Overview

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We aim to engineer Pseudomonas putida KT2440 for the biosynthesis of sandalwood oil components using PET monomers as a carbon source. Our project integrates metabolic engineering, synthetic biology, and computational modelling to create an innovative solution for plastic upcycling and high-value compound production. This antedisciplinary approach allows us to design around the challenges of metabolic engineering more efficiently than traditional experimental methods alone. By combining in silico predictions with wet lab techniques, we can explore possibilities and overcome obstacles that would be time-consuming or impractical to tackle solely through experiments, especially within the constraints of an iGEM project timeline.
The experimental aspects of our project include:

1. Chassis Selection: We chose P. putida KT2440 as our chassis organism due to its metabolic versatility, robustness, and ability to grow on various substrates. Specifically, we utilise the TA7-EG strain, engineered to metabolise both terephthalic acid (TPA) and ethylene glycol (EG) at neutral pH.
2. Pathway Engineering: Our strategy involves redirecting the carbon flux from PET monomers towards the native MEP pathway for terpenoid synthesis and introducing the santalol biosynthetic pathway. This consists of expressing three key enzymes from the Santalum album:
   1. Farnesyl pyrophosphate synthase (FPPS)
   2. Santalene synthase (SaSSy)
   3. Cytochrome P450 monooxygenase (CYP736A167) and its reductase partner (CPR1)

All these composite enzyme parts were designed to achieve modularity by using replaceable promoters and RBS sites, acting as a repository for future teams and learners to test further. This allows for fine-tuning the expression levels and testing the impact of different regulatory elements.

While the experimental approach, as seen above, is straightforward, our computational approach answers the working and optimization of our system as a whole.

1. Protein Engineering: A significant challenge in our project is the expression of the eukaryotic membrane-bound cytochrome P450 enzyme in our prokaryotic host. We have designed and ideated an innovative protein engineering strategy to overcome this. Our P450 engineering approach could address the challenges of expressing plant enzymes in bacteria. This strategy could apply to other systems involving eukaryotic P450s, broadening the impact of our project beyond sandalwood oil production.
   1. Transmembrane Domain Truncation: We identified and removed the hydrophobic N-terminal transmembrane domain of both CYP736A167 and CPR1. This modification aims to improve the solubility and expression of these enzymes in the bacterial cytosol.
   2. Fusion Protein Design: Inspired by self-sufficient bacterial P450s and previous studies, we designed a fusion protein combining the truncated CYP736A167 and CPR1, using a 36-amino acid linker (BC_linker) derived from a self-sufficient P450 from Bacillus cereus to join these two enzymes, this could enable the functional co-expression of P450-CPR.
2. Structural Modeling: We used AlphaFold2 to predict the structures of our engineered P450-CPR pair. With the predicted structures, we also looked at their binding energies with santalene to understand if they can effectively bind and catalyse santalene to santalol.
3. Metabolic Modeling: We utilise Flux Balance Analysis (FBA) to optimise growth conditions, analyse metabolic fluxes, and identify key regulatory points in the engineered pathway. This computational approach guides our experimental design and helps predict potential bottlenecks in the production process.

By integrating these approaches, we aim to create a microbial cell factory capable of upcycling PET waste into valuable sandalwood oil components. This project serves as a proof-of-concept for sustainable production of plant secondary metabolites from plastic waste. It could be extended to other high-value compounds with therapeutic or fragrant properties.

Chassis

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Why choose Pseudomonas putida KT2440 as a model organism?

Pseudomonas putida KT2440, a non-pathogenic soil bacterium, is renowned for biotechnological applications. Its unique metabolic pathways, rapid growth, high biomass yield, and low maintenance requirements make it valuable in industrial biotechnology. P. putida is increasingly recognized as a promising chassis for industrial biotechnology, particularly for producing fatty acids, alcohols, and other value-added chemicals. Its ability to grow on inexpensive renewable resources and its adaptability to industrial conditions make it a strong candidate for large-scale applications.

The organism's ability to adapt its metabolic fluxes allows for continuous growth without metabolic network restructuring ^[1]. P. putida strains excel in biocatalysis due to their high NADH regeneration capacity and low energy demands ^[2]. This efficiency is particularly advantageous for NADH-dependent processes ^[3]. Unlike other industrial hosts like E. coli, B. subtilis, and S. cerevisiae, P. putida KT2440 can break down carbon without producing byproducts, even at high rates. It also demonstrates remarkable resilience to disruptions in cellular ATP and NADH demands.

