1. New Parts

1.1 This could be New Information Learned from Literature

1.1.1 "Prenyltransferases catalyzing geranyldiphosphate formation in tomato fruit" (2020)

Research Background and Purpose

Monoterpenes are important contributors to tomato fruit aroma, but modern cultivated tomatoes have largely lost these volatile compounds. The immediate precursor for monoterpene biosynthesis is geranyl diphosphate (GPP), produced by geranyl diphosphate synthases (GPPS).

In many plants, GPPS exists as a heteromeric enzyme composed of a catalytic large subunit and a non-catalytic small subunit, which together influence the chain length of the product (GPP vs longer prenyl diphosphates)

This study aimed to identify and characterize enzymes in tomato that produce GPP, to understand why cultivated tomato fruits lack monoterpenes.

Experimental Methods

Researchers cloned and expressed a tomato prenyltransferase gene (LeGGPPS2) and conducted in vitro enzyme assays under various conditions to test its product profile. They also co-expressed LeGGPPS2 with GPPS small subunit proteins from other plants (e.g., Antirrhinum majus) and searched the tomato genome for any GPPS small subunit ortholog. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the terpene products, and gene expression data from cultivated and wild tomatoes were compared to see if differences in GPPS small subunit expression correlate with monoterpene content.

Key Findings

LeGGPPS2, previously known as a geranylgeranyl diphosphate synthase for carotenoid biosynthesis, was found to be remarkably flexible in its product outcome. In vitro, LeGGPPS2 could synthesize not only geranylgeranyl diphosphate (C₅₀) but also farnesyl diphosphate (FPP, C₁₅) and geranyl diphosphate (GPP, C₁₀), depending on assay conditions. Crucially, when LeGGPPS2 was combined with a GPPS small subunit (GPPS.SSU) - either from snapdragon or a newly discovered tomato GPPS.SSU - the enzyme's activity shifted to favor GPP formation over longer prenyl diphosphates. The team identified a GPPS.SSU gene in the genome of the tomato cultivar M82, but this small subunit is not expressed in M82 fruits, whereas equivalent GPPS.SSU genes are expressed in wild tomato relatives that accumulate monoterpenes. This indicates that domesticated tomatoes lack monoterpenes partly because they do not express the necessary GPPS small subunit to channel prenyl flux toward GPP.

Conclusions

The study concluded that the presence of a non-catalytic small subunit is key to enabling GPP production in tomato. LeGGPPS2 alone tends to produce longer-chain prenyl diphosphates (like FPP or GGPP), but in the presence of an appropriate GPPS.SSU partner, it preferentially generates GPP. The loss of monoterpene aroma in cultivated tomatoes appears to be due to the silencing or absence of GPPS.SSU expression in fruit, an unintended consequence of tomato domestication. By reintroducing or expressing a GPPS small subunit, it may be possible to restore GPP and monoterpene biosynthesis in tomato fruits.

Significance

This research provides insight into how GPP biosynthesis can be modulated by enzyme subunit composition. For synthetic biology, the finding suggests that co-expressing a GPPS large subunit with a suitable small subunit is an effective strategy to boost GPP formation and thereby monoterpene production. In the context of our project, this knowledge guided us to include a heteromeric GPPS system (i.e. “GPS”) to ensure a sufficient supply of geranyl diphosphate for downstream monoterpene synthesis. It also highlights a path to enhance flavor or fragrance traits in crops by reactivating dormant biosynthetic steps.

1.1.2 "A geraniol synthase regulates plant defense via alternative splicing in tea plants" (2023)

Research Background and Purpose

Geraniol is a floral-scent monoterpene abundant in tea (Camellia sinensis), and it also plays a role in the plant's defense against pathogens. Prior to this study, the specific enzyme responsible for geraniol biosynthesis in tea and the regulatory mechanisms controlling its activity were not fully understood. In particular, the potential role of alternative splicing in producing enzyme variants (isoforms) that could affect geraniol production and plant defense had not been explored. This study aimed to identify the geraniol synthase gene in tea and investigate how alternative splicing of this gene might influence geraniol formation and the plant's response to pathogen attack.

Experimental Methods

The researchers performed a combination of bioinformatic analysis and laboratory experiments. Using transcriptome databases (Tea Plant Information Archive) and gene expression data, they identified candidate terpene synthase genes and focused on one named CsTPS1 (a Camellia sinensis terpene synthase 1) as a likely geraniol synthase.

They discovered that CsTPS1 undergoes alternative splicing, producing two transcripts: the full-length CsTPS1 and a shorter splice isoform termed CsTPS1-AS. Enzyme assays were conducted by expressing both isoforms to test their activity in converting GPP to geraniol. For in planta functional analysis, tea plants were infected with fungal pathogens (Colletotrichum gloeosporioides and Neopestalotiopsis sp.) to observe changes in CsTPS1 and CsTPS1-AS expression. Additionally, gene-silencing experiments (VIGS or RNAi) were carried out to suppress either the full-length or the alternatively spliced isoform in tea plants, in order to compare their effects on geraniol production and disease resistance. The team also measured the expression of defense-related genes (like PR1, PR2, and salicylic acid pathway genes) and tested geraniol's antifungal activity in vitro.

Key Findings

Both the full-length CsTPS1 and the alternatively spliced CsTPS1-AS isoform encode functional geraniol synthases capable of converting GPP into geraniol in vitro. Interestingly, the expression of the alternatively spliced isoform CsTPS1-AS was found to increase significantly upon pathogen infection, more so than the full -length CsTPS1. When the researchers silenced CsTPS1-AS in tea plants, the geraniol content in those plants dropped and the plants became more susceptible to pathogen infection (showing more severe disease symptoms) compared to control plants. In contrast, silencing the full-length CsTPS1 did not significantly reduce geraniol levels or plant resistance under the same conditions. This implies that the CsTPS1-AS isoform plays a dominant role in producing geraniol during a defense response. Moreover, tea plants with CsTPS1-AS knocked down showed lower expression of key defense marker genes (PR1, PR2 and others in the salicylic acid defense pathway), correlating with the reduced resistance. The study also confirmed that geraniol itself has antifungal properties in vitro, inhibiting pathogen growth in a dose-dependent manner. Cellular localization experiments revealed that CsTPS1 and CsTPS1-AS localize to different subcellular compartments, suggesting they might have distinct roles despite producing the same compound.

