BR102018068380A2 - MICRO-ORGANISM WITH SYNTHETIC METABOLIC TRACKS OPTIMIZED FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR - Google Patents

Abstract

microorganism with synthetic metabolic pathways optimized for the production of l-methionine from multiple sources of carbon and sulfur the present invention describes a fermentative process for the production of amino acids, specifically l-methionine, using recombinant bacterial strains from alternative substrate , glycerin water, and reduced sources of sulfur, dimethylsulfoniopropionate. for this process, genetic modifications were carried out, such as insertion and deletion of genes, in microorganisms, preferably, but not exclusively escherichia coli, developing different metabolic pathways. with this, overproductive and excretory bacterial strains of l-methionine were obtained, reaching a higher concentration than with strains with only the deletion of the metj gene. through this technology it is possible to obtain a more sustainable and viable production of l-methionine, with low environmental impact.

Description

MICRO-ORGANISM WITH SYNTHETIC METABOLIC TRACKS OPTIMIZED FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR

FIELD OF THE INVENTION [1] The invention described here establishes a biotechnological process for the production of amino acids, specifically L-methionine, using strains modified preferentially, but not exclusively from Escherichia coli, the same strategy being applied to Corynebacterium glutamicum, Saccharomyces cerevisiae , among others. With the application of molecular biology and genetic engineering techniques, it was possible to produce L-methionine from biofuel residues as a carbon source, together with reduced sulfur sources. Through this process, super-producing and excretory L-methionine bacterial strains were obtained, making the production of the amino acid sustainable and of low environmental impact.

BACKGROUND OF THE INVENTION AND STATUS OF THE TECHNIQUE [2] Fermentative processes are based on the use of microorganisms for the production of different biomolecules of commercial interest. Currently, these processes are being carried out on an industrial scale for the production of amino acids, vitamins, nucleotides, essential oils, organic acids, enzymes, in addition to some biopolymers.

[3] The new techniques developed for genome analysis are responsible for ensuring a greater understanding of microorganisms. Enabling the use of metabolic engineering to carry out mutations in specific genes that act in the production of the metabolite of interest (D'Este et al., 2017). The application of genetic engineering tools avoids the realization of classic, random mutations, which can lead to an unwanted alteration in the microbial genome (Kumar and Gomes, 2005).

[4] Using the genomic information of microorganisms and the tools of genetic engineering it is possible to increase the expression of a given gene. For that different techniques can be used: the increase of copies of the gene in the chromosome; a greater number of copies of the vector; modification in the promoter region; application of a promoter that can be induced by providing a

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I 35 increase in the level of gene expression, either by attenuating or repressing the transcription of repressors (Makrides, 1996).

[5] Methionine is an essential amino acid and is necessary in human and animal nutrition. This amino acid can be found in two forms, L- and Dmethionine, being predominantly in nature in the L- form (Kumar and Gomes, 2005; Willke, 2014; Shim et al., 2016). Methionine has several industrial applications, such as in the food additive, pharmaceutical and cosmetic industry (Shim et al., 2016). In 2013, more than 600,000 tons of methionine were produced for animal feed alone (Willke, 2014).

[6] Methionine can be obtained by different processes, including the chemical process, generating a racemic mixture of its isomers; and the fermentative process, producing L-methionine (Kumar and Gomes, 2005; Willke, 2014; Shim et al., 2016; D'Este et al., 2017). The production of this amino acid chemically is increasingly undesirable, since dangerous chemical compounds, such as acrolein, ammonia and cyanide, are involved in the process. Therefore, more sustainable and less aggressive alternatives to the environment are being developed, including the production of methionine through biotechnological processes (Kumar and Gomes, 2005; Willke, 2014).

[7] The microorganisms most used today for the industrial production of amino acids by fermentative processes are the bacteria Escherichia coli and Corynebacterium glutamicum. Due to the study of metabolic engineering and the application of its tools, these microorganisms are capable of producing several amino acids with good yields (Shim et al., 2016; D'Este et al., 2017). Because they have well-described metabolic pathways, these bacteria are engineered to produce methionine (WO2007012078 A1; WO2007011939 A2; US20080311632; WO2009043372; WO2017118871 A1; EP 3296404 A1; Ferla and Patrick, 2014; Shim et al., 2016).

[8] Methionine biosynthesis from homoserine is carried out in three stages (acylation, sulfurylation and methylation), which are the target of study to ensure a good production of methionine. However, due to greater genetic information of microorganisms, new strategies for the production of methionine are being developed based on enzymes and intermediates alternative to the described route. Some marine bacteria are able to use methanethiol to synthesize

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I 35 methionine by a one-step process starting from O-acetylcheroserine (Kumar and Gomes 2005; Ferla and Patrick, 2014; Willke, 2014).

[9] The main problem related to methionine production is feedback inhibition, in addition to the sulfur source to be used. The process of reducing the sulfur source demands a large amount of energy from the microorganisms, therefore, more reduced sources of sulfur are being studied. Among them are thiosulfate, methanethiol and dimethyl disulfide (DMDS) (Kumar and Gomes, 2005; Bolten et al., 2010; Ferla and Patrick, 2014; Willke, 2014; WO2007011939 A2).

[10] When used, methanethiol presents a good yield of methionine, however, it is a toxic and explosive gas, causing new strategies to be generated for its replacement in the methionine production process. The dimethyl disulfide compound has an unpleasant aroma, similar to rotten cabbage, being toxic in nature due to the presence of sulfur. This compound is responsible for donating sulfur and the methyl radical incorporated during the production of L-methionine. Studies carried out with dimethyl disulfide as a sulfur source using genetically modified microorganisms, such as E.coli and Corynebacterium glutamicum, showed higher yields in the production of methionine, confirming the advantage in the production of the use of reduced sulfur sources (Kim et al., 2006; Bolten et al., 2010; WO2007011939 A2; Liang et al., 2015; Curson etal., 2017).

[11] This patent addresses the flexible use of reduced sulfur sources, including DMDS and dimethylsulfoniopropionate (DMSP). The assimilation of DMSP was possible using a biosynthetic pathway of L-methionine in E. coli and / or C. glutamicum based on genes from marine microorganisms capable of degrading DMSP in methanethiol, this compound being used for the direct production of methionine. DMSP is a tertiary sulfonic compound produced by some species of plants and marine algae, being responsible for promoting osmotic control (Kiene et al., 1999; Yoch, 2002).

[12] Some species of marine bacteria, such as Ruegeria pomeroyi, have been described as capable of degrading DMSP through two different metabolic pathways. The first route is called cleavage, in which the compounds: acrylate and DMS are obtained as by-products, and in the second route, called the demethylation route, by-products such as acetate and methanethiol are generated. Since methanethiol is capable of being used by the same bacteria for the production of L

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I 35 methionine (Reisch et al., 2011). Therefore, the present invention uses DMSP as a source of reduced sulfur that is non-toxic to human health, in addition to being naturally available in the environment and not harmful to it.

[13] The demethylation pathway begins with the conversion of DMSP to 5-methyltetrahydrofolate (THF) and methylmercaptopropionate (MMPA) using the enzyme DMSP demethylase (DmdA). The enzyme MMPA-CoA ligase (DmdB) acts on the substance MMPA, generating as product MMPA-coenzyme A (MMPA-CoA). MMPA-CoA is converted to methylthioacryloyl-coenzyme A (MTA-CoA) by the action of the enzyme MMPACoA dehydrogenase (DmdC). Finally, the formation of methanethiol and acetaldehyde occurs from the conversion of MTA-CoA by the enzyme methylthioacryloyl-CoA hydratase (DmdD) (Reisch et al., 2011). As much as this pathway produces methanethiol, a toxic gas, it is then used by microorganisms for the production of non-hazardous molecules.

[14] The present invention has a sustainable aspect which is the use of glycerinated water, an industrial residue obtained from the production of biodiesel, as an alternative substrate. The most important component of this residue is the glycerol that will be used as a carbon source in the fermentation process. As reported, the production of amino acids, especially L-methionine, requires a large amount of energy and the use of glycerol as a substrate can balance this cost, since glycerol has a high energy value, thus ensuring a more profitable production process. (Kaleta et al., 2013).

[15] Glycerol, also known as glycerin, corresponds to the largest by-product obtained in the biodiesel production process, more specifically in the transesterification of triglycerides. 10% of all biodiesel generated corresponds to the amount of crude glycerol produced. As an industrial by-product, crude glycerol has its composition varied depending on the method and substrate used for the production of biodiesel (Yang etal., 2012; Ondul and Dizge, 2014; Binhayeeding etal., 2017).

[16] Due to the high price of purification of crude glycerol for application in the pharmaceutical and food industry, biodiesel producers are looking for alternatives for disposal and reuse of this by-product. As a result, the use of crude glycerol can make biodiesel production feasible, adding more value to the by-product and decreasing production costs (Yang et al., 2012; Binhayeeding etal., 2017).

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I 35 [17] A strategy to be used to add value to crude glycerol is the biotransformation of this industrial by-product into several biomolecules that have added value. Because it is produced in large quantities, glycerol is an inexpensive substrate and is competitive with sugars for fermentation processes (Silva et al., 2009; Yang et al., 2012). Several microorganisms capable of using glycerol as a carbon source for the production of different biomolecules such as ethanol, organic acids, hydrogen, propionate, among others are described in the literature (Silva et al., 2009; Yang et al., 2012; Ondul and Dizge, 2014).

[18] Still, from an industrial point of view, it is important that strains used for the production of biomolecules are able to metabolize different sources of carbon. Thus, strains of E. coli are more advantageous than strains of C. glutamicum or S. cerevisiae, for example, because they are naturally capable of metabolizing arabinose, xylose, and other molecules as carbon sources, in addition to glucose and glycerol .

[19] In addition, the present invention describes the use of a microorganism capable of metabolizing other carbon sources besides glucose and glycerol. These alternative carbon sources, such as xylose and arabinose, for example, come from residues from the ethanol production industry from corn or sugarcane. Mainly E. coli strains are able to metabolize these sugars and use them as carbon sources, however, the present invention describes a process in which the strain used is able to metabolize several sources of carbon, including the sugars previously mentioned, simultaneously, so that the carbon sources available in the culture medium can be found in pure form, or in a mixture of substrates.

[20] The possibility of a microorganism using different carbon sources is an advantage mainly when industrial waste is used, which comprises a mixture of components, and not just the carbon source in its pure form. However, regardless of the microorganism used, genetic modifications are necessary for it to be able to metabolize different carbon sources simultaneously (Aidelberg et al., 2014, Desai and Rao, 2009).

[21] As described in the literature, the E. coli bacterium has an ABC system for the absorption of the amino acid methionine which is expressed by certain genes: metN, metI and metQ (Kadner, 1977; Merlin et al., 2002; Zhang et al. , 2003). However, the

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The mentioned system is not capable of operating in the direction of efflux of methionine, it is only absorbed by the ABC system. When genetically modifying a microorganism to produce a biomolecule, such as amino acids, it is necessary to consider the efflux system to be used for the excretion of the molecule, ensuring greater efficiency.

[22] Therefore, in the present invention, considering the case of the ABC system, new methionine efflux transporters have been studied and applied, seeking a higher excretion yield of L-methionine in microorganisms such as E. coli. In addition to the efflux system to be considered, the internal consumption of methionine by the microorganism itself can result in a decrease in the amount of methionine to be secreted into the culture medium. One of the strategies applied in order to solve this problem was the attenuation of the metK gene through the development of a metabolic valve that promotes the reduction of the expression of this gene from a certain moment in the fermentation process, so that the methionine produced is not consumed by itself microorganism and can be transported out of the cell with greater efficiency.

[23] The bacterium Corynebacterium glutamicum, presents an efflux transporter for different amino acids such as methionine, L-valine, L-leucine, among others. This transporter is called BrnFE, being expressed by the brnFE operon (Trotschel et al., 2005; Lange et al., 2012). According to Qin et al. (2015), when overexpressing the BrnFE transporter, a greater excretion of L-methionine produced by C. glutamicum was observed, with a concentration of 6.3 g / L obtained in 64 hours of fermentation.

[24] According to a report in the literature, E. coli presents an efflux system homologous to the BrnFE transporter, being expressed by the ygaZH genes, therefore, called the YgaZH transporter (Park et al., 2007). Park et al. (2007) described the YgaZH transporter as specific for the excretion of Lvalina, however, new research in E. coli indicates the use of the same transporter to improve the excretion of L-methionine. European patent application EP No. 3189150 reports the use of recombinant strains that have the E. coli ygaZ and ygaH homologous genes, these genes being overexpressed to ensure greater methionine excretion.

[25] Recent studies have characterized a new system of transporting amino acids out of the cell in the bacterium E. coli, being called yjeH.

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I 35

Carrier overexpression experiments carried out on the mutant strain of

E.coli, who has the deletion of the metJ gene, showed an increase in methionine excretion when performed in shaken flasks (Liu et al., 2015). The use of the yjeH gene is already claimed in United States patent application US No. 20090298135 for some applications. According to the authors, a production of up to 4.8 g / L of L-methionine was obtained when carried out in 2 L fermenters, with high aeration and cell density, using glucose as a carbon source.

[26] Canadian patent application CN No. 105886449 reports a production of L-methionine using recombinant E. coli in shaken flasks of up to 4,083 g / L and in fermenter, with 3 L of working volume, a production of up to 9,752 g / L. For this, the genes metJ, metI, lysA and / or thrB were deleted. And the metA * and yjeH genes were inserted into the bacterial strain.

[27] WO applications 20070777041, 2007012078, 2009043803 and US patents No. 7790424B2 and US9034611B2 report the use of genetic modifications in microorganisms for better yield in the production of Lmethionine, however, they do not describe the application of specific carriers for this, as reported in the present invention. In addition, the applications and patents mentioned above, from companies such as Metabolic Explorer and BASF, report the use of pure and plant-based carbon sources for the fermentation process, such as glucose, dextrose, among others.

[28] By using industrial waste from the production of biodiesel, glycerinated water, in different concentrations, in addition to a possible addition of a vegetable carbon source to accelerate production at the beginning of the process, the invention described here differs from the others cited. In addition, with the genetic modifications described in the present invention, microorganisms became able to consume different sources of carbon at the same time, without a pathway being inhibited by another substrate, thus, the modified microorganisms are multi feedstock, capable of metabolizing simultaneously mainly glucose, glycerol, arabinose, xylose, in addition to other molecules, as carbon sources.

[29] The company Metabolic Explorer holds several applications and patents granted regarding the production of methionine through fermentative processes using microorganisms with genetic modifications, most of which are used as a carbon source, glucose or sucrose (WO2009043803,

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US9034611B2, WO2007077041). The microorganisms used are preferably E.coli and C. glutamicum, with the following changes being made: increased expression of genes such as cysA cysU, cysT, cysM, cysL, cysC, cysE, cysl, cysJ, cysM, cysK, metE, metH , glyA, gvcTHP and metR; the deletion of the metA and metJ genes and the insertion of the metA gene with modification in the feedback response; alteration of pykA, pykF, lpd, serA, serB, serC, glyA, pntA, pntB, udhA, metF, thrA, thrA genes with alteration of feedback, purU and yncA; use of inductive promoters. These changes can be seen in patent applications WO No. 2007077041 and 2009043803 and in US patent No. 9034611B2.