These characteristics - energy stress resistance, low maintenance needs, and efficient catabolism of multiple carbon sources without byproducts - make P. putida KT2440 an excellent candidate for whole-cell bioprocesses. However, developing highly productive redox biocatalytic processes with this strain requires addressing competition for redox cofactors and complex metabolic balancing mechanisms ^[4].

P. putida's carbon uptake rate remains stable even when modified with recombinant NADH oxidase, unlike E. coli and S. cerevisiae. This resilience in harsh conditions is a notable advantage. Furthermore, P. putida can naturally grow on various substrates such as toluene, terephthalic acid, ethylene glycol, and many other organic compounds and sugars ^[5]. On top of that, it can be easily engineered to metabolize new sources. This capability makes it suitable for producing various biochemicals and biofuels from diverse feedstocks, including waste materials. In contrast, yeast typically has a more limited substrate range. This metabolic flexibility and robustness position P. putida as a superior candidate for biotechnological applications compared to existing model organisms ^[6].

Expression of different vectors in P.putida:

The genetic manipulation of Pseudomonas putida is facilitated by a diverse array of plasmids, with the pSEVA (Standard European Vector Architecture) collection standing out as particularly noteworthy. These plasmids are engineered to function effectively in P. putida and are distinguished by their design features that enhance stability and replication within P. putida.

A vital advantage of the pSEVA system is its modularity, allowing researchers to tailor plasmids for specific applications. This flexibility optimises transformation efficiency using replication origins specially adapted for P. putida. Additionally, the modular nature of pSEVA plasmids permits the simultaneous use of multiple compatible plasmids, expanding the toolkit for genetic engineering in this organism.

Specific pSEVA plasmids we will be using are:

1. pSEVA424: This plasmid typically carries a streptomycin/spectinomycin resistance marker and is often used for gene expression studies.
2. pSEVA631: This vector usually contains a gentamicin resistance gene and is frequently employed for gene knockouts or insertions.
3. pSEVA241: This plasmid commonly includes a kanamycin resistance marker and is helpful for various genetic modifications.

In addition to the pSEVA series, the pRGPDuo3 and pRGPDuo4 plasmids are notable tools for genetic manipulation in P. putida. These plasmids are part of a series designed for dual gene expression ^[7], allowing researchers to express two genes simultaneously under different promoters. This capability is beneficial for complex metabolic engineering projects or gene interaction studies.

pRGPDuo plasmids often incorporate features such as:

• Multiple cloning sites for easy insertion of genes of interest.
• Different antibiotic resistance markers for selection.
• Compatibility with other plasmids commonly used in P. putida.
• There are two different promoters for two different genes and hence, they can regulate individual expression levels.

The availability of such diverse and specialised plasmids significantly enhances our ability to harness P. putida for biotechnological applications, ranging from bioremediation to producing valuable compounds.

MEP Pathway:

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P. putida natively possesses the MEP pathway, which efficiently supplies the isoprenoid precursors IPP (Isopentenyl diphosphate) and DMAPP (Dimethylallyl diphosphate) from central metabolites pyruvate and glyceraldehyde-3-phosphate. This native pathway is well-suited for the host and can provide sufficient precursors to support terpenoid production without extensive engineering ^[8].

The endogenous regulation of the MEP pathway in P. putida is likely well-balanced to avoid the accumulation of toxic intermediates like HMBPP that can inhibit cell growth. Overexpression of the MVA pathway, on the other hand, can lead to imbalances and bottlenecks. P. putida exhibits high tolerance to the toxicity of terpenoids and other xenobiotics ^[9]. This makes it well-suited to handle the potential toxicity of terpenoid intermediates and products that accumulate when the MEP pathway is overexpressed for enhanced flux.

Several studies have successfully engineered the MEP pathway in P. putida to produce monoterpenoids like geranic acid and sesquiterpenoids like epi-isozizaene at notable titers. This demonstrates the feasibility and potential of the native MEP pathway as a starting point for further optimization. In contrast, while the MVA pathway (another terpenoid synthesis pathway) has been expressed in P. putida, the performance was par with that ofi E. coli, which had low productivity and titers, while overexpressing the MEP pathway was more efficient for producing terpenoids. Additionally, more extensive engineering is likely required to balance the heterologous MVA pathway in P. putida.

