Genetic engineering microorganism and method for producing succinate and method for preparing genetic engineering microorganism

Abstract

A genetically engineered microorganism for producing succinate includes: a host cell derived from a methylotrophic bacteria .The host cell has an ethylmalonyl-coenzyme A pathway. The activity of succinate dehydrogenase in the methylotrophic bacteria is reduced or inactivated, and the genetically engineered microorganism is capable of growing and producing succinate using methanol as the sole carbon source. Also provided herein are methods for producing succinate and methods for preparing genetically engineered microorganisms for producing succinate.

Description

Technical Field

[0001] This disclosure relates to genetically engineered microorganisms for the biosynthetic production of succinic acid and methods for producing succinic acid, as well as methods for manufacturing genetically engineered microorganisms for the production of succinic acid. Prior Technology

[0002] Succinic acid (also known as succinic acid) is a colorless crystal with a wide range of industrial applications, such as as a chemical intermediate in the manufacture of varnishes and perfumes, and as a flavoring agent, antibacterial agent, or neutralizing agent in the food industry. It also has specific applications in certain chemical markets, including the manufacture of coatings, surfactants, dyes, detergents, green solvents, biodegradable plastics, and growth promoters for plants and animals. Furthermore, succinic acid can be converted into other bulk chemicals, such as γ-butyrolactone, tetrahydrofuran, adipic acid, and 1,4-butanediol, a key precursor in tire manufacturing.

[0003] Previous chemical methods for producing succinic acid included paraffin oxidation, catalytic hydrogenation, and electrolytic reduction. Paraffin oxidation involves the oxidation of paraffin under calcium or manganese catalysis to produce a mixture of various carboxylic acids, which are then separated by steam distillation and crystallization to obtain succinic acid. Hydrogenation uses maleic anhydride or fumaric acid in the presence of hydrogen gas and catalyzed by nickel or a noble metal to produce succinic acid. Electrolytic reduction involves electrolyzing phthalic anhydride, sulfuric acid, and water in a 1:0.5:4 ratio in a ceramic electrolytic cell to form succinic acid.

[0004] However, the above methods generally have the following problems: (1) The reaction conditions are relatively harsh and the operation procedures are complex, requiring high temperature and high pressure environments. (2) The catalysts used (such as hydrogen) are somewhat dangerous. (3) There is a dependence on petrochemical raw materials, such as paraffin wax and maleic anhydride, which are petroleum derivatives and are not conducive to environmental protection.

[0005] Therefore, it is necessary to find green and environmentally friendly biotechnology to produce succinic acid in order to replace traditional chemical methods. Summary of the Invention

[0006] Several embodiments of this disclosure involve the biosynthesis of succinic acid and the development of related genetically engineered microorganisms.

[0007] Since the fermentation production of succinic acid using carbon sources such as sugars or glycerol poses concerns about competition with food and the use of petrochemical resources, in several embodiments of this disclosure, genetically engineered microorganisms using methanol as a carbon source are developed to biosynthesize succinic acid.

[0008] Several embodiments of this disclosure provide a genetically modified microorganism for producing succinic acid, comprising a host cell derived from methanolophilic bacteria. The host cell possesses the ethylmalonido-CoA pathway, the activity of succinate dehydrogenase in the methanolophilic bacteria is reduced or inactivated, and the genetically engineered microorganism is capable of growing and producing succinic acid using methanol as a single carbon source.

[0009] In some embodiments, the genetically engineered microorganisms that produce succinic acid also include: at least one heterologous gene encoding an enzyme for the glyoxylate cycle or an enzyme that converts isocitrate into glyoxylate and succinic acid.

[0010] In some embodiments, the at least one heterologous gene is located within a plastid.

[0011] In some embodiments, the at least one heterologous gene comprises: a gene encoding isocitrate lyase (aceA), a gene encoding malate synthase (aceB), or a combination thereof.

[0012] In some implementations, genetically engineered microorganisms are obtained through adaptive evolution.

[0013] In some implementations, the genetically engineered microorganism is strain Otter-1, with the registration number BCRC 911257.