[30] Patent application WO No. 2007012078 filed by BASF cites the modification of the metQ, metK and pepcK genes and the reinsertion of them in the microorganism. In addition to the change in the genes metX, metY, metB, metH, metF, metE, zwf, asK, horn, frpA, pyc, asd, cysE, cysK, cysM, cysZ, cysC, cysG, cysN, cysD and cysH. Thus, a production of up to 25 g / L of L-methionine was obtained in 72 hours of fermentation. The company Evonik, have a patent application WO No. 2002010209 in which the production of L-methionine has been optimized from the modifications in the genes metH, lysC, pgk, pyc, metA, metB, glyA, aecD, metY, thrB, thrC, ddh, aiming at the use of glucose as a carbon source.

[31] In the literature, it is possible to find processes that use glycerol as a substrate for the production of amino acids, including L-methionine. Patent application WO No. 2008002053 describes the use of E. coli with the inactivated galR and / or glpR genes for better production of threonine and methionine starting from glycerol as a substrate. The present invention differs from the process mentioned above due to different genetic changes carried out in E.coli. In addition, the fermentation process proposed in that invention opts for the fed batch strategy, the culture medium used being similar for the growth and production of Lmetionine, and by differentiated amino acid purification method.

[32] Patent application WO No. 2017118871 reports the production of L-methionine from the fermentation process and the recovery of the amino acid through clarification, addition of solvents for precipitation and subsequent separation of the precipitate. The invention described here differs from the process mentioned above by using another method of purifying L-methionine, involving the use of an ion exchange resin.

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I 35 [33] Bacteria of the genera Escherichia and Pantoea were used for the production of amino acids with glycerol present in the culture medium in US patent No. 7811798B2. In order to achieve this, the microorganisms underwent changes in the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase, with which L-phenylalanine, L-tyrosine and L-tryptophan were produced, with L-methionine being mentioned. The present invention is different from the patent cited for producing the amino acid Lmethionine using a bacterium with different genetic modifications as described in detail in the topic Description of the Invention.

[34] Patent application WO No. 2007011939 and US patent No. 9024063B2 describe the use of alternative sources of sulfur such as dimethyl disulfide (DMDS), metanetiol and dimethyl sulfide (DMS) for the production of methionine. In the case of WO No. 2007011939, DMDS was used as a donor of the methyl radical and sulfur for the methionine molecule when produced in C. glutamicum. DMDS, together with changes in microorganisms in the metF, metE and metH genes, enabled greater production of methionine. US patent No. 9024063B2 reports the use of methananiol and / or DMS associated with L-methionine precursors such as O-acetylchomoserin and Oscincinomerserine for the production of this amino acid by means of an enzymatic process. Therefore, the use of DMSP or its derivatives as a source of sulfur and methyl radical in a fermentation process using genetically modified strains, as described in the present invention, of bacteria such as E. coli and C. glutamicum is not reported in the literature.

[35] The present invention highlights that microorganisms will only be viable for the production of L-methionine using DMSP as a sulfur source if they use in their metabolism a biosynthetic pathway obtained through heterologous enzymes from marine bacteria, such as R. pomeroyi. The enzymes metX and metY of the bacterium C. glutamicum must be introduced in the metabolism of E. coli so that they present the system described as functional, being capable of converting homoserine into O-acetylcheroserine, and finally into L-methionine.

[36] The processes described in patent applications BR No. 10 2017 014956 0 and 10 2017 0174450 that precede this invention, are similar and served as the basis for it. Thus, new genetic changes were made and described, such as the development of metabolic valves for the threonine, lysine and attenuation of the metK gene, in addition to multi feedstock strains capable of

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I 35 metabolize different sources of carbon simultaneously, and the use of different sources of sulfur, generating differences between the previous processes and the one described here.

[37] US patent No. 8389250 describes a fermentative process for the production of methionine using modified microorganisms capable of improving cysteine production. The present invention chooses to make genetic modifications related to the serine production pathway in order to optimize it and guarantee higher yields of L-methionine.

[38] European patent application EP No. 3296404 reports the production of Lmethionine using recombinant microorganisms in which the production of at least one by-product, such as isoleucine, phenylalanine and threonine, has been attenuated, preferably abolished. In the case of the invention described here, metabolic valves are developed to control the production of lysine and threonine so as not to compromise cell growth at the beginning of fermentation and subsequently promote a greater production of L-methionine, attenuating the production of lysine and threonine.

DESCRIPTION OF THE INVENTION [39] Various strains of the E. coli bacterium can be used for the production of amino acids. Since the E. coli K-12 MG1655 strain had its genome fully sequenced and annotated (Blattner et al., 1997), in addition to several metabolic models already being available (Orth et al., 2011), this invention will make use of this as the preferred strain, which will be referred to from that point throughout the document as the chassis strain. However, all of the strategies mentioned here extend to C. glutamicum strains, as they have a methionine production pathway and metabolism of carbon sources similar to those of E. coli, respecting the due genes of each microorganism.

[40] E. coli K-12 MG1655 bacteria is defined by the chassis strain according to the genome sequenced by Blattner et al. (1997) and any spontaneous mutations that have been generated until the beginning of the genetic modification steps described below.

[41] In order to increase the metabolic flow for the production of Lmethionine, a series of gene changes were made in the MG1655 strain. The changes were made with the genomic editing system based on

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I 35 lambda recombinase and enzyme Cas9 according to Jiang et al., 2015. The system is based on the use of specific guide RNAs for target genes that will be recognized by the enzyme Cas9, generating a cut in the DNA in the desired region. In the presence of DNA containing the desired gene set to be inserted into the genome, flanked by regions of homology of approximately 400 base pairs for the upstream and downstream regions of the target, the desired gene set is incorporated by recombination in the region where the cut by enzyme Cas9, generating a deletion and at the same time an insertion of a gene cassette. Usually, the target genes chosen for the incorporation of a gene set are characterized by genes not important for the cell metabolism during the fermentation process described in this patent.

[42] In order to decrease the regulation of genes in the methionine production pathway, such as metA, metB, metC, metH, among others, the expression of the metJ gene is attenuated by means of complementary RNA. This strategy is characterized by the incorporation of a DNA sequence that codes for an RNA molecule complementary to the first 30 nucleotides of the metJ mRNA. The region subsequent to the metJ gene was selected as a target for the insertion of this complementary RNA gene, named r_metJ. The resulting strain was named NBmet001.

[43] In order to express a variant of the metA gene (metA *, containing a triple mutation that allows the protein resulting from its expression not to undergo allosteric regulation by L-methionine and S-adenosyl-methionine), the metA gene itself was selected as the target for the incorporation of metA * in strain NBmet001. In this case the expression of the native metA gene was interrupted, and replaced by the metA * gene. The expression of this gene is carried out under the control of the synthetic promoter J23118. The resulting strain was named NBmet002.

[44] In order to reduce the import of L-methionine by cells, the expression of the metI gene is attenuated by means of complementary RNA in the NBmet002 strain. This strategy is characterized by the incorporation of a DNA sequence that codes for an RNA molecule complementary to the first 30 nucleotides of the metI mRNA. The region subsequent to the metI gene was selected as a target for the insertion of this complementary RNA gene, named r_metI. The resulting strain was named NBmet003.

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I 35 [45] In order to decrease the metabolic competition generated by the production of Llisina, the expression of the lysA gene is attenuated by means of complementary RNA in the NBmet003 strain. This strategy is characterized by the incorporation of a DNA sequence that codes for an RNA molecule complementary to the first 30 nucleotides of the lysA mRNA. The region subsequent to the lysA gene was selected as a target for the insertion of this complementary RNA gene, named r_lysA. The resulting strain was named NBmet004.

[46] In order to decrease acetate production in the strain, the expression of the pta gene is attenuated by means of complementary RNA in the NBmet004 strain. This strategy is characterized by the incorporation of a DNA sequence that codes for an RNA molecule complementary to the first 30 nucleotides of the pta mRNA. The region subsequent to the pta gene was selected as a target for the insertion of this complementary RNA gene, named r_pta. The resulting strain was named NBmet005. The same applies to the ackA gene, also involved in the production of acetate. The resulting strain was named NBmet006, and the complementary RNA named r_ackA.

[47] In order to make the methionine-producing strain capable of withstanding more limited oxygenation conditions, the arcA gene encoding the arcA citric acid negative regulator was deleted from the genome of that strain. This gene was used as a target for the integration of other genes that are associated with the production of methionine, such as cysE and aspC. In the case of the cysE gene, a variant of the gene with mutations at positions T167A, G245S and M256I, was used in order to mitigate inhibition by the cysteine product. These modifications were performed on strain NBmet006, generating strain NBmet007. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve using the promoter responsive to oxygen is used to control the expression of a complementary RNA to the genes cysE and aspC, called r_EC. The expression of this RNA is induced in anaerobic conditions, so that the expression of said genes is attenuated, in order to generate a safety mechanism to prevent damage to cells by exacerbated metabolic flow if these conditions are reached during the process. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

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I 35 [48] In order to reduce the metabolic competition generated by the production of Ltreonin, the expression of the thrB gene is attenuated by means of complementary RNA in the NBmet007 strain. This strategy is characterized by the incorporation of a DNA sequence that codes for an RNA molecule complementary to the first 30 nucleotides of thrB mRNA. The region subsequent to the thrB gene was selected as a target for the insertion of this complementary RNA gene, named r_thrB. The resulting strain was named NBmet008.

[49] In order to make the strain capable of metabolizing different carbon sources simultaneously, the ptsG gene is deleted from the NBmet009 strain. This codes for the IIBC component of the specific glucose PTS enzyme, an enzyme involved in the process of assimilation of glucose as a carbon source. This domain, when phosphorylated, binds to the enzyme glycerol kinase, inhibiting its activity, allowing the strain not to regulate the metabolism pathways of carbon sources other than glucose. The deletion of the ptsG gene occurs through the insertion of the metF and metL genes, associated with the final production step of L-methionine, together with metH, and the formation of aspartate, respectively. The resulting strain is named NBmet009. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control the expression of a complementary RNA to the metF and metL genes, called r_FL. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[50] In order to increase the expression of the genes responsible for increasing the metabolic flow for glycerol metabolism, a second copy of the genes glpF (transporter) and glpK (glycerol phosphorylation in glycerol3-phosphate) is integrated into the chromosome of the NBmet009 strain. In addition, completing the gene set in the form of an operon, the metC and cysM genes were also inserted into the genome, aiming to increase the metabolic flow for the formation of methionine and cysteine, respectively. Therefore, the rbsA gene, which codes for a ribopyranose transporter, was selected as a target for the integration of the set glpF, glpK, metC and cysM, generating the strain NBmet010. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control

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I 35 expression of an RNA complementary to the genes glpF, glpK, metC and cysM, called r_FKCM. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[51] In order to increase the metabolic flow for the production of glycine and finally methionine, the gene set containing a second copy of the glyA and metB genes is integrated into the chromosome of the NBmet010 strain. For this, the arsB gene, which codes for an arsenite transporter, was selected as a target for the integration of the glyA, metB set, generating the NBmet011 strain. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control the expression of a complementary RNA to the glyA and metB genes, called r_AB. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[52] In order to increase L-methionine production in the final step of amino acid formation, a second copy of the metH and malY genes are incorporated into the chromosome of the NBmet011 strain. For that, the ldhA gene, associated with the production of lactic acid, was chosen as a target for the incorporation of the gene set metH and malY, generating the strain NBmet012. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control the expression of a complementary RNA to the metH and malY genes, called r_HY. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[53] In order to increase the metabolic flow for the production of L-methionine, the production of L-serine is necessary first. For this, a second copy of the serA, serB and serC genes is incorporated into the chromosome of the NBmet012 strain. For this, the lacZ gene, associated with lactose metabolism, was chosen as a target for the incorporation of the serA, serB and serC gene set, generating the strain

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I 35

NBmet013. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter.

[54] In order to increase the availability of NADPH for the production of methionine, the genes pntA, pntB and sthA, involved in the synthesis of NADH and NADPH, are incorporated into the chromosome of the strain NBmet013. Therefore, the rbsR gene is chosen as a target for the incorporation of the pntA, pntB and sthA gene set, generating the NBmet014 strain. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control the expression of a complementary RNA to the pntA, pntB and sthA genes, called r_ABA. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[55] In order to promote cell division even under conditions of high concentration of cells in the culture, a second copy of the ftsA and ftsZ genes is incorporated into the chromosome of the NBmet014 strain. For that, the region adjacent to the end of the ftsZ gene is chosen as a target for the insertion of the ftsA and ftsZ gene set, generating the NBmet015 strain. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control the expression of an RNA complementary to the ftsA and ftsZ genes, called r_AZ. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[56] In order to control the intrinsic consumption of L-methionine by the strain itself, the metK gene, which codes for the enzyme methionine adenosyltransferase, responsible for consuming L-methionine and forming the product S-adenosyl-methionine, is regulated modified in the strain NBmet015. Therefore, a metabolic valve system containing an RNA complementary to the first 30 nucleotides of the RNA referring to the metK gene is incorporated into the chromosome of the strain. This system is regulated by the pBAD arabinose sensitive promoter. In the event of adding arabinose to the system, the expression of the complementary RNA for metK is activated, so that the expression of the metK gene is attenuated. As it is a regulated system, it is possible to determine the specific moment when the amount of biomass

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The accumulated I 35 is sufficient for cell division to be compromised and methionine production increased due to the attenuated activity of the metK gene. The gene valve mentioned here is determined as r_metK, and this is incorporated into the region adjacent to the termination of the metK gene. The resulting strain is called NBmet016.

[57] Aiming at the formation of oxaloacetate directly from oxaloacetate, the pyc gene of Rhizobium etli is incorporated into the NBmet016 strain. In addition, the methionine transporter yJeH is incorporated into the chromosome of the same strain in operon together with the pyc gene. For this, the arcB gene was selected as a target for the integration of the pyc and yJeH gene set, thus generating the NBmet017 strain. The gene set mentioned above is integrated into the chromosome under the control of the J23118 promoter. In addition, a metabolic valve based on the nar promoter is used to control the expression of a complementary RNA to the pyc and yJeH genes, called r_py. As previously described, the expression of said genes is attenuated in possible conditions of anaerobiosis. The nar promoter and its complementary RNA are allocated downstream of the gene pool described above.

[58] In order to generate an alternative synthetic route for the incorporation of methyl radical and sulfur in a unique way for the production of L-methionine, the metX and metY genes of Corynebacterium glutamicum are incorporated into the genome of strain NBmet017. For that, the region adjacent to the end of the metH gene was chosen as a target for the integration of the metX and metY gene set. This gene pool allows the strain in question to be able to incorporate methyl sulfur groups (CH3-SH) in a unique way to methionine, constituting an alternative route of methionine production. In addition, the gene pool is regulated by a metabolic valve system, where the genes are under the control of the pBAD promoter, regulated by the presence or absence of arabinose in the system. The strain generated after this modification is called NBmet018.

[59] In order to generate an alternative synthetic pathway for the efficient incorporation of sulfate into L-methionine, the dmdA, dmdB, dmdC and dmdD genes of Ruegeria pomeroy are incorporated into the genome of strain NBmet018 under the control of a metabolic valve. This valve is based on the control of the expression of each of these genes also by arabinose, so that the gene set is under the control of the regulated promoter pBAD. This modification allows the strain

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I 35 in question is able to metabolize the substrate dimethylfulphoniopropionate (DMSP) as a source of sulfur and methyl radical, in addition to being able to metabolize dimethyldisulfide (DMDS) in a similar way. The strain generated after this modification is called NBmet019.

