Overview

CO₂ Emissions

It has been reported that air transportation and other modes of heavy-duty transport emitted 830 and 1060 Mt of CO₂ in 2014 respectively, accounting for 24% of the difficult-to-eliminate GHG emissions, and these numbers are projected to increase in the foreseeable future.

In today's world, the urgent need for biofuels is undeniable. With environmental concerns growing and fossil fuel reserves depleting, biofuels represent a sustainable and eco-friendly solution. They offer a path to reduce greenhouse gas emissions, enhance energy security, and foster a more sustainable future. Embracing biofuels is not just a choice; it's a necessity to address the pressing energy and environmental challenges of our time.

Jatropha curcas

Biofuels and blended fossil fuels are one of a kind and biodiesel is a renewable energy source that can replace fossil-based diesel and can reduce the drawbacks of diesel emission, produced using the transesterification or alcoholysis process. Trans-esterification is the process of turning triacylglycerides (TAGs) into methyl esters in the presence of a solvent, employing an alkali, acid, or enzyme as a catalyst.^[1]

Jatropha biodiesel (non-edible Jatropha curcas oil-based methyl ester) produced from transesterification, and mixed with ethanol, has been blended with conventional diesel in various compositions where it has been noticed that it is extremely stable at lower temperatures, making it an excellent choice for jet fuels. The most prevalent fatty acid in crude Jatropha curcas oil is oleic acid, followed by linoleic, palmitic, and stearic acid. Since it contains 80.9% unsaturated fatty acids, the oil has outstanding low-temperature properties (oleic and linoleic acids). Jatropha biodiesel is used because of its great blending capacity with diesel. Biodiesel is one of the most favorable and promising alternatives in the application of automobiles, boilers, gas turbines, etc. Both jatropha biodiesel and ethanol have high calorific value which is an important factor for engine power production. The performance analysis showed that the biodiesel blend of 98% diesel with 1.5% jatropha biodiesel and 0.5% of ethanol had a significant improvement in the engine performance than the conventional diesel.

Our Chassis

Why did we choose Yarrowia lipolytica?

Yeasts offer a number of advantages as expression systems for complex proteins. Being unicellular organisms, they present an ease of manipulation and a growth capacity similar to those of bacteria. Additionally, in contrast to prokaryotes, they possess a eukaryotic subcellular organisation able to perform the post-translational processing necessary for expression of complex proteins from “higher organisms”. In yeasts, secretion occurs via a complex multi-component apparatus, encompassing two major pathways: a post-translational and a co-translational one. This organisation allows proteolytic maturation, formation of disulfide bonds, N-linked and O-linked glycosylation of defined sites and other minor posttranslational modifications. Now, the question would be: why would one not use a conventional yeast such as Saccharomyces cerevisiae? First uses of the same revealed some limitations:

• low product yield
• poor plasmid stability
• difficulties in the scaling up of the production
• hyperglycosylation of recombinant proteins
• low secretion capacity

Subsequently, development of other expression systems occurred; this includes our chosen : the dimorphic Yarrowia lipolytica. Their performances frequently surpassed those of S. cerevisiae in terms of product yield, reduced hyperglycosylation and secretion efficiency, especially for large complex proteins^[13][14][15].

Described in ^[3], S. cerevisiae, H. polymorpha, K. lactis, S. pombe and Y. lipolytica were evaluated for their capacity to secrete active forms of six fungal enzymes. All of the examined alternative hosts performed better than S. cerevisiae, but their relative efficiency varied significantly with each heterologous protein. The most attractive host, especially in terms of performance reproducibility, was Y. lipolytica^[4]. Generally regarded as safe, (GRAS) this organism has a peculiarly interesting feature of being able to take up a lot of hydrophobic substrates (n-alkanes) with the help of protrusions on its surface that act like a channel and has become a model organism for numerous areas of research such as fatty acid metabolism.

Expression of vectors in Yarrowia lipolytica:

• Use of two types of shuttle vectors is prevalent in Yarrowia lipolytica - ARS and integrative vectors. The ARS vectors are not very attractive since the copy number in Y. lipolytica for ARS is extremely low (around 2-3); hence, we prefer to use the genome integrative vectors (based on homologous recombination)^[5]. Integration of exogenous DNA into the Y. lipolytica genome occurs almost exclusively by homologous recombination, with the notable exception of the zeta-based non-homologous integration system^[6].

