Recombinant Squalene synthase

What is recombinant squalene synthase and what reactions does it catalyze?

Squalene synthase (SQS) is a bifunctional enzyme that catalyzes two consecutive reactions: first, the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), and second, the rearrangement and NADPH-dependent reduction of PSPP to squalene . These reactions constitute the first committed steps in cholesterol biosynthesis in eukaryotes and hopanoid biosynthesis in some bacteria .

The recombinant form refers to the enzyme produced through genetic engineering techniques, where the gene encoding SQS is cloned into an expression vector and expressed in a host organism. This approach allows researchers to obtain pure enzyme for detailed structural and functional studies without the constraints of natural source limitations.

How is recombinant squalene synthase typically expressed in laboratory settings?

Recombinant squalene synthase is commonly expressed in Escherichia coli expression systems, though challenges with protein solubility often require optimization strategies. Based on published research, several approaches have proven effective:

• Truncation strategies: For plant squalene synthases like SmSQS2 from Salvia miltiorrhiza, truncating 28 amino acids from the carboxy terminus and expressing as a GST-Tag fusion protein in E. coli BL21 (DE3) significantly improves solubility .

• Buffer optimization: For bacterial SQS like that from Thermosynechococcus elongatus BP-1, soluble protein was obtained only when cells were disrupted and purified in buffers containing glycerol . Without such additives, the expressed proteins with His6 tags were found exclusively in inclusion bodies.

• Source-dependent protocols: While T. elongatus BP-1 SQS could be solubilized with optimized conditions, recombinant SQS from Bradyrhizobium japonicum and Zymomonas mobilis remained insoluble under all tested expression and purification conditions , highlighting the importance of source-specific protocol development.

What analytical methods are most effective for detecting and quantifying squalene synthase products?

Multiple analytical approaches are employed to characterize squalene synthase reaction products, with mass spectrometry-based techniques providing the most comprehensive data:

HPLC-Q-Orbitrap-MS/MS: This technique provides high-resolution detection and quantification of squalene synthase products. Typical parameters include:

• Atmospheric pressure chemical ionization (APCI) source

• 40 Arb sheath gas flow rate, 5 Arb aux gas flow rate

• 300°C capillary temperature

• 140,000 full MS resolution

• Collision energy of 30-35 in NCE mode

• 3.0 kV positive spray voltage

• Full scan between m/z 150-1500 in positive ion mode

This technique has successfully identified pseudomolecular ions ([M + H]+) at m/z 411.3985, 409.3829, 545.5081, and 547.5237 for squalene, dehydrosqualene, phytoene, and lycopersene respectively, with mass errors ≤2 ppm .

Gas Chromatography-Mass Spectrometry (GC-MS): Provides confirmation of squalene production in reaction mixtures through comparison with authenticated standards .

How does the substrate promiscuity of squalene synthase-like enzymes impact experimental design?

Recent research has revealed unexpected promiscuity in squalene synthase-like (SSL) enzymes, particularly dehydrosqualene synthase (CrtM) from Staphylococcus aureus. When designing experiments with SSL enzymes, researchers should consider:

• Multiple product formation: CrtM can produce not only its expected product dehydrosqualene (C30H48) but also squalene (C30H50) and phytoene (C40H64) both in vitro and in vivo . This necessitates comprehensive product analysis rather than targeting a single expected product.

• Substrate options: SSL enzymes can utilize both FPP and geranylgeranyl diphosphate (GGPP), requiring experiments to control for and potentially exploit this promiscuity .

• Quantification challenges: In experiments with CrtM expressed in Bacillus subtilis 168, the output of squalene, dehydrosqualene, and phytoene were measured at 147, 406, and 7 μg/L respectively . These varying yields require sensitive detection methods with appropriate dynamic range.

• Cofactor dependence: The ratio of products formed is significantly influenced by NAD(P)H availability, making cofactor concentration a critical experimental variable .

What mechanisms explain the catalytic versatility of squalene synthase-like enzymes?

The remarkable catalytic versatility of SSL enzymes stems from several structural and mechanistic features:

• Active site flexibility: Comparative studies between human SQS (HSQS) and S. aureus CrtM reveal structural similarities with an RMSD value of 2.5 Å between 253 Cα atoms, despite having different relative orientations of the pyrophosphate group in the bound PSPP intermediate .

• Conserved interaction patterns: The interactions (hydrogen bonds and salt bridges) between SSL enzymes and PSPP are primarily concentrated near the phosphate groups and magnesium ions, which is consistent with their essential role in the head-to-head condensation process .

• PSPP binding conformations: The conformation of PSPP displays remarkable similarity in its binding to SCrtM (PDB ID 3NPR), HSQS (PDB ID 3WEH), and BSQS (modeled through docking), particularly for the remaining two-thirds of the molecule apart from the pyrophosphate group .

• Reaction pathway flexibility: SSL enzymes demonstrate the ability not only to catalyze hydrogen transfer/dehydrogenation reactions (generating squalene or lycopersene) but also to facilitate dephosphorylation reactions (producing dehydrosqualene or phytoene) .

How do cofactors influence the regio- and stereochemistry of squalene synthase reactions?

Cofactors play a critical role in determining the outcome of squalene synthase-catalyzed reactions, particularly affecting regio- and stereochemistry:

• NADPH as a reductant: The presence of NADPH is essential for the reduction of PSPP to squalene. When recombinant SQS was incubated with FPP and NADPH, squalene was formed as the sole product .

