Genetically modified yeast and process for obtaining olefins using same

Abstract

The present description relates to a transgenic microorganism capable of converting vegetable oils into olefins. The present description also discloses a process for obtaining such hydrocarbons from triglycerides or free fatty acids of plant origin, which is carried out by the microorganism disclosed in the present description.

Description

[0001] Genetically modified yeast and the process of obtaining olefins using it.

[0002] DESCRIPTION FIELD

[0003]

[0001] The present description is in the field of genetically modified microorganisms. The present description is also in the field of obtaining hydrocarbons through synthesis processes carried out by microorganisms or enzymes.

[0004] FUNDAMENTALS OF DESCRIPTION

[0005]

[0002] Drop-in fuels are biofuels designed to be used directly in existing engines and combustion systems without the need for significant equipment modifications. They have physical and chemical properties similar to conventional fossil fuels such as gasoline, diesel, or kerosene, allowing them to replace or blend with traditional fuels without affecting performance. Produced from renewable sources such as biomass or waste, drop-in fuels offer a more sustainable alternative, helping to reduce greenhouse gas emissions. Their compatibility with current distribution infrastructure and existing engines facilitates the transition to cleaner fuels, making them a practical solution for various applications, including road transport, aviation, and shipping.

[0006]

[0003] Alkanes and alkenes are fundamental in the production of drop-in fuels due to their properties that allow for the direct replacement of fossil fuels in existing engines and combustion systems. Alkanes, being saturated hydrocarbons, offer high energy density and stability, desirable characteristics for fuels that need to guarantee performance and safety in various applications, such as road transport and aviation. Alkenes, or olefins, are unsaturated hydrocarbons that are added to fuels to modify their properties, such as freezing point and viscosity, adapting them to the specific needs of different types of engines.The ability of these compounds to be used without significant changes to existing systems facilitates the adoption of more sustainable fuels, contributing to the reduction of greenhouse gas emissions and offering a practical solution for the transition to a greener energy matrix.

[0007]

[0004] Drop-in fuels are produced through processes that convert renewable feedstocks, such as biomass, agricultural waste, or algae, into hydrocarbons that can be used directly in existing engines and combustion systems. Production generally involves two main steps: converting the feedstock into chemical intermediates, such as lipids, and transforming these intermediates into final hydrocarbons. These intermediates are then transformed into hydrocarbons through chemical processes such as hydrogenation, dehydrogenation, or Fischer-Tropsch synthesis. These hydrocarbons are formulated to have physicochemical properties similar to those of fossil fuels, such as energy density and flash point, allowing their use in existing engines and systems without the need for technical adjustments.This process facilitates the integration of more sustainable fuels into the current transportation and distribution infrastructure, contributing to the reduction of greenhouse gas emissions.

[0008]

[0005] The Fischer-Tropsch synthesis process, however, presents some significant drawbacks. Firstly, the high cost is a challenge, due to the price of the necessary catalysts and equipment, as well as the operational complexity involved. The process is also energy-intensive, requiring high temperatures and pressures, which can result in high energy costs and environmental impacts associated with energy generation. Additionally, the operation and maintenance of Fischer-Tropsch reactors are complex, requiring precise control of reaction conditions and management of byproducts and catalysts. Although it may be a solution for the production of drop-in fuels from alternative sources, the sustainability of the process depends on the origin of the synthesis gases; if these are derived from fossil sources, the contribution to the reduction of greenhouse gas emissions may be limited.Furthermore, the process can generate byproducts that require additional processing, further increasing economic and operational challenges. In this sense, it becomes necessary to find new alternatives that make the drop-in fuel production process even more sustainable and attractive to the transportation sector.

[0009]

[0006] It is known that certain microorganisms are capable of producing drop-in fuels, taking advantage of their natural ability to metabolize organic substrates to produce hydrocarbons. These microorganisms, often genetically modified, are engineered to convert sugars, lipids, or other organic compounds into hydrocarbon products with properties similar to those of fossil fuels. The microbial process is a promising alternative for the sustainable production of fuels, as it utilizes renewable materials and can be adjusted to maximize the production of desired hydrocarbons, facilitating integration with the current fuel infrastructure.