Its oleaginous nature:

• As an oleaginous yeast, Y. lipolytica can naturally accumulate lipids up to 30 to 50% of the cell dry weight (CDW), depending not only on each wild-type isolate genetic background but also on the carbon source used and the growth conditions. This lipid accumulation can reach up to 90% of CDW through genetic engineering, in obese Y. lipolytica cells.
• Lipid storage in Y. lipolytica results from an effective de novo synthesis pathway for triacylglycerols (TAG) when sugars or similarly catabolized compounds such as polysaccharides or glycerol are used as carbon sources. It is however more remarkable when hydrophobic substrates are used, benefiting then from both an efficient uptake of lipids from the medium and the efficient ex novo synthesis pathway (biomodification)^[7]. The storage lipids of Y. lipolytica consist mostly of TAG and sterol esters, more than the free fatty acids (FFA), and accumulate in a specialized subcellular compartment, the lipid body (LB). These lipids can comprise as high as 80% of unsaturated fatty acids, which present some valuable health benefits.

Lipid Profile of Y. lipolytica in comparison to other species

Fatty Acid Synthesis Pathway

Typically, fatty acid biosynthesis begins with acetyl-CoA, carboxylation produces the malonyl-CoA building blocks that are subsequently condensed and reduced in an iterative fashion until the fatty acid chain matures for use by the cell.

Malonyl‐CoA contains a 3‐carbon dicarboxylic acid, malonate, bound to Coenzyme A. Malonate is formed from acetyl‐CoA by the addition of CO₂ using the biotin cofactor of the enzyme acetyl‐CoA carboxylase.

Initiation

Fatty acid synthesis starts with acetyl‐CoA, and the chain grows from the “tail end” so that carbon 1 and the alpha‐carbon of the complete fatty acid are added last. The first reaction is the transfer of the acetyl group to a pantothenate group of acyl carrier protein (ACP), a region of the large mammalian Fatty Acid Synthase(FAS) protein. (The acyl carrier protein is a small, independent peptide in bacterial FAS, hence the name.) The pantothenate group of ACP is the same as is found on Coenzyme A, so the transfer requires no energy input:

In the preceding reaction, the S and SH refer to the thio group on the end of Coenzyme A or the pantothenate groups. The ∼ is a reminder that the bond between the carbonyl carbon of the acetyl group and the thio group is a “high energy” bond (that is, the activated acetyl group is easily donated to an acceptor). The second reaction is another transfer, this time, from the pantothenate of the ACP to cysteine sulfhydral (–SH) group on FAS.

The pantothenate –SH group is now ready to accept a malonyl group from malonyl-CoA:

Continuation

The FAS has now two activated substrates, the acetyl group bound on the cysteine –SH and the malonyl group bound on the pantothenate –SH. Transfer of the 2‐carbon acetyl unit from Acetyl∼S‐cysteine to malonyl‐CoA has two features:

• Release of the CO₂ group of malonic acid that was originally put on by acetyl‐CoA carboxylase.
• Generation of a 4‐carbon β‐keto acid derivative, bound to the pantothenate of the ACP protein.

The ketoacid is now reduced to the methylene (CH₂) state in a three‐step reaction sequence.

• Reduction by NADPH to form the β‐hydroxy acid derivative:

• Dehydration, that is, the removal of water to make a trans‐double bond:

• Reduction by NADPH to make the saturated fatty acid:

The elongated 4‐carbon chain is now ready to accept a new 2‐carbon unit from malonyl‐CoA. The 2‐carbon unit, which is added to the growing fatty acid chain, becomes carbons 1 and 2 of hexanoic acid (6‐carbons).

The cycle of transfer, elongation, reduction, dehydration, and reduction continues until palmitoyl‐ACP is made. Then the thioesterase activity of the FAS complex releases the 16‐carbon fatty acid palmitate from the FAS.

Note that fatty acid synthesis provides an extreme example of the phenomenon of metabolic channeling: neither free fatty acids with more than four carbons nor their CoA derivatives can directly participate in the synthesis of palmitate. Instead they must be broken down to acetyl‐CoA and reincorporated into the fatty acid.

