Method for producing acids with microbial cell strains

Description

1 / 34 METHOD FOR PRODUCING ACIDS WITH MICROBIAL CELL STRAINS (Divided from BR 11 2020 026031 2, filed on 06 / 17 / 2019) PRIORITY CLAIM

[001] This application claims priority to U.S. Patent Application No. 62 / 686,463 filed June 18, 2018, the full contents of which are incorporated herein by reference. TECHNICAL FIELD

[002] The revelation generally refers to strains of microbial cells that produce excess organic acid and methods of producing them. FUNDAMENTALS

[003] Organic acids refer to carbon-containing compounds that have acidic properties. Examples of organic acids include acetic acid, citric acid, gluconic acid, lactic acid, propionic acid, among many others. Because they are completely degradable, organic acids can be used in the production of biodegradable polymers. They also have other important industrial applications, including as food additives. SUMMARY

[004] The disclosure provides suitable microbial cell strains for industrial-scale production of organic acids and methods for producing and isolating such cell strains.

[005] In one aspect, a method of producing and isolating a microbial cell strain is provided, wherein the isolated microbial cell strain produces an excess of an organic acid compared to the parental microbial cell strain. The method uses serial passage of a parental strain in pH-controlled culture medium supplemented with the organic acid, preferably in non-immobilized culture, where the pH is controlled to a value above the pKa value of the organic acid. In some embodiments, the pH is preferably in the range between about 5.5 and about 7.5, more preferably at or near Petition 870260076101, dated 07 / 30 / 2026, page 9 / 52 2 / 34 to neutral, between about 6.0 and about 7.0, and most preferably around 7.0. In some embodiments, the culture medium is solidified. In some embodiments, the culture medium is supplemented with organic acid in a sufficient amount to inhibit the growth of normal microbial cells, for example, to reduce the doubling rate or growth rate, for example, by at least 5%, 10%, 20%, 30%, 40%, 50% or more. In some embodiments, the organic acid is supplemented in an amount that progressively increases in successive iterations of the serial passage. In some embodiments, the organic acid is supplemented in the same amount in successive iterations of the serial passage. In some embodiments, the organic acid is propionic acid, lactic acid, acetic acid, or butyric acid. In some embodiments, the organic acid is propionic acid, for example, the culture medium is supplemented with about 1.0%-3.0% propionic acid, for example, about 3.0% propionic acid. In some embodiments, the parent cell strain is a wild-type organism. In some embodiments, the parent cell strain is a microbial cell strain that is derived from single-celled microbes.

[006] In another aspect, a microbial cell strain that overproduces an organic acid is provided, wherein the microbial cell strain is produced and isolated using serial passage in pH-controlled culture media supplemented with the organic acid, wherein the pH is controlled to a value above the pKa value of the organic acid, preferably in the range between about 5.5 and about 7.5, more preferably at or near neutral, between about 6.0 and about 7.0, and most preferably at about 7.0.

[007] In another aspect, a microbial cell strain that overproduces an organic acid is provided, where the microbial cell strain has mutations that primarily alter, from Petition 870260076101, dated 07 / 30 / 2026, page 10 / 52 3 / 34 directly or indirectly, the structure, composition, and / or function of the cell envelope. Preferably, the microbial cell strain includes at least 2 genome mutations identified in Table 3 or analogous mutations. More preferably, the microbial cell strain includes all of the genome mutations identified in Table 3 or homologous mutations. In one embodiment, the microbial cell strain includes mutations in at least 2 genes identified in Table 3 or analogous mutations thereof. In another embodiment, the microbial cell strain includes mutations in all of the genes identified in Table 3 or their homologs (e.g., homologous genes in other species described herein). In another embodiment, the microbial cell strain includes a mutation in the antigen-O ligase domain-containing protein. In another embodiment, the microbial cell strain includes a mutation in the M18 aminopeptidase family.In another embodiment, the microbial cell strain includes a mutation in amino acid permease. In another embodiment, the microbial cell strain includes a mutation in adenine glycosylase.

[008] The microbe can be any microbe that produces an organic acid. In one embodiment, the microbe is of the genus Propionibacterium (Acidipropionibacterium), and more preferably the species P. acidipropionic. In another embodiment, the microbe is of the genus Lactobacillus, and more preferably the species L. acidophilus. In another embodiment, the microbe is of the genus Acetobacter. In another embodiment, the microbe is of the genus Gluconobacter. In another embodiment, the microbe is of the genus Clostridium, and more preferably the species C. butyricum. In some embodiments, the organic acid is propionic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is acetic acid. In some embodiments, the organic acid is butyric acid.

[009] Methods of producing acids are also provided here. Petition 870260076101, dated 07 / 30 / 2026, page 11 / 52 4 / 34 organic using the methods and microbes described here. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows the growth of wild-type and mutant P. acidipropionici in solid buffered medium with or without the addition of 1.0% PA, showing a 3-1 strain phenotype.

[0011] FIG. 2 shows the production of PA by a mutant strain of P. acidipropionici (strain 3-1) relative to the wild type in bioreactors using bleached American Beauty Cake Flour (WFM as bolus). Inoculation 1:200, working culture 1L, glucose equivalent 5% (weight / volume) WFM, 30oC, pH 7 (NaOH, 5M). DETAILED DESCRIPTION

[0012] The most common organic acids are carboxylic acids, whose acidity is associated with the carboxyl group (-COOH). They are generally weak acids with pKa values ​​between about 4-5. Propionic acid (PA), for example, is a carboxylic acid with the chemical formula C2H5COOH or C3H6O2. It is a colorless, oily liquid with a pungent odor (similar to Swiss cheese and sweat) and has physical properties between those of the smaller carboxylic, formic, and acetic acids, and the larger fatty acids. It has a molecular weight of about 74.1 g / mol, and a pKa of about 4.9, which means that in a solution having a pH of about 4.9, half of the PA is in the protonated (or undissociated), uncharged state (C2H5COOH), while the other half is in the deprotonated (or dissociated), negatively charged state (C2H5COO-), known as the propionate or propanoate ion, which can form salt or ester compounds.As the pH decreases (becoming more acidic), more active compounds are in the protonated, uncharged state; when the pH increases (becoming more basic), more active compounds are in the deprotonated, negatively charged state.

