Genetically engineered microorganisms for heterologous production of modified forms of surfactant protein Lv-ranaspumin-1 (LV-RSN-1), modified forms of this surfactant protein, synthetic genes encoding this surfactant protein, expression cassettes containing these synthetic genes, and expression vectors containing these synthetic genes

Abstract

The present invention relates to the heterologous production in microorganisms of modified predicted isoforms of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1), the sequence of which was deduced from analysis of protein extracts of nest foam from the Northeastern peppered frog (Raptodactylus bastus). More specifically, the present invention relates to two surfactant proteins consisting of modified predicted isoforms of Lv-Rsn-1, two synthetic genes each encoding one of these modified predicted isoforms of Lv-Rsn-1, two expression cassettes each containing one of the synthetic genes encoding one of the modified predicted isoforms of Lv-Rsn-1, two expression vectors each containing one of the synthetic genes encoding one of the modified predicted isoforms of Lv-Rsn-1, and two transgenic microorganisms, a bacterium and a yeast, each transformed with one of these synthetic genes and heterologously producing one of the modified predicted isoforms of Lv-Rsn-1. Lv-Rsn-1 possesses inter alia surface activity, emulsifying and dispersing properties, and its heterologous production allows its use in various applications and industrial products without the need to extract Lv-Rsn-1 from frog nest foam.

Description

[Technical Field]

[0001] This patent application relates to the heterologous production in microorganisms of a modified version of a predicted isoform of the surfactant protein Lv-ranaspumina-1 (also known as Lv-ranaspumin-1, Lv-Rsn-1), which was isolated and discovered in the foam nests of the Northeastern Pepper Frog (ra-pimenta-do-Nordeste, Leptodactylus vastus), and which cannot be obtained by extraction, preventing its use in various applications. More specifically, this patent application relates to the invention of two synthetic genes, each encoding a modified version of the predicted amino acid sequence of an Lv-Rsn-1 isoform; two expression cassettes each containing one of these synthetic genes; two expression vectors each containing one of these synthetic genes inserted into an expression cassette; two genetically engineered (recombinant) microorganisms (bacteria and yeast) that heterologously produce one of the modified forms of Lv-Rsn-1; and two proteins comprising modified forms of the Lv-Rsn-1 surfactant protein. Lv-Rsn-1 possesses, inter alia, surface-active, emulsifying, and dispersing properties, and its heterologous production enables its use in a variety of applications and products. [Background technology]

[0002] Surfactants constitute a class of amphiphilic molecules, i.e., their molecular structure simultaneously presents polar (hydrophilic and lipophobic) and non-polar (hydrophobic and lipophilic) parts, which allows these molecules to accumulate on surfaces and interfaces of systems and reduce their surface and interfacial free energy. In this context, "surface" usually refers to the boundary between liquid / solid or liquid / gas, and "interface" usually refers to the boundary between two immiscible liquids.

[0003] Surfactants have several applications in the agricultural, food, textile, pharmaceutical, cosmetic, household hygiene, oil and gas industries, among others. These applications exploit the different activities of these surface-active molecules. The main ones are emulsification, demulsification, solubilization and detergency (activities related to liquid-liquid interactions), wettability and dispersibility modification (activities related to solid-liquid interactions), and foaming (activities related to liquid-gas interactions).

[0004] In the oil and gas industry, surfactants can be used for purposes such as (1) bioremediation of contaminated areas when used to mobilize hydrocarbons and make them bioavailable to biodegradable organisms; (2) cleanup of oil spills when used to disperse oil slicks; (3) removal of oily residues from the interior of storage tanks; (4) control of harmful microorganisms when used as biocides and antifouling agents; (5) cleaning of filtration membranes when used to remove mineral deposits and biofilms impregnated within them; and (6) advanced oil recovery when used to mobilize residual oil in reservoirs and increase recovery.

[0005] So-called chemical surfactants are artificially synthesized from fossil precursors (petrochemicals) or plants (oleochemicals), in contrast to biosurfactants, which are synthesized by the metabolism of living organisms. Chemical surfactants currently dominate the surfactant market, but in recent years, the use of biosurfactants has been considered an increasingly attractive alternative due to their: (1) higher surface activity; (2) lower minimum concentration required to achieve maximum surface activity (i.e., low critical micelle concentration, or CMC); (3) lower ecotoxicity; (4) greater biodegradability; (5) increased surface stability over wider pH, salinity, and temperature ranges; (6) milder manufacturing conditions; and (7) the ability to be produced from renewable raw materials.

[0006] The global surfactants market was estimated at US$30.6 billion in 2016 and is forecast to increase to US$39.8 billion by 2021, a compound annual growth rate (CAGR) of 5.4% between 2016 and 2021. Within this sector, biosurfactants accounted for US$4.2 billion in 2017 and are forecast to grow to US$5.5 billion by 2022, a CAGR of 5.6% between 2017 and 2022.

[0007] Despite all the interesting properties and numerous advantages reported for chemical surfactants, the industrial production of biosurfactants still faces the challenge of high production costs, which is currently the biggest obstacle to the wider use of biosurfactants in various surfactant application fields. This high production cost is mainly due to the following reasons: (1) the high cost of raw materials, (2) the low yield of the production process, and (3) the costs associated with purifying biosurfactants.

[0008] Industrial production of biosurfactants can be achieved through two different approaches. The first approach is to use the species in which the biosurfactant was discovered as the production platform. This is the currently most widely adopted approach in the biosurfactant industry, which is dominated by two types of biosurfactants, both of which belong to the glycolipid class: (1) rhamnolipids (RL) produced by the bacterium Pseudomonas aeruginosa and (2) sophorolipids (SL) produced by the yeast Starmerella (Candida) bombicola. Two other types of biosurfactants of the glycolipid class are also commercially produced in smaller quantities: (1) mannosylerythritol lipids (MEL) produced by yeasts of the genus Pseudozyma, and (2) cellobiose lipids (CL) produced by yeasts of the species Ustilago maydis and Pseudozyma flocculosa. Finally, there is also commercial production of lipopeptide mixtures (e.g., fengicina, iturina, liquenisina, mycosubtilina, and surfactina) or isolated surfactins, both of which are produced by bacteria of the species Bacillus subtilis.