[60] Table 1 shows the strains constructed with their modifications.

Table 1 - Genetically modified strains for the production of L-methionine.

NBmet001 r metJ NBmet002 r metJ, metA: metA * NBmet003 r metJ, metA: metA *, r metI NBmet004 r metJ, metA: metA *, r metI, r lysA NBmet005 r metJ, metA: metA *, r metI, r lysA, r pta NBmet006 r metJ, metA: metA *, r metI, r lysA, r pta, r ackA NBmet007 r metJ, metA: metA *, r metI, r lysA, r pta, r ackA, arcA: cysE-aspC-r EC NBmet008 r metJ, metA: metA *, r metI, r lysA, r pta, r ackA, arcA: cysE-aspC-r EC, r thrB NBmet009 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r FL NBmet010 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r FL, rbsA: glpF-glpK-metC-cysM-r FKCM NBmet011 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r FL, rbsA: glpF-glpK-metC-cysM-r FKCM, arsB: glyA-metB -r AB NBmet012 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r HY NBmet013 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r HY, lacZ: serA-serB-serC NBmet014 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r HY, lacZ: serA-serB-serC, rbsR: pntA-pntB-sthA-r ABA NBmet015 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r_HY, lacZ: serA-serB-serC, rbsR: pntA-pntB-sthA-r_ABA, ftsA-ftsZr AZ NBmet016 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r_HY, lacZ: serA-serB-serC, rbsR: pntA-pntB-sthA-r_ABA, ftsA-ftsZr AZ, r metK NBmet017 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r_HY, lacZ: serA-serB-serC, rbsR: pntA-pntB-sthA-r_ABA, ftsA-ftsZr AZ, r metK, arcB: pyc-yJeH-r py

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I 35

NBmet018 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r_HY, lacZ: serA-serB-serC, rbsR: pntA-pntB-sthA-r_ABA, ftsA-ftsZr AZ, r metK, arcB: pyc-yJeH-r py, pBAD metX-metY NBmet019 r_metJ, metA: metA *, r_metI, r_lysA, r_pta, r_ackA, arcA: cysE-aspC, r_thrB, ptsG: metF-metL-r_FL, rbsA: glpF-glpK-metC-cysM-r_FKCM, arsB: glyAB-metB-rB_ , ldhA: metH-malY-r_HY, lacZ: serA-serB-serC, rbsR: pntA-pntB-sthA-r_ABA, ftsA-ftsZr_AZ, r_metK, arcB: pyc-yJeH-r_py, pBAD_metX-metY, dBDdm

[61] The terms increased gene expression, or overexpression of the gene, have similar meaning and are associated with a greater amount of proteins in the intracellular environment.

[62] In this patent, the increase in expression of said genes is related to: 1) incorporation of a second copy of the gene into the chromosome of said strains, 2) control of expression by a constitutive synthetic promoter and 3) inducible metabolic valve to regulate the expression of certain gene sets.

[63] The nucleotide sequences of the described genes have been chemically synthesized by experts in the art. In order to obtain the constructs of the gene sets for later incorporation into the chromosome, the gene groups and the different promoters were assembled using the Biobrick® method and previously described protocols (Sambrook, 1989).

L-METHIONINE PRODUCTION PROCESS [64] The amino acid L-methionine is produced as described in the invention by the bacterium Escherichia coli using residues from the biofuel industry as a carbon source, so the strategies mentioned here are not limited to this organism, and can also be applied to C. glutamicum or S. cerevisiae, with the necessary modifications. Additional substrates to carbon source such as simple, complex carbohydrates, starch, glycerol, fatty acids, among others can be added and combined with the residues. Other nutrients are added to the culture medium, as a source of nitrogen, and yeast extract, urea, corn steeping water, inorganic salts such as NH4NO3, KNO3, NaNO3 and NH4Cl can be used. The salts added to the medium as a sulfur source, can be Na2SO4, MgSO4, H2S, (NH4) 2SO4 or even Na2S2O3, not limited to these, so that other sources of sulfur, such as

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I 35 dimethylsulfoniopropionate (DMSP), dimethyl disulfide (DMDS) and methanethiol can be used in the process. Additional nutrients are required such as phosphorus, magnesium, calcium, molybdenum, boric acid, cobalt, copper, manganese, zinc, iron and B vitamins.

[65] The initial growth of the methionine-producing strain is carried out in a culture medium equal to or similar to the medium used during the large-scale fermentation process. Thus, the strain does not need an adaptation period for each reactor transfer, resulting in productivity gains due to the reduction of the lag phase of bacterial growth.

[66] The fermentation process takes place in an environment with a temperature between 30 ° C and 38 ° C, preferably 37 ° C. The pH of the medium is maintained between 6.8 to 7.2, with constant agitation. Preferably, the initial growth of the organism is carried out in reactors of smaller capacity with mechanical agitation and aeration with sterile air. Ideally, the system is constantly monitored, so that variables such as temperature, pH, dissolved oxygen and agitation are controlled during each stage of the process. Bacterial growth is monitored by optical density, viable cells and dry biomass.

[67] The pre-inoculum and inoculum phases last around 24 hours, since the accumulation of biomass is necessary for the process to be carried out on a large scale later. These steps can operate in batch or powered batch.

[68] Following the inoculations, the fermentation process is carried out in an aerated bioreactor (2 vvm - volume of air per volume of culture medium per minute) and mechanical agitation. The temperature and pH conditions follow the same standards as the conditions mentioned for the inoculum, and for maintaining the pH, the addition of sodium hydroxide solutions or similar bases, or hydrochloric acid or similar solution, is performed to maintain the stable pH.

[69] During the production of L-methionine, a 48- to 72-hour process, the reactor operates in a fed batch system, where the carbon source and other nutrients are added throughout the fermentation so that the microbial culture has constant availability of nutrients used to support their growth, survival and L-methionine production.

[70] In order to ensure the complete sterility of the environment where the fermentation process takes place, the culture medium is sterilized with steam inside the bioreactor itself. Regarding the raw material, it is previously sterilized

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I 35 by rapid heat and later added throughout the process. The concentration of carbon source during fermentation must not reach values higher than 100g / L in order to avoid repression of the growth of the microorganism by excess of substrate. However, in the pre-inoculum and inoculum stages, the carbon source is added in batches.

L-METHIONINE PURIFICATION PROCESS [71] After the production of L-methionine, this amino acid must be recovered from the fermented and purified culture medium in order to constitute a product with a high degree of purity.

[72] Initially, the fermented culture medium is sent to a lung tank where the cells are inactivated. Then, the biomass and other possible residual solids are subjected to a separation process, which is by centrifugation, filtration, microfiltration or ultrafiltration. The soluble fraction containing L-methionine is destined for the purification process while the biomass becomes a residue of the process.

[73] The purification of L-methionine is carried out by means of ion exchange where a cationic type resin is used. In this case, the pH of the solution must be lowered so that the pKa value of methionine is low enough that it is in acidic form, allowing its adsorption to the resin and subsequent elution in solution with alkaline pH. The process is carried out in a maximum of 120 minutes so that the adsorption is complete. For elution, an alkaline ammonium hydroxide solution is used.

[74] In order to make the final product more concentrated, the solution resulting from post-ion exchange column elution is evaporated, ultimately generating a pure and concentrated product available in liquid form.

[75] Methionine is obtained in solid form by spray drying. For this process to be carried out, the post-fermentation medium must contain a minimum amount of solids of 10 to 12%. For this amount to be reached, the medium can be evaporated or eluted from the ion exchange resin with concentrated base solutions.

[76] The process of purifying L-methionine is already described in patent application WO No.2013083934A1. The methodology described includes: a) clarification of the fermented broth and removal of insoluble and soluble solids by flocculation,

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I 35 sedimentation, microfiltration, ultrafiltration, nanofiltration, reverse osmosis and centrifugation; b) optionally, demineralization of the clarified broth in order to remove cations and anions; c) crystallization of the methionine from the broth. In the present invention, the process of separating biomass and insoluble solids is similar to that described in the patent. However, the process of obtaining L-methionine in solid form differs by using spray drying method instead of crystallization.

EXAMPLES

Example 1:

[77] The methionine-producing strains described in Table 1 were grown in Erlenmeyer flasks containing 50 mL of M1 culture medium (Table 2), at 37 ° C, for 48 h. After fermentation, the liquid fraction of the culture was separated from the solid fraction. The quantification of methionine present in the liquid fraction of culture was performed by analyzes of proton H ¹ and carbon C ¹³ by nuclear magnetic resonance (NMR) spectrometry. Table 03 presents the results for different strains that produce methionine.

Table 2 - M1 medium description

Compound Concentration (g L ^-1 ) KH2PO4 3 Na2HPO4-7H2O 12.8 MgSO4 0.24 Yeast extract 5 NaCl 0.5 CaCl2 0.011 NH4Cl 1 (NH4) 2SO4 16 Na2S2O3 1 Glycerol 20 Vitamin B1 0.001 Vitamin B7 0.001 B12 vitamin 0.001

Table 3 - Results obtained in shaken bottles.

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I 35

Strain DOe00 48 h L-methionine g / L 48 h NBmet007 8.3 ± 1 5.1 ± 0.06 NBmet012 7.9 ± 0.5 8.5 ± 0.2 NBmet019 6.9 ± 0.35 15 ± 0.46

Example 2:

[78] The best strain produced in agitated flasks, in this case NBmet019, was evaluated in a 5 L Minifors fermenter. The volume of medium (NB01) used was 1.6 L. The feeds were performed every 12 h using a concentrated solution (500 g L ^-1 of glycerol, ammonium sulphate 16 g L ^-1 , sodium thiosulfate 23.8 g L ^-1 , yeast extract 1 g L ^-1 , vitamins B1, B7 and B7 0.032 g L ^{- 1} , L-lysine 4.3 g L ^-1 ). The pH condition was maintained at 6.8 using ammonium hydroxide. The fermentation was maintained at 30 ° C, with an aeration rate of 3 L.min ^-1 , with agitation governed by cascade control associated with the concentration of dissolved oxygen, with a set point adjusted to 20%. Table 4 shows the results obtained in the described experiment.

Table 4 - Experiment results Example 2.

Strain Biomass Wet g / L (72 h) L-methionine g / L 72 h NBmet019 53 37 ± 0.83

DEFINITIONS [79] It is described here that the aforementioned "glycerol", "glycerin" and "glycerin water" are the carbon source used, varying only the concentration of C3H8O3 in its composition.

[80] The term "methionine" refers to the sulfur amino acid of the chemical formula HO2CCH (NH2) CH2CH2SCH3 and CAS number 59-51-8 or 63-68-3 specifically for the L-isomer.

[81] The term cited as “DMSP” characterizes the reduced source of sulfur and methyl radical used, of the molecular formula C5H10O2S, from micro and macroalgae, phytoplankton and plants, or any other biological source.

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I 35 [82] The term cited as “DMDS” characterizes the reduced source of sulfur and methyl radical used, of molecular formula C2H6S2 and CAS number 624-92-0.

[83] A microorganism can be modified to express exogenous genes if those genes are introduced into the microorganism with all the elements for its expression. Modification of the microorganism with exogenous DNA is a routine task for experts in the art.

[84] The term microorganism used here refers preferentially to E. coli.

[85] The terms downstream and purification are equivalent, referring to the methods, after fermentation, to obtain the product in its final form.

[86] The metJ gene defines the deoxynucleotide sequence identified in the genome of the chassis strain as b3938, among deoxynucleotides 4,128,078 to 4,128,395.

[87] A deoxynucleotide sequence is defined as any polynucleotide chain consisting of single-stranded or double-stranded deoxynucleotides that will be referred to in the invention as a DNA sequence only.

[88] The DNA sequence identified in the genome of the chassis strain as b4013 is defined by the metA gene, between nucleotides 4,214,280 to 4,215,209 according to the reference genome (Blattner et al., 1997).

[89] The variant with three point mutations in nucleotides C79T (cytosine at position 79 is replaced by a thymine), T887G and C893T is defined by metA ^fd . These three mutations lead to the exchange of amino acids R27C (arginine of position 27 of the polypeptide exchanged for a cysteine), I296S (isoleucine of position 296 for serine) and P298L (proline of position 298 for leucine) and desensitizes the enzyme MetA from feedback inhibition negative effect caused by L-methionine.

[90] The DNA sequence identified in the genome of the chassis strain as b2838, between nucleotides 2977637 to 2978899, is defined by the lysA gene according to the reference genome (Blattner et al., 1997).

[91] The DNA sequence identified in the genome of the chassis strain as b4401 is defined by the arcA gene, between nucleotides 4639590 to 4640306 according to the reference genome (Blattner et al., 1997).

[92] The DNA sequence identified in the genome of the chassis strain as b3210, between nucleotides 3350689 to 3353025, is defined by the arcB gene according to the reference genome (Blattner et al., 1997).

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I 35 [93] The DNA sequence identified in the genome of the chassis strain as b1101, between nucleotides 1157859 to 1159302, is defined by the ptsG gene according to the reference genome (Blattner et al., 1997).

[94] The DNA sequence identified in the genome of the chassis strain as b0003 is defined by the thrB gene, between nucleotides 2801 to 3733 according to the reference genome (Blattner et al., 1997).

[95] The DNA sequence identified in the genome of the chassis strain as b0004, between nucleotides 3734 to 5020, is defined by the thrC gene according to the reference genome (Blattner et al., 1997).

[96] The DNA sequence identified in the genome of the chassis strain as b2297, between nucleotides 2414747 to 2416891, is defined by the pta gene according to the reference genome (Blattner et al., 1997).

[97] The DNA sequence identified in the genome of the chassis strain as b2296 is defined by the ackA gene, between nucleotides 2413470 to 2414672 according to the reference genome (Blattner et al., 1997).

[98] The yjeH gene is defined as the sequence identified as b4141 and located between nucleotides 4369156 to 4370412 in the reference genome (Blattner et al., 1997).

[99] The sequence identified as b3941 and located between nucleotides 4132616 to 4133506 in the reference genome is defined as the metF gene (Blattner et al., 1997).

[100] The sequence identified as b4019 and located between nucleotides 4223828 to 4227511 in the reference genome is defined as the metH gene (Blattner et al., 1997).

[101] The sequence identified as b3940 and located between nucleotides 4129835 to 4132267 in the reference genome is defined as the metL gene (Blattner et al., 1997).

[102] The sequence identified as b3607 and located between nucleotides 3781741 to 3782562 in the reference genome is defined as the cysE gene (Blattner et al., 1997).

[103] The variant with three point mutations in nucleotides A721G (adenine of position 721 is replaced by a guanine), G727A (guanine of position 727 is replaced by an adenine) and G955A (guanine of position 955) are defined as the cysE ^fd gene. is replaced by an adenine). These mutations lead to the exchange of amino acids

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I 35

T167A (tyrosine of position 167 of the polypeptide exchanged for an alanine), G245S (glycine of position 245 of the polypeptide exchanged for a serine) and M254I (methionine of position 254 of the polypeptide exchanged for an isoleucine) and desensitizes the cysE enzyme for feedback inhibition negative effect caused by L-cysteine.

[104] The sequence identified as b2421 and located between nucleotides 2538672 to 2539583 in the reference genome is defined as cysM gene (Blattner et al., 1997).