[0013] Because PA inhibits the growth of mold and some bacteria at levels between 0.1 and 1% (weight / volume), PA and its salts Petition 870260076101, dated 07 / 30 / 2026, page 12 / 52 5 / 34 are used as preservatives in both animal feed and human food (such as baked goods). In the United States In the United States, PA is generally recognized as safe, or GRAS, by the Food and Drug Administration when used as a food additive. It is also approved for use as a food additive in Australia, New Zealand, and the EU. In addition, PA is an important intermediate in the synthesis of other chemicals, such as cellulose-derived plastics, pesticides, fruit flavorings, perfume bases, and pharmaceuticals.

[0014] Although they are widely distributed in nature, the commercial production of organic acids generally depends on chemical synthesis because it is more economically competitive. For example, PA is currently produced commercially almost exclusively through petrochemical processes. As crude oil and petrochemical prices rise, along with rapid development in the field of biotechnology, the economic gap between the manufacturing costs of PA through chemical synthesis and through microbial fermentation is narrowing. Associated with growing concerns about energy scarcity and environmental pollution, there has been increasing interest in the commercial-scale biosynthesis of organic acids, such as PA, from renewable resources.

[0015] The microbial production of organic acids by fermentation has been known and used for centuries. For example, Aspergillus niger and Yarrowia lipolytica have been used to produce citric acid; Lactobacillus has been used to produce lactic acid; Clostridium has been used to produce acetic acid; Aspergillus niger and Gluconobacter have been used to produce gluconic acid.

[0016] Propionibacterium is the microorganism most frequently used in the production of PA (as well as vitamin B12). Petition 870260076101, dated 07 / 30 / 2026, page 13 / 52 6 / 34 and Swiss cheese). Propionibacterium is a genus of Gram-positive, anaerobic, non-motile, non-spore-forming, rod-shaped bacteria that includes the species P. freudenreichii, P. acidifaciens, P. cyclohexanicum, P. australiense, P. acidipropionici, P. jensenii, P. thoenii, P. microaerophilum, P. olivae, P. damnosum, P. propionicum, P. acnes, P. avidum, P. granulosum, P. humerusii, and P. lymphophilum. For industrial production of PA, the most commonly used strain is P. acidipropionici. (A proposal has been made to reclassify the species within the genus Propionibacterium into three new genera: Acidipropionibacterium, Cutibacterium, and Pseudopropionibacterium (Scholz & Kilian 2016). However, Propionibacterium acidipropionici and Acidipropionibacterium acidipropionici are still used somewhat interchangeably. The ideal pH and temperature for Propionibacterium cell growth are approximately 6.0-7.0 and approximately 30-37°C, respectively (Ahmadi et al. 2017). Cell growth is inhibited at pH lower than approximately 5.0, although fermenters starting at neutral pH can reach pH 4.4 (Rehberger and Glatz 1998). Ahmadi et al. presents an overview of PA production on various carbon sources by several Propionibacterium species, as reported in the literature (Ahmadi et al. 2017), which is incorporated here by reference.

[0017] PA can also be produced by other anaerobic bacteria, such as certain species of Anaerovibrio, Bacteroides, Clostridium, Fusobacterium, Megasphaera, Propionispira, Selenomonas, and Veillonella.

[0018] There are several fermentation pathways that convert carbon sources into PA through a series of enzymatic reactions. The main fermentation pathway involved in PA production, especially in propionibacteria, is known as the Wood-Werkman cycle, which produces propionate from pyruvate, the terminal product of glucose, and involves many Petition 870260076101, dated 07 / 30 / 2026, page 14 / 52 7 / 34 intermediates, including oxaloacetate, malate, fumarate, succinate, succinyl-CoA, methylmalonyl-CoA, and propionyl-CoA, and many enzymes, including oxaloacetate transcarboxylase, biotin-dependent carboxytransferase, CoA transferase, fumarate hydrolase, lactate dehydrogenase, B12-dependent methylmalonyl-CoA mutase, malate dehydrogenase, and succinate dehydrogenase.

[0019] Although most pyruvate is converted to PA / propionate during fermentation, some is converted to acetate. The acetate formation pathway involves acetyl CoA and acetyl phosphate intermediates, and the pyruvate dehydrogenase, phosphotransacetylase, and acetate kinase enzyme complex.

[0020] Various carbon sources have been used for the production of microbial PA, including glucose, fructose, maltose, sucrose, xylose, lactose, glycerol, lactate, flour hydrolysate, molasses, whey, and a combination thereof. Various culture systems, such as batch, fed-batch, and continuous fermentation, have been used.

[0021] However, for commercial-scale microbial production of organic acids to be economically viable, the fermentation process must be able to convert carbon sources with a high yield (amount of organic acid produced from the carbon source, typically measured in g / g) and high productivity (rate of organic acid production, typically measured in g / Lh).

[0022] Various fermentation technologies, including fed-batch systems, continuous culture, multi-stage, cell immobilization, and extractive fermentation, have been explored to increase the yield of organic acid production. However, the modest increase in yield and productivity often obtained is offset by a significant increase in production costs.

[0023] For example, co-culture methods have been used Petition 870260076101, dated 07 / 30 / 2026, page 15 / 52 8 / 34 to produce PA using whey as feedstock (WO 85 / 04901; EP 0141642 A1). WO 85 / 04901 describes the use of Lactobacillus casei subspecies rhamnosus in the presence of Veillonella cricetid to interconvert lactate into propionate through a two-stage fermentation process. In the first stage, carbohydrates are converted into lactic acid by L. casei; in the second stage, lactic acid is fermented into propionate by V. cricetid. (Both the genera Lactobacillus and Veillonella belong to the phylum Firmicutes, while the genus Propionibacterium belongs to the phylum Actinobacteria.) EP 0141642 also describes the use of a mixed culture of lactic acid-producing bacteria (L. casei) and PA-producing bacteria (P. shermanii) to maximize fermentation yield. The co-culture systems of WO 85 / 04901 and EP 0141642 are reported as being very productive in terms of PA production from lactose, with final yields in the range of 20-100 g / L. However, such co-culture systems have considerable implications for process parameters. For example, they suffer from a lack of control regarding the growth and metabolic activity of each member of the system, which can lead to undergrowth of one member or contribute to the formation of the desired product. A lack of reproducibility is common with co-culture systems.