[0009] However, it is not always possible to use the same species of organism in which the biosurfactant was discovered; in this case, a second approach that may be adopted is the genetic transformation (recombination) of some microorganisms to heterologously produce the biosurfactant of interest. The microbial strains most used industrially as platforms for heterologous production of bioproducts in general are those for which large-scale industrial cultivation and genetic transformation techniques are already well mastered, mainly the following: Bacillus subtilis, Corynebacterium (Micrococcus) glutamicum and Escherichia coli species of bacteria, Candida albicans, Kluyveromyces lactis, Komagataella phaffii (Pichia pastoris), Saccharomyces cerevisiae and Yarrowia lipolytica. lipolytica yeasts and filamentous fungi of the species Aspergillus oryzae, Aspergillus niger, Penicillium chrysogenume, and Trichoderma reesei.

[0010] However, heterologous production of biosurfactants in these species presents challenges. Most scientifically known biosurfactants currently produced on an industrial scale belong to the classes of glycolipids and lipopeptides, which have a nonpolar portion composed of fatty acids and a polar portion composed of carbohydrates and peptides, respectively. The biosynthesis of these biosurfactants depends on a complex metabolic pathway involving several genes. To get an idea of ​​the complexity of this biosynthesis, the genes involved can be grouped into four major sets: (1) genes involved in the biosynthesis of the hydrophilic portion composed of peptides or carbohydrates; (2) genes involved in the biosynthesis of the hydrophobic portion composed of fatty acids; (3) genes involved in covalently linking the hydrophilic portion to the hydrophobic portion; and (4) genes involved in regulating the expression of genes belonging to the other three groups.

[0011] In light of this, the genetic and metabolic engineering challenges associated with the heterologous production of glycolipid- and lipopeptide-type biosurfactants in species traditionally used in biotechnological production processes may be even greater, since heterologous production in these species requires the insertion of numerous genes, precise regulation of the expression of the inserted genes, and an adequate supply of precursors to these metabolic pathways. These difficulties make the use of the above-mentioned species difficult, so industrial production of biosurfactants is currently limited to species that naturally produce these biosurfactants.

[0012] In addition to the limitation that biosurfactants must be produced by naturally synthesizing microorganisms, these biosurfactants face the additional challenge of being produced as mixtures of different isoforms (or homologs), which exhibit differences in their properties due to variations in the composition of both their hydrophobic and hydrophilic portions. For example, rhamnolipids may have one or two L-rhamnose-type sugars in their polar portion and one or two β-hydroxylated fatty acids containing an even number of carbons (8 to 16) in their nonpolar portion. The composition of biosurfactant mixtures, in terms of the proportion of each isoform, can vary considerably even between batches produced using the same microbial strain under the same culture conditions. Furthermore, because of the variability in the properties of different isoforms, the proportions of different isoforms present in the resulting mixture affect the properties of the final product, making its standardization difficult.

[0013] However, there is another class of biological surfactants, so-called surfactant proteins, whose members have the dual advantage of avoiding the need to insert several genes and making it easier to produce a single isoform.Surfactant proteins are polymers of amino acids coded by a single gene, and this gene is expressed by the classical pathway of transcribing DNA sequences into mRNA and translating the mRNA into protein on ribosomes according to the genetic code.In this way, a single gene can be inserted into the host microorganism (which facilitates genetic transformation), and thus heterologous production of a single isoform can be achieved (which facilitates standardization of the final product).

[0014] Among known natural sources of surfactant proteins, one that has been explored in recent years is frog nest foam, which is remarkable for its persistence in the environment. The first surfactant protein isolated from this source was produced by the ra-tungara frog (Physalaemus (Engystomops) pustulosus). Of the six proteins isolated from this frog nest foam (named ranaspumins and numbered 1–6), Ep-ranaspumin-2 (Ep-Rsn-2), consisting of 97 amino acids and with a molecular weight of 11.0 kDa (GenBank AAP48831), was the only protein that exhibited surfactant activity. Ep-Rsn-2 was able to reduce the water / air surface tension from 72 nM / m to 50 nM / m at a concentration of 10 μg / mL.

[0015] A second type of surfactant protein was isolated by the present applicants from the nest foam of the Northeastern peppered frog (Raptodactylus bastus). This protein was isolated from the foam fluid by chromatographic techniques, and in one of the two fractions obtained, a dense, distinct band was detected with a molecular weight of approximately 20 kDa, which showed emulsifying activity (emulsification index of 57%) at 0.1 mg / mL. This protein was designated Lv-ranaspumin-1 (Lv-Rsn-1).

[0016] The amino acid sequence of the Lv-Rsn-1 protein isolated from the foam fluid was inferred from the molecular weight of each band (23.5 kDa) obtained by denaturing gel electrophoresis, the sequence of 36 peptide fragments obtained by enzymatic digestion of this band, mass spectrometry sequencing of the fragments, and cross-referencing of the electron density map obtained by X-ray crystallography. This resulted in a predicted sequence of 217 amino acids. Two predicted three-dimensional structures of the crystals of this Lv-Rsn-1 protein have been submitted to the RCSB PDB (https: / / www.rcsb.org / ) under accession codes 4K82 and 4K83.

[0017] Comparing the two isoforms, Ep-Rsn-2 and Lv-Rsn-1, whose three-dimensional structures have already been published, reveals that they share only 38% amino acid sequence similarity and are completely different in three-dimensional structure. Lv-Rsn-1 has 13 α-helices and two antiparallel β-sheets forming a clamp, whereas Ep-Rsn-2 consists of four antiparallel β-sheets with an α-helix surrounding one of its faces perpendicularly. Therefore, although both are named ranaspumins because they are derived from frog nest foam, they are two distinct proteins.

[0018] Results obtained when L. vastus nest foam fractions containing Lv-Rsn-1 were analyzed by polyacrylamide gel electrophoresis and by sequencing of peptide fragments suggest the occurrence of other isoforms with amino acid sequences of different sizes and compositions. However, neither the L. vastus genome nor any genes encoding any of the isoforms of Lv-Rsn-1 have been sequenced to date, so the sequences of these other isoforms are still unknown.