[105] The sequence identified as b2942 and located between nucleotides 3086076 to 3087860 in the reference genome is defined as the metK gene (Blattner et al., 1997).

[106] The aspC gene is defined as the sequence identified as b0928 and located between nucleotides 984519 to 985709 in the reference genome (Blattner et al., 1997).

[107] The ftsA gene is defined as the sequence identified as b0094 and located between nucleotides 103982 to 105244 in the reference genome (Blattner et al., 1997).

[108] The ftsZ gene is defined as the sequence identified as b0095 and located between nucleotides 105305 to 106456 in the reference genome (Blattner et al., 1997).

[109] The sequence identified as b3927 and located between nucleotides 4117245 to 4118090 in the reference genome is defined as the glpF gene (Blattner et al., 1997).

[110] The sequence identified as b3926 and located between nucleotides 4115714 to 4117222 in the reference genome is defined as the glpK gene (Blattner et al., 1997).

[111] The variant with a point mutation in nucleotide G913A is defined as the glpK ^fd gene, generating the G305S substitution (glycine at position 305 replaced by a serine). This change allows the desensitization of this protein to enzymes of the glucose-specific phosphotransferase system, allowing the strain that contains this mutation to be able to metabolize glycerol and glucose simultaneously.

[112] The glyA gene is defined as the sequence identified as b2551 and located between nucleotides 2684254 to 2685507 in the reference genome (Blattner et al., 1997).

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I 35 [113] The malY gene is defined as the sequence identified as b1622 and located between nucleotides 1700957 to 1702129 in the reference genome (Blattner et al., 1997).

[114] The sequence identified as b3939 and located between nucleotides 4128672 to 4129832 in the reference genome is defined as the metB gene (Blattner et al., 1997).

[115] The sequence identified as b3008 and located between nucleotides 3152236 to 3153413 in the reference genome is defined as the metC gene (Blattner et al., 1997).

[116] The sequence identified as b1603 and located between nucleotides 1676371 to 1677903 in the reference genome is defined as the pntA gene (Blattner et al., 1997).

[117] The sequence identified as b1602 and located between nucleotides 1674972 to 1776360 in the reference genome is defined as the pntB gene (Blattner et al., 1997).

[118] The sequence identified as b3962 and located between nucleotides 4159390 to 4160790 in the reference genome is defined as the sthA gene (Blattner et al., 1997).

[119] The sequence identified as RHECIAT_CH0004290 and located between nucleotides 4367027 to 4370491 in the reference genome is defined as the pyc gene (Gonzalez et al., 2006).

[120] The deoxynucleotide sequence identified in the genome of R. pomeroyi strain ATCC 700808 is defined by the dmdA gene as SPO1913, among deoxynucleotides 2033982 to 2035076 in the reference genome (Moran et al., 2004).

[121] The deoxynucleotide sequence identified in the genome of strain R. pomeroyi ATCC 700808 is defined by the dmdB gene as SPO2045, among deoxynucleotides 2174586 to 2176205 in the reference genome (Moran et al., 2004).

[122] The deoxynucleotide sequence identified in the genome of strain R. pomeroyi ATCC 700808 is defined by the dmdC gene as SPO3804, among deoxynucleotides 4018434 to 4020200 in the reference genome (Moran et al., 2004).

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I 35 [123] The deoxynucleotide sequence identified in the genome of the R. pomeroyi strain ATCC 700808 is defined by the dmdD gene as SPO3805, among deoxynucleotides 4020260 to 4021063 in the reference genome (Moran et al.,

2004).

[124] The deoxynucleotide sequence identified in the genome of strain C. glutamicum ATCC 13032 as cg0754 is defined by the metX gene, among deoxynucleotides 666641 to 667774 in the reference genome (Kalinowski et al.,

2005).

[125] The deoxynucleotide sequence identified in the genome of the strain C. glutamicum ATCC 13032 is defined by the metY gene as cg0755, among deoxynucleotides 667918 to 669231 in the reference genome (Kalinowski et al., 2005).

[126] The serA gene is defined as the DNA sequence identified in the genome of the chassis strain as b2913, between nucleotides 3,057,178 to 3,058,410 according to the reference genome (Blattner et al., 1997).

[127] SerB gene is defined as the DNA sequence identified in the genome of the chassis strain as b4388, between nucleotides 4,624,895 to 4,625,853 according to the reference genome (Blattner et al., 1997).

[128] The serC gene is defined as the DNA sequence identified in the genome of the chassis strain as b0907, between nucleotides 957,653 to 958,741 according to the reference genome (Blattner et al., 1997).

[129] Genetic parts are defined as any sequence of DNA that can be joined to another part to generate a genetic device that aims to express a certain gene or regulate its expression.

SEQUENCES OF THE GENES CITED IN THIS PATENT

Following paragraph 1 (ASPC): atgtttgagaacattaccgccgctcctgccgacccgattctgggcctggccgatctgtttcgtgccgatgaacgtcccggcaaaattaacctcgggattggtgt ctataaagatgagacgggcaaaaccccggtactgaccagcgtgaaaaaggctgaacagtatctgctcgaaaatgaaaccaccaaaaattacctcggc attgacggcatccctgaatttggtcgctgcactcaggaactgctgtttggtaaaggtagcgccctgatcaatgacaaacgtgctcgcacggcacagactcc ggggggcactggcgcactacgcgtggctgccgatttcctggcaaaaaataccagcgttaagcgtgtgtgggtgagcaacccaagctggccgaaccata agagcgtctttaactctgcgggtctggaagttcgtgaatacgcttattatgatgcggaaaatcacactcttgacttcgatgcactgattaacagcctgaatgaa gctcaggctggcgacgtagtgctgttccatggctgctgccataacccaaccggtatcgaccctacgctggaacaatggcaaacactggcacaactctccg ttgagaaaggctggttaccgctgtttgacttcgcttaccagggttttgcccgtggtctggaagaagatgctgaaggactgcgcgctttcgcggctatgcataaa gagctgattgttgccagttcctactctaaaaactttggcctgtacaacgagcgtgttggcgcttgtactctggttgctgccgacagtgaaaccgttgatcgcgca ttcagccaaatgaaagcggcgattcgcgctaactactctaacccaccagcacacggcgcttctgttgttgccaccatcctgagcaacgatgcgttacgtgc gatttgggaacaagagctgactgatatgcgccagcgtattcagcgtatgcgt cagttgttcgtcaatacgctgcaagaaaaaggcgcaaaccgcgacttc

Petition 870180128975, of 9/11/2018, p. 39/52

I 35 agctttatcatcaaacagaacggcatgttctccttcagtggcctgacaaaagaacaagtgctgcgtctgcgcgaagagtttggcgtatatgcggttgcttctg gtcgcgtaaatgtggccgggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggtg

Sequence No. 2 (cysE *): atgtcgtgtgaagaactggaaattgtctggaacaatattaaagccgaagccagaacgctggcggactgtgagccaatgctggccagtttttaccacgcga cgctactcaagcacgaaaaccttggcagtgcactgagctacatgctggcgaacaagctgtcatcgccaattatgcctgctattgctatccgtgaagtggtgg aagaagcctacgccgctgacccggaaatgatcgcctctgcggcctgtgatattcaggcggtgcgtacccgcgacccggcagtcgataaatactcaaccc cgttgttatacctgaagggttttcatgccttgcaggcctatcgcatcggtcactggttgtggaatcaggggcgtcgcgcactggcaatctttctgcaaaaccag gtttctgtgacgttccaggtcgatattcacccggcagcaaaaattggtcgcggtatcatgcttgaccacgcgacaggcatcgtcgttggtgaagcggcggtg attgaaaacgacgtatcgattctgcaatctgtgacgcttggcggtacgggtaaatctggtggtgaccgtcacccgaaaattcgtgaaggtgtgatgattggcg cgggcgcgaaaatcctcggcaatattgaagttgggcgcggcgcgaagattggcgcaggttccgtggtgctgcaaccggtgccgccgcataccaccgcc gctggcgttccggctcgtattgtcagtaaaccagacagcgataagccatcaatggatatagaccagcatttcaacggtattaaccatacatttgagtatggg gatgggatctaa

Following paragraph 3 (cysM): gtgagtacattagaacaaacaataggcaatacgcctctggtgaagttgcagcgaatggggccggataacggcagtgaagtgtggttaaaactggaagg caataacccggcaggttcggtgaaagatcgtgcggcactttcgatgatcgtcgaggcggaaaagcgcggggaaattaaaccgggtgatgtcttaatcga agccaccagtggtaacaccggcattgcgctggcaatgattgccgcgctgaaaggctatcgcatgaaattgctgatgcccgacaacatgagccaggaac gccgtgcggcgatgcgtgcttatggtgcggaactgattcttgtcaccaaagagcagggcatggaaggtgcgcgcgatctggcgctggagatggcgaatc gtggcgaaggaaagctgctcgatcagttcaataatcccgataacccttatgcgcattacaccaccactgggccggaaatctggcagcaaaccggcgggc gcatcactcattttgtctccagcatggggacgaccggcactatcaccggcgtctcacgctttatgcgcgaacaatccaaaccggtgaccattgtcggcctgc aaccggaagagggcagcagcattcccggcattcgccgctggcctacggaatatctgccggggattttcaacgcttctctggtggatgaggtgctggatattc atcagcgcgatgcggaaaacaccatgcgcgaactggcggtgcgggaaggaatattctgtggcgtcagctccggcggcgcggttgccggagcactgcg ggtggcaaaagctaaccctgacgcggtggtggtggcgatcatctgcgatcgtggcgatcgctacctttctaccggggtgtttggggaagagcattttagcca gggggcggggatttaa

Sequence No. 4 (FTSA) atgatcaaggcgacggacagaaaactggtagtaggactggagattggtaccgcgaaggttgccgctttagtaggggaagttctgcccgacggtatggtc aatatcattggcgtgggcagctgcccgtcgcgtggtatggataaaggcggggtgaacgacctcgaatccgtggtcaagtgcgtacaacgcgccattgacc aggcagaattgatggcagattgtcagatctcttcggtatatctggcgctttctggtaagcacatcagctgccagaatgaaattggtatggtgcctatttctgaag aagaagtgacgcaagaagatgtggaaaacgtcgtccataccgcgaaatcggtgcgtgtgcgcgatgagcatcgtgtgctgcatgtgatcccgcaagagt atgcgattgactatcaggaagggatcaagaatccggtaggactttcgggcgtgcggatgcaggcaaaagtgcacctgatcacatgtcacaacgatatgg cgaaaaacatcgtcaaagcggttgaacgttgtgggctgaaagttgaccaactgatatttgccggactggcatcaagttattcggtattgacggaagatgaa cgtgaactgggtgtctgcgtcgtcgatatcggtggtggtacaatggatatcgccgtttataccggtggggcattgcgccacactaaggtaattccttatgctgg caatgtcgtgaccagtgatatcgcttacgcctttggcacgccgccaagcgacgccgaagcgattaaagttcgccacggttgtgcgctgggttccatcgttgg aaaagatgagagcgtggaagtgccgagcgtaggtggtcgtccgccacggagtctgcaacgtcagacactggcagaggtgatcgagccgcgctatacc gagctgctcaacctggtcaacgaagagatattgcagttgcaggaaaagcttcgccaacaag gggttaaacatcacctggcggcaggcattgtattaacc ggtggcgcagcgcagatcgaaggtcttgcagcctgtgctcagcgcgtgtttcatacgcaagtgcgtatcggcgcgccgctgaacattaccggtttaacgga ttatgctcaggagccgtattattcgacggcggtgggattgcttcactatgggaaagagtcacatcttaacggtgaagctgaagtagaaaaacgtgttacagc atcagttggctcgtggatcaagcgactcaatagttggctgcgaaaagagttttaa

Following paragraph 5 (FtsZ) atgtttgaaccaatggaacttaccaatgacgcggtgattaaagtcatcggcgtcggcggcggcggcggtaatgctgttgaacacatggtgcgcgagcgca ttgaaggtgttgaatttttcgcggtaaataccgatgcacaagcgctgcgtaaaacagcggttggacagacgattcaaatcggtagcggtatcaccaaagga ctgggcgctggcgctaatccagaagttggccgcaatgcggctgatgaggatcgcgatgcattgcgtgcggcgctggaaggtgcagacatggtctttattgc tgcgggtatgggtggtggtaccggtacaggtgcagcaccagtcgtcgctgaagtggcaaaagatttgggtatcctgaccgttgctgtcgtcactaagcctttc aactttgaaggcaagaagcgtatggcattcgcggagcaggggatcactgaactgtccaagcatgtggactctctgatcactatcccgaacgacaaactgc tgaaagttctgggccgcggtatctccctgctggatgcgtttggcgcagcgaacgatgtactgaaaggcgctgtgcaaggtatcgctgaactgattactcgtcc gggtttgatgaacgtggactttgcagacgtacgcaccgtaatgtctgagatgggctacgcaatgatgggttctggcgtggcgagcggtgaagaccgtgcgg aagaagctgctgaaatggctatctcttctccgctgctggaagatatcgacctgtctggcgcgcgcggcgtgctggttaacatcacggcgggcttcgacctgc gtctggatgagttcgaaacggtaggtaacaccatccgtgcatttgcttccgacaacgcgactgtggttatcggtacttctcttgacccggatatgaatgacgag ctgcgcgtaaccgttgttgcgacaggtatcggcatggacaaacgtcctgaaa tcactctggtgaccaataagcaggttcagcagccagtgatggatcgcta ccagcagcatgggatggctccgctgacccaggagcagaagccggttgctaaagtcgtgaatgacaatgcgccgcaaactgcgaaagagccgtcatgatgatcatgatgatcatgatgatcatgatgatgatgatgatgatgatgatgatgatgatgg

Following paragraph 6 (glpF) atgagtcaaacatcaaccttgaaaggccagtgcattgctgaatttctcggtaccgggttgttgattttcttcggtgtgggttgcgttgcagcactaaaagtcgctg gtgcgtcttttggtcagtgggaaatcagtgtcatttggggactgggggtggcaatggccatctacctgaccgcaggggtttccggcgcgcatcttaatcccgct gttaccattgcattgtggctgtttgcctgtttcgacaagcgcaaagttattccttttatcgtttcacaagttgccggcgctttctgtgctgcggctttagtttacgggcttt actacaatttatttttcgacttcgagcagactcatcacattgttcgcggcagcgttgaaagtgttgatctggctggcactttctctacttaccctaatcctcatatca attttgtgcaggctttcgcagttgagatggtgattaccgctattctgatggggctgatcctggcgttaacggacgatggcaacggtgtaccacgcggccctttg

Petition 870180128975, of 9/11/2018, p. 40/52

R 35 gctcccttgctgattggtctactgattgcggtcattggcgcatctatgggcccattgacaggttttgccatgaacccagcgcgtgacttcggtccgaaagtctttg cctggctggcgggctggggcaatgtcgcctttaccggcggcagagacattccttacttcctggtgccgcttttcggccctatcgttggcgcgattgtaggtgcat ttgcctaccgcaaactgattggtcgccatttgccttgcgatatctgtgttgtggaagaaaaggaaaccacaactccttcagaacaaaaagcttcgctgtaa