[0024] A major problem associated with microbial production of organic acids is the strong inhibitory effect of the end product on cell growth and the fermentation process, leading to low production yield and productivity. Acid tolerance has been presumed to be crucial for improving the yield and productivity of PA-producing strains (Rehberger and Glatz 1998). The high inhibitory effect of PA at pH 4.5-5.0 compared to lactic acid has been attributed to the fact that in this pH range, about half of PA (which has a pKa of about 4.9) would be present in the form Petition 870260076101, dated 07 / 30 / 2026, page 16 / 52 9 / 34 undissociated, protonated, and uncharged, while lactic acid (which has a pKa of about 3.1) would be primarily in the dissociated, deprotonated, and charged form. It was assumed that since undissociated acid could penetrate the cell wall and membrane more easily, more PA than lactic acid could enter the cell and exert its inhibitory effect. Enhancing acid tolerance was thus considered an effective strategy to alleviate end-product inhibition and improve PA production. Consequently, attempts were made to create “acid-tolerant” mutants of propionibacteria under high PA content and / or uncontrolled or low pH conditions.

[0025] For example, adaptive evolution via serial passage has been used to obtain P. acidipropionici mutant with improved acid tolerance (Woskow and Glatz 1991; Zhu et al. 2010). Serial passage is a method of growing microorganisms, such as bacteria, in two or more iterations in artificial environments, often created in a laboratory setting, to generate spontaneous mutations in the microorganisms as they develop throughout the course of the experiment to adapt to one or more novel environmental conditions designed for the experiment. For example, repeatedly subjecting microbes to extreme acidic conditions will lead to spontaneous mutations that allow the microbes to adapt to or tolerate such conditions.

[0026] In previous work, to create mutations that confer acid tolerance, mutant strains of P. acidipropionici were adapted to increase PA concentrations by repeated and serial transfers in selection media containing increased amounts of PA (from 0.5% to 5% (Woskow and Glatz 1991) or 1.5 g / L to 20 g / L (Zhu et al. 2010)) over a period of one year or more. Particularly, in these experiments, the pH in the selection media having increased amounts of PA was not Petition 870260076101, dated 07 / 30 / 2026, p. 17 / 52 10 / 34 controlled, presumably because it was assumed that the inhibitory effects on cell growth and PA production were caused by PA acidity.

[0027] P. acidipropionici mutant(s) with enhanced PA production have also been obtained by immobilization and adaptation in a fibrous bed bioreactor (Suwannakham and Yang 2005; Suwannakham 2005). The ability to obtain acid-tolerant mutant(s) in a fibrous bed bioreactor has been attributed to the high cell density and viability maintained in the bioreactor and the distinct physiology and survival capacity of immobilized cells as a result of their direct contact with each other and with a solid surface. The higher PA production has been attributed in part to the higher levels of oxaloacetate transcarboxylase and CoA transferase activity in the mutant(s). Despite the higher PA yield, in the high-density cell fibrous bed bioreactor, cell growth is limited. Furthermore, fibrous bed bioreactors are expensive and not scalable, and their uses are limited to small to medium-scale productions.

[0028] More recently, random mutagenesis strategies, such as genome shuffling, have been used to accelerate targeted microbial evolution. For example, Guan et al. reported the use of genome shuffling to generate an acid-tolerant mutant strain of P. acidipropionici (Guan et al. 2012). To obtain the strain, four successive cycles of genome shuffling via protoplast fusion were performed, and the acid-tolerant strain was selected using media supplemented with increased amounts of PA (from 5 to 20 g / L). Again, pH in the selection medium having increased amounts of PA was not controlled, presumably because it was assumed that the inhibitory effects on cell growth and PA production were caused by the acidity of PA.

[0029] Subsequent analyses identified 24 proteins that Petition 870260076101, dated 07 / 30 / 2026, page 18 / 52 11 / 34 differed significantly between the scrambled parental strains (Guan et al. 2014). The detected proteins were found to belong to four broad functional classes: cellular metabolism and energy production; DNA replication, RNA synthesis, and translation; post-translational modification, protein duplication, and chaperones; and hypothetical proteins of unknown function.

[0030] In another study, genome shuffling was used to generate acid-tolerant mutant strains of P. acidipropionici, P. intermedium, and P. jensenii (WO 2017 / 055932 A2). Three successive genome shuffling cycles were performed for each set of strains, each followed by selection of colonies from the acid side (pH 3) of pH / PA gradient plates prepared using agar culture medium supplemented with 5 g / L of PA at pH 3 or pH 6.5. Final individual recombinants were randomly selected after serial dilutions in culture medium plates and screened in a 96-well plate containing 100 μl of culture medium at pH 5 and 25 g / L of PA. The mutant strains were reported to have enhanced PA yields compared to native Propionibacterium and other known derived strains. Genomic analyses of one of the mutant strains of P.The researchers identified several modified genes, including those encoding the ABC polar amino acid transporter, the cytochrome C biogenesis protein, the ABC multiple sugar transporter, the large ribosomal RNA subunit, the long-chain acyl-CoA synthetase, and the cation diffusion facilitator. In addition, an extra copy of the complete ribosomal RNA gene and an extra copy of the arginine deiminase (ArgR) regulon with a point mutation were found in the mutant strain.

[0031] Targeted metabolic modification of propionibacteria has also been used to increase PA production. These studies generally target enzymes involved in pyruvate metabolism pathways to, for example, Petition 870260076101, dated 07 / 30 / 2026, p. 19 / 52 12 / 34 or inhibit the acetate formation pathway or enhance the PA formation pathway. For example, Yang and Suwannakham created strains of P. acidipropionici modified with genes encoding acetate kinase (which catalyzes the conversion of acetyl phosphate to acetate) and / or phosphotransacetylase (which catalyzes the conversion of acetyl CoA to acetyl phosphate) knockouts, with the aim of eliminating or reducing acetate formation and thus intensifying PA production (US 2011 / 0151529 A1; Suwannakham 2005).

[0032] Yang et al. created modified strains of P. acidipropionici and P. freudenreichii subsp. shermanii transformed with propionyl-CoA:succinate CoA transferase genes to increase PA production by overexpression of propionyl-CoA:succinate CoA transferase, which catalyzes the conversion of propionyl CoA to propionate (WO 2012 / 064883 A2). The resulting strains were reported to have increased PA production and PA resistance, as well as resistance to acidic pH in general. It is believed that the increased CoA transferase activity increases carbon flux through the PA formation pathway relative to the acetate formation pathway.