[0019] Lv-Rsn-1 is a surfactant protein with excellent potential for various applications, and enabling its industrial production is an obstacle that needs to be overcome to achieve its commercialization and application for various purposes. However, considering that Lv-Rsn-1 is a multicellular, sentient, and biosurfactant derived from animal organisms protected by environmental legislation, an approach using the producing organism itself is not feasible for both technical and economic reasons, as well as ethical and legal issues. For similar reasons, obtaining Lv-Rsn-1 by extraction from Northeastern peppered frog nest foam is also not feasible. As far as we can verify, there are no patents that refer to the heterologous production of surfactant proteins from frog nest foam of any species. Summary of the Invention [Problem to be solved by the invention]

[0020] In light of this disclosure, the present invention comprises two genetically modified microorganisms (bacteria and yeast) capable of heterologously producing modified forms of the Lv-Rsn-1 surfactant protein. In addition to these two genetically modified microorganisms, the present invention also contemplates the following: (1) two proteins consisting of modified versions of predicted isoforms of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1), (2) two synthetic genes whose sequences are optimized for expression in bacteria and yeast, respectively, and which encode one of the two modified versions of the predicted isoforms of the Lv-Rsn-1 surfactant protein, (3) two bacterial and yeast expression cassettes, each containing one of the synthetic genes encoding one of the modified versions of the predicted isoforms of the Lv-Rsn-1 surfactant protein, and (4) two expression vectors for expression in bacteria and yeast, respectively, containing one of the synthetic genes encoding one of the two modified versions of the predicted isoforms of the Lv-Rsn-1 surfactant protein. [Means for solving the problem]

[0021] The present invention is based on the identification of surfactant activity of the protein Lv-ranaspumin-1 (Lv-Rsn-1), which is produced by the Northeastern peppered frog (Raptodactylus bastus), and which has several potential industrial applications. Accordingly, the present invention contemplates two proteins, neither of which occurs in nature, consisting of modified versions of the predicted sequence of one of the isoforms of the Lv-Rsn-1 surfactant protein.

[0022] Another element of the present invention relates to two synthetic genes each encoding a modified version of the predicted sequence of an isoform of the Lv-Rsn-1 surfactant protein, the sequences of which have been optimized for codon usage to target expression in genetically engineered bacterial and eukaryotic microorganisms, respectively.

[0023] Another element of the present invention relates to two expression cassettes, each containing one of the synthetic genes encoding a modified version of the predicted sequence of the Lv-Rsn-1 surfactant protein operably linked to an active promoter, intended for insertion into expression vectors for replication and expression in genetically modified bacteria and yeast, respectively.

[0024] Another element of the present invention relates to two expression vectors, each containing one of the synthetic genes encoding a modified version of the predicted sequence of an isoform of the Lv-Rsn-1 surfactant protein, targeted for replication and expression in genetically engineered bacteria and yeast, respectively.

[0025] Another element of the present invention relates to two genetically modified microorganisms, a bacterium and a yeast, each transformed with a respective expression vector containing an expression cassette carrying a synthetic gene encoding a version of the predicted sequence of an isoform of the Lv-Rsn-1 surfactant protein, targeting the expression of this gene and the heterologous production of this surfactant protein.

[0026] Other objects, details and advantages of the present invention will become apparent from the following drawings, arrangements, descriptions and examples. [Brief explanation of the drawings]

[0027] [Figure 1]FIG. 1 shows a vector map for expression in bacteria pPBUFCBac-LvRsn1 (SEQ ID NO: 5) containing a gene (SEQ ID NO: 3) encoding a modified version of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein. The following are represented on the map: the T7 promoter sequence (T7 promoter); the lacO operator sequence (lac operator); two restriction enzyme sites for the NdeI endonuclease (NdeI) and one for the endonuclease EcoRI (EcoRI); a polyhistidine tag coding sequence (6xHis); a coding sequence for a cleavage site by the TEV protease (TEV site); a coding sequence for the Lv-ranaspumin-1 protein with codon frequency optimized for expression in E. coli (Lv-Rsn-1); a restriction enzyme site for the XhoI endonuclease; a T7 transcription termination sequence (T7 terminator); a promoter (AmpR promoter) and coding (AmpR) sequence for a marker gene for ampicillin resistance; the origin of replication sequence (ori) of the plasmid pBR322; the basis of mobilization the coding sequence for the primer repressor (rop) protein; and the promoter (lacI promoter) and coding (lacI) sequence for the lacI regulatory protein gene. [Figure 2]Figure 2 shows a vector map for expression in yeast pPBUFCYea-LvRsn1 (SEQ ID NO: 9) containing a modified gene (SEQ ID NO: 7) of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein. The following are represented on the map: the AOX1 promoter sequence (AOX1 promoter); a restriction enzyme site for the endonuclease SacI (SacI); a coding sequence for the alpha secretion factor (alpha secretion factor signal); a restriction enzyme site for the PstI endonuclease (PstI); the Lv-ranaspumin-1 protein coding sequence with codon frequency optimized for expression in K. phaffii (Lv-Rsn-1); a coding sequence for a cleavage site by the TEV protease (TEV). V site); restriction enzyme site for NotI endonuclease (NotI); c-Myc tag coding sequence (Myc); polyhistidine tag coding sequence (6xHis); transcription termination sequence AOX1 (AOX1 terminator); TEF1 promoter sequence (TEF1 promoter); EM7 promoter sequence (EM7 promoter); coding sequence for a marker gene for zeocin resistance (BleoR); CYC1 transcription termination sequence (CYC1 terminator); and pUC plasmid replication origin sequence (ori). [Figure 3] Figure 3 shows a photograph of protein electrophoresis in a polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE) performed to assess the presence of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein after resolubilization of inclusion bodies produced by the genetically engineered bacteria and purification on a nickel column. The "Post-Wash" sample refers to the insoluble fraction containing a buffer containing Triton-X-100 detergent and urea. The "Wash" sample refers to the wash of the unretained peak, and "MM" refers to the molecular weight marker. The arrow indicates the band corresponding to the Lv-Rsn-1 surfactant protein. [Figure 4] FIG. 4 shows a photograph of a water / kerosene emulsification assay using 0.1 mg / mL of a modified version of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein produced in E. coli. [Figure 5] Figure 5 shows a graph of the surfactant effect on water / air surface tension (unit: mN / m) promoted by a modified version of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in E. coli bacteria at different concentrations (unit: ppm). [Figure 6] Figure 6 shows a photograph of an oil dispersion assay in seawater with 1.0 mg / L of a modified version of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in bacteria (1.0 mg / L of bovine serum albumin protein - BSA was used as a reference). [Figure 7] Figure 7 shows a photograph of a wettability reversal assay of calcite powder impregnated with cyclohexanepentanoic acid by 50 mg / L of a modified form of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in bacteria. Seawater was used as a negative control, and 25 mg / L of Arquad C-50 cationic surfactant was used as a positive control. [Figure 8] Figure 8 shows photographs of tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) to assess the presence of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein in the supernatant and foam of a culture of genetically modified K. phaphii in a 5-liter bioreactor after 24, 48, 72, and 96 hours of addition of 0.5% (v / v) methanol. MM = molecular marker; C+ = the version of the Lv-Rsn-1 protein produced by E. coli. The red arrow indicates the band corresponding to one of the modified Lv-Rsn-1 protein isoforms produced by K. phaphii. [Figure 9] Figure 9 shows photographs of a water / kerosene emulsification assay using different concentrations of a modified version of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein produced in K. phagocytosis. The negative control consisted of BMMH medium at pH 6.0. [Figure 10]Figure 10 shows a graph of the surfactant effect on water / air surface tension (unit: mN / m) promoted by a modified version of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in K. phaphii yeast at different concentrations (unit: mg / mL). [Figure 11] Figure 11 shows a photograph of an inverse wettability assay of calcite powder impregnated with cyclohexanepentanoic acid by 50 mg / L of a modified form of one of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in bacteria. Seawater was used as a negative control, and 25 mg / L of the anionic surfactant sodium dodecyl sulfate (SDS) was used as a positive control.