Conclusions

This work revealed that CsTPS1 is the gene responsible for geraniol biosynthesis in tea, and more importantly, that its alternatively spliced form CsTPS1-AS is crucial for the plant's defense mechanism. The alternative splicing of a terpene synthase gene effectively creates a defense-responsive enzyme isoform (CsTPS1-AS) that boosts geraniol production when the plant is under pathogen attack. The full-length enzyme, while capable of making geraniol, appears less involved in the acute defense response. This discovery highlights alternative splicing as a novel regulatory layer in plant secondary metabolism: by producing a slightly different version of the geraniol synthase, the tea plant can ramp up production of a defensive metabolite (geraniol) without needing separate genes. The differential roles of CsTPS1 and CsTPS1-AS were clearly demonstrated by the silencing experiments - only the loss of the CsTPS1-AS isoform led to significant decreases in geraniol and increased disease susceptibility.

Significance

For the field of plant biotechnology and synthetic biology, this study provides a key insight: gene regulation via alternative splicing can significantly impact metabolic output and stress resistance. In practical terms, it means that engineering efforts to enhance the production of valuable metabolites (like geraniol) might need to consider not just the gene itself, but also its splicing variants. Our iGEM project benefits from this knowledge by emphasizing the use of the CsTPS1 gene (geraniol synthase) to produce geraniol, and recognizing that different isoforms or expression contexts could affect output. It also suggests that by mimicking what plants do - for instance, expressing a version of geraniol synthase optimized for stress conditions - we might improve yields of geraniol in microbial hosts. Furthermore, geraniol's demonstrated antifungal activity and the link to induced defense genes underscore its potential use as a biopesticide or antimicrobial agent. The findings from tea plants inspire future strategies to increase pest or pathogen resistance in crops through metabolic engineering of terpene pathways (e.g., by increasing the flux to geraniol or similar defense volatiles under inducible control).

1.1.3 "Tunable Production of (R)- or (S)-Citronellal from Geraniol via a Bienzymatic Cascade Using a Copper Radical Alcohol Oxidase and Old Yellow Enzyme" (2022)

Research Background and Purpose

(-)-Menthol is one of the most commercially important flavor compounds worldwide, and its industrial synthesis often relies on producing a key intermediate, (R)-citronellal. Traditionally, (R)-citronellal (a chiral monoterpene aldehyde) can be obtained by chemical hydrogenation of citral (a mixture of geranial and neral), but controlling the stereochemistry and dealing with the citral E/Z isomer mixture in biocatalysis is challenging. This study sought to develop a biocatalytic pathway to efficiently produce either enantiomer of citronellal directly from geraniol (an inexpensive, readily available precursor), thereby bypassing the problematic step of reducing citral's isomeric mixture. The goal was to achieve a one-pot enzymatic cascade that yields high conversion and enantioselectivity for either (R)- or (S)-citronellal by selecting appropriate enzymes.

Experimental Methods

The researchers designed a two-step enzyme cascade in a single pot. In the first step, a copper radical alcohol oxidase (CRO-AlcOx, specifically Cgr AlcOx from Coprinopsis sp.) oxidizes geraniol (a primary alcohol) to geranial (the corresponding aldehyde, also known as citral when combined with its isomer neral). In the second step, an old yellow enzyme (OYE, which is an NADPH-dependent enoate reductase) reduces geranial to citronellal. By choosing different OYE enzymes, they aimed to control the chirality of the produced citronellal. For example, using the enoate reductase OYE2 (from Saccharomyces cerevisiae) was expected to favor formation of (R)-citronellal, whereas using a different OYE (named GluER, an ene-reductase from Gluconobacter sp.) would favor (S)-citronellal. The cascade was tested in vitro by mixing geraniol with the oxidase and a chosen reductase, along with necessary cofactors (e.g. oxygen and possibly a cofactor regeneration system for NADPH). The reaction outcomes were analyzed by chiral gas chromatography to determine conversion rates and enantiomeric excess (ee) of the citronellal produced. Scale-up experiments were also performed to assess the yield and optical purity on a preparative scale.

Key Findings

The developed bienzymatic cascade successfully converted geraniol to citronellal with high efficiency and selectivity. Using OYE2 in the second step, the one-pot reaction achieved a 95.1% conversion of geraniol to (R)-citronellal, with an enantiomeric excess (ee) of 95.9% in favor of the desired (R) enantiomer. They were able to scale this reaction up to a 62 mg preparation, obtaining (R)-citronellal in high yield and with essentially the same high optical purity. Alternatively, by switching the second enzyme to GluER, the cascade produced the opposite enantiomer: (S)-citronellal, with 95.3% conversion and an even higher enantiomeric purity of 99.2% ee (almost enantiomerically pure). These results demonstrate that simply by choosing the appropriate reductase, the system can be tuned to yield either enantiomer of citronellal as the main product, without needing to separate mixtures of isomers post-reaction. Importantly, the use of geraniol as the starting material (instead of citral) and the tandem action of the copper oxidase and OYE overcome the issue of having to enzymatically reduce a mixture of E- and Z-citral isomers. The oxidase selectively produces geranial (the E-isomer), and the OYEs used are stereospecific in reducing the activated double bond to give predominantly one enantiomer of citronellal.

Conclusions

This study demonstrates an effective biocatalytic cascade to obtain chiral citronellal from a cheap substrate (geraniol) in just one pot. The key innovation is combining a radical alcohol oxidase with an ene-reductase, which streamlines the process and allows control over product chirality by enzyme selection. By achieving near-quantitative conversion and high enantioselectivity for both (R)- and (S)-citronellal, the authors provided a proof-of-concept for a tunable menthol precursor production system. The cascade addresses a major challenge in menthol synthesis - namely, the need for (R)-citronellal - in a greener, enzymatic way, avoiding harsh chemical catalysts or racemic mixtures. The successful scale-up to tens of milligrams suggests the approach could be further developed for industrial application.

Significance

For our iGEM project, this paper's findings are highly relevant because they highlight a strategy to produce valuable monoterpenoid aldehydes from geraniol using enzyme cascades. It reinforces the idea that geraniol can serve as a versatile branching point to different products depending on which downstream enzyme is applied. In our pathway, we include a geraniol-oxidizing enzyme (which we refer to as GeDH, geraniol dehydrogenase) to convert geraniol into an aldehyde product. While the specific enzyme we use might differ (e.g., a dehydrogenase that produces geranial (citral) rather than a chiral citronellal), the concept is similar: using oxidation of geraniol to broaden the product spectrum. The tunability demonstrated by the OYE variants suggests that, in the future, teams could plug in different reductases or additional enzymes to obtain various downstream products from a common intermediate. Moreover, this study exemplifies how coupling enzymes in one pot can circumvent equilibrium or selectivity issues - a lesson that guided us to consider enzyme compatibility and cofactor balance in our design. Overall, the cascade approach informs our contribution by showing a clear route to produce a high-value fragrance compound from a simple precursor with biological catalysts, supporting the feasibility of our engineered biosynthetic pathway.