[0010]

[0007] Although microorganisms engineered to produce drop-in fuels offer a promising solution for energy sustainability, they face several challenges. One is the efficiency of microorganisms in converting substrates into desired hydrocarbons, which may still be limited. Furthermore, competition with other undesirable metabolic products can reduce the quantity and purity of the hydrocarbons produced, requiring additional purification processes. The stability and consistency of microorganisms during large-scale production can also be problematic. Therefore, there is great potential in developing microorganisms capable of enabling the industrial production of biohydrocarbons and drop-in fuels.

[0011]

[0008] In addition to fuels, it should be noted that hydrocarbons such as alkenes, or olefins, are essential basic chemicals in the production of plastics, synthetic rubbers, solvents and resins, which expands the range of applications of biotechnological processes for the production of these compounds in a sustainable and renewable way.

[0012] STATE OF THE ART

[0013]

[0009] The state of the art comprises different processes for obtaining biohydrocarbons, especially bioderived alkenes.

[0014]

[0010] Patent document EP3317419, for example, discloses a method for producing medium-chain α-olefins using a genetically modified microorganism in the tricarboxylic acid cycle, in lipid synthesis, in metabolic intermediates or in reducing equivalents, wherein the modification comprises the insertion of a decarboxylase, a heterologous AMP-insensitive isocitrate dehydrogenase enzyme to reduce or eliminate the expression of acyl-CoA oxidases, and endogenous fatty alcohol dehydrogenases, and may also express genes encoding heterologous fatty acyl desaturases. The microorganism to be modified must be naturally oil-producing, such as Yarrowia lipolytica, and express at least one thioesterase. The recombinant microorganism is then placed in contact with a dual-substrate culture medium, composed of a fatty acid precursor and a single-carbon co-substrate.The microorganism will exhibit enhanced production of unsaturated fatty acids compared to a control microorganism without these modifications. Although Yarrowia lipolytica is a microorganism that, in theory, would be better suited for the purpose of biohydrocarbon production, its genetic manipulation to implement biotechnological improvements and direct its metabolic pathways to the product of interest is difficult, which leads to the search for other microorganisms as chassis for the construction of a highly effective strain for this purpose.

[0015]

[0011] One of these microorganisms that is most easily genetically manipulated is Saccharomyces cerevisiae, which is not naturally a producer of hydrocarbons. In EP3058078, a yeast S. cerevisiae is presented that has been genetically modified to produce fatty acid derivatives, including hydrocarbons and fatty alcohols. This yeast exhibits the absence or interruption of the gene encoding hexadecanal dehydrogenase (HFD1) and contains a heterologous gene encoding an acyl-Coenzyme A (CoA) reductase or an Acyl Carrier Protein (ACP) reductase. Additionally, the yeast may include heterologous genes from Synechococcus elongatus orf1594 or Acinetobacter baylyi Acr1, and a heterologous gene encoding a fatty aldehyde deformylating oxygenase, preferably from Synechococcus elongatus orf1593 or Nostoc puntiforme.Other yeast variants may also incorporate genes encoding cytosolic ferredoxin and NADP+ or NAD+ reductases, as well as additional genes such as Jeotgalicoccus spp Orf880, the LuxC, LuxD, and LuxE genes of Photorhabdus luminescens, and fatty aldehyde deformylating oxygenase genes from cyanobacteria. In some versions, the yeast may even have heterologous genes for carboxylic acid reductase from Mycobacterium marinum, CYP4G2 deformylating oxygenase from Musca domestica, and phosphopantetheine transferase from Aspergillus nidulans. Furthermore, the yeast may contain genes encoding cytosolic enzymes involved in fatty acid synthesis, such as acetyl-CoA C-acetyltransferase and other related enzymes, and may include additional reductases and thioesterases. The microorganism described in this document, however, uses the sphingolipid and SeFAR pathways to obtain fatty aldehydes, which are subsequently converted to alkanes and alkenes.Metabolic pathways involving the oxidation of fatty aldehydes, such as that described in EP3058078, however, exhibit lower efficiency in eukaryotes, as can be seen in the comparison of routes in Geng et al, 2023 (DOI: 10.18331 / BRJ2023.10.4.4), highlighting the need for a more refined solution to enable the biotechnological production of hydrocarbons.