This exploration of terpenoid production from the MEP pathway in Pseudomonas putida led to a strategic decision to utilize the organism's native MEP pathway as it best suits our objective. Through a combination of metabolic modeling approaches and comprehensive literature analysis, we realized that the initial two enzymatic reactions of the MEP pathway govern the rate-limiting steps in this biosynthetic route. Consequently, the genes encoding these first two reactions: 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) were identified as prime targets for overexpression to enhance the flux through the MEP pathway and potentially boost terpenoid yields.

Our Strain

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We strategically selected and optimized our microbial chassis for our PETal project to efficiently utilize PET monomers. Initially, we considered developing a strain that could metabolize both TPA and EG by introducing the tph operon from Comomonas sp: E6 and modifying genes in the Pentose Phosphate and Glycolysis pathways. However, through a literature review and interacting with Dr. Oliver, we learned about the specific work done on engineering P. putida KT2440 for TPA and EG uptake at neutral pH. This led us to adopt their strains: TA7 for TPA uptake and TA7-EG for both TPA and EG metabolism ^[10].

The TA7 strain was developed by introducing four genes (tphA1II, tphA2II, tphA3II, and tphBII) from the tphII operon of Comamonas sp. E6, enabling the conversion of TA to protocatechuate (PCA). Adaptive laboratory evolution (ALE) over seven days improved TA uptake at neutral pH. Whole-genome sequencing revealed key mutations: a missense mutation in the MhpT transporter (L374V) and another in a MarR-family transcriptional regulator (R51C), enhancing TA import.

The TA7-EG strain was created through further ALE, culturing P. putida TA7 in M9 minimal medium with 25 mM EG and 5 mM TA for eleven days. Whole-genome sequencing identified a nonsense mutation in the gclR gene, likely resulting in GclR loss of function. Normally, GclR is a transcriptional repressor of gcl and glxR, but with the mutation, it becomes inactive. Therefore, P. putida can utilise ethylene glycol as a carbon source through ALE when the gcl operon, which includes key enzymes such as glyoxylate carboligase (gcl) and tartronate semialdehyde reductase (glxR), is functionally expressed enabling the conversion of ethylene glycol into cellular biomass ^[16].

Growth experiments demonstrated that both TA7 and TA7-EG strains showed comparable growth in LB medium. However, in the M9 minimal medium supplemented with equimolar amounts of TA and EG, TA7-EG exhibited a shorter lag phase and higher final OD600 compared to TA7. Notably, TA7-EG also showed a slight growth advantage with TA as the sole carbon source. Cell dry weight measurements confirmed TA7-EG's improved capacity to accumulate biomass. By leveraging these pre-engineered strains with their enhanced abilities to metabolize PET monomers at neutral pH, we established a robust foundation for our project. This approach allowed us to focus on downstream pathway engineering for sandalwood oil production, aligning our goals with real-world applications within our project timeframe.

Our Engineered pathway:

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The following section describes how we incorporated the principles of synthetic biology to synthesize components of sandalwood oil, i.e., sesquiterpenoids which naturally occur as[(Z)-α-santalol (49%), (Z)-α-bergamotol (5%), (Z)-β-santalol(21%) and (Z)-epi-β-santalol (4%)]. We built our strategies to design the key gene constructs involved in Santalol biosynthetic pathway for its successful expression into our chassis.We planned to introduce genes coding for enzymes, namely Farnasyl pyrophosphate synthase(HQ343283.1), Santalene synthase(HQ343276.1), Cytochrome P450 monooxygenase i.e. CYP736A167(KU169302.1), and cytochrome P450 reductase i.e.CPR1( KC842187.1 ) from Santalum album downstream to MEP pathway in P.putida.

Fig.1 : Our engineered pathway in P.putida

Production of Santalene:

As shown in the pathway fig 1. Ethylene glycol and Terephthalic acid are been uptaken by P.putida TA7-EG. Modeling of their uptake and metabolism in-silico helped us to visualize and understand the route of these metabolites, their fluxes leading to MEP pathway and santalol synthesis considering all the enzymes involved in the process (refer to modeling page).