Figure 1: Engineered pathway for geranial (citral) production. Key enzymes contributed by our project include a geranyl diphosphate synthase (GPPS) for GPP supply, the tea plant geraniol synthase CsTPS1 for geraniol production, and a geraniol dehydrogenase (GeDH) for converting geraniol to an aldehyde. This figure illustrates how each enzyme sequentially acts on the metabolite to ultimately yield citral. Future teams can use this blueprint to produce other monoterpenoids by swapping or adding enzymes at these steps.

1.1.4 “Advances in mosquito repellents: effectiveness of citronellal derivatives in laboratory and field trials” (2022)

Research Background and Purpose

Citronellal is a principal component of citronella oil but its practical use is limited by high volatility and short protection time. This paper tested whether chemical modification of citronellal into hydroxylated cyclic acetals could lower volatility and extend protection while maintaining repellency against Aedes albopictus and Anopheles gambiae. The central question is directly relevant to our controlled-release strategy: can we secure DEET-like protection windows from citronellal-based actives by tuning physicochemical properties and formulation?

Experimental Methods

The authors synthesized a mixture of citronellal-glycerol acetals (1,3-dioxane/dioxolane isomers), characterized them by GC-MS and NMR, and ran human-bait laboratory assays to estimate percent protection over time, dose-response, and complete protection time. They then performed an 8-hour randomized latin -square human field trial in a temperate area with high A. albopictus density, comparing a 5% solution of the citronellal derivatives against commercial 20% DEET and 20% icaridin formulations. They also tested vanillin as a fixative co-additive.

Key Findings

In laboratory tests, the citronellal derivatives reached ≈95-100% protection at practical skin doses and maintained >90% protection for up to 8 h against A. albopictus; against A. gambiae, 90% efficacy lasted ≈7 h at lower dose. Adding 1% vanillin further extended high-level protection (≈>98% for 8 h). In field trials, 5% citronellal derivatives achieved ~99% protection at the start and ≈94% at 3.5 h, comparable to 20% icaridin at that time point; complete protection time ranked icaridin > citronellal derivatives ≈ DEET, with the citronellal derivatives performing competitively despite being used at one-quarter the active concentration of the synthetics. Volunteers reported weaker odor than neat citronellal, aiding acceptance.

Conclusions

Modifying citronellal to reduce volatility (and modestly increase hydrophilicity) yields repellents with substantially prolonged activity, approaching the durability of benchmark synthetics at much lower active loadings. Formulation with fixatives such as vanillin provides an additional, formulation-level lever to further extend protection times.

Significance for Our Project

These results justify our emphasis on near-body controlled release and fixative-assisted formulations: prolonged protection is achievable when citronellal's release rate is slowed—either by molecular modification or by materials engineering. We translate this into materials choices (microcapsules, gels, and solid matrices) that emulate the same kinetic goal (reduced evaporation, smoother flux) while retaining a “green” profile; our SOPs for release testing and our decision rules on crosslink density and oil-phase fraction are designed to target the multi-hour protection windows documented here.

1.1.5 “The Mosquito Repellent Activity of the Active Component of Air Freshener Gel from Java Citronella Oil (Cymbopogon winterianus)” (2020)

Research Background and Purpose

Citronella (Java type) contains citronellal, citronellol, and geraniol. While whole-oil repellency is well known, the relative activities of individual components and the performance of consumer-relevant formats (e.g., gels) are not always clear. This paper isolated the main constituents and evaluated their repellency against Aedes aegypti, linking composition, format, and efficacy—evidence directly applicable to our “non-spray, near-body” concept and our component-level selection of citronellal as an active.

Experimental Methods

Java citronella oil was produced by steam distillation and then fractionated by batch vacuum distillation to enrich citronellal, citronellol, and geraniol. The team formulated air-freshener type gels with defined loads of each component and assessed repellency in human-volunteer laboratory assays against A. aegypti, recording percent protection over time. The article reports comparative protection profiles of the individual actives in a low-cost, solid-format matrix.

Key Findings

All three components—citronellal, citronellol, and geraniol—exhibited measurable repellency in short-term tests, with geraniol and citronellol often scoring slightly higher than citronellal at matched conditions; nonetheless, citronellal contributed substantially to early-time protection. The gel format provided usable release but, as expected for volatile monoterpenes, protection declined over time without fixatives or barrier materials, underscoring the need for release-rate control. (The paper's DOI and abstracted records confirm the component-wise testing against A. aegypti and the formulation context.)

Conclusions

Component-resolved testing shows citronellal is a valid mosquito-repellent active, though performance depends strongly on format and release kinetics. Simple gels can deliver short-term protection; extending duration requires either co-formulants that curb volatility or material architectures that slow evaporation.

Significance for Our Project

These data support our selection of citronellal as a core active while also validating our engineering focus on controlled release rather than sprays or naked application. Our microcapsule/gel/solid-block carriers, plus fixative options (e.g., vanillin) and fabric-friendly near-body placement, are designed specifically to overcome the volatility-limited protection windows that Eden et al. observed in simpler gels. This aligns with our wiki SOPs on headspace sampling and our design rule that “slower flux → longer protection,” the same principle that extended efficacy in Iovinella et al. via molecular acetals.

1.2 This could be New Data Collected from Laboratory Experiments

In the present study, we opted to reconstitute a three-enzyme cascade reaction pathway—specifically the conversion sequence "geranyl pyrophosphate (GPP) → geraniol → citronellal"—using *E. coli* BL21 as the host strain. For this purpose, the genes GPS, CsTPS1, and GeDH were individually cloned into the vectors pET28a, pET21a, and pBAD23, respectively. This cloning strategy yielded a compatible multi-plasmid co-expression system, where each plasmid carries a unique antibiotic resistance marker.

Such a system not only simplifies the screening and selection of positive transformants but also supports dual induction and dosage control through the use of IPTG (isopropyl β-D-1-thiogalactopyranoside) and L-arabinose. A key focus of this design is the incorporation of modular, interchangeable expression cassettes; this feature allows for flexible modulation of metabolic flux distribution and enables subsequent optimization of culture conditions, such as adjusting induction intensity or cultivation temperature.

Figure 2: Plasmid Design

To begin with, we acquired the sequences of the three target genes and performed codon optimization tailored for E. coli. The optimized gene sequences were synthesized by Sangon Biotech (Shanghai) and provided as fragments inserted into pUC19 plasmids. Both the target gene fragments and the backbone vectors (sourced from the laboratory stock) were amplified using PCR, and their authenticity was confirmed through agarose gel electrophoresis analysis (as shown in Figure 3).

Figure 3: M: Marker; 1: GeDH; 2: CsTPS1; 3: GPS; 4: pET28a; 5: pET21a; 6: pBAD33

Subsequently, the gene fragments were integrated into the respective vectors via Gibson assembly, and the resulting constructs were introduced into BL21 competent cells. Positive bacterial transformants were successfully obtained (Figure 4).