[0016]

[0012] As alternatives, there are those based on microbial cytochrome P450 fatty acid decarboxylases. Document EP3317419, for example, discloses a method for the production of C7 to Cn alpha-olefins, which involves the culture of recombinant microorganisms expressing certain decarboxylases. In this case, the preferred substrate for the decarboxylase enzyme is a C8 to C12 free fatty acid. The invention is based, at least in part, on the discovery that certain genomic sequences of Alicyclobacillus acidocaldarius and Staphylococcus massiliensis encode enzymes with free fatty acid decarboxylase activity, neither of which organisms had previously been reported as producers of terminal olefins. Specific polypeptides with decarboxylase activity on medium-chain free fatty acids, especially C8 to C12 and particularly C12, have been identified, resulting in the production of medium-chain alpha-olefins, such as C7 to Cn.Examples include an olefin-producing enzyme (Sm46) identified in Staphylococcus massiliensis, the fatty acid hydroxylase P450Bs (Bs168) from Bacillus subtilis, and the decarboxylase enzyme from Alicyclobacillus acidocaldarius, all with specific activity on C12 fatty acids, thus producing Cn alpha-olefins. The method may include genetic engineering of host cells to produce or overproduce free fatty acids in the C8 to C12 range, and incorporation of nucleotides encoding a thioesterase specific for acyl-ACP. Additionally, an oligomerization reaction can be performed using the obtained alpha-olefins to produce poly-alpha-olefins such as C33 poly-alpha-olefins, which can be subsequently hydrogenated. It is known, however, that some OleTs may be unstable or perform suboptimally when expressed in heterologous systems.The expression of OleTs in recombinant host cells can result in low enzyme stability, reduced activities, or problems with protein solubility. This can affect the efficient production of olefins and require further optimizations. Furthermore, OleTs often require specific cofactors, such as peroxides or NADPH, to function. The need for these cofactors can increase the complexity of the modifications required in the microorganism chassis, especially if it is necessary to add or regenerate these cofactors, which can affect cell viability.

[0013] Along the same lines, patent document EP3061827 refers to a genetically modified microbial cell for the production of at least one terminal alkene from short-chain fatty acids, specifically C4-C10.This cell has at least two genetic modifications: the first activates the expression of an enzyme (E1) from the CYP152 peroxygenase family, such as CYPSPa (E1a), CYPBSB (E1b), or OleT (E1c), with OleT being preferentially chosen and having at least 60% sequence identity with SEQ ID NO:1 described in the document. The second mutation activates the expression of at least one NAD(P)+ oxidoreductase (E2) and the corresponding mediator protein, which may be, for example, ferredoxin reductase (E2a) with ferredoxin or putidaredoxin reductase (E2b) with putidaredoxin, with E2 having 60% sequence identity with SEQ ID NO:2 and the mediator protein having 60% identity with SEQ ID NO:3 described in the document. Additionally, the cell may contain a third mutation that increases the expression of an enzyme (E3) responsible for the regeneration of NAD(P)H, selected from glucose dehydrogenases, phosphite dehydrogenase, and formate dehydrogenase.The cell may also exhibit a reduced capacity for fatty acid degradation compared to the wild-type cell, achieved by deleting genes encoding fatty acid importers, fatty acid-CoA ligases, acyl-CoA dehydrogenases, among others. The cell may be from a prokaryotic or lower eukaryotic organism. The described method involves contacting the recombinant cell with a medium containing the short-chain fatty acid to produce the desired terminal alkene. The production mechanism based on the ferredoxin reductase (E2a) system with ferredoxin or putidaredoxin reductase (E2b), however, is a disadvantage of this invention. The article by Florian et al., 2020 (https: / / doi.org / 10.1016 / j.mec.2019.e00111) shows that the action of oxidation-reduction partners limits the activity of P450 enzymes such as OleT, risking not achieving 100% of the production potential of the biohydrocarbons of interest.