As mentioned in the previous section, P.putida TA7-EG is engineered to grow on PET monomers TPA and EG and has a native MEP pathway for terpenoid synthesis. Hence the possible route that we modeled shows that EG and TPA are being converted to Pyruvate and Glyceraldehyde-3-phosphate, which will be catalyzed by enzymes DXS and DXR to produce MEP. These two enzymes, if overexpressed, were shown to increase flux of terpenoid precursors by Dr. Jordi ^[8] and validated by us through FBA (refer to modelling page). Further, MEP is converted to five carbon compounds IPP/DMAPP catalyzed by enzymes IspD, IspE, IspF, IspG,IspH^[12]. IPP molecules will combine to form a 15-carbon compound called Farnasyl pyrophosphate(FPP). The enzyme responsible for this is FPPS(FPP synthase). Although FPPS is present in P.putida, we are introducing FPPS gene from santalum album as well. This will ensure it’s enough availability and controlled expression under the inducible promoter. FPP is one of the end products of the MEP pathway and is the precursor of Santalene. Santalene synthase(SaSSy) acts on FPP to synthesize a variety of isomers i.e. endo-bergamotene,beta-santalene, (Z)--farnesene, epi--santalene, and-santalene.The ability of SaSSy to produce multiple isomers is attributed to its substrate flexibility and the presence of different geometric isomers of FPP, which serve as the precursor for santalene synthesis ^[17]. Expression of FPPS and SaSSy has been previously demonstrated in E.coli ^[11]; hence we will be procuring the same plasmid pcdf-duet-SaSSy-FFPS (refer to results) used by Dr. Bohlman and try to express it in our chassis P.putida. Since the pcdf-duet is not specifically designed or optimized for our chassis, we simultaneously started working on constructing our own inserts to address the uncertainty regarding its successful expression. We designed a system of bicistronic inserts with appropriate promoters,RBS ,inter-ribosomal entry site and spacer sequences which will be cloned with pSEVA backbones for stable expression. While doing so, our PI, Dr. Vijay, and Prof. Lorenzo guided us when required and validated our constructs (refer to iHP page).

The details about the basic parts we designed containing enzymes FPPS (BBa_K5181005), SaSSy(BBa_K5181004), promoter araBAD with RBS (BBa_K5181006), and IRES(BBa_K5181013) to form the composite part araBAD_SaSSy-FPPS(BBa_K5181014). The schematic representation of the design strategy is shown below (refer to parts description page).

Fig 2: araBAD_SaSSy-FPPS(BBa_K5181014)

Please refer to the results section for documentation of the working of this part and it’s successful expression.

Santalol synthesis:

The conversion of isomers of santalene to ‘santalol’, the major component of natural sandalwood oil, is a critical reaction in our project. This transformation necessitates the introduction of two essential enzymes: cytochrome P450 monooxygenase(SaCYP736A167) and cytochrome P450 reductase(CPR1), both sourced from Santalum album.
Reconstitution of plant biosynthetic pathways in microbial cell factories, allows the production of high-value natural products without the problems associated with the native hosts such as seasonal nature of the production, low amounts of the product per plant, and tedious extraction from plant material ^[13].
Expressing eukaryotic (plant) membrane-bound cytochrome P450 enzymes in prokaryotic systems like Pseudomonas putida presents several significant challenges:
Many plant P450s are membrane-bound and have a hydrophobic nature complicating their expression in bacterial systems due to differences in membrane composition ^[18]. These enzymes often fail to anchor properly to the bacterial membrane, resulting in aggregation rather than functional proteins. P450s require cofactors like NAD(P)H/FAD whose availability The of plant P450s, their requirement for plant-specific redox partners, and the lack of optimal expression conditions in bacterial hosts make the functional expression of these enzymes challenging ^[14],[15].
To overcome these challenges, we adopted a few strategies as mentioned previously by Dr Poborsky ^[13], i.e., transmembrane domain truncation improved the product titres 2- to 170-fold. This suggests that the transmembrane domain truncation is a versatile strategy to improve the functional expression of plant P450s in bacterial hosts.

Our approach for P.putida:

In literature we observed that P.putida has its own naturally occurring P450 enzyme(P450 CAM:1IWJ) as shown in Fig.3,which is a well-studied enzyme that plays a crucial role in the hydroxylation of camphor. The knowledge about structure and function of P450 CAM helped us to find similarity with Santalum album CYP736A167 to achieve it’s bacterial expression.