[0014] In some embodiments, the genetically engineered microorganism has: enhanced expression of at least one gene for methanol metabolism; enhanced expression of at least one gene for the serine cycle; reduced expression of at least one gene for the citrate cycle pathway; and enhanced expression of at least one gene for the ethylmalondiamide-CoA pathway.

[0015] In some embodiments, the at least one gene of the methanol metabolism pathway comprises a methanol dehydrogenase gene (mxaF); the at least one gene of the serine cycle pathway comprises a serine hydroxymethyltransferase gene (glyA); the at least one gene of the citrate cycle pathway comprises citrate synthase (gltA); and the at least one gene of the ethylmalondiamide-CoA pathway comprises: acetylacetyl-CoA reductase (phaB) or crotonyl-CoA carboxylase / reductase (ccr), or a combination thereof.

[0016] Multiple embodiments of this disclosure provide a method for producing succinic acid, comprising: culturing a strain for producing succinic acid, comprising: culturing the genetically engineered microorganism for producing succinic acid in a mineral culture medium, wherein the single carbon source in the mineral culture medium is methanol.

[0017] In some embodiments, the strain that produces succinic acid is cultured using single-batch fermentation or continuous batch fermentation.

[0018] In some embodiments, the method of producing succinic acid further includes: supplementing a nitrogen source to the mineral culture medium.

[0019] Multiple embodiments of this disclosure provide a method for preparing genetically engineered microorganisms, comprising: reducing or inactivating the activity of succinate dehydrogenase (Sdh) in methanolophilic bacteria via a first gene editing technique, wherein the methanolophilic bacteria contain the ethylmalonido-CoA pathway; and obtaining a strain that produces succinic acid using methanol as a single carbon source via a second gene editing technique or an adaptive evolutionary process.

[0020] In some embodiments, via this second gene editing technology, the succinic acid-producing strain expresses a heterologous glyoxylate cycle enzyme or an enzyme that converts isocitrate into glyoxylate and succinic acid.

[0021] In some embodiments, via the second gene editing technique or the adaptive evolutionary process, the succinic acid-producing strain has increased carbon flux in methanol metabolism, reduced in the citrate pathway, and increased in the ethylmalonidate-CoA pathway. Simple Explanation of the Diagram

[0022] To make the above and other objects, features, advantages and embodiments of this disclosure more apparent and understandable, please refer to the accompanying drawings, which are described below. Figure 1 illustrates a biosynthetic pathway from methanol to succinic acid according to some embodiments of this disclosure. Figure 2 shows a central metabolic diagram of methanolophilic bacteria according to some embodiments. Figure 3 is a bar chart based on an experimental example, comparing the growth of strains. Figure 4A is a graph showing the growth of a strain and the production of succinic acid based on an experimental example. Figure 4B is a growth curve based on a strain from an experimental example. Figure 4C is a graph showing the yield of succinic acid based on an experimental example. Figure 5 is a bar chart based on an experimental example, testing the effects of adding different compounds on strain growth and succinic acid production. Figure 6A shows a central metabolic diagram of a genetically engineered microorganism according to some embodiments. Figure 6B shows the design of a plastid based on an experimental example. Figure 6C shows the design of a plastid based on an experimental example. Figure 6D is a bar chart based on an experimental example, showing the growth and succinic acid production of the strain cultured in a mineral medium containing methanol after transfection with plastids from Figures 6B and 6C, respectively. Figure 7A illustrates the operational flow of adaptive laboratory evolution based on an experimental example. Figure 7B is a graph based on an experimental example, comparing the growth of different strains in adaptive laboratory evolution. Figure 8 is a bar chart based on an experimental example, showing the expression of some genes in a strain derived from adaptive laboratory evolution with methanol as the sole carbon source. Figure 9 is a graph based on an experimental example, showing the growth of the strain and the yield of succinic acid using methanol as the sole carbon source. Figure 10 is a graph based on an experimental example, showing the growth of the strain and the yield of succinic acid in a batch fermentation using methanol as the sole carbon source. Figures 11A to 11C are growth curves based on an experimental example of the strain. Figures 11A and 11B show the effect of different concentrations of CoCl2 in the culture medium on the growth of the strain. Figure 11C shows the effect of adding 4 μM CoCl2 and 4 μM B vitamins to the culture medium on the growth of the strain. Figures 11D through 11F show the succinic acid yield curves under the test conditions described in Figures 11A through 11C, respectively. Figure 12 shows a graph based on an experimental example, testing the toxicity of different sodium ion concentrations to the strain. Figures 13A and 13B show the growth curves and succinic acid yields of the strains when NaOH, KOH, and NH4OH were used as neutralizing agents in the culture, respectively, based on an experimental example. Figure 14 is a bar chart based on an experimental example, showing the residual methanol concentration measured after the opening of a fermentation shake flask was covered with different caps, methanol aqueous solution was added, and the flask was shaken for 12 hours. Figures 15A to 15C respectively show the methanol concentration variation curve, succinic acid production curve, and strain growth curve based on an experimental example, testing the effect of covering the fermentation shake flask with different caps on the methanol concentration, succinic acid production, and strain growth in the culture medium. Implementation