[0014] The expression of genes encoding P450 enzymes in S. cerevisiae, in particular, is reported. Patent document US101 13208 discloses a modification of yeast to produce terminal alkenes via a P450 fatty acid decarboxylation pathway. The yeast was genetically modified with the insertion of heterologous fatty acid decarboxylase genes, the deletion of specific genes (FAA1, FAA4, CTT1, CTA1, and CCP1), and the overexpression of the HEM3 gene. With these modifications, the yeast became capable of producing hydrocarbons such as 1-undecene, 1-tridecene, among others. The invention also addresses methods for producing these alkenes, which involve the cultivation and fermentation of the modified yeast under controlled conditions. Furthermore, it describes metabolic engineering methods to optimize alkene production in yeast by adjusting conditions such as temperature, dissolved oxygen concentration, and pH control.These combined strategies resulted in a significant increase in alkene production compared to unmodified yeasts, reaching an increase of up to 67.4 times in production. A drawback of this invention, however, is the fact that it relies on the excretion of peroxisome peroxide, which they produce via the degradation of fatty acyl-CoA, and mitochondria, so that the peroxide meets the needs of the OleTs. This excretion is not, in fact, intended to occur into the cytosol, given that peroxide is extremely reactive and can be damaging to other cellular structures.

[0017]

[0015] In WO2014 / 102201, a specific bacterial fatty acid decarboxylase is described that can be inserted into S. cerevisiae or an oilseed species. The production of 15- to 19-carbon alkenes is predicted, and the host cell may possess disruptions in the elo3, faa1, faa4, adh, mitochondrial dehydrogenase, and acetaldehyde-to-ethanol conversion genes. A drawback in the case of S. cerevisiae, however, lies in the fact that S. cerevisiae is a sugar-to-ethanol converter. Disrupting this and certain other pathways proposed in the document reduces cellular robustness. BRIEF DESCRIPTION OF THE INVENTION

[0018]

[0016] The present invention relates to a genetically modified microorganism to produce alkenes from a culture medium containing triglycerides or free fatty acids as a carbon source, wherein alkene production occurs in the peroxisome. Concomitantly, in the peroxisome, a portion of the fatty acids is degraded to produce hydrogen peroxide (H2O2), an essential cosubstrate for the activity of OleTs enzymes. The objective of the invention is to disclose a transgenic microorganism capable of converting vegetable oils into olefins, as well as to describe a process for obtaining these hydrocarbons from vegetable-derived triglycerides.

[0019]

[0017] The objectives of this description are achieved by a genetically modified yeast:

[0020]

[0018] (i) by inserting a gene encoding a decarboxylase from the CYP152 family selected from the group comprising amino acid sequences SEQ ID: 01, SEQ ID: 02 or SEQ ID: 03;

[0021]

[0019] (ii) by deletion of the genes that encode two enzymes Acyl-coenzyme A Synthetases FAA2 and FAA4;

[0022]

[0020] (iii) by deletion of the gene encoding the peroxisomal Catalase A enzyme CTA1;

[0023]

[0021] (iv) by deletion of the gene encoding the HAP1 transcription factor, responsive to heme and oxygen levels;

[0024]

[0022] (v) by inserting the gene that codes for porphobilinogen deaminase HEM3, which is limiting in the production of heme groups.

[0025]

[0023] The transgenic yeast may also comprise the insertion of a gene encoding a lipase selected from the group comprising the amino acid sequences SEQ ID: 04, SEQ ID: 05 and SEQ ID: 06, but not limited to.