[0028] A brief description of the biological sequence listing SEQ ID NO: 1 is the predicted amino acid sequence of one of the Lv-Rsn-1 surfactant protein isoforms present in the nest foam of the Northeastern peppered frog (Raptodactylus bastus). SEQ ID NO:2 is the nucleotide sequence resulting from the back translation of SEQ ID NO:1. SEQ ID NO:3 is the nucleotide sequence obtained from SEQ ID NO:2 after having codon usage optimized for expression in bacteria, using E. coli as the reference species. SEQ ID NO:4 is a nucleotide sequence comprising SEQ ID NO:3 with the addition of a restriction enzyme site for an endonuclease, a coding sequence for a polyhistidine tag, and a TEV protease cleavage site coding sequence. SEQ ID NO:5 is the nucleotide sequence of a vector for expression in bacteria, designated pPBUFCBac-LvRsn1, which contains an expression cassette comprising SEQ ID NO:3. SEQ ID NO:6 is the amino acid sequence of a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein encoded by SEQ ID NO:4. SEQ ID NO:7 is the nucleotide sequence obtained from SEQ ID NO:2 after having codon usage optimized for expression in yeast, using Komagataella phagocytosis (Pichia pastoris) as the reference species. SEQ ID NO: 8 is a nucleotide sequence comprising SEQ ID NO: 7 to which is added a coding sequence for secretory factor α, a coding sequence for a TEV protease cleavage site, a coding sequence for a Myc tag, and a coding sequence for a polyhistidine tag. SEQ ID NO:9 is the nucleotide sequence of a vector for expression in yeast, designated pPBUFCYea-LvRsn1, which contains an expression cassette comprising SEQ ID NO:7. SEQ ID NO:10 is the amino acid sequence of a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein encoded by SEQ ID NO:8. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention relates to the construction of two genetically engineered microorganisms, one bacterial and the other yeast, that heterologously produce modified forms of one of the predicted isoforms of the surfactant protein Lv-Rsn-1. Additionally, the present invention relates to two modified forms of Lv-Rsn-1, two synthetic genes encoding each of these modified forms of Lv-Rsn-1, two expression cassettes each containing the synthetic genes encoding these modified forms of Lv-Rsn-1, and two expression vectors each containing one of these synthetic genes encoding these modified forms of Lv-Rsn-1.

[0030] The amino acid sequence used in this study (SEQ ID NO: 1) corresponds to the predicted sequence of the Lv-Rsn-1 protein isoform based on analysis of protein extracts from foamy Raptodactylus bathus nests by polyacrylamide gel electrophoresis and peptide fragment sequencing. The predicted sequence of this isoform of the Lv-Rsn-1 surfactant protein, with 216 amino acids and a molecular weight of 23.3 kDa, was reverse-translated using the Reverse Translation tool of the Sequence Manipulation Suite (http: / / www.bioinformatics.org / sms2 / rev_trans.html) to obtain the nucleotide sequence encoding the amino acid sequence (SEQ ID NO: 2).

[0031] The nucleotide sequences thus obtained were then optimized for codon usage using the Codon Optimization Tool from Integrated DNA Technologies (http: / / www.idtdna.com) using Escherichia coli as the reference species for expression in bacteria (SEQ ID NO: 3) and Komagataella phaphi (Pichia pastoris) as the reference species for expression in yeast (SEQ ID NO: 7).

[0032] Using a nucleotide sequence (SEQ ID NO: 3) optimized for expression in bacteria, a gene was designed with additional nucleotides downstream and upstream of SEQ ID NO: 3 consisting of sites for an endonuclease (to facilitate insertion of the nucleotide into an expression vector) and sequences encoding additional amino acid sequences (to facilitate purification of the expressed protein).

[0033] This gene was artificially synthesized, and both the synthetic gene and a vector for expression in bacteria were digested with two endonucleases (ensuring that the gene was inserted in the correct orientation relative to the promoter in the expression cassette) and then ligated with DNA ligase. The resulting expression vector contains an expression cassette with the synthetic gene operably linked to a promoter active in bacteria, and this expression cassette can be removed from this vector and inserted into another vector for expression in bacteria by digestion with endonucleases and ligation with DNA ligase.

[0034] A bacterial strain was genetically transformed with an expression vector containing a synthetic gene encoding a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein. The genetically modified bacteria were grown in an appropriate culture medium for their replication and expression of this version of Lv-Rsn-1.

[0035] For yeast, a nucleotide sequence optimized for expression in yeast (SEQ ID NO: 7) was used to design a gene with additional nucleotides downstream and upstream of SEQ ID NO: 3 consisting of sites for an endonuclease (to facilitate insertion of the nucleotide into an expression vector) and sequences encoding additional amino acid sequences (to facilitate purification of the expressed protein).

[0036] This gene was artificially synthesized, and both the synthetic gene and a vector for expression in yeast were digested with two endonucleases (ensuring that the gene was inserted in the correct orientation relative to the promoter in the expression cassette) and then ligated with DNA ligase. The resulting expression vector contains an expression cassette with the synthetic gene operably linked to a promoter active in bacteria, and this expression cassette can be removed from this vector and inserted into another vector for expression in yeast by digestion with endonucleases and ligation with DNA ligase.