Figure 4: Bacterial Transformants

To confirm the proper construction of the engineered bacterial strain, we designed specific specific primer sets for each exogenous gene (GPS, CsTPS1, and GeDH) to conduct genotypic verification. These primers were designed to target both internal conserved sequences of the genes and flanking regions of the vectors, a strategy aimed at enhancing detection specificity and ensuring accurate confirmation of insert orientation.
Two separate single colonies were selected from the transformation plates and directly used as templates for colony PCR. Agarose gel electrophoresis analysis revealed amplification bands at the anticipated sizes for all three genes, with no presence of non-specific amplification products or primer dimers (Figure 5). These findings served as initial evidence that all three genes had been correctly inserted into the host strain with the proper orientation, thereby confirming the success of the transformation process.

Figure 5: M: Marker; 1,2: GeDH; 3,4: CsTPS1; 5,6: GPS

To mitigate false positives from colony PCR (e.g., due to template contamination or homologous amplification), the two positive clones were sent to Sangon Biotech for Sanger sequencing. Forward and reverse reads covered the insertions and vector junctions. The sequences matched the designed sequences exactly.

1.3 Summary

Based on the five papers and our completed construction and verification, the evidence chain from mechanism to realization can be connected in more detail as follows: In terms of precursor supply and chain length regulation, tomato research revealed that the heterologous complex of large/small subunits of GPPS has a decisive effect on product chain length. The large subunit alone (such as LeGGPPS2) may be biased towards FPP/GGPP production in vitro, while pairing with the non-catalytic small subunit can stably direct the flux to GPP, which directly suggests that "heterologous complex" or functionally equivalent GPP synthesis modules should be given priority in the heterologous chassis to avoid the substrate being taken away by endogenous IspA or downstream FPP pathways; on the product generation and regulation side, the study of tea tree CsTPS1 not only clarified the core enzyme that directs GPP to geraniol, but also found through alternative splicing that the defensive isozyme CsTPS1-AS dominates product generation under stress conditions, indicating that the same TPS family has differences in expression context, positioning and timing. Abnormalities can significantly affect flux and homeostasis, providing two engineering insights for microbial expression: first, codon optimization and expression intensity stratification of TPS to reduce misfolding and metabolic burden; second, the introduction of "defense/inducible" expression strategies to mimic plant stress responses to enhance transient flux when needed. Regarding downstream selectivity, OYE (named GeDH in our project) produces citronellal with high conversion and high enantioselectivity in a one-pot system.

On the end-use application side, two mosquito repellent studies demonstrated that molecular modifications such as acetalization of citronellal combined with a small amount of a fixative (such as vanillin) can significantly extend the duration of protection, approaching the durability of DEET/icaridin. Furthermore, they also pointed out that while solid-phase formats such as simple gels can achieve rapid onset of action, they are limited by volatilization rates and exhibit significant degradation of protection over time. This reinforces the engineering direction of controlled release through materials science rather than simply increasing concentration. Based on this external evidence, we experimentally reconstructed the "GPP→geraniol →citronellal" three-stage pathway using E. coli BL21(DE3): GPS, CsTPS1, and GeDH were cloned into pET28a, pET21a, and pBAD23, respectively, to form a multi-plasmid system with complementary resistance, replicon compatibility, and independently regulated induction mode. All three genes were codon-optimized for E. coli and synthesized by the supplier. Inserts were amplified by PCR, assembled with the vector by isothermal Gibson, and transformed into BL21. Single clones were picked for colony PCR and 1.0% agarose gel electrophoresis to obtain bands consistent with the expectations. These were then sent for Sanger sequencing, covering the entire insert length and the vector linker region, and were completely consistent with the design, confirming the coexistence and correct orientation of the three plasmids at the genetic level.

1.4 Reference

[1]Hivert G., et al. Prenyltransferases catalyzing geranyldiphosphate formation in tomato fruit. Plant Science 2020;296:110504.
[2]Jiang H., Zhang M., Yu F., et al. A geraniol synthase regulates plant defense via alternative splicing in tea plants. Horticulture Research 2023;10(10):uhad184.
[3]Ribeaucourt D., Höfler G. T., Yemloul M., Bissaro B., Lambert F., Berrin J-G., Lafond M., Paul C. E. Tunable production of (R)- or (S)-citronellal from geraniol via a bienzymatic cascade using a copper radical alcohol oxidase and old yellow enzyme. ACS Catalysis 2022;12(2):1111-1116.
[4]Iovinella I., Pelosi P., Conti B., et al. Advances in mosquito repellents: effectiveness of citronellal derivatives in laboratory and field trials. Pest Management Science 2022;78(12).
[5]Kim J-K., Kang C-S., Lee J-K., Kim Y-R., Han H-Y., Yun H-K. Evaluation of repellency effect of two natural aroma mosquito repellent compounds, citronella and citronellal. Entomological Research 2005;35(2):117-120.

2. Document Troubleshooting that would be Helpful to Future Teams

2.1 Problem Background

Our multi-plasmid, dual-induction pathway (GPS-CsTPS1-GeDH) was genetically correct by colony PCR and Sanger sequencing, yet the effective carbon flux toward citronellal still depended on balanced transcription, translation, and process conditions. The design inherently risked copy-number imbalance, antibiotic selection drift, and resource competition, any of which could depress expression of one module and create a kinetic bottleneck. In parallel, product detection could be confounded by intracellular-extracellular partitioning and by volatility losses, while single-channel analytics risked method-specific bias. Finally, closely sized proteins and shared epitope tags could complicate Western blot interpretation, masking true expression states and delaying root-cause identification.

2.2 Experimental Methods

We standardized chassis health checks by running uninduced growth curves for engineered, empty-vector, and wild-type strains under identical inoculum, selection, temperature, and shaking conditions (Figure 6).

Figure 6 Group 1: Engineered bacteria + LB + antibiotics; Group 2: Wild-type bacteria + LB; Group 3: Empty vector bacteria + LB + antibiotics

We fixed induction at mid-log phase and used calibrated IPTG and L-arabinose doses to minimize variability, deliberately employing TB medium with glycerol to avoid catabolite repression of PBAD. Transcriptional activation was quantified by qPCR using 16S rRNA as the internal reference and 2^-ΔΔCt analysis with amplicons of ~200 bp (Figure 7).

Figure 7: Relative expression level of GPS, CsTPS1, and GeDH

While translation was verified by SDS-PAGE and His-tag Western blot, with attention to gel resolution around 40-65 kDa to separate GPS and GeDH (Figure 8).