[0026]

[0024] The objectives of the present description are also achieved by a process for obtaining bioderived hydrocarbons comprising cultivating the genetically modified yeast of the present description in a medium containing fatty acids or, alternatively, in a medium containing triglycerides and at least one added lipase enzyme.

[0027]

[0025] The genetic modifications described herein enable the microorganism to perform the transformation into product within the peroxisome, eliminating the need for a disruption step in the ethanol pathway, which would reduce cellular robustness. The modifications described herein are advantageous because they utilize OleTs compatible with the cellular environment (low salt requirement and activity at neutral pH), factors that are generally required by enzymes of this type. This is important because the cell interior is not a salt-rich environment. Furthermore, OleTs have the ability to convert unsaturated fatty acids into olefins, which are the most abundant in nature, especially in oils from plant sources.Thus, the genetically modified microorganism described herein is capable of converting vegetable oils, including residual vegetable oils, into olefins, olefins being important intermediates in the synthesis of aviation fuels and other chemical derivatives.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029]

[0026] The present invention is illustrated in the embodiments represented in figures, as briefly described below.

[0030]

[0027] Figure 1 is a schematic representation of the modified metabolic pathway in the microorganism of the present description to allow the production of olefins from vegetable oils or free fatty acids, according to an embodiment of the present description.

[0031]

[0028] Figure 2 is a schematic representation of the modified metabolic pathway in the microorganism to allow the production of heme groups to increase the activity of P450 decarboxylase enzymes.

[0032]

[0029] Figure 3A is a chromatogram obtained by gas chromatography showing confirmation of the peak corresponding to the 1,9-heptadecadiene product produced from oleic acid, the most abundant fatty acid. Figure 3B shows the optical density (OD) at 600 nm of the 96-hour culture of strains that overexpressed OleTPRN and OleTco. The control refers to the strain transformed with the empty p426 vector.

[0033]

[0030] Figure 4 is a bar graph showing the production of olefins with the modified microorganism in a medium containing lipids as the sole carbon source.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035]

[0031] The present description refers to a genetically modified microorganism capable of converting vegetable oils into olefins. The present description also refers to a process for obtaining such bioderived hydrocarbons from vegetable triglycerides.

[0036]

[0032] The objectives of the present description are achieved by a genetically modified yeast:

[0037]

[0033] (i) by inserting a gene encoding a cytochrome P450 class decarboxylase of the CYP152 family selected from the group comprising amino acid sequences SEQ ID: 01, SEQ ID: 02 or SEQ ID: 03;

[0038]

[0034] (ii) by deletion of the genes that encode the two enzymes Acyl-coenzyme A Synthetases FAA2 and FAA4;

[0039]

[0035] (iii) by deletion of the gene encoding the peroxisomal Catalase A enzyme CTA1;

[0040]

[0036] (iv) by deletion of the gene encoding the HAP1 transcription factor, responsive to heme group and oxygen levels;

[0041]

[0037] (v) by inserting the gene that codes for porphobilinogen deaminase HEM3, which is limiting in the production of heme groups.

[0042]

[0038] In one embodiment, a cytochrome P450 class decarboxylase from the CYP152 family originates from Mycobacterium abscessus, is called OleTMA and defined as SEQ ID: 01.

[0043]

[0039] In one embodiment, a cytochrome P450 class decarboxylase from the CYP152 family originates from bacteria of the genus Rothia nasimurium, is called OLeTPRN and defined as SEQ ID: 02.

[0044]

[0040] In one embodiment, a cytochrome P450 class decarboxylase of the CYP152 family with peroxidase activity originates from Corynebacterium doosanense, is named OleTco and defined as SEQ ID: 03.

[0045]

[0041] A person skilled in the art who is familiar with the genetic transformation tools available in the state of the art will know how to define the means for gene insertion and deletion and employ appropriate techniques.

[0046]

[0042] In one embodiment, the genetically modified yeast of the present description is also optionally modified by the insertion of at least one gene encoding a lipase.