[0033] The table below describes a list of genes that have been manipulated using recombinant DNA. These genes constitute conventional genetic targets where regulatory mutations can be expected to increase PA yields. TABLE 1 Gene(s) Organism Effect Reference OtsA (trehalose biosynthesis) P. acidipropionici Artificially overexpressed Jiang et al. 2015 Several genes in arginine deaminase and glutamate decarboxylase systems P. jensenii Artificially overexpressed Guan et al. 2016 Petition 870260076101, dated 07 / 30 / 2026, page 20 / 52 13 / 34 PropionylCoA:succinate CoA transferase P. acidipropionici P. shermanii Artificially overexpressed Wang et al. 2015 WO 2012 / 064883 A2 Acetate kinase P. acidipropionici Artificial knockout Suwannakham et al. 2006 Suwannakham 2005 US 2011 / 0151529 A1 Phosphotransacetylase P. acidipropionici Artificial knockout US 2011 / 0151529 A1

[0034] Targeted genetic modification in propionibacteria, however, is challenging. As an initial issue, the effect of acid alteration and stress on bacterial physiology is complex and not well understood, making it difficult to improve tolerance to organic acids through the manipulation of specific genes. In fact, despite knowledge about the identity of the intermediates and enzymes in the Wood-Werkman pathway that form PA in propionibacteria, genetic manipulations of genes in this pathway have not increased PA yields to a significant extent.

[0035] Furthermore, the high GC content in propionibacteria makes it difficult to identify individual gene locations and all coding regions in the genome, which complicates genetic manipulation. In addition, there are only a small number of cloning vectors available for introducing recombinant DNA into propionibacteria cells, which are known to have low transformation efficiency. The selection of transformants is also complicated by the ability of propionibacteria to rapidly develop spontaneous resistance to antibiotic markers.

[0036] In addition to these challenges, the use of recombinant DNA to produce microbial cell strains is incompatible with the development of an organic food ingredient such as PA. At least in the United States, PA or other organic acids produced by genetically modified microbes cannot be labeled as organic or “natural preservatives,” which is Petition 870260076101, dated 07 / 30 / 2026, page 21 / 52 14 / 34 especially important in the food industry. Therefore, there remains a need for new microbial strains suitable for industrial-scale production of organic acids and methods for producing and isolating such strains.

[0037] The toxicity of organic acids to microbes is not well understood despite its relevance in the food and chemical industries that use fermentation for organic acid production. Despite knowledge about the identity of the intermediates and enzymes in the Wood-Werkman pathway that forms PA in propionibacteria, genetic manipulations of the genes in this pathway have not increased PA yields to a significant extent. One reason could be that these genes do not limit PA formation. Therefore, altering their sequence or expression would not change PA levels. Instead, it is argued here that other cellular targets control PA yields, but their identities could not be predicted based on current knowledge. The unknown process is what limits PA formation. Since this process is unknown, the genes involved in this process cannot be predicted.

[0038] Previous efforts in creating PA-resistant bacteria through serial passage or genome shuffling have generally used media with increased amounts of PA, but either without pH control or at a pH significantly below the pKa of PA. This is based on the idea that toxicity, and therefore resistance, arises from the concentration of the organic acid. However, this approach does not consider the mechanism of organic acid uptake by the cell, which involves the transport system, which depends on the nature of the transporter and the membrane or envelope in which it is located.

[0039] Organic acids are weak acids with pKa values ​​generally between about 4-5. The relationship between pH and pKa is described by the Henderson-Hasselbalch equation: pH = pKa + log10([A-] / [HA]); Petition 870260076101, dated 07 / 30 / 2026, page 22 / 52 15 / 34 where [HA] is the concentration of the protonated, undissociated, and uncharged weak acid, and [A-] is the concentration of the deprotonated, dissociated, and negatively charged conjugate base. In a typical fermentation process, the pH of the microbial culture, when the organic acid reaches its maximum concentration, is approximately at the pKa of the organic acid without the use of a buffering agent. A solution having a pH of about 4-5 is not as acidic in relation to the known pH tolerance of acid-producing bacteria. Most of these bacteria grow at pH values ​​in this range, although the optimum pH for cell growth is typically around 6-7.

[0040] Intracellular transport of organic acids can be achieved through diffusion or through the action of membrane transport protein systems depending on whether the organic acids are charged or uncharged. When organic acids are not deprotonated or dissociated, they are not charged. In this state, they can diffuse across cell membranes without relying on transport systems. Charged molecules, however, always require a transport system to be translocated across membranes.

[0041] At a pH value that equals its pKa value, half of the organic acid is in the protonated (or undissociated), uncharged form, while the other half is in the deprotonated (or dissociated), negatively charged form. At pH values ​​below their pKa values, organic acids would be mostly uncharged because their carboxyl groups would be protonated. At pH values ​​above their pKa values, organic acids would be mostly deprotonated or dissociated and therefore negatively charged.

[0042] With high concentrations of organic acid, the pH is relatively low, and the organic acid would be primarily in the uncharged state and could diffuse into the cell in its acidic form. This is the basis for previous efforts to Petition 870260076101, dated 07 / 30 / 2026, page 23 / 52 16 / 34 Isolate organic acid-resistant microbes either without pH control or at a pH significantly below the pKa of the organic acid. The approach, in theory, could generate cell strains with mutations that produce resistance due to the entry of organic acid into the cell based on diffusion. It is assumed that uncharged organic acid diffuses through the cell membrane into the cytoplasm and releases protons due to the relatively alkaline pH inside the cell; the increase in intracellular acidity inhibits cell growth and organic acid formation. In other words, it was presumed that organic acids in their uncharged state limited their own production. Despite the published literature and patents, in the applicants' experience, this approach does not effectively generate resistant microbes and may require years of progress to become effective.

[0043] The applicants hypothesized that it was not the acidity of the organic acid that was toxic, as previously assumed by others. Rather, it was the deprotonated, negatively charged form or the neutral salt of the organic acid (propionate) that was toxic, and would be more effective as a selection agent to recover spontaneous resistance mutations.

[0044] Unlike previous efforts, the applicants hypothesized that using pH control at a value above the pKa value of the organic acids to be produced, and preferably at least 1 unit above, would ensure that most of the organic acids remained in a charged and deprotonated form. In this form, they would remain dependent on protein transport systems for intracellular uptake. This would prevent the recovery of cell strains with mutations that produced resistance due to diffusion-based organic acid entry into cells, should such mutations be discovered.