Figure 8: The Western blot results of GPS, CsTPS1, and GeDH.

For product-level validation, we sampled both supernatant and cell lysate from induced fermentations, quantified citronellal by HPLC using an external standard curve and defined retention time, and cross-validated the same samples by UV-Vis to test orthogonal agreement (Figure 9).

Figure 9 HPLC of TB fermentation supernatant.

Throughout, we constrained inoculation, induction timing, and antibiotic concentrations across runs to suppress noise from plasmid loss or resource competition, and we interpreted merged WB bands using expected molecular weights and band morphology rather than presence/absence alone.

2.3 Findings and Results

Uninduced growth trajectories of engineered, empty-vector, and wild-type strains overlapped in lag duration, exponential slope, and stationary-phase OD₆₀₀, indicating that multi-plasmid carriage did not impose a measurable basal burden and allowing downstream differences to be attributed to pathway activity rather than chassis sickness. Upon dual induction, qPCR demonstrated robust transcriptional upregulation for all three genes, with CsTPS1 and GPS showing slightly higher fold changes than GeDH, revealing a likely terminal-step bottleneck at the RNA level and suggesting that expression balance rather than genetic integrity limited flux. Western blots confirmed protein-level expression at the expected windows for GPS (~46.9 kDa), CsTPS1 (~63.7 kDa), and GeDH (~41.6 kDa); partial overlap between GPS and GeDH appeared as a thicker composite band but was resolved by size expectations and gel conditions. HPLC quantified citronellal at ~0.9 g/L in both supernatant and lysate, and statistical testing found no significant difference between compartments, establishing that broth sampling alone is representative for future assays. UV-Vis measurements on the same samples aligned with HPLC quantitation, providing orthogonal confirmation of the production level and excluding method-specific artifacts.

2.4 Significance of Troubleshooting

This troubleshooting closed the loop from “chassis health → transcriptional activation → protein expression → product formation,” converting genetic correctness into process-credible, measurement-robust evidence that the pathway is functionally active. By proving negligible basal burden, we removed growth defects as a confounder; by pairing qPCR with WB, we localized the principal constraint to terminal-step expression rather than miscloning or translation failure; and by demonstrating intracellular-extracellular equivalence and HPLC -UV agreement, we established a streamlined, defensible quantification workflow that future cycles can apply at scale. Practically, these results de-risk subsequent optimization: the next DBTL round can focus on balancing expression (e.g., promoter/RBS tuning or staggered induction to elevate GeDH), refining inducer set-points via response-surface designs, and coupling fermentation parameters to controlled-release materials without re-interrogating construct integrity or assay validity. As a reusable template for future teams, the method emphasizes standardized pre-induction growth checks, multi-level expression validation, dual-channel analytics, and early mapping of product partitioning—four elements that together transform pathway activation from a qualitative claim into quantitatively reproducible practice.

2.5 Recommendations

Future teams should institutionalize a short “pre-fermentation validation” routine that always includes an uninduced growth check, a dual-induction qPCR snapshot, and a single Western blot lane per target around the expected molecular-weight window; locking these three gates before product assays prevents wasted runs and cleanly localizes bottlenecks. Expression balance should be treated as a tunable design variable rather than a fixed property: consider a staggered-induction scheme (upstream first, then GeDH after 30-90 min), test a small matrix of promoter/RBS strengths for the terminal step, and—if transcription remains limiting—raise GeDH dosage via copy number or promoter swap while monitoring chassis burden with the same growth assay you trust. For analytics, maintain an external-standard HPLC method with a documented retention-time tolerance window and re-verify the standard curve each batch; preserve an orthogonal readout (UV-Vis or GC-MS spot checks) and decide early, based on an intracellular/extracellular partitioning test, whether supernatant alone is a faithful proxy to streamline throughput. On the process side, keep glycerol-based rich media for PBAD friendliness, fix induction at mid-log (OD₆₀₀≈0.6) unless data justify change, and if volatility becomes a constraint at longer durations, introduce a simple overlay or closed-cap sampling regime to prevent evaporative loss before concluding that titers have plateaued.

Reproducibility hinges on disciplined controls and metadata: always run wild-type and empty-vector baselines under identical selection pressure, record inoculum density, induction time stamps, and antibiotic lots, and save raw Ct tables, gel images, chromatograms, and calibration files alongside analysis notebooks so that effect sizes (e.g., 2^-ΔΔCt and g L⁻¹) can be recomputed independently. When scaling, change one factor at a time and carry over the entire three-gate validation; if a change breaks performance, revert to the last validated state and only then explore multivariate optimization (e.g., response-surface tuning of IPTG/L -arabinose and temperature). Finally, align wet-lab outputs with the intended product format by planning downstream materials tests early; pairing a stable biological titer with a controlled-release matrix and a headspace assay closes the loop between pathway flux and end-use efficacy, converting bench-level gains into claims that withstand wiki and review scrutiny.

3. Proposed Combined Approach (Response Surface and Model) to Improve Fermentation Yield

3.1 Response Surface Methodology for Process Optimization

To elevate citronellal production from a merely capable level to a truly high-yield process, we applied a Response Surface Methodology (RSM) approach using a Box-Behnken experimental design. This design systematically varied three key induction factors - temperature, IPTG (isopropyl-β-D-thiogalactopyranoside) concentration, and L-arabinose concentration - each at three levels. The goal was to capture not only individual effects on citronellal titer, but also factor interactions and nonlinear (quadratic) effects, which simple one-factor-at -a-time experiments would miss. The Box-Behnken design provides evenly distributed design points without extreme combinations, requiring only 15 experimental runs to fit a full quadratic model (including main effects, two-factor interactions, and squared terms).

Factors and Levels: We tested induction temperature at 20°C, 28°C, 36°C; IPTG at 0.10, 0.50, 0.90 mM; and L -arabinose at 0.02%, 0.11%, 0.20% (w/v). These levels were chosen to span a broad range while focusing on practical conditions. Each run of the 15 shake-flask fermentations used a unique combination of these factor levels as dictated by the design, and the resulting citronellal concentration (g/L) was measured for each.

This RSM approach allowed us to model the fermentation yield as a function of the three induction parameters and identify optimal conditions without performing an exhaustive number of experiments. By using a statistical design with built-in replication and center points, we also ensured that the model's lack-of-fit could be assessed for reliability.

3.2 Dry Lab Rational Design Targeting the Rate-Limiting Enzyme

In parallel with the experimental design, we pursued a computational “dry lab” rational design focusing on the pathway's known rate-limiting enzyme, GeDH (geraniol dehydrogenase). While RSM optimized the process conditions, this modeling effort aimed to optimize the biocatalyst itself. The rationale was to enhance the enzyme's performance (stability and affinity) for the pathway intermediate, thereby boosting overall flux to citronellal. Our dry lab workflow (Figure 10) involved several cutting-edge tools in sequence:

Figure 10 Dry lab roadmap

Structure Prediction (Chai1): We first generated a high-confidence 3D structure model of GeDH to understand its folding and active site configuration.