Fig3: P450 CAM

Fig 4: Similarity between Santalum album P450 and P.putida P450 CAM

Hence we performed homology studies between Santalum album CYP736A167 and P450 CAM as both are a part of same family as shown in Fig 4. Interestingly their core domains matched even though they had very low sequence similarity(30.5%).When we aligned it with EMBOSS Needle and they had RMSD difference of(3.647) as shown in the Fig.5.

Fig 5: Alignment

We adopted following strategies for expression of our plant genes in bacterial system.

1. Protein Engineering: Strategies like transmembrane domain truncation and fusion with membrane-anchoring proteins can enhance the expression and solubility of membrane-bound P450s in bacterial systems.
2. Redox Partner Engineering: Optimizing the interaction between P450s and their redox partners, such as cytochrome P450 reductase, can enhance electron transfer and improve overall catalytic efficiency.

N terminal truncation and Designing a fusion protein

The Santalum album being a Eukaryotic plant P450 had 27 amino acids extension in its N-terminal end which is hydrophobic in nature and corresponded to an alpha helix (as predicted by alphafold2) shown in Fig.6., which we considered as a transmembrane domain.Thus we concluded that this P450 could fold properly in the bacterial cytosolic environment even if their transmembrane region is truncated by visualizing them in PyMOL (refer to Fig.7).

Fig 6: Native Cytochrome P450 monooxygenase

Fig 7:Truncated Cytochrome P450 monooxygenase

P450 is not self sufficient and needs native CPR1 (Fig.8)as its redox partner, which again is a membrane-bound protein. Hence we truncated the transmembrane domain of CPR as well as shown in the Fig.9.

Fig 8: Native Sandalwood Cytochrome P450 Reductase

Fig 9: Truncated Sandalwood Cytochrome P450 reductase

Moreover these two proteins need to be present in close proximity to shuttle their FADH which is possible in their native cellular conditions as they are guided by the translocation signal in their N-terminal domain. But in our case, the transmembrane truncated plant P450-CPR pair requires a functional architecture to be expressable in bacterial cytosol.
While overcoming this challenge we came across a self-sufficient bacterial P450 from Bacillus cereus(The alphafold2 predicted structure was obtained from UniProt id:A0A6M3ZBL7; publication: source of the nucleotide sequence Dragos et.al 2020 ) shown in Fig.10. To obtain more information on the protein origin we blasted the amino acid sequence obtained from UniProt in the ‘nr’ database of NCBI. Surprisingly we got only 1 match with 100% query coverage and sequence similarity, the publication obtained under ^[19] Where they characterized a self sufficient P450 of Bacillus cereus. Thus, this predicted protein’s architecture was followed to build our own P450, The predicted protein would be called pP450 to avoid confusion.

Fig 10: Bacillus cereus bifunctional P450

When we structurally aligned saCYP736A167 and saCPR1 on pP450, and observed that saCYP736A167 superimposed on pP450 with an RMSD difference of 2.163Å , and saCPR1 on pP450 with an RMSD difference of saCPR1 and pP450 was 1.506Å. This showed the self-sufficient P450 has both an oxidase and reductase domain which are also very similar to the eukaryotic counterparts, without their transmembrane domain. Thus, following the architecture of pP450, we used the previously truncated versions saCYP736A167 and saCPR1 to design the fusion protein.

Fig 11:The BC linker

Moreover we also found a 36 amino acid long loop connecting the oxidase and reductase domain, The loop connected the C-terminal of its oxidase domain to the N-terminal of its reductase domain as shown in Fig.11. Both the reductase domain has a characteristic structural similarity in their N-termini where there are 4 parallel beta-sheets and the linker connects to the exact structural region same region where the transmembrane region of CPR1 connects to the core body of the enzyme. This 36 amino acid region were used to join the truncated saCYP736A167 and saCPR1 and finally it joined in the exact manner it joined the domains of the bifunctional P450 to form our fusion protein. The 36 amino acid sequence is referred to as BC_linker in this study.

Fig 12: Our fusion protein

As shown in the Fig.12 the P450-CPR fusion protein connected by BC linker under the control of suitable promoter will be ready to be experimentally tested.