[0047]

[0043] In one embodiment, the lipase enzyme is as defined by an amino acid sequence selected from the group comprising SEQ ID: 04, SEQ ID: 05 or SEQ ID: 06, not limited to.

[0048]

[0044] In an optional embodiment, the lipase is as defined by SEQ ID: 04 and originates from Janthinobacterium lividum.

[0049]

[0045] In an optional embodiment, the lipase is as defined by SEQ ID: 05 and originates from Penicilliopsis zonata.

[0050]

[0046] In an optional embodiment, the lipase is as defined by SEQ ID: 06 and originates from Capronia epimyces.

[0051]

[0047] In one embodiment, a process for obtaining olefins comprises cultivating the genetically modified microorganism in a medium containing triglycerides and at least one heterologous lipase enzyme.

[0052]

[0048] In one embodiment, the heterologous lipase added to the medium is a commercially available lipase.

[0053]

[0049] In one embodiment, heterologous lipase is secreted by the genetically modified yeast of the present description, according to an optional embodiment of the present description.

[0054]

[0050] In one embodiment of the present description, a process for obtaining olefins comprises cultivating the genetically modified microorganism in a medium containing free fatty acids.

[0055]

[0051] In one embodiment, the medium containing triglycerides or fatty acids is a synthetic medium containing a source of vitamins, a source of nitrogen, and a source of triglycerides.

[0056]

[0052] In one embodiment, a source of vitamins may be a yeast extract, and a source of nitrogen may be ammonium sulfate.

[0057]

[0053] In one embodiment, the triglyceride medium contains between 1% and 10% of a vegetable oil.

[0058]

[0054] In one embodiment, the triglycerides in the medium are derived from a low-cost triglyceride source, such as by-products and residues from the agro-industrial processing of vegetable oils, including effluents rich in free fatty acids.

[0059]

[0055] Figure 1 is a schematic representation of the modified metabolic pathway in the genetically modified yeast of the present description to enable the production of olefins from vegetable oils, according to an embodiment of the present description.

[0060]

[0056] Lipase hydrolyzes extracellular lipids, releasing fatty acids, such as oleic acid.

[0061]

[0057] CYP152 family decarboxylase enzymes with peroxidase activity, abbreviated as OleTs, convert fatty acids into olefins in peroxisomes. However, the OleTs described here are capable of converting oleic acid, the most abundant fatty acid in plant lipids, without inhibition.

[0062]

[0058] The fatty acid degradation pathway (beta-oxidation) generates hydrogen peroxide (H2O2). H2O2 acts as a co-substrate for OleT.

[0063]

[0059] The FAA2 and FAA4 genes encode the S. cerevisiae acyl-CoA synthetases named Faa2p and Faa4p, respectively. Deletion of only FAA2 and FAA4 reduces conversion to acyl-CoA in the cytosol and peroxisome. Thus, substrate is ensured for both the beta-oxidation pathway (acyl-CoA) and the OleT enzyme (free fatty acid).

[0060] The CTA1 gene encodes catalase A, a peroxisomal enzyme. Its deletion prevents the conversion of hydrogen peroxide (H2O2) to water.

[0064]

[0061] Deletion of HAP1, which encodes a heme group sensor, along with overexpression of the rate-limiting gene for this biosynthesis pathway (HEM3), stimulates the constitutive production of this cofactor essential for OleT activity.

[0065]

[0062] In one embodiment, in the process carried out by the genetically modified S. cerevisiae yeast described herein, the yeast is able to convert free fatty acids present in the medium into olefins within the peroxisome, eliminating the need to perform a genetic modification step to disrupt the ethanol pathway to divert the production of fatty acids in a native manner.

[0066]

[0063] In one embodiment, in the process carried out by the genetically modified S. cerevisiae yeast described herein, the yeast is able to convert plant triglycerides present in the medium into an internalizable substrate, subsequently transforming them into olefins within the peroxisome, eliminating the need to perform a genetic modification step to disrupt the ethanol pathway for native diversion of fatty acid production.