Mutational Scanning (FuncLib): Using the predicted structure, FuncLib was employed to explore networks of beneficial mutations. It provided mutation suggestions along with thermostability scores, helping us identify mutations (or combinations of mutations) that could improve enzyme stability without compromising function.

Docking Simulations (Rosetta): Candidate mutant enzyme structures were then subjected to molecular docking simulations with the substrate/product. Rosetta was used to predict how mutations affected the binding of the enzyme to its substrate (citronellol or the corresponding aldehyde). Each variant received a docking score, indicating predicted binding affinity and positioning.

Molecular Dynamics (GROMACS): Top-scoring variants were further evaluated by running molecular dynamics simulations of the enzyme-substrate complex. This step tested the stability of the enzyme-ligand interaction over time and allowed calculation of binding free energies. By observing the dominant enzyme conformations during MD (Fig. 5.4), we ensured the mutations did not disrupt the active site and that the substrate remained snugly bound.

Using this multi-step in silico approach, we filtered a large space of possible mutations down to a few promising GeDH variants for experimental testing. Early on, single, double, and triple mutants were generated in silico, but interestingly, we found that quadruple mutants generally achieved better scores in both stability and docking. This suggested a synergistic effect where multiple mutations together produced a more robust and active enzyme.

3.3 In Silico Screening and Selection of GeDH Mutants

Thousands of GeDH variant sequences were scored computationally. Stability scores (from FuncLib) and docking scores (from Rosetta) were combined to rank the mutants. We observed a clear trend: variants carrying four mutations tended to outperform those with fewer changes, indicating that a coordinated set of mutations was beneficial. For instance, mutant 54883 had one of the best combined scores (approximately -1280), whereas the wild-type GeDH by comparison scored around -983 - a much less favorable score indicating poorer predicted stability and binding. From the comprehensive in silico analysis, four top mutant sequences were chosen, alongside the wild-type, for more detailed evaluation via molecular dynamics. The MD simulations confirmed that these mutants maintained stable structures and proper active-site geometry over time. For example, one candidate showed that the mutated enzyme aligns very well with the wild-type conformation, and it keeps the ligand tightly bound in the correct orientation. Another candidate (mutant 54456) also bound the ligand in the expected pocket with a very favorable binding free energy, indicating strong affinity. Based on these dry lab results, we designed a quadruple mutant GeDH carrying the specific amino acid substitutions Q60R, L125P, F147Y, and A300D. This combination was predicted to substantially enhance the enzyme's thermostability and catalytic efficiency for converting the intermediate (citronellol or related alcohol) to citronellal. To test this experimentally in the next step, we constructed a new variant of our production strain in which only the GeDH expression cassette was replaced with the gene for this mutant enzyme (the chassis and all upstream pathway enzymes were kept unchanged; see the Model section for details on the mutation design). This approach isolates the effect of the enzyme mutation on the pathway flux without other confounding changes.

3.4 Experimental Execution of the Combined Strategy

Following the design phase, we moved to the Build and Test phases. According to the Box-Behnken RSM plan, we conducted 15 shake-flask fermentations corresponding to the planned combinations of temperature, IPTG, and L-arabinose. During each run, cultures were induced with both IPTG and L-arabinose (dual induction) at the specified concentrations and grown at the set temperature. After induction and an appropriate cultivation period, samples were taken and the citronellal concentration in the supernatant was quantified (primarily by HPLC with an external standard calibration, supplemented by UV absorbance methods for quick estimates). Each experiment provided a data point for the response surface model. The citronellal titers we observed spanned a wide range, from as low as ~0.43 g/L in unfavorable conditions up to ~0.96 g/L in the best condition tested. This data was then used to fit a quadratic model and perform ANOVA to decipher the effects:

ANOVA Results: The RSM model was statistically significant (F = 8.90, p = 0.0134), indicating that the chosen factors reliably explain variations in yield. There was no significant lack-of-fit (p = 0.0868), so the quadratic model adequately fits our experimental data.

Main Effects: Induction temperature emerged as the most influential single factor (F = 42.18, p = 0.0013). Lowering the temperature dramatically increased yield, suggesting that slower growth or enhanced protein folding at 20°C helped the pathway produce more citronellal.

Interaction Effects: Notably, the interaction between IPTG and L-arabinose was significant (p = 0.0422). This indicates the two inducers must be co-optimized - simply adding a high amount of one inducer will not maximize production unless the other is appropriately set. In practice, it meant that our dual-induction system required careful balancing; for example, too much IPTG with too little arabinose (or vice versa) could lead to suboptimal enzyme expression balance in the pathway.

Quadratic Effects: The IPTG squared term (B²) was highly significant (p = 0.0069). This reveals that IPTG has an optimal concentration window; beyond a certain point, increasing IPTG gives diminishing returns or even negative effects (perhaps due to metabolic burden or plasmid stress). Thus, there is an optimal IPTG concentration rather than “more is better.”

Crucially, the RSM model yielded a set of predicted optimal conditions for induction: 20 °C, 0.46 mM IPTG, and 0.20% (w/v) L-arabinose. These conditions were essentially in line with the best experimental runs (with the IPTG optimum refining to ~0.46 mM, slightly below the tested 0.50 mM level).

3.5 Fermentation Results Under Optimal Conditions

With the optimal process conditions identified, we next evaluated the fermentation performance under these conditions, both for the original strain and for the new GeDH mutant strain. Fermentations were carried out at 20 °C with 0.46 mM IPTG and 0.20% L-arabinose induction, and citronellal production was measured by HPLC:

Baseline (Wild-Type Enzyme): Under the RSM-predicted optimal induction conditions, the original strain (with wild-type GeDH enzyme) produced approximately 1.01 g/L of citronellal in the culture supernatant. This confirmed that our statistically optimized conditions indeed boosted the titer beyond what any of the unoptimized conditions achieved in the earlier RSM runs (compare to the previous max ~0.96 g/L).

Engineered Enzyme Variant: Strikingly, the strain expressing the mutant GeDH (Q60R/L125P/F147Y/A300D) reached a citronellal titer of about 1.36 g/L under the same optimal induction regimen. This is a substantial improvement over the wild-type, on the order of a 34% increase in yield. The higher titer directly validates our rational design strategy: by enhancing the catalytic efficiency at the pathway's terminal step (oxidation of citronellol to citronellal), we successfully increased the overall flux of the pathway to produce more final product.