Sequence No. 7 (glpK ^d) atgactgaaaaaaaatatatcgttgcgctcgaccagggcaccaccagctcccgcgcggtcgtaatggatcacgatgccaatatcattagcgtgtcgcagc gcgaatttgagcaaatctacccaaaaccaggttgggtagaacacgacccaatggaaatctgggccacccaaagctccacgctggtagaagtgctggcg aaagccgatatcagttccgatcaaattgcagctatcggtattacgaaccagcgtgaaaccactattgtctgggaaaaagaaaccggcaagcctatctata acgccattgtctggcagtgccgtcgtaccgcagaaatctgcgagcatttaaaacgtgacggtttagaagattatatccgcagcaataccggtctggtgattg acccgtacttttctggcaccaaagtgaagtggattctcgaccatgtggaaggctctcgcgagcgtgcacgtcgtggtgaattgctgtttggtacggttgatacg tggcttatctggaaaatgactcagggccgtgtccatgtgaccgattacaccaacgcctctcgtaccatgttgttcaacatccataccctggactgggacgaca aaatgctggaagtgctggatattccgcgcgagatgctgccagaagtgcgtcgttcttccgaagtatacggtcagactaacattggcggcaaaggcggcac gcgtattccaatctccgggatcgccggtgaccagcaggccgcgctgtttggtcagttgtgcgtgaaagaagggatggcgaagaacacctatggcactggc tgctttatgctgatgaacactggcgagaaagcggtgaaatcagaaaacggcctgctgaccaccatcgcctgcggcccgactggcgaagtgaactatgcg ttggaaagtgcggtgtttatggcaggcgcatccattcagtggctgcgcgatgaaatg aagttgattaacgacgcctacgattccgaatatttcgccaccaaa gtgcaaaacaccaatggtgtgtatgtggttccggcatttaccgggctgggtgcgccgtactgggacccgtatgcgcgcggggcgattttcggtctgactcgtg gggtgaacgctaaccacattatacgcgcgacgctggagtctattgcttatcagacgcgtgacgtgctggaagcgatgcaggccgactctggtatccgtctg cacgccctgcgcgtggatggtggcgcagtagcaaacaatttcctgatgcagttccagtccgatattctcggcacccgcgttgagcgcccggaagtgcgcg aagtcaccgcattgggtgcggcctatctcgcaggcctggcggttggcttctggcagaacctcgacgagctgcaagagaaagcggtgattgagcgcgagtt the ccgtccaggcatcgaaaccactgagcgtaattaccgttacgcaggctggaaaaaagcggttaaacgcgcgatggcgtgggaagaacacgacgaata

Following paragraph 8 (glyA) atgttaaagcgtgaaatgaacattgccgattatgatgccgaactgtggcaggctatggagcaggaaaaagtacgtcaggaagagcacatcgaactgatc gcctccgaaaactacaccagcccgcgcgtaatgcaggcgcagggttctcagctgaccaacaaatatgctgaaggttatccgggcaaacgctactacgg cggttgcgagtatgttgatatcgttgaacaactggcgatcgatcgtgcgaaagaactgttcggcgctgactacgctaacgtccagccgcactccggctccca ggctaactttgcggtctacaccgcgctgctggaaccaggtgataccgttctgggtatgaacctggcgcatggcggtcacctgactcacggttctccggttaac ttctccggtaaactgtacaacatcgttccttacggtatcgatgctaccggtcatatcgactacgccgatctggaaaaacaagccaaagaacacaagccga aaatgattatcggtggtttctctgcatattccggcgtggtggactgggcgaaaatgcgtgaaatcgctgacagcatcggtgcttacctgttcgttgatatggcgc acgttgcgggcctggttgctgctggcgtctacccgaacccggttcctcatgctcacgttgttactaccaccactcacaaaaccctggcgggtccgcgcggcg gcctgatcctggcgaaaggtggtagcgaagagctgtacaaaaaactgaactctgccgttttccctggtggtcagggcggtccgttgatgcacgtaatcgcc ggtaaagcggttgctctgaaagaagcgatggagcctgagttcaaaacttaccagcagcaggtcgctaaaaacgctaaagcgatggtagaagtgttcctc gagcgcggctacaaagtggtttccggcggcactgataaccacctgttcctggttgatc tggttgataaaaacctgaccggtaaagaagcagacgccgctct gggccgtgctaacatcaccgtcaacaaaaacagcgtaccgaacgatccgaagagcccgtttgtgacctccggtattcgtgtaggtactccggcgattacc cgtcgcggctttaaagaagccgaagcgaaagaactggctggctggatgtgtgacgtgctggacagcatcaatgatgaagccgttatcgagcgcatcaaa ggtaaagttctcgacatctgcgcacgttacccggtttacgcataa

Following paragraph 9 (Maly) atgttcgatttttcaaaggtcgtggatcgtcatggcacatggtgtacacagtgggattatgtcgctgaccgtttcggcactgctgacctgttaccgttcacgatttc agacatggattttgccactgccccctgcattatcgaggcgctgaatcagcgcctgatgcacggcgtatttggctacagccgctggaaaaacgatgagtttct cgcggctattgcccactggttttccacccagcattacaccgccatcgattctcagacggtggtgtatggcccttctgtcatctatatggtttcagaactgattcgtc agtggtctgaaacaggtgaaggcgtggtgatccacacacccgcctatgacgcattttacaaggccattgaaggtaaccagcgcacagtaatgcccgttgc tttagagaagcaggctgatggttggttttgcgatatgggcaagttggaagccgtgttggcgaaaccagaatgtaaaattatgctcctgtgtagcccacagaat cctaccgggaaagtgtggacgtgcgatgagctggagatcatggctgacctgtgcgagcgtcatggtgtgcgggttatttccgatgaaatccatatggatatg gtttggggcgagcagccgcatattccctggagtaatgtggctcgcggagactgggcgttgctaacgtcgggctcgaaaagtttcaatattcccgccctgacc ggtgcttacgggattatagaaaatagcagtagccgcgatgcctatttatcggcactgaaaggccgtgatgggctttcttccccttcggtactggcgttaactgc ccatatcgccgcctatcagcaaggcgcgccgtggctggatgccttacgcatctatctgaaagataacctgacgtatatcgcagataaaatgaacgccgcgt ttcctgaactcaactggcagatcccacaatccacttatc tggcatggcttgatttacgtccgttgaatattgacgacaacgcgttgcaaaaagcacttatcgaa caagaaaaagtcgcgatcatgccggggtatacctacggtgaagaaggtcgtggttttgtccgtctcaatgccggctgcccacgttcgaaactggaaaaag gtgtggctggattaattaacgccatccgcgctgttcgttaa

Sequence No. 10 (Goal ^fd) atgccgattcgtgtgccggacgagctacccgccgtcaatttcttgcgtgaagaaaacgtctttgtgatgacaacttcttgtgcgtctggtcaggaaattcgtcca cttaaggttctgatccttaacctgatgccgaagaagattgaaactgaaaatcagtttctgcgcctgctttcaaactcacctttgcaggtcgatattcagctgttgc gcatcgattcccgtgaatcgcgcaacacgcccgcagagcatctgaacaacttctactgtaactttgaagatattcaggatcagaactttgacggtttgattgta actggtgcgccgctgggcctggtggagtttaatgatgtcgcttactggccgcagatcaaacaggtgctggagtggtcgaaagatcacgtcacctcgacgct gtttgtctgctgggcggtacaggccgcgctcaatatcctctacggcattcctaagcaaactcgcaccgaaaaactctctggcgtttacgagcatcatattctcc atcctcatgcgcttctgacgcgtggctttgatgattcattcctggcaccgcattcgcgctatgctgactttccggcagcgttgattcgtgattacaccgatctggaa attctggcagagacggaagaaggggatgcatatctgtttgccagtaaagataagcgcattgcctttgtgacgggccatcccgaatatgatgcgcaaacgct

Petition 870180128975, of 9/11/2018, p. 41/52

35 I

Following paragraph 11 (metB) atgacgcgtaaacaggccaccatcgcagtgcgtagcgggttaaatgacgacgaacagtatggttgcgttgtcccaccgatccatctttccagcacctataa ctttaccggatttaatgaaccgcgcgcgcatgattactcgcgtcgcggcaacccaacgcgcgatgtggttcagcgtgcgctggcagaactggaaggtggt gctggtgcagtacttactaataccggcatgtccgcgattcacctggtaacgaccgtctttttgaaacctggcgatctgctggttgcgccgcacgactgctacgg cggtagctatcgcctgttcgacagtctggcgaaacgcggttgctatcgcgtgttgtttgttgatcaaggcgatgaacaggcattacgggcagcgctggcaga aaaacccaaactggtactggtagaaagcccaagtaatccattgttacgcgtcgtggatattgcgaaaatctgccatctggcaagggaagtcggggcggtg agcgtggtggataacaccttcttaagcccggcattacaaaatccgctggcattaggtgccgatctggtgttgcattcatgcacgaaatatctgaacggtcact cagacgtagtggccggcgtggtgattgctaaagacccggacgttgtcactgaactggcctggtgggcaaacaatattggcgtgacgggcggcgcgtttga cagctatctgctgctacgtgggttgcgaacgctggtgccgcgtatggagctggcgcagcgcaacgcgcaggcgattgtgaaatacctgcaaacccagcc gttggtgaaaaaactgtatcacccgtcgttgccggaaaatcaggggcatgaaattgccgcgcgccagcaaaaaggctttggcgcaatgttgagttttgaac tggatggcgatgagcagacgctgcgtcgtttcctgggcgggctgtcgttgtttacg ctggcggaatcattagggggagtggaaagtttaatctctcacgccgc aaccatgacacatatccaggcatggcaccagaagcgcgtgctgccgccgggatctccgagacgctgctgcgtatctccacggggaaatggga

Following paragraph 12 (metC) atggcggacaaaaagcttgatactcaactggtgaatgcaggacgcagcaaaaaatacactctcggcgcggtaaatagcgtgattcagcgcgcttcttcgc tggtctttgacagtgtagaagccaaaaaacacgcgacacgtaatcgcgccaatggagagttgttctatggacggcgcggaacgttaacccatttctccttac aacaagcgatgtgtgaactggaaggtggcgcaggctgcgtgctatttccctgcggggcggcagcggttgctaattccattcttgcttttatcgaacagggcg atcatgtgttgatgaccaacaccgcctatgaaccgagtcaggatttctgtagcaaaatcctcagcaaactgggcgtaacgacatcatggtttgatccgctgat tggtgccgatatcgttaagcatctgcaaccaaacactaaaatcgtgtttctggaatcgccaggctccatcaccatggaagtccacgacgttccggcgattgtt gccgccgtacgcagtgtggtgccggatgccatcattatgatcgacaacacctgggcagccggtgtgctgtttaaggcgctggattttggcatcgatgtttctatt caagccgccaccaaatatctggttgggcattcagatgcgatgattggcactgccgtgtgcaatgcccgttgctgggagcagctacgggaaaatgcctatct gatgggccagatggtcgatgccgataccgcctatataaccagccgtggcctgcgcacattaggtgtgcgtttgcgtcaacatcatgaaagcagtctgaaag tggctgaatggctggcagaacatccgcaagttgcgcgagttaaccaccctgctctgcctggcagtaaaggtcacgaattttggaaacgagactttacaggc agcagcgggctattttcctttgtgcttaagaaaaaactcaataatgaa gagctggcgaactatctggataacttcagtttattcagcatggcctactcgtgggg cgggtatgaatcgttgatcctggcaaatcaaccagaacatatcgccgccattcgcccacaaggcgagatcgattttagcgggaccttgattcgcctgcatat tggtctggaagatgtcgacgatctgattgccgatctggacgccggttttgcgcgaattgtataa

Following paragraph 13 (metformin) atgagcttttttcacgccagccagcgggatgccctgaatcagagcctggcagaagtccaggggcagattaacgtttcgttcgagtttttcccgccgcgtacca gtgaaatggagcagaccctgtggaactccatcgatcgccttagcagcctgaaaccgaagtttgtatcggtgacctatggcgcgaactccggcgagcgcg accgtacgcacagcattattaaaggcattaaagatcgcactggtctggaagcggcaccgcatcttacttgcattgatgcgacgcccgacgagctgcgcac cattgcacgcgactactggaataacggtattcgtcatatcgtggcgctgcgtggcgatctgccgccgggaagtggtaagccagaaatgtatgcttctgacct ggtgacgctgttaaaagaagtggcagatttcgatatctccgtggcggcgtatccggaagttcacccggaagcaaaaagcgctcaggcggatttgcttaatc tgaaacgcaaagtggatgccggagccaaccgcgcgattactcagttcttcttcgatgtcgaaagctacctgcgttttcgtgaccgctgtgtatcggcgggcat tgatgtggaaattattccgggaattttgccggtatctaactttaaacaggcgaagaaatttgccgatatgaccaacgtgcgtattccggcgtggatggcgcaa atgttcgacggtctggatgatgatgccgaaacccgcaaactggttggcgcgaatattgccatggatatggtgaagattttaagccgtgaaggagtgaaaga tttccacttctatacgcttaaccgtgctgaaatgagttacgcgatttgccatacgctgggggttcgacctggtttataa

Sequence No. 14 (meth) gtgagcagcaaagtggaacaactgcgtgcgcagttaaatgaacgtattctggtgctggacggcggtatgggcaccatgatccagagttatcgactgaacg aagccgattttcgtggtgaacgctttgccgactggccatgcgacctcaaaggcaacaacgacctgctggtactcagtaaaccggaagtgatcgccgctatc cacaacgcctactttgaagcgggcgcggatatcatcgaaaccaacaccttcaactccacgaccattgcgatggcggattaccagatggaatccctgtcgg cggaaatcaactttgcggcggcgaaactggcgcgagcttgtgctgacgagtggaccgcgcgcacgccagagaaaccgcgctacgttgccggtgttctcg gcccgaccaaccgcacggcgtctatttctccggacgtcaacgatccggcatttcgtaatatcacttttgacgggctggtggcggcttatcgagagtccacca aagcgctggtggaaggtggcgcggatctgatcctgattgaaaccgttttcgacacccttaacgccaaagcggcggtatttgcggtgaaaacggagtttgaa gcgctgggcgttgagctgccgattatgatctccggcaccatcaccgacgcctccgggcgcacgctctccgggcagaccaccgaagcattttacaactcatt gcgccacgccgaagctctgacctttggcctgaactgtgcgctggggcccgatgaactgcgccagtacgtgcaggagctgtcacggattgcggaatgctac gtcaccgcgcacccgaacgccgggctacccaacgcctttggtgagtacgatctcgacgccgacacgatggcaaaacagatacgtgaatgggcgcaag cgggttttctcaatatcgtcggcggctgctgtggcaccacgccacaacatattgcagcgatgagt cgtgcagtagaaggattagcgccgcgcaaactgcc ggaaattcccgtagcctgccgtttgtccggcctggagccgctgaacattggcgaagatagcctgtttgtgaacgtgggtgaacgcaccaacgtcaccggtt ccgctaagttcaagcgcctgatcaaagaagagaaatacagcgaggcgctggatgtcgcgcgtcaacaggtggaaaacggcgcgcagattatcgatat caacatggatgaagggatgctcgatgccgaagcggcgatggtgcgttttctcaatctgattgccggtgaaccggatatcgctcgcgtgccgattatgatcga ctcctcaaaatgggacgtcattgaaaaaggtctgaagtgtatccagggcaaaggcattgttaactctatctcgatgaaagagggcgtcgatgcctttatccat cacgcgaaattgttgcgtcgctacggtgcggcagtggtggtaatggcctttgacgaacagggacaggccgatactcgcgcacggaaaatcgagatttgcc gtcgggcgtacaaaatcctcaccgaagaggttggcttcccgccagaagatatcatcttcgacccaaacatcttcgcggtcgcaactggcattgaagagca caacaactacgcgcaggactttatcggcgcgtgtgaagacatcaaacgcgaactgccgcacgcgctgatttccggcggcgtatctaacgtttctttctcgttc