[0037] Yeast strains were genetically transformed with an expression vector containing a synthetic gene encoding a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein. The genetically modified yeast was grown in an appropriate culture medium for its replication and expression of this version of Lv-Rsn-1. [Example]

[0038] The present invention may be better understood by the following examples: It should be noted that the present invention is not limited to the examples mentioned, but can be used in all the applications described or in any other equivalent variants.

[0039] Example 1: Obtaining a vector for the production of Lv-Rsn-1 in bacteria Nucleotides were added to a nucleotide sequence (SEQ ID NO: 3) encoding the amino acid sequence of an isoform of the Lv-Rsn-1 surfactant protein, optimized for bacterial expression, to obtain a synthetic gene (SEQ ID NO: 4) having the following composition, when read from 5' to 3': (1) a restriction enzyme site sequence for EcoRI endonuclease with a thymine added at the 3' end (5'-GAATTCT-3'); (2) a sequence encoding a TEV protease cleavage site (ENLYFQGS) (5'-GAATTCT-3'); '-GAAAACTTGTATTTCCAGGGCAGC-3'); (3) restriction enzyme site sequence for NdeI endonuclease (5'-CATATG-3'); (4) start codon (5'-ATG-3'); (5) sequence encoding the amino acid sequence for the predicted isoform of Lv-Rsn-1 surfactant protein with codon frequency optimized for expression in bacteria (SEQ ID NO: 3); (6) stop codon (5'-TAA-3'); and (7) restriction enzyme site sequence for XhoI endonuclease (5'-CTCGAG-3').

[0040] This synthetic gene (SEQ ID NO: 4) and a vector for expression in bacteria were both digested with EcoRI and XhoI endonucleases and then ligated with DNA ligase. The final sequence of the expression vector, designated pPBUFCBac-LvRsn1 (SEQ ID NO: 5), is shown in Figure 2.

[0041] The modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein, encoded by the gene present in the expression vector pPBUFCYea-LvRsn1 (SEQ ID NO: 5), has 236 amino acids and a molecular weight of 25.8 kDa (SEQ ID NO: 6), and differs from the predicted isoform by the addition of a 20-amino acid sequence at its N-terminal end, which contains a polyhistidine tail (HHHHHH) and a cleavage site for TEV protease (ENLYFQG). When this modified version of the Lv-Rsn-1 surfactant protein isoform is digested by TEV protease, it differs from the predicted isoform by the presence of five amino acids (GSHMM) at its N-terminal end.

[0042] Example 2: Obtaining genetically modified bacteria An E. coli bacterial strain was genetically transformed with the pPBUFCBac-LvRsn1 vector (SEQ ID NO: 5) by electroporation. The strain used was derived from E. coli K-12, which: (1) is auxotrophic for the amino acid leucine and sensitive to the antibiotic kanamycin, (2) harbors mutations in the glutathione reductase (gor) and thioredoxin reductase (trxB) genes, and (3) harbors genes for tetracycline antibiotic resistance.

[0043] E. coli cells were washed three times with 10% (m / v) glycerol solution to make them electrocompetent, and then 1.0 μL of this solution was added with the pPBUFCBac-LvRsn1 vector (50 μg / μL). The cell mixture with the vector was kept on ice for 1 minute, then transferred to a 0.2 cm electroporation cuvette and subjected to a 2.5 kV shock. After electroporation, 960 μL of SOC medium (20 g / L tryptone, 5 g / L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added, and the mixture was incubated at 150 rpm and 37°C for 1 hour. After the incubation period, 50 μL of the culture was inoculated into a Petri dish containing LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar).

[0044] The resulting colonies were then cultured in LB medium supplemented with 12.5 μg / mL tetracycline (to select only cells of this strain and prevent the growth of contaminating bacteria) and 100 μg / mL ampicillin (to select only transformed cells and prevent the growth of non-transformed cells).

[0045] The genetic transformation of the resulting colonies with the expression vector pPBUFCBac-LvRsn1 was evaluated by extracting DNA from these colonies, digesting the extracted DNA with the restriction enzymes EcoRI and XhoI, and electrophoresing it on a 1% (m / v) agarose gel to confirm the presence of the expected 692-bp fragment. The genetic transformation of E. coli with the expression vector pPBUFCBac-LvRsn1 was confirmed by the replication and stable inheritance of pPBUFCBac-LvRsn1 over several colonies and generations.

[0046] Example 3: Production of Lv-Rsn-1 by genetically modified bacteria E. coli cells transformed with the pPBUFCBac-LvRsn1 vector were grown in LB medium supplemented with isopropyl-β-D-1-thiogalactopyranoside (IPTG), a structural analog of allolactose that interacts with the lacI protein, displacing it from the lacO operator and thus inducing expression of the gene encoding a modified version of the Lv-Rsn-1 surfactant protein isoform.

[0047] An isolated colony was transferred to 10 mL of LB broth supplemented with two antibiotics (12.5 μg / mL tetracycline and 100 μg / mL ampicillin) and then incubated at 250 rpm and 37°C for 16 hours to obtain a pre-inoculum of genetically transformed cells. A 1 mL volume of the culture was then transferred to a flask containing 100 mL of LB medium supplemented with 100 μg / mL ampicillin and incubated at 37°C and 250 rpm until the cells reached the logarithmic phase of growth (optical density at 600 nm of 0.5-0.7). The flask was left at room temperature for 15 minutes. IPTG was then added to a final concentration of 0.5 mM, and the culture was incubated at 200 rpm and 37°C for 3 hours. After this period, the culture was centrifuged at 7,800 g for 13 min at 4°C, the supernatant was discarded, and the pellet was washed with 0.1 M NaCl and centrifuged at 7,800 g for 1 h at 4°C.

[0048] To purify a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein, expression of which is induced by IPTG, cells were resuspended in 5 mL of resuspension buffer (50 mM Tris-HCl pH 8.0 and 150 mM NaCl) and sonicated in an ice bath for 5 min at 40% amplitude for three cycles. After sonication, the cells were centrifuged at 15,000 g for 45 min at 4°C to separate the soluble (supernatant) and insoluble (precipitate) fractions.