[0067]

[0064] The genetic modifications achieved in the present description have the advantage of eliminating the genetic modification step for disruption of the ethanol pathway, since the ethanol pathway is necessary for cell survival.

[0068] EXAMPLES OF IMPROVEMENT OF THE INVENTION

[0069]

[0065] In what follows, exemplary realizations of the object described here are presented, in a non-restrictive manner, illustrating results and advantages achieved by it.

[0070]

[0066] Example 1: Selection of orthologs from OleTPRN:

[0071]

[0067] One of the main challenges in the production of alkenes from renewable sources via enzymatic routes is the discovery of new enzymes capable of efficiently converting oleic acid into petrochemical-like building blocks, and to date only the OleTPRN enzyme demonstrates this characteristic. To identify new P450 decarboxylases of the CYP152 family with similar activity, the web tool “Enzyme Function Initiative-Enzyme Similarity Tool” was used, which allowed the construction of a Sequence Similarity Network (SSN) based on the OleTPRN sequence. The initial analysis, with an E-value of 1 e-10 and a minimum sequence identity of 90%, was refined by applying a 50% threshold, resulting in the definition of four isofunctional groups, visualized by the Cytoscape software.Among these groups, an isofunctional cluster that includes OleTPRN showed potential for oleate decarboxylation, leading to the selection of five genes for yeast screening, with codon optimization and cloning into the p426-HXT7t vector with a tag in the C-terminal portion for enzymatic translocation to the peroxisome.

[0072]

[0068] Example 2: Construction of strains and screening of OleTs:

[0073]

[0069] For the construction of strains and screening of OleTs, S. cerevisiae (CEN.PK 2-1 C) was initially transformed with recombinant vectors containing the selected OleTs and cultured in SCURA- medium (complete synthetic medium) with 5% oleic acid. The E. coli DH5a strain was used for plasmid propagation and vector cloning, being cultured at 37°C in LB medium with antibiotics for plasmid selection. OleTPRN (ORC20205) and OleTcü (WP_018021878) were obtained with codon optimization for S. cerevisiae and amplified by PCR using Phusion DNA polymerase. Plasmids were constructed using the Gibson assembly method and transformed into E. coli for positive recombination screening. After screening, the plasmids were used to transform S. cerevisiae using the lithium acetate method. The strain is CEN.PK 2-1 C was cultivated using YPD medium for general propagation and SC medium (complete synthetic medium) supplemented with 5% oleic acid for selection and alkene production. After 96 hours of cultivation, the cultures were centrifuged, and both the pellet and the supernatant were analyzed by gas chromatography.

[0074]

[0070] For supernatant analysis, hydrocarbons were extracted with 1 mL of chloroform and analyzed directly. For pellet analysis, cells were disrupted with 500 µL of chloroform, 250 µL of methanol, 200 µL of glass microspheres, and the internal standard 1-tetradecene (final concentration of 0.98 mg / mL) by agitation for 5 minutes. The organic phase was then collected and derivatized using the methanol-HCl method due to the high fatty acid content, which could clog the chromatograph. In summary, 400 pL of the organic phase was transferred to a 2 mL glass vial for the derivatization step, in which 25 pL of HCl (2 M in methanol) and 75 pL of a second internal standard solution (1-hexadecene, final concentration of 50 pg / mL in methanol) were added. The sample was incubated at 50 °C for 30 minutes and then cooled with 40 pL of NaOH solution (1 M in methanol).500 µL of water were added for phase separation, and after centrifugation at 5000 rpm for 5 minutes, the organic phase was analyzed by gas chromatography. GC analysis was conducted using a 7890A gas chromatograph (Agilent Technologies, Santa Clara, USA), employing an RTx-5MS column (30 m x 0.25 mm x 0.25 µm), detector temperature of 290 °C, injector temperature of 230 °C, split ratio of 1:10, and oven temperature: 30 °C for 2 minutes, increasing to 220 °C at a rate of 10 °C / min, increasing to 240 °C at a rate of 2 °C / min, and increasing to 350 °C at 50 °C / min. To identify the peaks, the retention time was compared with authentic standards of alkene products. The quantification of the compounds was performed by adding internal standards to each sample (1-tetradecene), and the exact quantification of the added internal standards was done using calibration curves for each internal standard (1-hexadecene).

