 6xHis or Strep-tag II can be incorporated to facilitate purification while preserving enzymatic activity.

How can site-directed mutagenesis be used to alter the substrate specificity of P. luminescens ACS?

Engineering the substrate specificity of ACS enzymes through site-directed mutagenesis has been successfully demonstrated with related enzymes. The methodology involves:

• Computational modeling: Create a structural model of P. luminescens ACS based on homologous enzymes with known tertiary structures

• Binding pocket identification: Identify residues that form the carboxylate binding pocket, which determines substrate specificity

• Targeted mutagenesis: Systematically substitute specific residues to alter the size and physico-chemical properties of the binding pocket

• Functional validation: Test the mutant enzymes for altered substrate preferences using enzymatic assays

Research with related ACS enzymes has demonstrated that targeted modification of as few as four specific residues can dramatically shift substrate specificity from exclusively acetate to longer-chain carboxylates (up to hexanoate) or branched substrates like methylvalerate . This approach allows rational design of new biocatalysts without requiring experimentally determined tertiary structures of the target enzyme .

What role does ACS play in secondary metabolite production in P. luminescens?

Acetyl-CoA synthetase likely plays a significant role in P. luminescens secondary metabolism by providing acetyl-CoA, a crucial building block for numerous secondary metabolites. The connectivity between primary and secondary metabolism in P. luminescens is particularly evident in:

• Stilbene production: P. luminescens produces 3,5-dihydroxy-4-isopropylstilbene (ST), an antibiotic that requires acetyl-CoA for biosynthesis. The biosynthetic pathway involves:

  • Phenylalanine conversion to cinnamic acid via phenylalanine ammonia-lyase (encoded by stlA)

  • Further metabolism that likely incorporates acetyl-CoA-derived components

• Regulation by global factors: Secondary metabolism in P. luminescens is controlled by global regulators including:

  • HexA, a LysR-type transcriptional repressor that regulates stilbene production

  • When hexA is knocked out, there is dramatic upregulation of biosynthesized small molecules

• Metabolic switching: Acetyl-CoA sits at a junction between primary metabolism and specialized secondary metabolism. The TCA cycle status influences this metabolic switching, as evidenced by the mdh mutant which cannot produce secondary metabolites .

The connection between ACS activity and specialized metabolite production makes it a potential target for engineering enhanced production of valuable compounds.

How do regulatory factors affect the expression and activity of ACS in P. luminescens?

P. luminescens employs complex regulatory networks that influence metabolic enzyme expression and activity, including ACS. Key regulatory mechanisms include:

• Growth phase-dependent regulation: Secondary metabolism in P. luminescens is tightly linked to growth phase, with most secondary metabolites being produced during the post-exponential phase . ACS activity is likely regulated in a similar manner to support this metabolic shift.

• Global regulators:

  • HexA functions as a repressor of secondary metabolism while bacteria dwell within their nematode host

  • Disruption of hexA leads to derepression of antibacterial activities and increased production of secondary metabolites

• Quorum sensing:

  • LuxS-type signaling mechanisms influence metabolite production in P. luminescens

  • LuxS has been shown to repress certain gene clusters (like cpm) at the end of exponential growth phase

• Nutritional signals:

  • L-proline from insect hemolymph serves as both a nutrient signal and electron source

  • High concentrations of L-proline trigger upregulation of secondary metabolite production

  • This likely influences ACS expression or activity to channel acetyl-CoA toward secondary metabolism

A comprehensive understanding of these regulatory mechanisms provides opportunities to manipulate ACS expression for enhanced production of valuable metabolites or to study metabolic flux control.

What techniques are most effective for assessing the enzymatic activity of recombinant P. luminescens ACS?

Several complementary techniques can be employed to comprehensively characterize recombinant P. luminescens ACS activity:

• Spectrophotometric coupled assays:

  • Measure AMP production by coupling to additional enzymes (adenylate kinase and pyruvate kinase)

  • Monitor NADH oxidation at 340 nm as a proxy for ACS activity

  • Advantages: Real-time kinetics, high-throughput capability

  • Limitations: Potential interference from coupling enzymes

• Direct product analysis:

  • HPLC quantification of acetyl-CoA formation

  • LC-MS/MS for precise identification and quantification

  • Advantages: Direct measurement, specificity

  • Limitations: More time-consuming, requires specialized equipment

• Isotope-based methods:

  • Use [14C]-labeled acetate or [32P]-ATP to track substrate conversion

  • Measure incorporation of radiolabel into products

  • Advantages: High sensitivity, ability to track metabolic fate

  • Limitations: Radiation hazards, specialized disposal requirements

• Pyrophosphate release assays:

  • Measure PPi release using pyrophosphatase and a colorimetric phosphate detection system

  • Advantages: Simple, adaptable to microplate format

  • Limitations: Indirect measurement

For kinetic characterization, systematic variation of substrate concentrations (acetate, ATP, and CoA) should be performed while monitoring initial reaction rates to determine key parameters (Km, Vmax, kcat) using appropriate enzyme kinetic models.

How can metabolic flux analysis be used to understand the impact of ACS on P. luminescens metabolism?

Metabolic flux analysis (MFA) offers powerful approaches to understand how ACS influences carbon flow through P. luminescens metabolic networks:

• 13C-MFA methodology:

  • Culture P. luminescens with 13C-labeled substrates (e.g., [1-13C]acetate, [U-13C]glucose)

  • Harvest cells at defined time points

  • Extract and analyze isotope enrichment patterns in metabolites using GC-MS or LC-MS

  • Calculate flux distributions using computational models

• Key experimental design considerations:

  • Compare wild-type with ACS knockout or overexpression strains

  • Analyze flux during different growth phases (exponential vs. post-exponential)

  • Examine effects of environmental perturbations (oxygen levels, carbon sources)

• Target metabolites for analysis:

  • TCA cycle intermediates (citrate, succinate, malate)

  • Amino acids derived from acetyl-CoA

  • Fatty acids and lipids

  • Secondary metabolite precursors

• Computational approaches:

  • Use isotopomer balancing and metabolic network models

  • Incorporate transcriptomic and proteomic data for comprehensive analysis

  • Perform sensitivity analysis to identify key control points

MFA studies with P. luminescens would be particularly valuable to understand how metabolic flux redistributes during the transition from pathogenesis to mutualism, and how ACS activity influences this metabolic switch. This approach could reveal how the TCA cycle acts as a metabolic sensor that determines lifestyle decisions in this bacterium .

How does P. luminescens ACS compare to homologous enzymes from other bacteria in terms of structural and functional properties?

Comparative analysis of P. luminescens ACS with homologous enzymes provides insights into its unique characteristics:

While the specific structural features of P. luminescens ACS haven't been experimentally determined, computational modeling using homologous enzymes as templates can predict its structure with reasonable accuracy . Such models suggest that, like other ACS enzymes, the carboxylate binding pocket would determine substrate specificity, and targeted mutations could potentially alter this specificity .

The reaction mechanism is conserved across ACS enzymes, involving:

• Formation of an acyl-adenylate intermediate

• Reaction with CoA to form the final acyl-CoA product

Understanding these similarities and differences facilitates rational enzyme engineering and provides evolutionary insights.

What challenges exist in crystallizing recombinant P. luminescens ACS for structural studies?

Obtaining high-quality crystals of recombinant P. luminescens ACS for X-ray crystallography presents several challenges:

• Protein stability issues:

  • ACS enzymes often have flexible domains that move during catalysis

  • This conformational heterogeneity can inhibit crystal formation

  • Solution: Consider co-crystallization with substrates, products, or substrate analogs to stabilize specific conformations

• Purification challenges:

  • Membrane association may require careful detergent selection

  • Aggregation propensity at high concentrations needed for crystallization

  • Solution: Screen multiple buffer conditions and additives that promote monodispersity

• Crystal optimization strategies:

  • Employ surface entropy reduction (SER) by mutating surface-exposed lysine and glutamate residues

  • Use truncation constructs removing flexible regions

  • Try fusion proteins like T4 lysozyme to provide crystal contacts

• Alternative structural approaches:

  • Cryo-electron microscopy for proteins recalcitrant to crystallization

  • NMR for dynamic structural analysis of smaller domains

  • Small-angle X-ray scattering (SAXS) for envelope structure in solution

While no specific crystallization reports for P. luminescens ACS appear in the search results, computational modeling based on related structures offers a viable alternative for understanding structure-function relationships .

How can recombinant P. luminescens ACS be applied to metabolic engineering for production of valuable compounds?