[0049] The predicted modified isoforms of Lv-Rsn-1 surfactant protein were purified from the insoluble fraction (the precipitate containing inclusion bodies) using a resolubilization and renaturation protocol. The insoluble fraction was resuspended in wash buffer (100 mM Tris-HCl pH 8.0, 5 mM EDTA, 5 mM DTT, 2 M urea, and 2% Triton X-100) and centrifuged at 22,000 g for 30 min at 4°C. This preparation was analyzed by 15% SDS-PAGE to confirm whether two washes with a buffer containing Triton X-100 detergent and urea were sufficient to extract most of the predicted modified isoforms of Lv-Rsn-1 surfactant protein from the inclusion bodies.

[0050] Purification of the modified predicted isoform of the Lv-Rsn-1 surfactant protein from the supernatant of the Triton X-100 and urea wash was performed by dialysis against ultrapure water followed by application to a nickel column containing silica resin pre-equilibrated with 50 mM Tris-HCl pH 8.0 and 100 mM NaCl. Lv-Rsn-1 was eluted with an elution buffer equivalent to 50 mM Tris-HCl pH 8.0, 500 mM NaCl, and 250 mM imidazole.

[0051] Analysis by SDS-PAGE demonstrated that the modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein was indeed expressed in E. coli. Figure 3 shows the results of electrophoresis of the protein extract after resolubilization of inclusion bodies and a nickel column purification process (due to interaction with the polyhistidine tail present in the modified version of Lv-Rsn-1). "Post-wash" refers to the insoluble fraction using a buffer containing Triton-X-100 detergent and urea, "wash" refers to the washing of the unretained peak, and "MM" refers to the molecular tag. The arrow indicates the band corresponding to Lv-Rsn-1.

[0052] Cultivation of bacteria genetically transformed with the pPBUFCBac-LvRsn1 vector (SEQ ID NO: 5) demonstrated that this vector was stably inherited over several generations of cells, which replicated the vector and, in the presence of IPTG, began to express a gene encoding a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein, which was concentrated in inclusion bodies.

[0053] From the above disclosure, it is clear that the present invention technically enables the heterologous production in bacteria of a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein and represents a solution to the current state of the art, since it allows the production of this surfactant protein of animal origin without the need to collect and extract foam from nests of Northeastern peppered frogs (Raptodactylus bastus) and all the costs and environmental impacts that result from this collection and extraction.

[0054] Example 4: Activity of bacterially produced Lv-Rsn-1 Modified versions of predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in genetically transformed Escherichia coli cells were purified and subjected to various assays to evaluate their emulsification, water / air surface tension reduction activity, oil dispersion, and wettability reversal.

[0055] The emulsification effect of modified versions of the predicted isoform of the Lv-Rsn-1 surfactant protein expressed in E. coli at concentrations of 0.01 to 10.0 mg / mL on a water / kerosene mixture is shown in Figure 4.

[0056] Reduction of water / air surface tension by modified predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in E. coli. Lv-Rsn-1 at a concentration of 0.15 mg / mL reduces the surface tension of water from 72 mN / m to 39 mN / m, as shown in Figure 5.

[0057] The dispersal of an oil spill in seawater by a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein expressed in E. coli at a concentration of 1.0 mg / L is shown in Figure 6. The same Figure 6 also shows the absence of dispersant activity in the oil spill before the addition of Lv-Rsn-1 and in the bovine serum albumin (BSA) protein used as a reference, also at a concentration of 1 mg / L.

[0058] The reversal of wettability of calcite powder (CaCO3) impregnated with cyclohexanepentanoic acid by a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein expressed in E. coli at a concentration of 50 mg / L is shown in Figure 7. The reversal of wettability is measured by the presence of a greater amount of calcite powder at the bottom of the test tube compared to the negative control (seawater).

[0059] From the above disclosure, it is clear that modified versions of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in E. coli act as surfactants, emulsifiers, dispersants, and wettability reversal agents and can be applied to a variety of purposes related to these activities.

[0060] Example 5: Obtaining a vector for the production of Lv-Rsn-1 in yeast Nucleotides were added to the nucleotide sequence (SEQ ID NO: 7) optimized for expression in yeast, resulting in a synthetic gene (SEQ ID NO: 8) with the following composition, when read in the 5' to 3' direction: (1) a restriction enzyme site for PstI endonuclease (5'-CTGCAG-3'); (2) two nucleotides (5'-GN-3') to place the Lv-Rsn-1 coding sequence in the same translation frame as the α-secretion factor; (3) a sequence encoding the Lv-Rsn-1 surfactant protein with codon frequency optimized for yeast expression (SEQ ID NO: 7); (4) a sequence encoding a TEV protease cleavage site (ENLYFQG) (5'-GAGAACCTTTACTTTCAGGGA-3'); and (5) a restriction enzyme site for NotI endonuclease (5'-GCGGCCCC-3').

[0061] This synthetic gene (SEQ ID NO:79) and a vector for bacterial expression were both digested with PstI and NotI endonucleases and then ligated with DNA ligase. The final sequence of the expression vector, designated pPBUFCYea-LvRsn1 (SEQ ID NO:9), is shown in Figure 3.

[0062] The modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein encoded by the gene present in the expression vector pPBUFCYea-LvRsn1 (SEQ ID NO: 9) has 341 amino acids and a molecular weight of 36.7 kDa (SEQ ID NO: 10) and differs from the predicted isoform by the addition of secretory factor alpha plus two amino acids at its N-terminal end, a TEV protease cleavage site plus six amino acids at its C-terminal end, a c-Myc tag plus five amino acids, and a polyhistidine tail. When this modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein is digested with TEV protease, it differs from the predicted isoform by the presence of 91 amino acids at its N-terminal end and six amino acids at its C-terminal end.

[0063] Example 6: Obtaining genetically modified yeast A yeast strain of the species Komagataella phaphii was genetically transformed with the vector pPBUFCYea-LvRsn1 (SEQ ID NO: 9) by electroporation. The strain used was Komagataella phaphii GS115 / ATCC 20864, ​​which lacks the histidine amino acid (HIS4). - ), and the active genes for AOX1 and AOX2 (Mut + ) and can therefore use methanol as a nutrient.