[0023] The spirit of this disclosure will be clearly illustrated below with detailed descriptions and diagrams. It should be understood that this disclosure can have various variations in different forms, but none of them depart from the scope of this disclosure, and the descriptions and diagrams therein are for illustrative purposes only and not for limiting the scope of this disclosure.

[0024] Currently, there are three main pathways for the microbial synthesis of succinic acid: (1) Utilizing the natural tricarboxylic acid cycle (TCA) reduction pathway (also known as the fermentation pathway) in organisms, phosphoenolpyruvate (PEP) is sequentially converted into oxaloacetate (OA), malic acid, and fumarate under anaerobic conditions, finally forming succinic acid. (2) Utilizing the natural tricarboxylic acid cycle (TCA) oxidation pathway in organisms, acetyl-CoA and OAA are synthesized into citrate under aerobic conditions, and then converted into succinyl-CoA through isocitrate, α-ketoglutarate, and succinyl-CoA. (3) Using the glyoxylate pathway, isocitrate lyase (AceA) is expressed under aerobic conditions to cleave isocitrate into glyoxylate and succinate. Then, glyoxylate is synthesized into malic acid via malate synthase (AceB) and acetyl-CoA.

[0025] Previous technologies have utilized strains that naturally produce succinic acid, such as *Actinobacillus succinogenes*, *Anaerobiospirillum succiniciproducens*, *Mannheimia succiniciproducens*, *Clostridium thermossuccinogenes*, and *Cytophagasuccinicans*, to produce succinic acid using sugars or glycerol as carbon sources. Additionally, previous technologies have employed genetic engineering to modify common model organisms (such as *E. coli* and *Saccharomyces cerevisiae*) and use sugars or glycerol as carbon sources to produce succinic acid. However, these methods have drawbacks, including the potential competition with food crops and the risk of using petrochemical resources when using these carbon sources for fermentation.

[0026] Therefore, in several embodiments of this disclosure, another renewable resource, methanol, is used as a carbon source for the bioconversion method to produce succinic acid.

[0027] Methanol is an electron-rich organic C1 carbon source. Previously, methanol was mainly derived from coal, natural gas, and biomass. Recently, with the gradual industrialization of carbon fixation technology for synthesizing green methanol via carbon dioxide hydrogenation, methanol has become a sustainable, environmentally friendly, and renewable emerging raw material. Furthermore, methanol can also be obtained through methane conversion, giving it flexible and diverse sources. It is projected that global methanol production capacity will grow to 300 million tons by 2030. Therefore, using methanol as a non-food-based fermentation feedstock to synthesize succinic acid within microorganisms can reduce the succinic acid market's dependence on petrochemical feedstocks and sugars, and, combined with the development of green methanol, enhance the reuse value of carbon dioxide.

[0028] This disclosure discloses several embodiments that utilize techniques such as metabolic engineering, synthetic biology, and microbiology to design non-naturally occurring microorganisms to biosynthesize succinic acid from methanol. Metabolic engineering improves the production efficiency and sustainability of high-value compounds by modifying the metabolic pathways of microorganisms. Synthetic biology applies techniques such as gene editing and synthetic genomes to achieve specific biosynthetic goals. Microbiology studies the growth and metabolism of microorganisms, providing theoretical and experimental support for these technologies.