Petition 870180128975, of 9/11/2018, p. 42/52

R 35 cgtggcaacgatccggtgcgcgaagccattcacgcagtgttcctctactacgctattcgcaatggcatggatatggggatcgtcaacgccgggcaactggc gatttacgacgacctacccgctgaactgcgcgacgcggtggaagatgtgattcttaatcgtcgcgacgatggcaccgagcgtttactggagcttgccgaga aatatcgcggcagcaaaaccgacgacaccgccaacgcccagcaggcggagtggcgctcgtgggaagtgaataaacgtctggaatactcgctggtca aaggcattaccgagtttatcgagcaggataccgaagaagcccgccagcaggctacgcgcccgattgaagtgattgaaggcccgttgatggacggcatg aatgtggtcggcgacctgtttggcgaagggaaaatgttcctgccacaggtggtcaaatcggcgcgcgtcatgaaacaggcggtggcctacctcgaaccgt ttattgaagccagcaaagagcagggcaaaaccaacggcaagatggtgatcgccaccgtgaagggcgacgtccacgacatcggtaaaaatatcgttgg tgtggtgctgcaatgtaacaactacgaaattgtcgatctcggcgttatggtgcctgcggaaaaaattctccgtaccgctaaagaagtgaatgctgatctgattg gcctttcggggcttatcacgccgtcgctggacgagatggttaacgtggcgaaagagatggagcgtcagggcttcactattccgttactgattggcggcgcga cgacctcaaaagcgcacacggcggtgaaaatcgagcagaactacagcggcccgacggtgtatgtgcagaatgcctcgcgtaccgttggtgtggtggcg gcgctgctttccgatacccagcgtgatgattttgtcgctcgtacccgcaaggagtacgaaaccgtacgtattcagcacgggcgcaagaa accgcgcacac caccggtcacgctggaagcggcgcgcgataacgatttcgcttttgactggcaggcttacacgccgccggtggcgcaccgtctcggcgtgcaggaagtcg aagccagcatcgaaacgctgcgtaattacatcgactggacaccgttctttatgacctggtcgctggccgggaagtatccgcgcattctggaagatgaagtg gtgggcgttgaggcgcagcggctgtttaaagacgccaacgacatgctggataaattaagcgccgagaaaacgctgaatccgcgtggcgtggtgggcct gttcccggcaaaccgtgtgggcgatgacattgaaatctaccgtgacgaaacgcgtacccatgtgatcaacgtcagccaccatctgcgtcaacagaccga aaaaacaggcttcgctaactactgtctcgctgacttcgttgcgccgaagctttctggtaaagcagattacatcggcgcatttgccgtgactggcgggctggaa gaggacgcactggctgatgcctttgaagcgcagcacgatgattacaacaaaatcatggtgaaagcgcttgccgaccgtttagccgaagcctttgcggagt atctccatgagcgtgtgcgtaaagtctactggggctatgcgccgaacgagaacctcagcaacgaagagctgatccgcgaaaactaccagggcatccgt ccggcaccgggctatccggcctgcccggaacatacggaaaaagccaccatctgggagctgctggaagtggaaaaacacactggcatgaaactcaca gaatctttcgccatgtggcccggtgcatcggtttcgggttggtacttcagccacccggacagcaagtactacgctgtagcacaaattcagcgcgatcaggtt gaagattatgcccgccgtaaaggtatgagcgttaccgaagttgagcgctggctggcaccgaatctggggtatgacgcggactg The

Following paragraph 15 (METL) atgagtgtgattgcgcaggcaggggcgaaaggtcgtcagctgcataaatttggtggcagtagtctggctgatgtgaagtgttatttgcgtgtcgcgggcattat ggcggagtactctcagcctgacgatatgatggtggtttccgccgccggtagcaccactaaccagttgattaactggttgaaactaagccagaccgatcgtct ctctgcgcatcaggttcaacaaacgctgcgtcgctatcagtgcgatctgattagcggtctgctacccgctgaagaagccgatagcctcattagcgcttttgtca gcgaccttgagcgcctggcggcgctgctcgacagcggtattaacgacgcagtgtatgcggaagtggtgggccacggggaagtatggtcggcacgtctga tgtctgcggtacttaatcaacaagggctgccagcggcctggcttgatgcccgcgagtttttacgcgctgaacgcgccgcacaaccgcaggttgatgaagg gctttcttacccgttgctgcaacagctgctggtgcaacatccgggcaaacgtctggtggtgaccggatttatcagccgcaacaacgccggtgaaacggtgct gctggggcgtaacggttccgactattccgcgacacaaatcggtgcgctggcgggtgtttctcgcgtaaccatctggagcgacgtcgccggggtatacagtg ccgacccgcgtaaagtgaaagatgcctgcctgctgccgttgctgcgtctggatgaggccagcgaactggcgcgcctggcggctcccgttcttcacgcccgt actttacagccggtttctggcagcgaaatcgacctgcaactgcgctgtagctacacgccggatcaaggttccacgcgcattgaacgcgtgctggcctccgg tactggtgcgcgtattgtcaccagccacgatgatgtctgtttgattgagttt caggtgcccgccagtcaggatttcaaactggcgcataaagagatcgaccaa atcctgaaacgcgcgcaggtacgcccgctggcggttggcgtacataacgatcgccagttgctgcaattttgctacacctcagaagtggccgacagtgcgct gaaaatcctcgacgaagcgggattacctggcgaactgcgcctgcgtcaggggctggcgctggtggcgatggtcggtgcaggcgtcacccgtaacccgct gcattgccaccgcttctggcagcaactgaaaggccagccggtcgaatttacctggcagtccgatgacggcatcagcctggtggcagtactgcgcaccgg cccgaccgaaagcctgattcaggggctgcatcagtccgtcttccgcgcagaaaaacgcatcggcctggtattgttcggtaagggcaatatcggttcccgttg gctggaactgttcgcccgtgagcagagcacgctttcggcacgtaccggctttgagtttgtgctggcaggtgtggtggacagccgccgcagcctgttgagcta tgacgggctggacgccagccgcgcgttagccttcttcaacgatgaagcggttgagcaggatgaagagtcgttgttcctgtggatgcgcgcccatccgtatg atgatttagtggtgctggacgttaccgccagccagcagcttgctgatcagtatcttgatttcgccagccacggtttccacgttatcagcgccaacaaactggcg ggagccagcgacagcaataaatatcgccagatccacgacgccttcgaaaaaaccgggcgtcactggctgtacaatgccaccgtcggtgcgggcttgcc gatcaaccacaccgtgcgcgatctgatcgacagcggcgatactattttgtcgatcagcgggatcttctccggcacgctctcctggctgttcctgcaattcgac ggtagcgtgccgtttaccgagctggtggat caggcgtggcagcagggcttaaccgaacctgacccgcgtgacgatctctctggcaaagacgtgatgcgc aagctggtgattctggcgcgtgaagcaggttacaacatcgaaccggatcaggtacgtgtggaatcgctggtgcctgctcattgcgaaggcggcagcatcg accatttctttgaaaatggcgatgaactgaacgagcagatggtgcaacggctggaagcggcccgcgaaatggggctggtgctgcgctacgtggcgcgttt cgatgccaacggtaaagcgcgtgtaggcgtggaagcggtgcgtgaagatcatccgttggcatcactgctgccgtgcgataacgtctttgccatcgaaagc cgctggtatcgcgataaccctctggtgatccgcggacctggcgctgggcgcgacgtcaccgccggggcgattcagtcggatatcaaccggctggcacag ttgttgtaa

Sequence No. 16 (pntA) atgcgaattggcataccaagagaacggttaaccaatgaaacccgtgttgcagcaacgccaaaaacagtggaacagctgctgaaactgggttttaccgtc gcggtagagagcggcgcgggtcaactggcaagttttgacgataaagcgtttgtgcaagcgggcgctgaaattgtagaagggaatagcgtctggcagtca gagatcattctgaaggtcaatgcgccgttagatgatgaaattgcgttactgaatcctgggacaacgctggtgagttttatctggcctgcgcagaatccggaatt aatgcaaaaacttgcggaacgtaacgtgaccgtgatggcgatggactctgtgccgcgtatctcacgcgcacaatcgctggacgcactaagctcgatggc gaacatcgccggttatcgcgccattgttgaagcggcacatgaatttgggcgcttctttaccgggcaaattactgcggccgggaaagtgccaccggcaaaa gtgatggtgattggtgcgggtgttgcaggtctggccgccattggcgcagcaaacagtctcggcgcgattgtgcgtgcattcgacacccgcccggaagtgaa agaacaagttcaaagtatgggcgcggaatttctcgagctggattttaaagaggaagctggcagcggcgatggctatgccaaagtgatgtcggacgcgttc atcaaagcggaaatggaactctttgccgcccaggcaaaagaggtcgatatcattgtcaccaccgcgcttattccaggcaaaccagcgccgaagctaatt acccgtgaaatggttgactccatgaaggcgggcagtgtgattgtcgacctggcagcccaaaacggcggcaactgtgaatacaccgtgccgggtgaaatc ttcactacggaaaatggtgtcaaagtgattggttataccgatcttccgggccgtctgccgacgcaa tcctcacagctttacggcacaaacctcgttaatctgct gaaactgttgtgcaaagagaaagacggcaatatcactgttgattttgatgatgtggtgattcgcggcgtgaccgtgatccgtgcgggcgaaattacctggcc

Petition 870180128975, of 9/11/2018, p. 43/52

R 35 ggcaccgccgattcaggtatcagctcagccgcaggcggcacaaaaagcggcaccggaagtgaaaactgaggaaaaatgtacctgctcaccgtggcg taaatacgcgttgatggcgctggcaatcattctttttggctggatggcaagcgttgcgccgaaagaatttcttgggcacttcaccgttttcgcgctggcctgcgtt gtcggttattacgtggtgtggaatgtatcgcacgcgctgcatacaccgttgatgtcggtcaccaacgcgatttcagggattattgttgtcggagcactgttgcag attggccagggcggctgggttagcttccttagttttatcgcggtgcttatagccagcattaatattttcggtggcttcaccgtgactcagcgcatgctgaaaatgtt ccgcaaaaattaa

Sequence No. 17 (pntB) atgtctggaggattagttacagctgcatacattgttgccgcgatcctgtttatcttcagtctggccggtctttcgaaacatgaaacgtctcgccagggtaacaact tcggtatcgccgggatggcgattgcgttaatcgcaaccatttttggaccggatacgggtaatgttggctggatcttgctggcgatggtcattggtggggcaattg gtatccgtctggcgaagaaagttgaaatgaccgaaatgccagaactggtggcgatcctgcatagcttcgtgggtctggcggcagtgctggttggctttaaca gctatctgcatcatgacgcgggaatggcaccgattctggtcaatattcacctgacggaagtgttcctcggtatcttcatcggggcggtaacgttcacgggttcg gtggtggcgttcggcaaactgtgtggcaagatttcgtctaaaccattgatgctgccaaaccgtcacaaaatgaacctggcggctctggtcgtttccttcctgct gctgattgtatttgttcgcacggacagcgtcggcctgcaagtgctggcattgctgataatgaccgcaattgcgctggtattcggctggcatttagtcgcctccat cggtggtgcagatatgccagtggtggtgtcgatgctgaactcgtactccggctgggcggctgcggctgcgggctttatgctcagcaacgacctgctgattgtg accggtgcgctggtcggttcttcgggggctatcctttcttacattatgtgtaaggcgatgaaccgttcctttatcagcgttattgcgggtggtttcggcaccgacgg ctcttctactggcgatgatcaggaagtgggtgagcaccgcgaaatcaccgcagaagagacagcggaactgctgaaaaactcccattcagtgatcattact ccggggtacggcatggcagtcgcgcaggcg caatatcctgtcgctgaaattactgagaaattgcgcgctcgtggtattaatgtgcgtttcggtatccacccg gtcgcggggcgtttgcctggacatatgaacgtattgctggctgaagcaaaagtaccgtatgacatcgtgctggaaatggacgagatcaatgatgactttgct gataccgataccgtactggtgattggtgctaacgatacggttaacccggcggcgcaggatgatccgaagagtccgattgctggtatgcctgtgctggaagt gtggaaagcgcagaacgtgattgtctttaaacgttcgatgaacactggctatgctggtgtgcaaaacccgctgttcttcaaggaaaacacccacatgctgttt ggtgacgccaaagccagcgtggatgcaatcctgaaagctctgtaa