A Shuttle System

Fatty acids are generated cytoplasmically while acetyl‐CoA is made in the mitochondrion by pyruvate dehydrogenase.This implies that a shuttle system must exist to get the acetyl‐CoA or its equivalent out of the mitochondrion. The shuttle system operates in the following way: acetyl‐CoA is first converted to citrate by citrate synthase in the Tricarboxylic Acid(TCA)‐cycle reaction. Then the citrate is transferred out of the mitochondrion by either of two carriers, driven by the electroosmotic gradient: either a citrate/phosphate antiport or a citrate/malate antiport. After it is in the cytosol, citrate is cleaved to its 2‐ and 4‐carbon components by citrate lyase to make acetyl‐CoA and oxaloacetate. Citrate lyase requires ATP.

Fatty acid biosynthesis (and most biosynthetic reactions) requires NADPH to supply the reducing equivalents. Oxaloacetate is used to generate NADPH for biosynthesis in a two‐step sequence. The first step is the malate dehydrogenase reaction found in the TCA cycle. This reaction results in the formation of NAD from NADH (the NADH primarily comes from glycolysis). The malate formed is a substrate for the malic enzyme reaction, which makes pyruvate, CO₂, and NADPH. Pyruvate is transported into the mitochondria where pyruvate carboxylase uses ATP energy to regenerate oxaloacetate.

Palmitate is the starting point for other fatty acids that use a set of related reactions to generate the modified chains and head groups of the lipid classes. Microsomal enzymes primarily catalyze these chain modifications. Desaturation uses O₂ as the ultimate electron acceptor to introduce double bonds at the nine, six, and five positions of an acyl‐CoA.

Why express the Thioesterases?

In bacteria, plants, and algae the different enzymes that catalyze the FAS cycle are expressed as discrete proteins (type II), whereas non-plant eukaryotes use an FAS in which all functionality is supplied by multi-domain megasynthases.

• Both type I and type II FAS rely on a small protein, the acyl carrier protein (ACP), to shuttle the fatty acid cargo from enzyme to enzyme. ACPs transport and present the growing acyl chain to appropriate reaction partners for elongation and fatty acid production.
• Malonyl-CoA ACP transacylase (MCAT) catalyzes the initiation of fatty acid biosynthesis by conversion of malonyl-CoA to malonyl-ACP, the key building block in fatty acid synthesis.
• KSs catalyze the chain extension step in FAS, via Claisen condensation to form the carbon-carbon bond.
• After the initial reduction of ketone to alcohol by KR, the β-hydroxyacyl alcohol is dehydrated to an enoyl moiety by DH enzymes via the elimination of water.
• After chain elongation, fatty acid biosynthesis is terminated either by offloading fatty acids from ACP by acyl-ACP TEs, releasing free fatty acids, or by direct transesterification onto a lipid by acyltransferases (AT). In general, prokaryotes utilize ATs whereas eukaryotes make use of dedicated TEs.

In Plants

In plants, fatty acid synthesis starts in the plastid by the fatty acid synthase (FAS) complex, which usually generates palmitoyl-ACP (16:0-ACP) and stearoyl-ACP (18:0-ACP) through continuous elongation of fatty acid chains in a 2 carbon increase for each cycle^[8].

Further, plastidial acyltransferases can terminate de novo fatty acid synthesis, and the acyl group of acyl-acyl carrier protein (acyl-ACP) can be used to produce glycerolipids in plastids (prokaryotic pathway) or, alternatively, acyl-ACP thioesterases (FATs) can release free fatty acids and ACP by hydrolyzing acyl-ACP.

Therefore, FATs are the key enzymes in de novo synthesis of free fatty acids in higher plant plastids and play an important role in the distribution of de novo synthesized free fatty acids between the prokaryotic and eukaryotic pathways . Due to the distinct substrate specificity of different FATs, FAT can influence the chain length and saturation degree of fatty acids, and the composition of fatty acids in various organs of higher plants^[9]..

Developing a single cell producing oil organism (SCO)

In ^[10], the same thioesterases that we wanted to express, had been so in E. coli and A. thaliana results of which show that JcFATA and JcFATB have chloroplastial localisation and modified embryos show increased growth (increased seed weight, width). In addition to that, considerable phenotypic changes in the seeds of the JcFATA and JcFATB ectopic expression lines were observed (that is, the contents of 18:1, 18:2, and 18:3 were increased by 69–95%, 58–68%, and 52–70%, respectively, in the JcFATA lines compared with the wild type and saturated fatty acids 16:0, 18:0, 20:0, and 22:0 were significantly increased by 84–108%, 124–145%, 65–92%, and 96–170%, respectively, in the JcFATB lines compared with the wild type).