• Effects of non-reactive NADPH analogues: When recombinant SQS was incubated with FPP in the presence of dihydroNADPH (NADPH3, an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products were formed instead of just squalene:

  • Dehydrosqualene (DSQ), a C30 analogue of phytoene

  • 10(S)-hydroxysqualene (HSQ), a hydroxy analogue of squalene

  • Rillingol (ROH), a cyclopropylcarbinyl alcohol

• Enhanced stereocontrol: The binding of the cofactor analogue NADPH3 substantially enhances the ability of SQS to control the regio- and stereochemistry of PSPP rearrangements .

• NAD(P)H availability as a limiting factor: Despite enhancing FPP availability through upstream gene upregulation in engineered B. subtilis strains, there was no statistically significant increase in squalene production (P = 0.104) without additional supplementation of NAD(P)H , highlighting the cofactor's role as a potential bottleneck.

What are the comparative yields of different products when using dehydrosqualene synthase versus traditional squalene synthase?

Recent research has revealed that dehydrosqualene synthase (CrtM) from S. aureus can produce more squalene than traditional squalene synthase under certain conditions. The comparative yields observed in different experimental setups are summarized in the table below:

These findings demonstrate that despite SCrtM exhibiting a distinct preference for dehydrosqualene synthesis in terms of selectivity, its squalene synthesis efficiency (630 μg/L) was superior to that of SQS from B. megaterium (260 μg/L) . This suggests the potential to replace traditional SQS with CrtM in squalene biosynthetic cell factories for higher squalene production.

How can researchers optimize solubility of recombinant squalene synthases?

Obtaining soluble, active recombinant squalene synthase remains challenging. Successful strategies include:

• Truncation of C-terminal regions: For SmSQS2 from Salvia miltiorrhiza, deleting 28 amino acids from the carboxy terminus significantly improved solubility when expressed as a GST-Tag fusion protein .

• Buffer optimization: For T. elongatus BP-1 SQS, soluble recombinant protein was obtained only after extensive optimization using buffers containing glycerol .

• Source-specific approaches: While T. elongatus SQS could be solubilized under optimized conditions, B. japonicum and Z. mobilis SQSs remained insoluble under all tested conditions , indicating that source-specific approaches are necessary.

• Expression system selection: While E. coli is the most common expression host, organisms like Saccharomyces cerevisiae may offer advantages for eukaryotic SQS expression, particularly for achieving proper folding and post-translational modifications.

• Fusion partners: GST-Tag fusion proteins have shown success in improving solubility of plant-derived SQS , suggesting that other fusion partners like MBP or SUMO may also be worth exploring.

What in vitro reaction conditions maximize squalene production with recombinant enzymes?

Optimal in vitro reaction conditions for squalene production with recombinant squalene synthase include:

• Substrate concentrations: Effective reactions have been conducted with 20 mM FPP and 0.5 mM NAD(P)H .

• Temperature and duration: Reactions are typically performed at 37°C for 15 minutes, followed by termination and extraction .

• Extraction protocol: Products are extracted using 3 mL of petroleum ether, followed by washing the organic layer with 2 mL of Milli-Q water. After removing the aqueous phase, samples are dried using nitrogen and dissolved in 250 μL of isopropanol-acetonitrile (7:3, v/v) for analysis .

• Cofactor requirements: NAD(P)H is essential for squalene formation, with its concentration potentially limiting the reaction yield .

• Magnesium ions: Mg²⁺ is critical for the head-to-head condensation process, including participation in dephosphorylation , and should be included in the reaction buffer.

What mutagenesis strategies might enhance squalene production in SSL enzymes?

Based on current understanding of SSL enzymes, several mutagenesis strategies could potentially enhance squalene production:

• Targeting phosphate group interactions: Since the synthetic selectivity of SSL enzymes may be significantly influenced by the conformation of phosphate groups and the positions of magnesium ions , mutations affecting these interactions could alter product specificity.

• Exploiting natural CrtM efficiency: Given that CrtM naturally produces more squalene than some native SQS enzymes, structure-guided mutations could potentially enhance this catalytic preference further .

• Enhancing NAD(P)H binding: Since NAD(P)H availability affects squalene synthesis, mutations that enhance cofactor binding or utilization efficiency could increase squalene yields .

• Active site engineering: Previous investigations have leveraged the flexibility of CrtM's active site, enabling the production of diverse long-chain terpenoids alongside dehydrosqualene . Similar approaches could be directed specifically toward enhancing squalene production.

• Cross-species chimeras: Creating chimeric enzymes combining domains from CrtM and traditional SQS could potentially combine the higher synthesis efficiency of CrtM with the product selectivity of SQS.

How might recombinant SSL enzymes be leveraged for novel terpenoid biosynthesis?

The remarkable promiscuity of SSL enzymes offers opportunities for novel terpenoid biosynthesis applications:

• Diverse product spectrum: SSL enzymes can catalyze the formation of multiple products including squalene, dehydrosqualene, phytoene, and lycopersene , suggesting potential for further expansion of this product range through enzyme engineering.

• Substrate flexibility: The ability of CrtM to utilize both FPP and GGPP indicates potential for accepting other non-native substrates to produce novel terpenoids.

• Alternative reduction pathways: The observation that SQS can produce different products depending on the presence of NADPH or its analogues suggests opportunities to exploit this mechanistic flexibility for novel compound synthesis.

• Combining SSL enzymes with other enzymes: Integration of SSL enzymes into artificial biosynthetic pathways could enable production of novel terpenoid structures through sequential enzymatic transformations.