Following paragraph 18 (PYC) ttgcccatttccaagatactcgttgccaatcgctctgaaatagccatccgcgtgttccgcgcggccaacgagcttggaataaaaacggtggcgatctgggcg gaagaggacaagctggcgctgcaccgcttcaaggcggatgagagttatcaggtcggccgcggtccgcatctggcccgcgatctcgggccgatcgaga gctatctgtcgatcgacgaggtgatccgggtcgccaagctttcgggtgccgacgccattcaccccggttacggcctcttgtccgaaagcccggaatttgtcg atgcctgcaacaaggccgggatcatcttcatcggcccgaaggccgatacgatgcgccagctcggcaacaaggtcgcggcgcgcaatctggcgatctcg gtcggcgtgcccgtcgtgccggcgaccgagccgctaccggacgatatggccgaagtggcgaagatggcggcagcaatcggctatcccgtcatgctgaa agcctcctggggcggcggcggccgcggcatgcgcgtcatccgcgccgaagccgatctcgcccgcgaggtgacggaggccaagcgcgaggcgatgg ccgccttcggcaaggacgaggtctatctggaaaagctggtcgagcgcgcccgccacgtcgaaagccagatcctcggcgacacacacggcaatgtcgt gcatctgttcgagcgcgactgctcgatccagcgccgcaaccagaaggtcgtcgagcgcgcgcccgcgccctacctctcagaggcgcagcgccaggaa ctcgccgcctattcgctgaagatcgcagcggcgaccaactatatcggcgccggcaccgtcgaatatctgatggatgccgataccggcaaattctacttcat cgaggtcaatccgcgcatccaggtcgagcacacggtgaccgaagtcgtcaccggtatcgacatcgtcaaggcgcagatcc acatcctcgacggcgctg cgatcggcacgccggaatcgggcgttcccgctcaggccgatatccggctcaacggccatgcgctgcaatgccgcatcaccaccgaagatcccgaaca caatttcattccggactacggccgcatcaccgcctatcgctcggcttccggcttcggcatccggcttgacggcggcacctcctattccggcgccatcattacc cgttattatgatccgctgctcgtcaaggtcacggcctgggcgccgaacccgtccgaagcgatttcccgtatggaccgggcgctgcgcgaatttcgcatccgc ggcgtcgccaccaacctgaccttcctcgaagcgatcatcggccatccgaagttccgcgacaacagctacaccacccgcttcatcgacaccacgccgga actcttccagcaggtcaagcgccaggaccgcgcgacgaagctcttgacctatctcgccgatgtcaccatcaatggccatcccgaggccaaggacaggc cgaagcccctcgaaaacgccgccaagccggtggtgccctatgccaatggcaacggggtcaaggatggcacgaagcagctgctcgacacgctcggcc cgaagaaattcggcgaatggatgcgcaatgagaagcgcgtgcttctgaccgatacgacgatgcgcgacgcccaccagtcgctgctcgccactcgcatg cgcacctatgacatcgccaggatcgccggcacctatgcgcacgcgctgccgaatctcttgtcgctcgaatgctggggtggcgccaccttcgacgtctccat gcgcttcctcaccgaagatccgtgggagcggctggcgctgatccgcgagggcgcgccgaacctgctcctgcaaatgctgttgcgcggcgccaacggcgt cggctacaccaactatcccgacaatgtcgtcaaatatttcgtccgccaggcggccagaggcggcatcgatcttttccgcg tcttcgactgcctgaactgggtc gagaacatgcgggtgtcgatggatgcgattgccgaggagaacaagctctgcgaggcggcgatctgctacaccggcgatatcctcaattccgcccgcccg aaatacgacctgaaatactataccgaccttgccgtcgaactcgagaaggccggcgcccatatcatcgcagtcaaggatatggcgggtctgttgaagccg gcggcggcgaaggtgctgttcaaggcgctgcgcgaagcgaccggcctgccgatccacttccacacgcatgacacttcgggcatcgcggccgcgaccgt ccttgccgcggtcgaagccggtgtcgatgccgtcgatgcggcgatggatgcgctttccggcaatacctcgcagccttgtctcggctcgatcgtcgaggcgct ctccggctccgagcgcgatccgggcctcgatccggaatggattcgccgtatctcgttctattgggaagcggtgcgcaaccagtatgccgccttcgaaagcg acctcaaggggccggcctcggaagtctatctgcatgaaatgccgggcggccagttcaccaatctcaaggagcaggcccgctcgctcggtctcgagaccc gctggcatcaggtggcgcaggcctatgccgacgccaaccagatgttcggcgatatcgtcaaggtgacgccctcctccaaggtggtcggcgacatggcgc tgatgatggtgtcccaggatctgacggtcgccgacgtcgtcagccccgagcgcgaagtctccttccccgaatcggtggtgtcgatgctgaagggcgatctc ggccagccgccgccgggatggccggcagcacttcagaaaaaggcactgaagggcgaaaagccctatacggtgcgtcccggctcgctgctgaaggaa gccgatctcgatgccgagcgcaaggtcatcgagacgaagctggagcgcgaggtcagcgacttcgaatttgcct cctatctgatgtatccgaaggtcttcac cgactttgcgctcgcctccgatacctatggcccggtctcggtgctgccgacgcctgcctatttctacgggctggccgacggcgaggagctgtttgccgatatc gaacgaggcaagacgctcgtcatcgtcaatcaggcaatgagcgccaccgacagccagggcatggtcaccgtcttcttcgaactcaacggccagccgc gccgcatcaaggtgccggaccgggcccatggggcgacgggtgcggccgtgcgccgcaaggccgagcccggtaatggtgctcatgtcggtgcgccgat gcccggcgtcatcagccgcgtcttcgcctcatccggccaagccgtcagcgccggtgatgtgctcgtctccatcgaagcgatgaagatggaaacggcgatc catgcggaaaaggatggaacggttgccgaaattctcgtcaaggccggcgatcagattgatgccaaggacctgcttgtcgtctacgccgcttga

Following paragraph 19 (stha)

Petition 870180128975, of 9/11/2018, p. 44/52

R 35 atgccacattcctacgattacgatgccatagtaataggttccggccccggcggcgaaggcgctgcaatgggcctggttaagcaaggtgcgcgcgtcgca gttatcgagcgttatcaaaatgttggcggcggttgcacccactggggcaccatcccgtcgaaagctctccgtcacgccgtcagccgcattatagaatttaatc aaaacccactttacagcgaccattcccgactgctccgctcttcttttgccgatatccttaaccatgccgataacgtgattaatcaacaaacgcgcatgcgtcag ggattttacgaacgtaatcactgtgaaatattgcagggaaacgctcgctttgttgacgagcatacgttggcgctggattgcccggacggcagcgttgaaaca ctaaccgctgaaaaatttgttattgcctgcggctctcgtccatatcatccaacagatgttgatttcacccatccacgcatttacgacagcgactcaattctcagc atgcaccacgaaccgcgccatgtacttatctatggtgctggagtgatcggctgtgaatatgcgtcgatcttccgcggtatggatgtaaaagtggatctgatca acacccgcgatcgcctgctggcatttctcgatcaagagatgtcagattctctctcctatcacttctggaacagtggcgtagtgattcgtcacaacgaagagtac gagaagatcgaaggctgtgacgatggtgtgatcatgcatctgaagtcgggtaaaaaactgaaagctgactgcctgctctatgccaacggtcgcaccggta ataccgattcgctggcgttacagaacattgggctagaaactgacagccgcggacagctgaaggtcaacagcatgtatcagaccgcacagccacacgttt acgcggtgggcgacgtgattggttatccgagcctggcgtcggcggcctatgaccaggggcgcattgc cgcgcaggcgctggtaaaaggcgaagccac cgcacatctgattgaagatatccctaccggtatttacaccatcccggaaatcagctctgtgggcaaaaccgaacagcagctgaccgcaatgaaagtgcca tatgaagtgggccgcgcccagtttaaacatctggcacgcgcacaaatcgtcggcatgaacgtgggcacgctgaaaattttgttccatcgggaaacaaaag agattctgggtattcactgctttggcgagcgcgctgccgaaattattcatatcggtcaggcgattatggaacagaaaggtggcggcaacactattgagtactt cgtcaacaccacctttaactacccgacgatggcggaagcctatcgggtagctgcgttaaacggtttaaaccgcctgttttaa

Sequence No. 20 (MAD) atggccagcatcttcccgtcccgccgggtccggcgcacacctttttccgccggtgtcgaggcggccggggtcaagggctataccgtctacaatcacatgttg ctgcccacggtgttcgacagcctgcaggccgattgcgcccatctgaaggaacatgtgcaggtctgggacgtggcctgcgagcggcaggtcagcatccag gggcccgacgcgctgcggctgatgaagctgatcagcccgcgcgacatggaccggatggccgatgaccagtgttactacgtgcccacggtcgatcatcgt ggcggcatgctgaacgatccggtggcggtgaaactggccgccgatcattactggctgtcgctggccgatggcgacctgctgcaattcgggctggggattg cgatcgcccggggcttcgatgtcgagatcgtcgaacccgatgtctcgccgctggccgtgcagggacccagggccgacgatctgatggcgcgggtctttgg cgaggcggtgcgcgatatccgctttttccgctacaagcggctggcctttcagggagtcgagcttgtggtggcgcgctcaggctggtcgaaacagggcggct tcgagatctatgtcgagggttcggaactgggcatgccgctgtggaacgcgctgtttgccgccggtgcggacctgaacgtgcgcgcgggttgccccaacaa tatcgagcgcgtcgagagcgggttgttgagctatggcaacgacatgacccgcgagaacacgccgtatgaatgcggcctgggtaagttctgcaattcgccc gaggactatatcggcaaggcagcactggccgaacaggccaagaacggaccggcgcgccagatccgggcactggtgatcggtggcgagattccgccc tgtcaggatgcctggccgctgctggccgacggtcgccaggtggggcaggtggggtcagcgatccattcc cctgaattcggcgtgaatgtcgcgatcggca tggtggatcgcagccattgggcgccgggcaccgggatggaagtggaaacgcccgacggcatgcggccggttacggtgcgcgagggttt

SEQ ID NO: 21 (DMDB) atgcttggacagatgatgtatcagccgctgctgatctcgtcgctgatagatcacgccgcgcgctatcacggcgaggcgcagatctggtcggtcagcaccga aggcggggtcgaggagaccaactgggcggggatcgccgacaatgcccgccgcctgggctcggtcctgaccgatgcagggctggcgccgcaatcgcg tgttgccacgctggcctggaacaaccggcggcatctggagatctattacggggtgtcgggcgccggctttgttctgcacacgatcaacccgcgcctgttccc cgaacagctggtctatatcctcaaccatgccgaggatcgcatcctgttcttcgatgccactttcctgccgctggtcgaggggattcgcccgcatctgaccacc gttgaacggctggttctgatggggccgcgcgacgaggcggcggcagcgcgcatcgaggggctggaattctacgacgagtttgtcgccaccggggatgc gggttttgactggcccgatcttgacgagcgcacggcttcgagcctgtgctatacctcggggaccacgggcaatcccaagggcgtactttattcgcaccgttc caccgtgctgcacagtttcggctcgaacacccgcgattgcatcggtttttcggcgcgcgatgtggtgatgccggtggtgccgatgttccatgtcaacgcctgg ggcacgccctatgcctgtgcgatgtctggatcgtgcatggtgctgccggggcccgatctgcatggcgaggcgctggttggtctgatcgaccgctatcgggtc accatcgcgctgggggtgccgaccatctggcaggggctgttggccaccgcccgcgccaagggcagcacgctggaaagcctgacgcgcacggtgatc ggcggcgcggcctgtccgccctcgatgatcgccgagttccgcgaccgctatggtgtcgataccg tccatgcctggggcatgtccgagatgagcccgctgg gcaccaccaaccagccgctggccaagcacggcgccctgccgatcgaggcgcagcacaagctgcgcgagaaccagggccgcccgccctatggggt ggagctgaagatcgtcgatgacgatggcaacacgttgcccaatgacggtcagacccagggcgaccttatggtgcgcggccattgggtgctggacagcta tttccagttgcaggaccagccgatcctgtcggatggctggttcgccaccggcgatgtggcgacgctggaccgcgacggctatatgacgatccgggaccgg tccaaggatatcatcaagtcgggcggcgaatggatcagctcggtcgagctggagaatatcgcggttgcgcatccgaaactggccaccgccgcggtgatc ggggtgccgcatccgaaatgggatgaacgcccgcttttggtggcggtcaaggccgagggcgagacgcctgacgaggcggagttgctggcgttctttgac ggcaagatcgcgaaatggcaggtgcctgaccgggtcgtgtttgtcgaggcgctgccgctgaatgccaccggcaaggtgctgaaacggacccttcgcga acagttcagggatgttctgaccgggtga

Sequence No. 22 (DMDC) atgacctatcaggcgccggtccgtgacatcatgttcgccatcgaacacctttcgcaatggccccaagtcgaggcgctgcaaacctattccgagatcgaact ggacgatgcccgcgccgcgctggaggaattcggccgtttctgcggcgagatgatcgcccccctgtccaccatcggcgataccgaaggcgcccggctgg aaaatgggcgcgtcgtcctgcccgagggctacaagaccgcctatgaccagttcgtcgatatggggtggcaaagcctcagtcacccggccgaacatggc ggcatgggcctgcccaaggttgttggcgccgccgcgaccgaaatcgtcaacagcgccgatatgagctttggcctctgcccgttgttgaccaacggcgcca tcgacgcgctgtcgatcaccgggtccgatgcgcaaaaggccttctatctggacaagctgatcaccgggcgctggtcgggcaccatgaacctgaccgagc cgcaggcgggcagcgacctcagccgcgtgcgctgcacggcggtgccgcaggatgacggcacctatgcgatcagcggcaccaagatcttcatcaccttc ggcgaacatgacctgtcggaaaacatcgtgcatctggtgctggcccgcacccccgacgcacccgaaggggtgcgcgggctgagcctgttcgtggtgcc caagctgcttgcgggcgagggcggcgaaacctcgcagcgcaatacgctgggctgcgtcagcctggaacacaagctgggcgtgcgcgccagcccgac cgcggtgatggaatatgacaatgccaccggctatctggtgggcgaggaaaacagcggcctgcgctacatgttcatcatgatgacctcggcccgctatgcg gtgggtgtgcagggtgtggcgattgccgaacgcgcctatcagcatgcgctgtcctatgcccgtgaccggatacaaagcc gacccgtagacggctcggcc caggacgcggtgccgatcatccagcaccccgatgtccgccggatgctgctcaggatgcgcgcgctgaccgaaggcgggcgggcgcttgccatcgcca cgggcggctggctcgacctggccgaacatgggcccgaagaggcccgcgccgaggcgcaatcaatggccgaattccttgtgccgctggtcaaggggttc

Petition 870180128975, of 9/11/2018, p. 45/52

R 35 tgcaccgagcgcgcggtcgaggtcgcctcgctaggtgtccagatccacggcggcatgggtttcatcgaggaaaccggcgtggcccagttctatcgcgatg cccgcatcctgccgatctacgagggcaccaccgccattcaggccaatgacctgttgggtcgcaaggtgctgcgcgatggcggtcgcaccgcccgccgct ttgccgagatgatcgccgcaaccgagggtgagctgtccaagggtggcgcggcggcgcaacggatcgcccagcgcctggccgaagcgcgcgcggcct ttgccgccggcctcgaccatctgctggcaaccgccgggcaggaccccaaccgcgcctatgccggcagcgtgcccttcctgatgctgaccggcaatctgg ccacaggctggcagcttggcctgtcggcgctggccgccgaggccgagctggccaagggcggcgatgccgagttcctgcaggccaagatcgcaaccgc cgacatctttgcccagcaggtgctggtcgaatgttcggccgagcacagccgcatcaccgataccggcgacagcctgctgaccgcatcgctctga

Sequence No. 23 (dmdD) gtgacccaggatgtaacctccggctacagcaatctcgaccttgatctgcgcgacaatggcgtctgcgttgtgactctgaaccgcccggacaagcgcaacg cgctggatgtcgccaccatcgaggaactcgtcaccttcttcagcaccgcccatcgcaagggcgtgcgcgccgtggtgctgaccggcgcgggtgatcatttc tgcgccggccttgacctggtcgaacactggaaggccgaccgcagcgcggatgatttcatgcatgtctgcctgcgctggcacgaggcgttcaacaagatgg aatatggcggtgtgccgatcatcgccgcgcttcggggcgcggttgtcggcggcggtctggaactggccagcgccgcccacctccgggtcatggaccaga gcacctatttcgccctgcccgaaggccagcgcggcatcttcaccgggggcggcgccaccatccgcgtgtcggacatgatcggcaaataccggatgatcg acatgatcctgaccggtcgtgtctatcaggggcaagaggccgccgatctgggcctggcgcaatacatcaccgaaggatccagctttgacaaggcgatgg agctggccgacaagatcgccagcaacctgccgctgaccaacttcgccatctgttcggccatcagccacatgcagaacatgtcgggtctggacgccgcct atgccgaggcctttgtcggcggcatcgtcaacacccagcccgccgcccgcgaacggctcgaggccttcgccaacaagaccgcagcgcgcgtgcgccc caacagctga