Three classes of hydrocarbons can be biologically synthesized: those derived from fatty acids, isoprenoids, and polyketides, respectively. Further, for the production of hydrocarbons from fatty acids - two biochemical pathways exist:

• The two-step cyanobacterial pathway involving acyl-ACP reduction and aldehyde decarbonylation (enzymes involved are the pair formed by the acyl-ACP reductase (AAR) and the decarbonylating aldehyde-deformylating oxygenase (ADO)).
• The one-step decarboxylation reaction from free fatty acids (FFAs) (enzyme involved - algal photo-enzyme glucose–methanol–choline (GMC) oxidoreductase termed fatty acid photo-decarboxylase (FAP)).

Both of these have been expressed in the two widely known model organisms E. coli and S. cerevisiae.

The production of 10.87 mg/L and 58.7 mg/L alka(e)nes were achieved in the presence of light with batch and fed-batch fermentation modes, respectively, in Yarrowia lipolytica ^[2].

Our Strain

W29, the most ubiquitous strain of Y. lipolytica available worldwide, originally isolated from the 1970 sewers of Paris, was used to produce benefitting genetically modified strains that have been used the most for heterologous protein expression. The favorable genetic background of W29 for protein secretion has prompted the design at INRA France of a series of GM derivatives for applications in the domain of heterologous protein production, first equipped with a ura3-302 allele (URA3 disrupted by ScSUC2 cassette) that provided both an auxotrophy and the ability to use sucrose as sole carbon source. Then, a series of “Po1” GM strains were derived, by addition of leucine auxotrophy, by deletion of the major protease (AEP) or of both extracellular proteases (AEP and AXP) and finally by complementation of one or of both auxotrophy^[11].

Further engineering to change the only yeast derived sequences in Po1 strains brought in bacterial docking sites in the genome, and thus, the strain Po1g has been equipped with an integrated bacterial-derived sequence, namely a pBR322 docking platform, in order to facilitate the further integration of pBR322-based expression vectors. This easy-to-use integration system takes benefit of the large region of homology between these vectors and the docking platform to obtain very high transformation efficiencies (in the range of 104 to 105 transformants per gram of DNA) and a high percentage of targeted integration (in the range of 80–90%) despite the high level of non-homologous end joining (NHEJ) in Y. lipolytica cells^[12].

This strain retains only a leucine auxotrophy, allowing its transformants to be prototrophs. The YLEX Yeast Expression kit, that we sourced from Yeastern Taiwan Biotech Co. directly, was aimed at easy and rapid testing of heterologous production of a given protein in Y. lipolytica and has also been demonstrated to be particularly adapted to enzyme engineering, notably through directed mutagenesis.

Performing Deletions to Aid in Gene Integrations

In contrast to S. cerevisiae, Y. lipolytica uses mainly non-homologous end-joining (NHEJ), and not homologous recombination (HR), for repairing DNA double-strand breaks (DSB). Consequently, targeted integration of exogenous DNA by single crossover can occur at acceptable rates (up to 80%, but seemingly locus and strain dependent) only if flanking homologous regions of at least 0.5 kb, and preferably 0.75–1 kb, are present. Most eukaryotic microorganisms use NHEJ as the main DSB repair pathway, and knocking out or destroying NHEJ-related genes is an effective strategy to increase the efficiency of gene targeting. However, many studies also showed that disrupting the NHEJ pathway can make the cells prone to mutation, which is not conducive to industrial application_[16]. Moreover, the cell growth can be severely affected under non-optimal conditions, such as high temperature, ultraviolet irradiation and the presence of chemical DNA-damaging agents^[17]. In addition, in strains lacking components of the NHEJ pathway, the integration efficiency is highly dependent on the targeted gene locus.

Consequently, strengthening the HR repair is also an effective approach for improving the gene targeting efficiency. The HR repair pathway is mediated by a class of conserved enzymes called DNA recombinase enzymes, notably the RAD51/RAD52 complex, a major participant in the targeted integration of foreign DNA in eukaryotes. The heterologous expression of S. cerevisiae HR related genes such as ScRad51 and ScRad52, has been shown to greatly increase the efficiency of HR and reduce the off target effects. Thus, we decided to express ScRAD52, that would aid in knock outs of the genes (FAA1 and Alk2) further.