These results demonstrate the power of a combined approach. The process optimization (lower temperature, balanced dual inducer levels) created a more favorable environment for production, while the enzyme optimization removed a bottleneck at the pathway's end. Together, they resulted in a significantly higher citronellal yield than either approach alone could likely achieve.

3.6 Key Engineering Insights and Future Directions

The Learnings from this part can be summarized as follows:

Induction Temperature is a Critical Lever: Lowering the induction temperature emerged as the most powerful lever for increasing citronellal yield. Cooler temperatures likely improve protein folding and reduce metabolic stress, thereby enhancing the production of our target compound.

Dual Induction Requires Balance: When using two inducers (IPTG and L-arabinose), their coordination is crucial. We discovered that IPTG has an optimal range - more is not always better beyond a certain point. Thus, proper tuning of both inducer concentrations is necessary for maximizing yield; high doses of IPTG need sufficient L-arabinose to be effective (and vice versa).

Enzyme Enhancement Boosts Pathway Flux: Re-engineering the pathway's terminal enzyme (GeDH) proved highly effective. The rationally designed mutant significantly increased the flux through the final oxidation step, leading to higher product titers. This, combined with process tweaks, yielded significant gains in production.

As a result of these findings, we have updated our standard protocol for citronellal production. The induction conditions of 20°C, 0.46 mM IPTG, 0.20% L-arabinose are now established as the benchmark for our experiments moving forward. We also refined our product quantification strategy, relying on direct supernatant sampling and HPLC with external standards as the primary measurement method (with rapid UV spectroscopy assays as a supplemental tool for quick checks). On the molecular side, the success with GeDH has encouraged us to extend this enzyme improvement strategy to other steps in the pathway. We plan to apply saturation mutagenesis and combinatorial mutation approaches to upstream enzymes, followed by similar computational screening, to systematically remove bottlenecks throughout the pathway. Looking ahead, the next cycle of optimization will tackle factors we have not yet explored. We intend to incorporate additional process parameters such as dissolved oxygen levels, pH control, and induction timing into the RSM design for scale-up trials. Including these factors will help us statistically optimize the fermentation for even higher volumetric yields and improve batch-to-batch consistency in larger bioreactors. Together, these improvements provide both process-level and molecular-level levers to drive further enhancements. By iteratively refining conditions and enzyme performance, we are steadily reducing production costs and pushing the citronellal biosynthesis process toward pilot-scale feasibility. This combined approach of RSM optimization and rational enzyme design has proven to be a powerful framework for improving fermentation yields in our project, and it will continue to guide our engineering efforts in subsequent cycles.

4. Add information to an existing Part

4.1 New Information Learned from Literature

4.1.1 "Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation" (2014)

Research Background and Purpose

This study investigates the role of collagen-binding mutations in the A3 domain of von Willebrand Factor (VWF) in thrombus formation. VWF is a key protein in the coagulation process, binding to exposed subendothelial collagen to initiate platelet adhesion and thrombus formation. The main goal is to analyze these collagen-binding mutants' effects on VWF function through mouse models and in vitro experiments.

Experimental Methods

Researchers created five loss-of-function mutants and one gain-of-function mutant in the VWF cDNA, including p.S1731T, p.W1745C, p.S1783A, p.H1786D, p.L1757A, and one A3 domain deletion mutant. These mutants were analyzed through collagen binding assays, platelet adhesion assays, and flow chamber assays under high shear conditions.

Key Findings

Loss-of-function mutants showed significantly reduced collagen binding capacity: ELISA experiments revealed that most loss-of-function mutants had markedly reduced binding to type I and III collagen, especially p.W1745C and p.S1783A, with collagen-binding activities only 10%-30% of the wild type.

The gain-of-function mutant p.L1757A exhibited significantly enhanced collagen binding capacity: This mutant showed increased collagen binding in vitro, which was confirmed under high shear conditions, suggesting a stronger prothrombotic effect in thrombus formation.

The p.H1786D mutant demonstrated significantly weakened thrombus formation ability in vivo: In a ferric chloride-induced mouse model, the p.H1786D mutant showed delayed platelet adhesion and reduced thrombus formation, while the p.L1757A gain- of-function mutant accelerated thrombus formation.

Conclusions

The study indicates that VWF's binding to collagen plays a crucial role in thrombus formation. Different VWF mutants display varying degrees of functional deficiency or enhancement, significantly affecting the speed and extent of thrombus formation. The study also suggests that GPVI and α2β1 receptors are important in the direct binding of platelets to collagen, although other pathways can partially compensate for platelet adhesion and thrombus formation in the absence of VWF.

Significance

This research provides new insights into the specific mechanisms of VWF in thrombus formation, particularly how collagen-binding mutations affect platelet adhesion and thrombus formation. These findings may have important implications for diagnosing and treating von Willebrand disease and could provide a basis for developing new antithrombotic therapeutic strategies [1]

4.1.2 "Novel Likely Pathogenic Variant in the A3 Domain of von Willebrand Factor Leading to a Collagen-Binding Defect" (2021)

Research Background

This study explores a newly discovered likely pathogenic variant in the A3 domain of von Willebrand Factor (VWF) that causes a collagen -binding defect. Von Willebrand disease (VWD) is a common congenital bleeding disorder primarily caused by mutations in the VWF gene, leading to quantitative or qualitative abnormalities in VWF. The 2M subtype of VWD is particularly associated with mutations in the A1 or A3 domains, with A3 domain mutations typically resulting in decreased collagen-binding activity to type I and III collagen.

Research Methods

The researchers analyzed the cases of two siblings with a bleeding tendency. These siblings had significantly reduced VWF collagen-binding activity, while other VWF parameters and multimer analysis results were normal. Next-generation sequencing (NGS) identified a heterozygous nonsynonymous single-nucleotide variant (nsSNV) in exon 30 of the VWF gene, leading to the substitution of serine with leucine at position 1731 (p.Ser1731Leu). This serine residue was previously shown to be critical for VWF collagen binding.

Key Findings

Case Analysis: The patients' VWF values were extremely low, at 0.09 and 0.11 U/mL, compared to the normal range of 0.6-1.5 U/mL. Genetic Mutation: The researchers discovered the same p.Ser1731Leu mutation in both patients, located in the A3 domain, a major collagen-binding site. In vitro functional tests indicated that this mutation caused a significant defect in VWF collagen binding. Genetic Analysis: In vitro pathogenicity predictions using multiple bioinformatics tools (e.g., SIFT, MutationTaster, and PolyPhen2) consistently suggested that this mutation might be pathogenic.

Conclusions

This study is the first to describe the p.Ser1731Leu mutation in the A3 domain of VWF, which causes a collagen-binding defect, suggesting it is a new likely pathogenic variant that may lead to the development of the 2M subtype of VWD. This discovery provides new perspectives on the molecular mechanisms of VWF dysfunction and may have important implications for the diagnosis and classification of VWD.