Recombinant P. luminescens ACS offers several applications in metabolic engineering:

• Enhanced production of natural products:

  • Overexpression of engineered ACS could increase acetyl-CoA availability for secondary metabolite production

  • P. luminescens produces valuable bioactive compounds including carbapenem-like antibiotics and stilbene derivatives

  • Targeted upregulation could improve yields of these compounds

• Expanding substrate scope through enzyme engineering:

  • Structural redesign of the carboxylate binding pocket can create enzymes that activate alternative carboxylic acids

  • This approach enables incorporation of non-natural building blocks into polyketides and other acetyl-CoA-derived products

  • Successful examples include engineering ACS to accept longer-chain (hexanoate) or branched (methylvalerate) substrates

• Pathway optimization strategies:

  • Co-expression with other key enzymes to create artificial metabolic channels

  • Fine-tuning expression levels to balance pathway flux

  • Coupling to NADPH-regenerating systems for sustained activity

• Integration with regulatory engineering:

  • Manipulation of global regulators like HexA could enhance secondary metabolism

  • Uncoupling ACS expression from native regulatory networks through synthetic biology approaches

These applications could lead to improved production systems for antibiotics, insecticides, and other bioactive compounds naturally produced by P. luminescens or engineered pathway products.

How does heterologous expression of P. luminescens ACS affect host cell metabolism?

Heterologous expression of P. luminescens ACS can significantly impact host cell metabolism in several ways:

Understanding these effects is crucial for optimizing expression systems and harnessing the full potential of recombinant P. luminescens ACS for biotechnological applications.

What techniques are available for investigating the in vivo function of ACS in P. luminescens during different lifecycle stages?

Investigating ACS function during P. luminescens lifecycle stages requires specialized techniques:

• Genetic approaches:

  • Creation of conditional knockdowns using inducible antisense RNA

  • CRISPR interference (CRISPRi) for tunable gene repression

  • Fluorescent transcriptional reporters to monitor expression dynamics

  • Challenge: Maintaining genetic stability of manipulated strains, as demonstrated by difficulties in obtaining stable hexA knockouts

• In vivo activity measurements:

  • Isotope tracing experiments during insect infection and nematode colonization

  • Development of activity-based protein profiling probes specific for ACS

  • Metabolomics comparison of wild-type and ACS-modified strains during lifecycle transitions

• Lifecycle-specific considerations:

  • Methods for isolating bacteria from different stages (insect hemolymph vs. nematode gut)

  • Co-culturing systems with nematode hosts to simulate mutualistic phase

  • Insect infection models for pathogenic phase studies

• Integration with global analyses:

  • Quantitative proteomics to measure ACS abundance across lifecycle stages

  • RNA-seq for transcriptional profiling

  • Chromatin immunoprecipitation (ChIP) to identify regulatory factors controlling ACS expression

These approaches would be particularly valuable in understanding how ACS contributes to the metabolic switch between pathogenesis and mutualism, a transition that appears to be regulated through TCA cycle function .

How can systems biology approaches integrate ACS function into the broader metabolic network of P. luminescens?

Systems biology offers powerful frameworks to understand ACS's role in P. luminescens metabolism:

• Genome-scale metabolic modeling:

  • Construction of a comprehensive metabolic network incorporating ACS reactions

  • Flux balance analysis to predict metabolic states under different conditions

  • In silico gene knockout simulations to predict phenotypic consequences

  • Integration of transcriptomic and proteomic data to create context-specific models

• Multi-omics integration strategies:

  • Correlation analysis between transcriptome, proteome, and metabolome datasets

  • Time-course experiments capturing dynamic responses to environmental changes

  • Network analysis to identify regulatory hubs connecting ACS to other metabolic processes

• Key biological questions addressable through systems approaches:

  • How acetyl-CoA availability influences secondary metabolite production

  • Metabolic rewiring during transition between pathogenic and mutualistic lifestyles

  • Identification of bottlenecks in metabolic pathways that could be engineering targets

• Experimental validation methods:

  • Targeted metabolomics focusing on acetyl-CoA-dependent pathways

  • Isotope labeling experiments to validate predicted flux distributions

  • Creation of synthetic gene circuits to test hypothesized regulatory mechanisms

These integrative approaches would help contextualize how ACS functions as part of the broader metabolic network that enables P. luminescens to navigate its complex lifecycle involving both pathogenic and mutualistic interactions .