Project Design


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:

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:

Its oleaginous nature:

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.


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:


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:

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

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.

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:

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


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

Transformation into the Chassis

Proof of Concept