Significance

The study highlights the importance of molecular diagnostics in identifying VWD subtypes and developing personalized treatment plans. Future research may further reveal clinical differences among patients with these mutations, helping to optimize diagnostic and therapeutic strategies.[2]

4.1.3 "Coarse-Grain Modeling of Shear-Induced Binding between von Willebrand Factor and Collagen" (2018)

Research Background and Purpose

This study aims to simulate the shear-induced binding mechanism between von Willebrand Factor (VWF) and collagen using a coarse-grained molecular model. VWF is a multimeric protein that plays a critical role in blood coagulation. When blood vessels are damaged, increased blood flow causes VWF to extend, exposing binding sites for platelets and collagen. The study uses Brownian dynamics simulations to explore VWF's binding behavior to exposed collagen on injured arterial surfaces.

Model and Methods

The study utilized Brownian dynamics simulations and a coarse-grained molecular model, focusing on VWF's behavior on static surfaces and under shear flow conditions. VWF molecules bind to collagen via reversible ligand-receptor-type bonds, following Bell model kinetics. The model was modified by introducing an additional binding criterion, ensuring binding inhibition at low shear rates and increased binding at higher shear rates.

Key Findings

Binding Depends on Shear Rate: Simulations showed that VWF binding to collagen was less likely at lower shear rates but significantly increased at higher shear rates, consistent with experimental observations of VWF unfolding and enhanced binding under high shear conditions. Effectiveness of Model Improvement: To better match experimental results, the study introduced a new binding criterion: requiring sufficient stretching of the A2 domain adjacent to the binding site before binding occurs. This modification allowed the model to exhibit shear-induced binding behavior within reasonable parameter ranges, especially under high binding energy conditions.

Biological Significance

The simulation results support the idea that VWF regulates its binding to collagen through structural changes (such as A2 domain stretching) under shear forces. This finding is significant for understanding VWF's role in thrombus formation and may provide new insights into treating related diseases.

Conclusions

The study demonstrates that introducing an A2 domain stretching criterion into the VWF molecular model can effectively simulate VWF binding behavior under high shear conditions. This model improvement provides a deeper understanding of how VWF regulates its binding to collagen in blood flow environments, explaining the observed biological activity of VWF under high shear forces. [3]

4.1.4 "The Role of the von Willebrand Factor Collagen-Binding Assay (VWF) in the Diagnosis and Treatment of von Willebrand Disease (VWD) and Way Beyond: A Comprehensive 36-Year History" (2023)

Research Background and Purpose

This article provides a detailed overview of the von Willebrand Factor collagen-binding assay (VWF) in the diagnosis of von Willebrand disease (VWD) and its development over the past 36 years. VWD is the most common inherited bleeding disorder, and the VWF assay is an important tool for assessing VWF's collagen-binding capacity. The article also discusses the assay's applications in diagnosing other diseases, such as acquired von Willebrand syndrome (AVWS).

Research Methods

The article reviews the history of the VWF assay, analyzing its development and application since its first report in 1986. The study summarizes different laboratory methods and evaluates their effectiveness in diagnosing VWD and other thrombotic diseases.

Key Findings

The Role of VWF in VWD Diagnosis: Since its first application in 1986, VWF has become one of the important tools in diagnosing VWD. The study shows that optimized VWF assays can effectively help identify different subtypes of VWD, particularly types 2A and 2B.

Diversity in Testing Methods: Due to different laboratories using collagen from different sources and methods, the VWF assay results vary. The article points out that optimized VWF methods are crucial for obtaining accurate results.

Broad Application of VWF:CB: In addition to diagnosing VWD, VWF is also used to assess ADAMTS13 activity, AVWS caused by mechanical circulatory support devices, and certain thrombotic diseases (such as thrombotic thrombocytopenic purpura caused by COVID-19). Challenges and Limitations of VWF: While VWF is a powerful diagnostic tool, its use in the U.S. is limited by FDA regulations, restricting its application in certain regions.

Conclusions

VWF plays a crucial role in diagnosing and treating VWD, and its application range is expanding as testing technology improves. The article emphasizes the historical significance of VWF and its potential in diagnosing various thrombotic diseases.

Significance

This article provides a comprehensive historical review of VWF for professionals engaged in hematology research and clinical diagnosis, highlighting its key role in diagnosing VWD and other related diseases. This summary helps researchers and clinicians better understand and apply this assay method. [4]

4.1.5 "Increased Binding of von Willebrand Factor to Sub-Endothelial Collagen May Facilitate Thrombotic Events Complicating Bothrops lanceolatus Envenomation in Humans" (2023)

Research Background

Envenomation by the Bothrops lanceolatus snake can cause severe thrombotic events in humans. Von Willebrand Factor (VWF) plays a key role in thrombus formation, particularly by mediating platelet adhesion through its binding to collagen when blood vessels are damaged. This study investigates how B. lanceolatus venom affects VWF's binding to collagen and its potential role in promoting thrombus formation.

Research Methods

The research team conducted in vitro experiments to assess the effects of B. lanceolatus venom on VWF's binding activity to different collagen types (types I, III, and VI). Additionally, the study examined the ability of Bothrofav anti-venom serum to reverse these effects.

Key Findings

Inhibition of Collagen-Binding Activity: High concentrations of B. lanceolatus venom completely inhibited VWF's binding activity to type I and III collagen. This suggests that components in the venom may block VWF's A3 domain, preventing its binding to collagen.

Enhanced Binding Activity to Type VI Collagen: Conversely, low concentrations of B. lanceolatus venom significantly enhanced VWF's binding activity to type VI collagen, which may be related to the venom's potential enhancement of VWF A1 domain binding to collagen.

Effect of Anti-Venom Serum: Bothrofav anti-venom serum was able to completely reverse the inhibitory effect of B. lanceolatus venom on VWF binding to type I and III collagen, but its protective effect was reduced at higher venom concentrations.

Changes in VWF Antigen Levels: At low concentrations, B. lanceolatus venom increased VWF antigen levels, likely due to proteolysis induced by the venom, while at high concentrations, it reduced VWF antigen levels.

Conclusions

This study indicates that B. lanceolatus venom may promote thrombotic events by inhibiting VWF's binding to type I and III collagen while enhancing its binding to type VI collagen. This finding underscores the importance of the rapid use of anti-venom serum following envenomation to prevent severe thrombotic complications.

Significance

The study reveals the complex regulatory effects of snake venom on VWF function and suggests that specific interventions targeting different collagen types should be considered in clinical treatment to reduce the risk of thrombus formation following envenomation. [5]

References