[0045] Specifically, the process used was the serial passage of the initial microbial cell strain (usually, but not Petition 870260076101, dated 07 / 30 / 2026, page 24 / 52 17 / 34 necessarily, a wild type) in free cell culture (i.e., not immobilized or planktonic) in a bacteriological culture medium supplemented with organic acid of interest in an amount sufficient to inhibit normal microbial growth (either in progressively increasing amounts or in the same amount for all passages) under conditions of continuous pH control to a specific pH that is above the pKa value of the organic acid. The pH is controlled to a value above the pKa value of the organic acid, preferably in the range between about 5.5 and about 7.5, more preferably at or near neutral, between about 6.0 and about 7.0, and most preferably at about 7.0. Although the present examples describe the use of Propionibacterium, other microbes may be used that are fermentative organisms that excrete organic acids, for example, Lactobacillus, Acetobacter, Gluconobacter, or Clostridium. The organic acid used may be, for example, PA, lactic acid, acetic acid, or butyric acid. In some embodiments, the microbe is of the genus Propionibacterium (Acidipropionibacterium), and more preferably the species P. acidipropionici, and the organic acid is PA. In some embodiments, the microbe is of the genus Lactobacillus, and more preferably the species L. acidophilus, and the organic acid is lactic acid. In some embodiments, the microbe is of the genus Acetobacter or the genus Gluconobacter, and the organic acid is acetic acid. In some embodiments, the microbe is of the genus Clostridium, and more preferably the species is C. butyricum, and the organic acid is butyric acid.

[0046] Using their pH-controlled serial passage method, the applicants were able to create and isolate a new microbial strain having increased organic acid production compared to the parental strain in less than two weeks, much faster than using the conventional serial passage method described in Woskow and Glatz 1991, which generally takes Petition 870260076101, dated 07 / 30 / 2026, page 25 / 52 18 / 34 at least one year. The applicants' method is also much less complex and more easily scalable than other random mutagenesis methods, such as genome shuffling and cell immobilization in a fibrous bed bioreactor or targeted genetic modification. Organic acids produced by mutant cell strains created and isolated using serial passage with pH control can be labeled as organic or natural preservatives, which is especially important in the food industry.

[0047] The same method of serial passage with pH control can be used to produce and isolate a variety of microbes, including but not limited to propionibacteria, lactobacilli, acetic acid bacteria, and clostridia, which overproduce various organic acids, including but not limited to PA, lactic acid, acetic acid, and butyric acid. All charged molecules depend on transport systems and their associated membranes / envelopes to function. Alterations in these cellular components would achieve the same result as described here for propionate for other organic acids.

[0048] The same screening method (i.e., use of bacteriological culture medium supplemented with organic acid of interest in an amount sufficient to inhibit normal microbial growth under conditions of continuous pH control at a pH that is above the pKa value of the organic acid) can be used in screening microbial libraries generated from genome shuffling or other random mutagenesis methods for isolates that exhibit tolerance and increased organic acid production.

[0049] Using this pH control method, applicants were able to target unique resistance mechanisms that depended on transport and / or unpredictable intracellular targets, including those involved in regulation and metabolism. Then, genome resequencing was used to Petition 870260076101, dated 07 / 30 / 2026, page 26 / 52 19 / 34 identify the critical genes through their mutational changes that caused genetic resistance to high concentrations of organic acids.

[0050] The resulting mutations generally affected the functions of the cell envelope, as shown in Table 2. TABLE 2. ENVELOPE AND ASSOCIATED CATEGORIES FUNCTIONS OF THE ENVELOPE: Membrane transporters / proteins (10 ORFs affected): Major facilitator superfamily proteins, amino acid permeases, hypothetical membrane protein, LemA membrane protein, intramembrane metalloproteinase, AAA ATPase, sodium-proton antiporter. Gain of function in penicillin-binding protein and amino acid permease Cell wall / peptidoglycan synthesis: Penicillin-binding proteins, proteins containing the antigen-O ligase domain (many mutations) ENVELOPE MODIFICATION FUNCTIONS: Oxidation / reduction: Flavin reductase, alpha / beta hydrolase, pyruvate carboxylase, MocA oxidoreductase, protophyringene oxidase, KGD Glycosyltransferases / hydrolases: Glycosyltransferase, glycosyl hydrolase, adenine glycosylase

[0051] These mutations primarily altered the structure, composition, and function of the cell envelope, which consists of the cell wall and membrane(s), including the cytoplasmic membrane. A complete list of the identified mutations is provided in Table 3. The applicants did not observe any mutations in genes that were targeted for metabolic modification and manipulated using recombinant DNA as previously reported (see Table 1). Mutations in multiple genes appear to be necessary for Petition 870260076101, dated 07 / 30 / 2026, page 27 / 52 20 / 34 produce the mutant phenotype (such as increased growth in media supplemented with organic acid and / or overproduction of organic acid compared to the initial microbial cell strain). This is in direct contrast to previous knowledge, where single genes were manipulated to try to change PA yields.

[0052] According to the present invention, other conventional techniques of microbiology, molecular biology, recombinant DNA and biochemistry can be used. Such techniques are fully explained in the literature and within the competence of those skilled in the art. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of the matter described in the claims. EXAMPLES Example 1 Isolation of strain 3-1

[0053] A P. acidipropionici (ATCC 25562) was cultured at high cell density in 10 mL M24 + 2.0% glucose medium. Serial dilutions of this culture (100 to 10-3) were then plated in solid M24 + 2.0% glucose medium, solidified with agar, supplemented with 1.0%, 2.0%, and 3.0% (w / v) PA, all neutralized to pH 7.0 using sodium hydroxide. Cells were also plated in solid M24 + 2.0% glucose medium without any additional PA.

[0054] Strain 3-1 was deposited under the name NFS-2018 on July 10, 2019, in the American Type Culture Collection (10801 University Blvd. Manassas, Virginia 20110-2209) and received the accession number ATCC PTA-125895).

[0055] After a 5-day anaerobic incubation at 30°C, colony growth at different PA concentrations was evaluated. Three colonies grew on the 3% PA plate placed on the plate with undiluted cells; no colonies grew on the 3% PA plates placed on the plate with diluted cells. The Petition 870260076101, dated 07 / 30 / 2026, page 28 / 52 21 / 34 Three colonies were isolated and re-seeded onto plates with no PA, PA at 2.0%, and PA at 3.0% PA (all neutralized to pH 7.0 using sodium hydroxide), along with freshly cultured wild-type P. acidipropionici cells.

[0056] After a second 5-day anaerobic incubation at 30°C, colony growth at different PA concentrations was again evaluated. All three isolates, but not the wild type, were able to grow on the 1.0% PA plate (Fig. 1). Only isolate #1 was able to grow on the PA plates at 2.0% and 3.0%. This isolate was named strain 3-1 (3 denotes PA a 3.0%, and 1 denotes isolate #1). Isolate #1 was inoculated in mL of M24 liquid medium + 2.0% glucose and cultured to high cell density, and frozen permanent strains of these cells were produced.