[0064] K. phaffii cells were prepared by inoculating 5.0 mL of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) with the isolated colony and incubating them at 250 rpm and 30°C for 16 hours. Next, 250 μL of the culture was transferred to 500 mL of YPD broth in a 2.0 L container and incubated at 250 rpm and 30°C for approximately 16 hours until an optical density at 600 nm of 1.3–1.6 was obtained. The culture was then centrifuged at 1,500 g for 5 minutes at 4°C. The supernatant was discarded, and the cell-containing pellet was gently resuspended in 500 mL of ice-cold ultrapure water. The cells were then centrifuged again, and the pellet was resuspended in 250 mL of ice-cold ultrapure water. The cells were centrifuged, and the pellet was resuspended in 20 mL of ice-cold 1.0 M D-sorbitol. Finally, the cells were centrifuged and resuspended in 1.0 ml of ice-cold 1.0 M D-sorbitol, thereby obtaining electrocompetent cells, which were used for gene transformation.

[0065] In preparation for genetic transformation, the vector pPBUFCYea-LvRsn1 (SEQ ID NO: 9) was linearized with SacI endonuclease, and the linearized vector was then purified using a potassium acetate precipitation protocol. To this end, 3.0 M potassium acetate, pH 5.5, was added to a final concentration of 0.3 M, followed by two volumes of 100% ethanol and incubation at -20°C for 30 minutes. The mixture was then centrifuged at 12,000 g for 10 minutes at 4°C, and the precipitate was washed with 70% ethanol and centrifuged again. The plasmid was dried at 37°C for 10 minutes, after which it was resuspended in ultrapure water, quantified by spectrophotometry, and stored in a freezer at -20°C.

[0066] For genetic transformation, a 90 μL volume of solution containing electrocompetent K. phaffii cells was mixed with 5–10 μg of linearized pPBUFCYea-LvRsn1 vector DNA (SEQ ID NO: 9) in a 600 μL microtube, and the final volume was transferred to a 0.2 cm electroporation cuvette pre-chilled on ice. The cuvette containing the cells and linearized vector was incubated on ice for 5 min and then subjected to a 2.5 kV pulse in an electroporator. Immediately, 1.0 mL of ice-cold 1.0 M D-sorbitol was added to the cuvette, and the contents were then transferred to a 15 mL tube. The tube was incubated at 30 °C for 2 h without agitation. A 200 μL volume of the contents of the tube was then inoculated onto a plate containing YDPS agar medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 182.2 g / L D-sorbitol, and 15 g / L agar) supplemented with 100 μg / mL Zeocin® antibiotic. The plate was incubated in an oven at 30°C for 3 to 10 days until colonies appeared. Transformed clones were cultured in YPD medium, supplemented with 20% (v / v) glycerol, and stored at -80°C. The transformed clones were sequentially subjected to increasing concentrations of Zeocin® (500 μg / mL, 1000 μg / mL, and 2000 μg / mL) in YPD agar medium to select for transformants containing multiple copies of the pPBUFCYea-LvRsn1 vector (SEQ ID NO: 9).

[0067] To confirm the success of genetic transformation, genomic DNA was extracted from colonies of cells resistant to 2,000 μg / mL Zeocin® using a cetyltrimethylammonium bromide (CTAB) protocol. At the end of the extraction, the resulting DNA was eluted in 50 μL of 10 mM Tris-HCl pH 8.0 supplemented with 20 μg / μL ribonuclease. Samples were quantified and evaluated by absorbance measurements at 230 nm, 260 nm, and 280 nm in a spectrophotometer and then stored in a freezer at -20°C. Detection of the presence of a gene encoding a modified form of the Lv-Rsn-1 surfactant protein in transformed clones was performed using polymerase chain reaction (PCR) technology with primers AOX1-fwd (5'-GACTGGTTCCAATTGACAAGC-3') and AOX1-rev (5'-GCAAATGGCATTCTGACATCC-3'). PCR reactions were performed in a final volume of 25 μL containing 50 ng of genomic DNA, 20 mM Tris-HCl (pH 8.4), 3.0 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of each primer, and 1.0 unit of Taq DNA polymerase. The reactions were performed in a thermocycler programmed for an initial denaturation step (94°C for 5 min), followed by 30 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1.5 min. The final cycle was followed by a final extension at 72°C for 10 min. PCR products were visualized by 1.0% (m / v) agarose gel electrophoresis. The gel was stained with SYBR Green and contained a 1 kb marker as a reference. Genetic transformation of K. phaphii with the expression vector pPBUFCYea-LvRsn1 (SEQ ID NO: 9) and stable inheritance of this transformation across generations were confirmed.

[0068] Example 7: Confirmation of Lv-Rsn-1 production in yeast K. phaffii cells transformed with the pPBUFCYea-LvRsn1 vector (SEQ ID NO: 9) were cultured on plates containing YPD agar medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar) supplemented with 100 μg / mL of the antibiotic Zeocin® at 30° C. For this inoculum precursor, a pure colony was cultured in 200 mL of BMGH medium (13.4 g / L yeast extract with ammonium sulfate and no amino acids, 10 mL / L glycerol, 100 mL / L phosphate buffer 1.0 M pH 6.0, 2.0 mL / L 0.02% biotin, 10 mL / L 0.4% histidine, and 100 μg / L Zeocin®) at 250 rpm for 16 hours at 29° C. until an optical density at 600 nm of approximately 2.0 was achieved. The culture was then centrifuged at 3,500 rpm for 5 min, and the pellet was washed twice with BMGH medium (13.4 g / L yeast extract containing ammonium sulfate and no amino acids, 100 mL / L 1.0 M phosphate buffer pH 6.0, 2.0 mL / L 0.02% biotin, 10 mL / L 0.4% histidine, and 0.5% v / v methanol) to remove glycerol present in the BMGH medium used for the inoculation precursor.

[0069] After this step, the cells were resuspended and inoculated into a 500 mL flask containing 50 mL of BMMH to an optical density at 600 nm of 1.0. The culture was incubated at 29°C with agitation at 250 rpm for 96 hours, and the optical density at 600 nm was monitored every 24 hours, at which time the culture was supplemented with 0.5% (v / v) methanol. At the end of 96 hours, the culture was centrifuged at 3,000 g for 5 minutes.