When Y. lipolytica grows in an oily environment, a large battery of extracellular lipases (TGL4) produce fatty acids out of the oils, which will be rapidly incorporated into the cell. In the cytosol, these free fatty acids can be activated by FAA1 (fatty acyl-CoA synthetase, YALI0D17864g) to produce acyl-CoA. So we chose to delete FAA1 in order to curb the degradation of free fatty acids, by conferring hygromycin resistance by inserting the E. coli hph gene.

Due to the expression of CvFAP, we expected a very high amount of hydrocarbons in the chassis and in Y. lipolytica, the presence of n-alkanes leads to transcriptional activation of alkane-degrading enzymes. The main monooxygenase (ALK1) responsible for hydrocarbon degradation, was chosen to be deleted, by conferring nourseothricin resistance by inserting the Streptomyces noursei nat1 gene. The following gene cassettes were formed^[18]:

Designing the Gene Fragments:

Choosing the right promoter - In Y. lipolytica, the first strong promoters to be isolated and characterized were: the promoter from the XPR2 (pXPR2) gene, which codes for an alkaline extracellular protease^[19], and the constitutive promoter of TEF (pTEF), which codes for translation elongation factor-1 ^[20]. pTEF has been shown to be the most active promoter and thus was chosen to be the promoter for our fragments (apart from the hp4d promoter in pYLEX).

Wet Lab Plan

Overview

For the production of our desired biodiesel (the revered oil of Jatropha curcas) we express its plastidial thioesterases JcFATA and JcFATB in the oleaginous yeast Yarrowia lipolytica. Further, for turning this into an oil drop system we express the photodecarboxylase(fatty acid to alkane) CvFAP from the algae Chlorella variabilis. To achieve this, we started with the following steps that can be divided into three major parts:

Gene Fragment Assembly and Amplification

• We ordered the gene fragments from Twist Bioscience and did gradient PCR, subsequent PCRs for amplification of each of these fragments to a desired concentration (synthesized primers for the same which can be found here).
• The gene assembly was done with the help of a procedure called NEBuilder® HiFi DNA Assembly, a cloning procedure to bring together two or more gene fragments without using restriction digestion and ligation.
• Along with our 4 fragments (3 genes) and our bacterial cloning vector pUC19, we performed the NEBuilder® HiFi DNA Assembly procedure to make our recombinant plasmid.
• The recombinant plasmid (hereafter named pUC19-JC) is used to transform E. coli again; we used blue-white screening using IPTG and X-gal to screen for colonies that we require.
• After plasmid miniprep, we ran the products on confirmatory gels to see if we have the required plasmid in our colonies. We also ran a confirmatory test by doing a sequential double digest of pUC19-JC with SalI and BamHI. For the final verification we sent our plasmid for sequencing.
• The protocols for the procedures we performed can be found here.

Transformation into the Chassis

• For the expression of our protein, we used the YLEX Yeast Expression kit where, based on INRA INAPG licensed patent, it provides an easy approach for cloning and expressing a gene of interest in the yeast Yarrowia lipolytica.
• Using this kit, heterologous protein may be expressed intracellularly or secreted from the cell into the medium by selecting respectively the supplied expression vector pYLEX1 or pYLSC1.
• The heterologous protein expression is driven by a strong hybrid promoter (hp4d) carrying four tandem copies of an upstream activator sequence (UAS1B) from pXPR2 and a minimal pLEU2 fragment.
• To achieve expression in yeast, pYLEX1 containing a cloned gene of interest is linearized by a selected restriction enzyme to produce an expression cassette that can integrate with high efficiency into the Y. lipolytica genome by homologous recombination with an integrated pBR platform.
• A leucine gene (LEU2) in pYLEX1 provides for selection of yeast containing an integrated expression cassette by allowing their growth on leucine-free minimal medium.(We use the nitrogen deficient fatty acid accumulation inducing YNB medium).
• For ligation into the vector, we needed to reconstitute the promoter by adding AATG base sequence into the gene of interest (because of the PmlI digestion site, the promoter in the original vector has been made into a fusion protein) and thus had to use PCR to remove the insert from pUC19 and ligate it into pYLEX.
• Although the ligation didn’t work, we successfully integrated pYLEX and our insert by doing a NEBuilder® HiFi DNA Assembly of it again. Transformation was performed using the YLOS protocol given in the manual.