Sequence No. 24 (metX) atgcccaccctcgcgccttcaggtcaacttgaaatccaagcgatcggtgatgtctccaccgaagccggagcaatcattacaaacgctgaaatcgcctatc accgctggggtgaataccgcgtagataaagaaggacgcagcaatgtcgttctcatcgaacacgccctcactggagattccaacgcagccgattggtggg ctgacttgctcggtcccggcaaagccatcaacactgatatttactgcgtgatctgtaccaacgtcatcggtggttgcaacggttccaccggacctggctccat gcatccagatggaaatttctggggtaatcgcttccccgccacgtccattcgtgatcaggtaaacgccgaaaaacaattcctcgacgcactcggcatcacca cggtcgccgcagtacttggtggttccatgggtggtgcccgcaccctagagtgggccgcaatgtacccagaaactgttggcgcagctgctgttcttgcagtttct gcacgcgccagcgcctggcaaatcggcattcaatccgcccaaattaaggcgattgaaaacgaccaccactggcacgaaggcaactactacgaatccg gctgcaacccagccaccggactcggcgccgcccgacgcatcgcccacctcacctaccgtggcgaactagaaatcgacgaacgcttcggcaccaaag cccaaaagaacgaaaacccactcggtccctaccgcaagcccgaccagcgcttcgccgtggaatcctacttggactaccaagcagacaagctagtaca gcgtttcgacgccggctcctacgtcttgctcaccgacgccctcaaccgccacgacattggtcgcgaccgcggaggcctcaacaaggcactcgaatccatc aaagttccagtccttgtcgcaggcgtagataccgatattttgtacccctaccaccagcaagaacacctct ccagaaacctgggaaatctactggcaatggc aaaaatcgtatcccctgtcggccacgatgctttcctcaccgaaagccgccaaatggatcgcatcgtgaggaacttcttcagcctcatctccccagacgaacaacccttcgacctacatttt

Sequence No. 25 (Mety) atgccaaagtacgacaattccaatgctgaccagtggggctttgaaacccgctccattcacgcaggccagtcagtagacgcacagaccagcg cacgaaaccttccgatctaccaatccaccgctttcgtgttcgactccgctgagcacgccaagcagcgtttcgcacttgaggatctaggccctgttt actcccgcctcaccaacccaaccgttgaggctttggaaaaccgcatcgcttccctcgaaggtggcgtccacgctgtagcgttctcctccggaca ggccgcaaccaccaacgccattttgaacctggcaggagcgggcgaccacatcgtcacctccccacgcctctacggtggcaccgagactcta ttccttatcactcttaaccgcctgggtatcgatgtttccttcgtggaaaaccccgacgaccctgagtcctggcaggcagccgttcagccaaacacc aaagcattcttcggcgagactttcgccaacccacaggcagacgtcctggatattcctgcggtggctgaagttgcgcaccgcaacagcgttcca ctgatcatcgacaacaccatcgctaccgcagcgctcgtgcgcccgctcgagctcggcgcagacgttgtcgtcgcttccctcaccaagttctaca ccggcaacggctccggactgggcggcgtgcttatcgacggcggaaagttcgattggactgtcgaaaaggatggaaagccagtattcccctac ttcgtcactccagatgctgcttaccacggattgaagtacgcagaccttggtgcaccagccttcggcctcaaggttcgcgttggccttctacgcgac accggctccaccctctccgcattcaacgcatgggctgcagtccagggcatcgacaccctttccctgcgcctggagcgccacaacgaaaacgc catcaaggttgcagaattcctcaacaaccac gagaaggtggaaaaggttaacttcgcaggcctgaaggattccccttggtacgcaaccaagg aaaagcttggcctgaagtacaccggctccgttctcaccttcgagatcaagggcggcaaggatgaggcttgggcatttatcgacgccctgaagc tacactccaaccttgcaaacatcggcgatgttcgctccctcgttgttcacccagcaaccaccacccattcacagtccgacgaagctggcctggc acgcgcgggcgttacccagtccaccgtccgcctgtccgttggcatcgagaccattgatgatatcatcgctgacctcgaaggcggctttgctgca atctag

Sequence No. 26 (sera) atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgcaccaaaaggcgctggaaagccttcgtgcagctg gttacaccaacatcgaatttcacaaaggcgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctgcgatccc gtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctattggctgtttctgtatcggaacaaaccaggttgatctggatgc ggcggcaaagcgcgggatcccggtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcgaactgctgctgctattgc gcggcgtgccggaagccaatgctaaagcgcaccgtggcgtgtggaacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgg gtatcatcggctacggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttacttttatgatattgaaaataaactgccgc tgggcaacgccactcaggtacagcatctttctgacctgctgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaa tatgatgggcgcgaaagaaatttcactaatgaagcccggctcgctgctgattaatgcttcgcgcggtactgtggtggatattccggcgctgtgtga tgcgctggcgagcaaacatctggcgggggcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacctctccgctgt

Petition 870180128975, of 9/11/2018, p. 46/52

R 35 gtgaattcgacaacgtccttctgacgccacacattggcggttcgactcaggaagcgcaggagaatatcggcctggaagttgcgggtaaattga tcaagtattctgacaatggctcaacgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgtcgtctgatgcacatccac gaaaaccgtccgggcgtgctaactgcgctgaacaaaatcttcgccgagcagggcgtcaacatcgccgcgcaatatctgcaaacttccgccc agatgggttatgtggttattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatgaaagctattccgggtaccattcgcgc ccgtctgctgtactaa

Sequence No. 27 (Serb) atgcctaacattacctggtgcgacctgcctgaagatgtctctttatggccgggtctgcctctttcattaagtggtgatgaagtgatgccactggattac cacgcaggtcgtagcggctggctgctgtatggtcgtgggctggataaacaacgtctgacccaataccagagcaaactgggt gcggcgatggtgattgttgccgcctggtgcgtggaagattatcaggtgattcgtctggcaggttcactcaccgcacgggctacacgcctggccc acgaagcgcagctggatgtcgccccgctggggaaaatcccgcacctgcgcacgccgggtttgctggtgatggatatggactccaccgccatc cagattgaatgtattgatgaaattgccaaactggccggaacgggcgagatggtggcggaagtaaccgaacgggcgatgcgcggcgaactc gattttaccgccagcctgcgcagccgtgtggcgacgctgaaaggcgctgacgccaatattctgcaacaggtgcgtgaaaatctgccgctgatg ccaggcttaacgcaactggtgctcaagctggaaacgctgggctggaaagtggcgattgcctccggcggctttactttctttgctgaatacctgcg cgacaagctgcgcctgaccgccgtggtagccaatgaactggagatcatggacggtaaatttaccggcaatgtgatcggcgacatcgtagacg cgcagtacaaagcgaaaactctgactcgcctcgcgcaggagtatgaaatcccgctggcgcagaccgtggcgattggcgatggagccaatg acctgccgatgatcaaagcggcagggctggggattgcctaccatgccaagccaaaagtgaatgaaaaggcggaagtcaccatccgtcacg ctgacctgatgggggtattctgcatcctctcaggcagcctgaat cagaa gtaa

Sequence No. 28 (serC) atggctcaaatcttcaattttagttctggtccggcaatgctaccggcagaggtgcttaaacaggctcaacaggaactgcgcgactggaacggtct tggtacgtcggtgatggaagtgagtcaccgtggcaaagagttcattcaggttgcagaggaagccgagaaggattttcgcgatcttcttaatgtcc cctccaactacaaggtattattctgccatggcggtggtcgcggtcagtttgctgcggtaccgctgaatattctcggtgataaaaccaccgcagatt atgttgatgccggttactgggcggcaagtgccattaaagaagcgaaaaaatactgcacgcctaatgtctttgacgccaaagtgactgttgatggt ctgcgcgcggttaagccaatgcgtgaatggcaactctctgataatgctgcttatatgcattattgcccgaatgaaaccatcgatggtatcgccatc gacgaaacgccagacttcggcgcagatgtggtggtcgccgctgacttctcttcaaccattctttcccgtccgattgacgtcagccgttatggtgtaa tttacgctggcgcgcagaaaaatatcggcccggctggcctgacaatcgtcatcgttcgtgaagatttgctgggcaaagcgaatatcgcgtgtcc gtcgattctggattattccatcctcaacgataacggctccatgtttaacacg ccgccgacatttgcctggtatctatctggtctggtctttaaatggctgaaagcgaacggcggtgtagctgaaatggataaaatcaatcagcaaaa agcagaactgctatatggggtgattgataacagcgatttctaccgcaatgacgtggcgaaagctaaccgttcgcggatgaacgtgccgttcca gttggcggacagtgcgcttgacaaattgttccttgaagagtcttttgctgctggcctt catgcactgaaaggtcaccgtgtggtcggcggaatgcg cgcttctatttataacgccatgccgctggaaggcgttaaagcgctgacagacttcatggttgagttcgaacgccgtcacggttaac

Claims (24)

1. MICRORGANISM WITH SYNTHETIC METABOLIC TRACKS OPTIMIZED FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR characterized by being a fermentative process for the production of amino acids, specifically Lethionine, through the use of different carbon sources , such as glucose, glycerol, xylose and arabinose, separately or simultaneously. 2. MICRO-ORGANISM WITH SYNTHETIC OPTIMIZED METABOLIC TRACKS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claim 1, characterized by the application of genetic engineering techniques to obtain a superproductive bacterial strain of L-methionine from the carbon sources mentioned above. 3. MICRORGANISM WITH SYNTHETIC OPTIMIZED METABOLIC PATHWAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claim 2, characterized by the attenuation of the metJ gene by means of complementary RNA, reducing the negative regulation of this gene on genes producing L-methionine. 4. MICRO-ORGANISM WITH SYNTHETIC METABOLIC TRACKS OPTIMIZED FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 and 3, characterized by the fact that it is the attenuation of the metI gene by means of complementary RNA, decreasing the expression of said Lmethionine importer. 5. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONINE FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 4, characterized by the fact that it is the attenuation of the lysA gene by means of complementary RNA, decreasing the production of L-lysine and increasing the metabolic flow for the production of L-methionine. 6. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC PATHWAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 5, characterized by the attenuation of the pta and ackA genes by means of complementary RNA, decreasing the production of acetic acid and increasing the metabolic flow for the production of L-methionine. 7. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE Petition 870180128975, of 9/11/2018, p. 48/52 2 I 5 SOURCES OF CARBON AND SULFUR according to claims 2 to 6, characterized by being a metabolic valve system based on the nar promoter to control the expression of the cysE and aspC genes inserted in the chromosome of that strain, increasing the metabolic flow for the production of cysteine and aspartate and attenuating its expression in situations anaerobia so that there is no metabolic overload in the occurrence of such events. 8. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONINE FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 7, characterized by the fact that it is the attenuation of the thrB gene by means of complementary RNA, decreasing the production of threonine and increasing the metabolic flow for the production of L-methionine. 9. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC TRACKS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 8, characterized by being a metabolic valve system based on the nar promoter to control the expression of the metF and metL genes inserted in the chromosome of that strain, increasing the metabolic flow for the production of 5.10 tetrahydrofolate and homoserine, and attenuating its expression in anaerobic situations so that there is no metabolic overload in the occurrence of such events. 10. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 9, characterized by being a metabolic valve system based on the nar promoter for the control of the expression of the glpK, glpF, metC and cysM genes inserted in the chromosome of that strain, increasing the metabolic flow for glycerol consumption and homocysteine production and cysteine, and attenuating its expression in anaerobic situations so that there is no metabolic overload in the occurrence of such events. 11. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 10, characterized by being a metabolic valve system based on the nar promoter to control the expression of the glyA and metB genes inserted in the chromosome of that strain, increasing the metabolic flow for the production of glycine and cystathionine, and attenuating its expression in situations anaerobia so that there is no metabolic overload in the occurrence of such events. 12. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC PATHWAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLES Petition 870180128975, of 9/11/2018, p. 49/52 3 I 5 SOURCES OF CARBON AND SULFUR according to claims 2 to 11, characterized by being a metabolic valve system based on the nar promoter to control the expression of the metH and malY genes inserted in the chromosome of that strain, increasing the metabolic flow for the production of methionine and homocysteine, and attenuating its expression in situations anaerobia so that there is no metabolic overload in the occurrence of such events. 13. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 12, characterized by the insertion of the serA, serB and serC gene set under the control of a constitutive synthetic promoter in the region corresponding to the lacZ gene, increasing the metabolic flow for the production of L-serine and consequently L-methionine. 14. MICRO-ORGANISM WITH SYNTHETIC OPTIMIZED METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 13, characterized by being a metabolic valve system based on the nar promoter to control the expression of the pntA, pntB and sthA genes inserted in the chromosome of that strain, increasing the production of NADH and NADPH, and attenuating its expression in situations of anaerobia so that there is no metabolic overload in the occurrence of such events. 15. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC TRACKS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 14, characterized by being a metabolic valve system based on the nar promoter for the control of the expression of the ftsA and ftsZ genes inserted in the chromosome of that strain, promoting cell division even at high cell density, and attenuating its expression in anaerobic situations so that there is no metabolic overload in the occurrence of such events. 16. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC TRACKS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 15, characterized by being a metabolic valve system based on the nar promoter to control the expression of the pyc and yJeH genes inserted in the chromosome of that strain, promoting the production of oxaloacetate and increasing the export of methionine to the extracellular medium, and attenuating its expression in anaerobic situations so that there is no metabolic overload in the occurrence of such events. Petition 870180128975, of 9/11/2018, p. 50/52 4 I 5 17. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC PATHWAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 16, characterized by the insertion of regulation regulation of the metK gene expression through a metabolic valve induced by arabinose, where the regulation of metK occurs through the controlled expression of a complementary RNA, attenuating the expression of that gene and thus reducing the conversion from L-methionine to S-adenosylmethionine. 18. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC PATHWAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 to 17, characterized by being a fermentative process for the production of amino acids, specifically L-methionine, through the use of different sources of sulfur, reduced or not, such as dimethyl disulfide (DMDS), ammonium sulfate, dimethylsulfoniopropionate (DMSP) , taurine, alkanes sulfonates, thiosulfate. 19. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC TRACKS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 2 and 18, characterized by the arabinose-controlled metabolic valve system that controls the expression of the metory and metY gene set of Corynebacterium glutamicum inserted in the chromosome of that strain, which is able to incorporate methanethiol and dimethyl disulfide (DMDS) into a methionine alternative regulated for the production of said amino acid. 20. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC TRACKS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claim 2 and 19, characterized by a metabolic valve system controlled by arabinose that controls the expression of the dmdA gene from Ruegeria pomeroyi inserted in the chromosome of that strain, which is capable of converting dimethylsulfoniopropionate into methylmercaptopropionate. 21. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claim 2 and 20, characterized by a metabolic valve system controlled by arabinose that controls the expression of the dmdB gene from Ruegeria pomeroyi inserted in the chromosome of that strain, which is capable of converting methylmercaptopropionate into MMPA-coenzyme A. 22. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claim 2 and Petition 870180128975, of 9/11/2018, p. 51/52 5 I 5 21, characterized by a metabolic valve system controlled by arabinose that controls the expression of the dmdC gene from Ruegeria pomeroyi inserted in the chromosome of that strain, which is able to convert MMPAcoenzyme A into methylthioacryloyl-coenzyme A. 23. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC WAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claim 2 and 22, characterized by a metabolic valve system controlled by arabinose that controls the expression of the dmdD gene from Ruegeria pomeroyi inserted in the chromosome of that strain, which is capable of converting methylthioacryloyl-coenzyme A into methanethiol. 24. MICRO-ORGANISM WITH OPTIMIZED SYNTHETIC METABOLIC PATHWAYS FOR THE PRODUCTION OF L-METHIONIN FROM MULTIPLE SOURCES OF CARBON AND SULFUR according to claims 1 and 2, characterized by being a genetically modified microorganism capable of metabolizing variable carbon sources residues from the production of biofuels or from another process that results in the formation of a residue containing glucose, glycerol, xylose and / or arabinose.