[0057] After the resistance phenotype to 3.0% PA in solid media was confirmed for strain 3-1, PA production in 10 mL continuous cultures and 1 L bioreactor cultures of this strain was compared with its parental P. acidipropionici (ATCC 25562) cells by HPLC in a wide range of media and culture conditions. Example 2 PA production by strain 3-1 and wild-type P. acidipropionici

[0058] Wild-type P. acidipropionici (ATCC 255562) and strain 3-1 were cultured from a frozen permanent strain at 30°C under anaerobic conditions in M24 medium supplemented with 2% glucose. Cells were subcultured every 48 h in fresh M24 medium starting with 10 mL and then 50 mL to use as seed for the 1 L bioreactor vessels.

[0059] For the preparation of wheat flour medium, 75 g of American-style cake flour were added to 1 L of ddH2O in a sterile 2 L flask while mixing. One mL of Enzenco alpha-amylase and 500 mL of 50 ppm CaCl2 were added. Petition 870260076101, dated 07 / 30 / 2026, page 29 / 52 22 / 34 to the mixture to hydrolyze cake flour. The pH was adjusted to 6.0 by adding 5 mL of 5M NaOH and the temperature was maintained at 90°C for 1 hour. The mixture was allowed to cool, then incubated at 37°C overnight. After overnight incubation, the temperature was increased to 60°C and the pH adjusted to 7.0 by adding 2 mL of 5M NaOH. To release glucose, 1 mL of Enzenco glucoamylase, 0.05 g of protease, 0.4 g of MgSO4, and 10 g of Ohly KAT yeast extract were added to the mixture while stirring. The mixture was maintained at 60°C for 2 hours. The mixture was allowed to cool, then added to a glass-jacketed bioreactor vessel and then sealed. Before autoclaving, the pH was calibrated.

[0060] Fermentations were carried out in 1 L volumes in 3 L bioreactor vessels. The temperature was maintained at 30°C, the pH was maintained at 7.0 using 5M NaOH, and the cultures were stirred at 200 rpm. 3 mL of filtered sterile trace element solution were added to the bioreactor before inoculation. The glucose concentration was determined using a YSI 2900 analyzer. 1 L of wheat flour medium was seeded with 5% inoculum. Samples were removed every 24 hours for PA analysis by HPLC. The results are shown in Fig. 2.

[0061] Both strain 3-1 and the parental wild-type strain reached maximum PA concentration in about 120 hours. The maximum PA concentration produced by strain 3-1 is about 36 g / L, compared to about 30 g / L by the parental wild-type strain.

[0062] Additional experiments were performed under 5-6 different conditions, 3-4 times each, to compare PA production by strain 3-1 and wild-type P. acidipropionici. Similar results to those shown in Fig. 2 were obtained. There is a minimum 15% increase in PA production by strain 3-1 compared to the wild type after 60 hours of cultivation. Example 3 Petition 870260076101, dated 07 / 30 / 2026, page 30 / 52 23 / 34 genomic analyses of strain 3-1

[0063] Resequencing of the 3-1 strain genome was used to identify critical genes through their mutational changes that caused resistance to high concentrations of organic acids.