[0070] Expression of the predicted Lv-Rsn-1 surfactant protein isoform by K. pfaffii was monitored by Tricine-SDS-PAGE. Before loading onto the electrophoresis gel, a sample from the supernatant was precipitated with acetone. A sample (0.3 mg) from the precipitate was mixed with 0.3 mL of sample buffer (2 mL SDS, 1.2 mL glycerol, 0.2 mL β-mercaptoethanol, 1.0 mg Coomassie Brilliant Blue G-250, and 0.5 mL Tris-HCl pH 6.8 for a final volume of 10 mL) and incubated at 100°C for 10 min. The results demonstrated that the predicted Lv-Rsn-1 surfactant protein isoform was expressed by K. pfaffii and maintained this characteristic over generations.

[0071] Subsequently, the same K. phaffii cells transformed with the pPBUFCYea-LvRsn1 vector were cultured in a 5.0 L bioreactor. For the inoculum precursor, pure colonies were cultured in six 2.0 L flasks containing 200 mL of BMGH medium supplemented with 100 μg / mL ampicillin at 250 rpm and 29°C for 16 hours until an optical density at 600 nm of approximately 2.0 was reached. These cultures were pooled, and the final volume was centrifuged at 3,000 rpm for 5 minutes. The pellet was washed twice with BMMH expression medium to remove residual glycerol. The cells were then resuspended in 100 mL of BMMH medium, and this volume was transferred to the bioreactor, resulting in an optical density at 600 nm of approximately 1.5.

[0072] The culture was performed in batch mode at 29°C, 500 rpm, and 1 vvm aeration using a maximum volume of 3.0 L of BMMH expression medium supplemented with 100 μg / mL Zeocin®. The initial optical density at 600 nm was recorded, and the culture was fed with 0.5% (v / v) methanol. This procedure was repeated every 24 hours for up to 96 hours. Every 24 hours, the overflow foam volume was collected in a Mariotte flask. This Mariotte flask was connected to the bioreactor to collect the overflow foam from the bath. The liquefied foam was centrifuged at 9,000 rpm and 4°C for 10 minutes. The foam protein concentration was quantified, and the protein profile was analyzed by Tricine-SDS-PAGE 15%. The results, as shown in Figure 8, indicated that the modified form of Lv-Rsn-1 produced by K. phaphii in the 5.0 L bioreactor was concentrated in the foam.

[0073] Example 8: Activity of Lv-Rsn-1 produced by yeast Modified versions of predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in genetically transformed K. phaffii cells cultured in a 5.0 L bioreactor were purified and subjected to various assays to evaluate their emulsifying activity, water / air surface tension reduction, and wettability reversal.

[0074] The emulsifying effect of modified versions of the predicted isoform of the Lv-Rsn-1 surfactant protein expressed in K. phaphii at concentrations of 0.03–0.24 mg / mL on water / kerosene mixtures is shown in Figure 9 .

[0075] Reduction of water / air surface tension by modified versions of the predicted isoform of the Lv-Rsn-1 surfactant protein expressed in K. phaphii. Lv-Rsn-1 at a concentration of 0.24 mg / mL reduces the surface tension of water from 72 mN / m to 35 mN / m, as shown in Figure 10.

[0076] Figure 11 shows the reversal of wettability of calcite powder (CaCO3) impregnated with cyclohexanepentanoic acid by a modified version of the predicted isoform of the Lv-Rsn-1 surfactant protein expressed in K. phaphii at a concentration of 50 mg / L. The reversal of wettability is measured by the presence of a greater amount of calcite powder at the bottom of the test tube compared to the negative control (seawater). As a positive control, sodium dodecyl sulfate (SDS) was used at a concentration of 2,500 mg / L.

[0077] From the above disclosure, it is clear that modified versions of the predicted isoforms of the Lv-Rsn-1 surfactant protein expressed in K. phaphii act as surfactants, emulsifiers, dispersants, and wettability reversal agents and can be applied for a variety of purposes related to these activities.

[0078] Those skilled in the art will understand that numerous variations are possible within the scope of protection of the present application, which reinforces the fact that the present invention is not limited to the specific configurations and embodiments described above.

[0079] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 18

Table 19

Claims

1. A polynucleotide encoding the predicted sequence of an isoform of surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1), and consisting of SEQ ID NO:

2.

2. A polynucleotide encoding the predicted sequence of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1), having a codon frequency optimized for expression in bacteria, and consisting of SEQ ID NO:

3.

3. A polynucleotide encoding the predicted sequence of an isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1), having a codon frequency optimized for expression in yeast, and consisting of SEQ ID NO:

7.

4. A polypeptide which is a modified isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and consists of sequence number 6.

5. A polypeptide which is a modified isoform of the surfactant protein Lv-ranaspumin-1 (Lv-Rsn-1) and consists of SEQ ID NO:

10.

6. An expression cassette comprising the polynucleotide of claim 2 operably linked to a promoter directing expression in bacteria.

7. An expression cassette comprising the polynucleotide of claim 3 operably linked to a promoter directing expression in a fungus.

8. An expression vector comprising the expression cassette of claim 6.

9. An expression and transformation vector comprising the expression cassette of claim 7.

10. A genetically modified microorganism, characterized in that it is a bacterium that produces a protein, wherein the coding sequence of said protein comprises the expression cassette of claim 6.

11. A genetically modified microorganism, characterized in that it is a yeast fungus that produces a protein, the coding sequence of which comprises the expression cassette of claim 7.

12. A method for producing a genetically modified organism, comprising providing the bacterium of claim 10, a) transforming a bacterial strain with the expression cassette of claim 6; b) selecting the transformed bacteria; A method comprising:

13. A method for producing a genetically modified organism, comprising providing the yeast according to claim 11, a) transforming a yeast strain with the expression cassette of claim 7; b) selecting the transformed yeast; A method comprising:

14. A composition comprising the polypeptide of claim 4.

15. A composition comprising the polypeptide of claim 5.

16. An advanced oil recovery method using a biological surfactant protein obtained from a genetically modified organism described in claim 10 or 11, characterized in that the genetically modified organism is capable of synthesizing the biological surfactant protein Lv-ranaspumin-1.

17. A method for bioremediation of oil using a biological surfactant protein obtained from a genetically modified organism described in claim 10 or 11, characterized in that the genetically modified organism is capable of synthesizing the biological surfactant protein Lv-ranaspumin-1.

18. A method for cleaning tanks in the oil and gas industry using a biological surfactant protein obtained from a genetically modified organism described in claim 10 or 11, characterized in that the genetically modified organism is capable of synthesizing the biological surfactant protein Lv-ranaspumin-1.

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