[0075]

[0071] The S. cerevisiae strain was engineered with the deletion of the FAA2 and FAA4 genes to reduce the activation of free fatty acids (FFA), and CTA1 to prevent H2O2 detoxification in the peroxisome. The activation of FFA by S. cerevisiae to form its CoA conjugate is a signal for the utilization of fatty acids within the cell. S. cerevisiae possesses four genes responsible for this activation: FAA1, FAA2, FAA3, and FAA4. The deletion of all four acetyl-CoA synthetase genes is lethal, as the fatty acid conjugate is crucial for the construction of the cell membrane and other cellular structures. Furthermore, the FAA1 enzyme has been shown to be important not only for FA-CoA conjugation but also for the transport of FFA into the cell. On the other hand, FAA3 acts primarily on FFA with 22 carbons or more, which does not apply in our case. Therefore, only the FAA2 and FAA4 genes were deleted to increase the pool of FFA within the cell. The recombinant strain (CEN.PK 2-1 C; Afaa2; Afaa4; Actal ) was generated using the CRISPR-Cas9 method.

[0076]

[0072] This recombinant strain was transformed with vectors containing OleTs and cultured (initial OD at 600 nm of 2) in SCURA-, using 5% oleate as the sole carbon source for 96 hours. 1,8-heptadecadiene (A-C17:2) was detected in strains overexpressing OleTPRN and an OleT homolog from Corynebacterium doosanense, as shown in Figure 3A. Therefore, this result demonstrated that [3-peroxisomal oxidation] can, in fact, be coupled to fatty acid decarboxylation to produce alkenes. Surprisingly, despite the putative inhibitory effect triggered by hydrocarbons, yeast strains overexpressing OleTs did not exhibit growth deficiency compared to the empty vector control, as shown in Figure 3B. Olefin production was higher in these strains, as shown in Figure 4.

[0077]

[0073] Although exemplary embodiments of the processes and products described have been presented in this report, the scope of protection is not intended to be limited to the literal description thereof. Therefore, the description should be interpreted not as limiting, but merely as examples of particular embodiments that retain the inventive concept presented herein. A person skilled in the art may readily apply the teachings presented herein to analogous solutions arising therefrom, limited only by the scope of the claims in this application.

Claims

CLAIMS 1. Genetically modified yeast characterized by being modified: (i) by inserting a gene encoding a decarboxylase from the CYP152 family selected from the group comprising amino acid sequences SEQ ID: 01, SEQ ID: 02 or SEQ ID: 03; (ii) by deletion of the genes that encode two Acyl-coenzyme A synthetase enzymes, FAA2 and FAA4; (iii) by deletion of the gene encoding the peroxisomal catalase A enzyme CTA1; (iv) by deletion of the gene encoding the HAP1 transcription factor; (v) by inserting the gene that codes for porphobilinogen deaminase HEM3.

2. Yeast, according to claim 1, characterized by further comprising: (i) the insertion of a gene encoding a lipase preferentially selected from the group comprising the amino acid sequences SEQ ID: 04, SEQ ID: 05 and SEQ ID:

06.

3. Yeast, according to either of claims 1 and 2, characterized by being a Saccharomyces cerevisiae yeast.

4. Process for obtaining olefins characterized by cultivating yeast as defined in claims 1 and 2 in a medium comprising vegetable triglycerides or free fatty acids.

5. Process according to claim 4, characterized in that the medium comprises from 1% to 10% vegetable oil.

6. Process, according to any one of claims 4 to 5, characterized in that the medium further comprises at least one lipase enzyme.

7. Process according to any one of claims 4 to 6, characterized in that the yeast is Saccharomyces cerevisiae.

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