[0064] 65 loss-of-function mutations in 29 genes were identified. The mutations generally affected cell envelope functions, as shown in Table 2. These mutations primarily alter the structure, composition, and function of the cell envelope, which consists of the cell wall and membrane(s), including the cytoplasmic membrane. A complete list of the mutations identified in strain 3-1 is presented in Table 3. TABLE 3. GENOME MUTATIONS OF STRAIN 3-1 ORF Coordinates SEQ ID NO. Change Non-synonymous 130744-130746 ASQ49_RS00690 SAM-dependent methyltransferase class I 1 Arg ^ His 130744-130746 ASQ49_RS00695 MFS transporter 2 Thr ^ Pro 130748 ASQ49_RS00690 SAM-dependent methyltransferase class I 1 Pro ^ Leu 130752 ASQ49_RS00695 MFS transporter 2 Arg ^ Gly 181601 (80% confidence) ASQ49_RS00915 LemA family protein 3 Insertion (no frameshift) 181607-181609 ASQ49_RS00915 LemA family protein 3 Gln ^ Leu Petition 870260076101, dated 07 / 30 / 2026, page 31 / 52 24 / 34 ORF Coordinates SEQ ID NO. Change 240311 ASQ49_01155 Flavin reductase 4 Pro ^ His 240440 ASQ49_01155 Flavin reductase 4 Ala ^ Val 281222 ASQ49_RS01330 Hypothetical protein ('hit' BLAST for MFS transporter) 5 Trp ^ STOP 344598 ASQ49_RS01635 MFS transporter 6 Thr ^ Pro 525954 ASQ49_RS02385 Glycosyltransferase family 1 7 Ala ^ Glu 548143-548147 ASQ49_RS02475 Hypothetical protein ('hits' BLAST for antigen-O ligase and membrane protein) 8 Ala ^ Gly Gly ^ Leu 548153-548156 ASQ49_RS02475 Hypothetical protein (Strong 'hits' BLAST for antigen-O ligase and membrane protein) 8 Ala ^ Val Gly ^ Leu 548162 ASQ49_RS02475 Hypothetical protein (Strong 'hits' BLAST for antigen-O ligase and membrane protein) 8 Ala ^ Val 558158-558160 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 His ^ Gly Petition 870260076101, dated 07 / 30 / 2026, page 32 / 52 25 / 34 ORF Coordinates SEQ ID NO. Change 558181 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Gln A His 558228 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Ala A Thr 558252 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Ser A Pro 558258 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Ser A Pro 558266 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Arg A Leu 558273 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Ser A Ala 558279 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Gln Glu A 558282 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Glu A Gln 558288-588290 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Leu A Pro 558291-558293 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 Glu A Val Petition 870260076101, dated 07 / 30 / 2026, page 33 / 52 26 / 34 ORF Coordinates SEQ ID NO. Change 558306 ASQ49_RS02520 Protein containing antigen-O domain ligase 9 Pro ^ Ala 558308-558310 ASQ49_RS02520 Protein containing antigen-O domain ligase 9 Thr ^ Ser 562835 ASQ49_RS02535 Protein containing antigen-O domain ligase 10 Val ^ Leu 562840 ASQ49_RS02535 Protein containing antigen-O domain ligase 10 Ala ^ Val 562843 ASQ49_RS02535 Protein containing antigen-O domain ligase 10 Gly ^ Ala 566353 (*50% frequency) ASQ49_RS02550 Penicillin-binding protein 11 Lys ^ Gln 566356 (*50% frequency) ASQ49_RS02550 penicillin-binding protein 11 Ala ^ Ser 618017-618019 ASQ49_RS02820 Phosphotransferase 12 Glu ^ Ala 738302 ASQ49_RS03340 Alpha / beta hydrolase 13 Thr ^ Ala Petition 870260076101, dated 07 / 30 / 2026, page 34 / 52 27 / 34 ORF Coordinates SEQ ID NO. Change 742073 ASQ49_RS03360 Hypothetical protein ('Hits' BLAST for intramembrane metalloprotease) 14 Ile ^ Leu 1176596 ASQ49_RS05220 gfo / Idh / MocA family oxidoreductase 15 Ala ^ Val 1279986 ASQ49_RS05630 Alpha / beta hydrolase 16 Ile ^ Val 1331356 ASQ49_RS05840 Amino acid permease 17 Gly ^ Ser 1331366 ASQ49_RS05840 Amino acid permease 17 Arg ^ His 1521847 ASQ49_RS06625 Hypothetical protein ('Hits' BLAST for protoporphyrinogen oxidase) 18 Thr ^ Ala 1816621 ASQ49_RS07985 Adenine glycosylase 19 Ser ^ Ala 1816687 ASQ49_RS07985 Adenine glycosylase 19 Insertion in the table (1 amino acid) 1817191 ASQ49_RS07985 Adenine glycosylase 19 Gly ^ Glu 1817202 ASQ49_RS07985 Adenine glycosylase 19 Glu ^ Ala Petition 870260076101, dated 07 / 30 / 2026, page 35 / 52 28 / 34 ORF Coordinates SEQ ID NO. Change 1854503 SQ49_RS08150 Hypothetical protein ('hit' BLAST for sodium-proton antiporter) 20 Lys ^ Arg 1854520 SQ49_RS08150 Hypothetical protein ('hit' BLAST for sodium-proton antiporter) 20 Ile ^ Met 2679601 ASQ49_12020 Multifunctional oxoglutarate decarboxylase / thiamine pyrophosphate oxoglutarate dehydrogenase binding subunit / succinyltransferase dihydrolipolysis residue (kgd) 21 His ^ Asp 2927020 ASQ49_RS13125 Amino acid permease 22 Insertion into the frame (4 amino acids) 2927030 ASQ49_RS13125 Amino acid permease 22 Gly-Ser ^ Ala-Ala 2928883 ASQ49_RS13130 ​​Hypothetical protein (glycosyl hydroxylase family) 23 Asn ^ Tyr 3517645 (*50% frequency) ASQ49_RS15965 M18 family aminopeptidase 24 Thr ^ Arg Petition 870260076101, dated 07 / 30 / 2026, page 36 / 52 29 / 34 ORF Coordinates SEQ ID NO. Change 3517646 (*50% frequency) ASQ49_RS15965 M18 family aminopeptidase 24 Thr ^ Ser 3517648 (*50% frequency) ASQ49_RS15965 M18 family aminopeptidase 24 Ser ^ Thr 3517649 (*50% frequency) ASQ49_RS15965 M18 family aminopeptidase 24 Ser ^ Gly 3517652 ASQ49_RS15965 M18 family aminopeptidase 24 Ser ^ Tyr 3517655 ASQ49_RS15965 M18 family aminopeptidase 24 Ser ^ Asn FRAME SHIFTS 558244 ASQ49_RS02520 Protein containing domain of Antigen-O ligase 9 558246 ASQ49_RS02520 Protein containing antigen-O ligase domain 9 2867178 ASQ49_RS12835 AAA ATPase 25 FRAME DISPLACEMENT REPAIRS Petition 870260076101, dated 07 / 30 / 2026, page 37 / 52 30 / 34 ORF Coordinates SEQ ID NO. Change 448285 ASQ49_RS02075 DUF1116 domain-containing protein 26 561527 ASQ49_RS02530 Glycosyltransferase 27 900222 ASQ49_RS03980 Acetyl-CoA carrier protein subunit Carboxyl biotin carboxylase 28 919056 ASQ49_RS04070 Penicillin-binding protein 29 1330401 ASQ49_RS05840 Amino acid permease 17 1330407 ASQ49_RS05840 Amino acid permease 17

[0065] Mutations in these genes (or their homologs in other species described herein) likely confer genetic resistance to high concentrations of organic acids by altering membrane transport protein systems and / or previously unknown intracellular targets involved in regulation and / or metabolism.

[0066] Multiple mutations in the same gene imply that the gene is very important for the trait and requires multiple changes to contribute to the trait. Notably, several genes had three or more mutations, which may indicate their critical roles in limiting organic acid formation. These include genes encoding: Protein containing antigen-O ligase domain (15 mutations in ASQ49_RS02520; 3 mutations in Petition 870260076101, dated 07 / 30 / 2026, page 38 / 52 31 / 34 ASQ49_RS02535; and 3 mutations in ASQ49_RS02475 (Hypothetical protein with strong BLAST 'hits' for antigen-O ligase and membrane protein); M18 aminopeptidase family (6 mutations in ASQ49_RS15965); amino acid permease (4 mutations in ASQ49_RS05840); and adenine glycosylase (4 mutations in ASQ49_RS07985). REFERENCES

[0067] 1. Woskow S.A., B.A. Glatz. 1991. “Propionic acid production by a propionic acid-tolerant strain of Propionibacterium acidipropionici in batch and semicontinuous fermentation”. Applied and Environmental Microbiology 57:28212828. [00 68] 2. Zhu Y., J. Li, M. Tan, L. Liu, L. Jiang, J. Sun, P. Lee, G. Du, J. Chen. 2010. “Optimization and scale-up of propionic acid production by propionic acid-tolerant Propionibacterium acidipropionici with glycerol as the carbon source”. Bioresource Technology 101:8902-8906.

[0069] 3. Wang Z., S.-T. Yang. 2013. “Propionic acid production in glycerol / glucose co-fermentation by Propionibacterium freudenreichii subsp. Shermanii”. Bioresource Technology 137:116-123.

[0070] 4. Zhuge X., L. Liu, H.-d. Shin, J. Li, G. Du, J. Chen. 2014. “Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy”. Bioresource Technology 152:519-525.

[0071] 5. Zhuge X., J. Li, H.-d. Shin, L. Liu, G. Du, J. Chen. 2015. “Improved propionic acid production with metabolically engineered Propionibacterium jensenii by an oxidoreduction potential-shift control strategy”. Bioresource Technology 175:606-612.