Proof of Concept

Thin Layer Chromatography(TLC)

• For the first qualitative analysis, we performed TLC, where we tried to find and compare the intensities of the C16 and C18 fatty acids in our samples.
• TLC is used to separate compounds based on their affinity towards the stationary and the mobile phase. We used a silica-based TLC plate where the mobile phase used is 60% ethyl acetate in hexane. Less polar compounds tend to move faster as compared to more polar ones. Here in the TLC, the C18 fatty acids would move faster and their spots will have a higher Rf value than C16. This is what was observed as the C18 standard was clearly above the C16 standard.
• For our samples, we got spots corresponding to C16 & C18 but we couldn’t resolve them in the TLC. The intensity of the G1 spot was slightly higher than the control but we couldn’t draw any inferences except for the fact that we indeed had C16 and C18 FFA in our samples.
• We also cannot resolve C18:1 and C18:2 from C18:0 using a TLC. So we decided to run an LC-HRMS (Liquid Chromatography coupled with High Resolution Mass Spectrometry) to get a quantitative estimation of the FFA profiles present in our samples.

Western Blot

• Western blot was used to check the expression of JcFATB (48.6 kDa), CvFAP (62.9 kDa) and JcFATA (42.9 kDa) proteins. We used FLAG tag to check the expression of JcFATB protein and 6X His tag to check the expression of CvFAP and JcFATA proteins. Empty pYLEX transformed Po1g was used as a control colony and pYLEX-JC (recombinant plasmid) transformed Po1g was used as the experimental colony. We detected the band at 48.6kDa for JcFATB in G1 and G3 (Po1g transformed with pYLEX-JC) and 62.9kDa for CvFAP in G1 and G3, but the control (Po1g transformed with empty pYLEX) also showed the same bands. We observed the presence of non-specific bands, apart from the desired 48.6kDa band and 62.9kDa band in both the western blots.
• These observations for western blots might have occurred due to the contamination in the control sample due to pipetting error or due to spillover from the wells of the gel from the experimental samples G1 or G3 (Po1g transformed with pYLEX-JC). Also, the pYLEX vector may have sequences that may be interfering with the JcFATB and CvFAP expression in the background, giving the non-specific bands at 48.6kDa and 62.9kDa bands in the control.
• However, we didn’t observe any band for JcFATA at 42.9kDa in G1 and G3. It may be a binding error with 6X His antibody due to its low specificity.
• Possible solutions that could be implemented are trying to minimize the chances of contamination in the control sample or to check the protein profile after 6X His tag incubation for untransformed Po1g to check if the Y.lipolytica expresses proteins of similar sizes natively and get a confirmation of whether the bands are appearing due to pYLEX vector background expression. However, we couldn’t do this troubleshooting due to time constraints.

Liquid Chromatography - High Resolution Mass Spectrometry(LC-HRMS)

• To test for the change and increase in fatty acid profile of the transformed strain that we expected, we conducted LC-HRMS analysis of the fatty acid profiles. We looked for the peaks corresponding to 16:0(palmitic), 18:0(stearic), 18:1(oleic) and 18:2(linoleic) fatty acids.
• Since we also ran a standard along with each sample, we conducted quantitative measurements and compared the overall fatty acid content as well as compositions of the 4 types of fatty acids we are targeting.
• We also ran LC-HRMS for our deletion strain, in which we had deleted the beta-oxidation gene FAA1. We got strong results, showing a significant increase in the total fatty acid content for both the strain transformed with the Jatropha curcas thioesterases(using pYLEX-JC) as well as the deletion strain. Further, in the pYLEX-JC transformed strains, the composition of the fatty acids altered significantly to give us an intermediate between that of Jatropha oil and the empty pYLEX strain.

Gas Chromatography - Mass Spectrometry(GC-MS)

• For detecting the presence of hydrocarbons, we decided to proceed with GC-MS (Gas Chromatography coupled with Mass Spectrometry). We incubated the transformed and the control yeast in blue light for 72 hours to check for the activation of the CvFAP gene in the transformed strain, normalized the OD₆₀₀ values, pelleted the cells, and extracted the hydrocarbons using hexane. The sample was then analyzed by GC-MS. We expected to get a sharp peak for C15 and C17 straight-chain alkanes, but we did not get any peak. It is possible that since we didn’t perform the Alk2 deletion, the alkanes decomposed to fatty alcohols by its native metabolic pathway as a substrate. By the time we could figure out what went wrong and troubleshoot, we ran out of time.