Following figure summarises the plan:

Fig.13: Summary of the plan

Hence we designed a composite part with appropriate replaceable promoter and the fusion protein sequence optimised for P. putida.
tet-P450-CPR_fusion protein:BBa_K5181015 -
It is a composite part comprising of basic parts - Self-sufficient tet_RBS (BBa_K5181009) and P450-CPR_fusion protein (BC_linker): BBa_K5181008 - This part is an artificial fusion protein of CYP450_truncated and CPR_truncated with BC linker.

Fig.14: tet-P450-CPR_fusion protein: BBa_K5181015

Along with the BC linker we are designing P450-CPR fusion protein with other linkers such as BM linker and GST linker.Please refer to the results page to know more about the expression of these four genes namely FPPS,SaSSy, P450-CPR discussed in the above section so far.This part design caters to the expectation of an ideal modular part where the promoter, RBS is a replaceable insert that will help check the expression of our GOI under different inducible promoters and suitable RBS.
The P450-CPR fusion protein (BC linker) is depicted below:

Lab Plan

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To harness the sandalwood oil components synthesis pathway from Santalum album into Pseudomonas putida, we aimed to engineer the same in our KT2440 TA7-EG strain for the biosynthesis of santalene and santalol. This metabolic engineering approach involved the heterologous expression of three key enzymatic components:

1. Farnesyl pyrophosphate synthase (FPPS)
2. Santalene synthase (SaSSy)
3. A cytochrome P450 monooxygenase (CYP450) and its cognate cytochrome P450 reductase (CPR) as an oxo-reductase enzyme pair

The experimental plan to optimize the bacterial growth on TPA & EG and further introduction of the genes associated with the pathway, confirmation of santalene and santalol synthesis has been divided into three major phases:

Optimisation of the strain on the PET monomers:

• After procuring the strains, we tried to grow the strains on the M9 media and TPA + EG as a carbon source to see how efficiently the bacteria grow in that media.
• Initially, the goal was to optimize the M9 media for our chassis, as the pH and concentration of every element in the media can affect bacterial growth. Our bacteria finally started growing, but it took almost 48 hours.
• Once the media optimization was done, we wanted our bacteria to grow within at least 12 to 16 hours; we performed the method suggested by Dr. Oliver and Dr. Yashraj to give a serial dilution of the glucose with TPA and EG starting from 5%, reaching to complete TPA and EG media solution.
• We saw that with this method, the bacteria started to grow within 14 hours and had a better growth rate than the previous.

Expression of SaSSy-FPPS into the chassis for the synthesis of santalene:

• For the expression of santalene, we tried expressing the pCDFduet, which we obtained from Dr Bohlmann in the competent cells of the P.putida TA7-EG.
• After inducing the protein, we performed the Ni2+ NTA Nickel, Bradford assay, and coomassie gel to analyze whether the protein was expressed. As a result, we got a thick band at 65 kDa, which constitutes the SaSSy gene.
• Chemical analysis was confirmed by performing Gas chromatography-mass spectrometry (GC-MS). A sample of transformed bacteria with pCDFDuet was given. To confirm the presence of santalene, we gave an untransformed bacterial pellet sample as a control. In the report, there was a significant peak of 204 m/z, corresponding to the molecular weight of santalene, and in the control, there was no peak at the same retention time as the transformed one.
• However, in the report, the abundance of the santalene is very low, and we need to make a controlled expression for SaSSy-FPPS in our chassis, so we optimized the SaSSy-FPPS gene constructs codon for our bacteria.
• We assembled all the fragments once the gene constructs were in the lab. We checked them in the agarose gel to confirm if they were assembled.
• After the gel confirmation, we performed PCR for all our assembled SaSSy-FPPS fragments and restricted digested with the vector pSEVA 241 for cloning.

Expression of CPR- P450 into the chassis for the synthesis of Santalol:

• As mentioned earlier, the oxidoreductase P450-CPR is a membrane-bound enzyme; this expression is challenging in prokaryotes. By adopting different engineering strategies, the promoter-gene inserts were synthesized as fragments.
• The P450-CPR fragments were assembled with the help of Gibson assembly to confirm the assembly, and an agarose gel experiment was performed.
• We performed a PCR reaction and amplified the insert part to ligate the gene part to the vector pSEVA241 to have enough gene concentration.
• The vector pSEVA241 and the assembled insert P450-CPR were restricted digested for the conventional cloning as the concentration of both insert and vector was less to perform a Gibson assembly for ligation.
• The ligation was performed overnight, and the same was transformed into electro-competent cells.