[0072] 6. Coral J. 2008. “Propionic acid production by Propionibacterium sp. using low-cost carbon sources in submerged Petição 870260076101, de 30 / 07 / 2026, pág. 39 / 52 32 / 34 fermentation”. Dissertation. Federal University of Parana.

[0073] 7. Zhang A., J. Sun, Z. Wang, S.-T. Yang, H. Zhou. 2015. “Effects of carbon dioxide on cell growth and propionic acid production from glycerol and glucose by Propionibacterium acidipropionici”. Bioresource Technology 175:374-381.

[0074] 8. Wang Z., M. Lin, L. Wang, E. M. Ammar, S.-T. Yang. 2015. “Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing three biotin-dependent carboxilases”. Process Biochemistry 50:194-204.

[0075] 9. Suwannakham S., Y. Huang, S.-T. Yang. 2006. “Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation”. Biotechnology and Bioengineering 94:383-95.

[0076] 10. Suwannakham S., S.-T. Yang. 2005. “Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor”. Biotechnology and Bioengineering 91:325-337.

[0077] 11. Suwannakham S. 2005. “Metabolic engineering for enhanced propionic acid fermentation by Propionibacterium acidipropionici”. Dissertation. Ohio State University.

[0078] 12. Tufvesson P., A. Ekman, R. R. R. Sardari, K. Engdahl, L. Tufvesson. 2013. “Economic and environmental assessment of propionic acid production by fermentation using different renewable raw materials”. Bioresource Technology 149:556-564.

[0079] 13. Thierry A., S.-M. Deutsch, H. Falentin, M. Dalmasso, F. J. Cousin, G. Jan. 2011. “New insights into physiology and metabolism of Propionibacterium freudenreichii”. International Journal of Food Microbiology 149:19-27.

[0080] 14. Scholz C.F.P., M. Kilian. 2016. “The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacterium to the Petição 870260076101, de 30 / 07 / 2026, pág. 40 / 52 33 / 34 proposed novel genera Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov”. International Journal of Systematic and Evolutionary Microbiology 66:4422-4432.

[0081] 15. Rehberger J.L., B.A. Glatz. 1998. “Response of cultures of Propionibacterium to acid and low pH: tolerance and inhibition”. Journal of Food Production 61:211-216.

[0082] 16. Ahmadi N., K. Khosravi-Darani, A.M. Mortazavian. 2017. “An overview of biotechnological production of propionic acid: From upstream to downstream processes”. Electronic Journal of Biotechnology 28:67-75.

[0083] 17. Guan N., L. Liu, X. Zhug, Q. Xu, J. Li, G. Du, J. Chen. 2012. “Genome-shuffling improves acid tolerance of Propionibacterium acidipropionici and propionic acid production”. Advances in Chemistry Research 15:143-152.

[0084] 18. Guan N., H. Shin, R. R. Chen, J. Li, L. Liu, G. Du, J. Chen. 2014. “Understanding of how Propionibacterium acidipropionici respond to propionic acid stress at the level of proteomics”. Scientific Reports 4:6951.

[0085] 19. Guan N., H.D. Shin, G. Du, J. Chen, L. Liu. 2016. “Metabolic engineering of acid resistance elements to improve acid resistance and propionic acid production of Propionibacterium jensenii”. Biotechnology and Bioengineering 113:1294-304.

[0086] 20. Jiang L., H. Cui, L. Zhu, Y. Hu, X. Xu, S. Li, H. Huang. 2015. “Enhanced propionic acid production from whey lactose with immobilized Propionibacterium acidipropionici and the role of trehalose synthesis in acid tolerance”. Green Chemistry 15:250-259.

[0087] 21. EP 0141642.

[0088] 22. WO 85 / 04901.

[0089] 23. US 2011 / 0151529 A1.

[0090] 24. WO 2012 / 064883 A2. Petição 870260076101, de 30 / 07 / 2026, pág. 41 / 52 34 / 34

[0091] 25. WO 2017 / 055932 A2. Petição 870260076101, de 30 / 07 / 2026, pág. 42 / 52

Claims

1 / 2 CLAIMS 1. A method for producing an organic acid selected from propionic acid, lactic acid, acetic acid, butyric acid, or combinations thereof, characterized by comprising: providing a microbial cell of P. acidipropionici that produces the organic acid in excess compared to the parental cell strain of P.acidipropionici, wherein the isolated cell strain comprises one or more loss-of-function mutations in genes encoding the DUF1116 domain-containing protein (A5Q49_R502075; SEQ ID NO: 26) and the M18 family aminopeptidase (A5Q49_RS15965; SEQ ID NO: 24), wherein one or more loss-of-function mutations in a gene encoding the DUF1116 domain-containing protein (A5Q49_R502075; SEQ ID NO: 26) occur at coordinate 448285 in the genome, and wherein one or more loss-of-function mutations in a gene encoding the M18 family aminopeptidase (A5Q49_RS15965; SEQ ID NO: 24) occur at one or more coordinates in the genome, selected from the coordinate group consisting of 3517645, 3517646, 3517648, 3517649, 3517652 and 3517655; and cultivate said microbial cell in a culture medium.

2. A method for producing an organic acid selected from propionic acid, lactic acid, acetic acid, butyric acid, or combinations thereof, characterized by comprising: providing the microbial cell of P. acidipropionici comprising one or more loss-of-function mutations in a gene encoding a protein selected from the group consisting of a protein containing an O-antigen ligase domain comprising the amino acid sequence SEQ ID NO: 9, an amino acid permease comprising the amino acid sequence SEQ ID NO: 17, a protein containing a DUF1116 domain comprising the amino acid sequence SEQ ID NO: 26, an aminopeptidase of the M18 family comprising the amino acid sequence SEQ ID NO: 24 and adenine glycosylase comprising the amino acid sequence SEQ ID NO: 19; and cultivate the P. acidipropionici microbial cell in a culture medium.

3. Method according to claim 1 or 2, characterized in that the culture medium is maintained at a pH of 5.5 to 7.

5.

4. Method, according to any one of claims 1 to 3, characterized in that the culture medium is maintained at a pH of 6.0 to 7.

0.

5. Method according to claim 4, characterized in that said culture is carried out at 30°C for 60 to 120 hours.

6. A method according to any one of claims 3 to 5, characterized in that the organic acid is propionic acid.

7. Method, according to any one of claims 3 to 6, characterized in that the amount of organic acid produced by the method after 60 hours of cultivation is at least 15% greater compared with the amount of organic acid produced under the same conditions using wild-type P. acidipropionici. Petition 870260076101, dated 07 / 30 / 2026, pp. 44 / 52

Images