[0029] In this article, the term "endogenous" refers to the activity of a nucleic acid sequence or gene product that is present in the host microorganism.

[0030] In this article, the term "exogenous" means that the activity of a nucleic acid sequence or a gene product is introduced into the host microorganism.

[0031] In this article, the term "homologous" refers to the activity of a nucleic acid sequence or gene product derived from a host microorganism.

[0032] In this document, the term "heterogeneous" means that a substance is not naturally present in the host cell. For example, a nucleic acid sequence is heterogeneous to a host cell if at least one of the following is true: (a) the nucleic acid is not naturally found in that cell (i.e., it is an "exogenous" nucleic acid); (b) the nucleic acid is naturally found in a given host cell (i.e., "endogenous"), but the nucleic acid or the RNA or protein produced by transcription and translation of this nucleic acid is produced or present in the host cell in a non-natural (e.g., greater or less than naturally present) amount; (c) the nucleic acid contains a nucleotide sequence encoding an endogenous protein of the host cell, but which differs in sequence from the endogenous nucleotide sequence encoding the same protein (having the same or substantially the same amino acid sequence), thereby generally resulting in the protein being produced in the cell in a larger amount, or, in the case of an enzyme, in a mutant form with altered (e.g., higher or lower or different) activity; and / or (d) the nucleic acid contains two or more nucleotide sequences that are not found in the host cell in the same way.

[0033] In this document, enhanced or decreased gene expression in genetically engineered microorganisms refers to gene expression compared to a strain that has not undergone a specific genetic engineering of the microorganism. Enhanced enzyme activity refers to an increase of at least 150% in protein activity compared to a suitable control strain. Decreased enzyme activity refers to less activity of the enzyme product compared to a strain that has not undergone genetic engineering of the microorganism, at least a reduction of 75%. Preferably, the activity reduction reaches at least 80%, 85%, 90%, or 95%, and in a more preferred embodiment, the activity is eliminated (100%).

[0034] In several embodiments, transcription, translation, protein stability, protein function, or the like may be modulated and optimized through the use of genetic engineering and appropriate culture conditions. In several embodiments, metabolic flux may be optimized by reducing, disrupting, or knocking out competitive metabolic pathways that compete with intermediates leading to the desired pathway. In some embodiments, some of the genes involved in biosynthesis are endogenous. In some embodiments, some of the genes involved in biosynthesis are foreign to the host, or may be heterologous to the host microorganism due to modification via mutagenesis, recombination, and / or association with heterologous expression control sequences in endogenous host cells.

[0035] In several embodiments of this disclosure, methanol-loving microorganisms, such as methylotrophic bacteria, which possess a natural ethylmalonyl-CoA synthesis pathway (EMCP), are utilized to metabolize methanol into succinic acid. In some embodiments, the host of the genetically engineered microorganisms used is derived from methanolotrophic bacteria, such as *Methylobacterium*, *Methylophilus*, *Methylomonas*, or similar species.

[0036] See Figures 1 and 2. Figure 1 illustrates the metabolic pathway from acetyl-CoA to succinic acid production. Figure 2 shows a schematic diagram of the central metabolism of methanolophiles, including methanol metabolism, the serine cycle, the tricarboxylic acid cycle (TCA cycle), and the acetyl-CoA pathway.

[0037] In the metabolic pathway of methanolophiles, methanol can be converted to formaldehyde via methanol dehydrogenase MxaF, and a portion of the formaldehyde is spontaneously converted to methylene-THF, which is then converted to serine via GlyA and enters the serine cycle. Acetyl-CoA produced in the serine cycle can then enter the ethylmalonido-CoA pathway. Figure 1 shows various enzymes involved in the ethylmalonyl-CoA pathway, including: β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), crotonase (CroR), crotonyl-CoA carboxylase / reductase (Ccr), ethylmalonyl-CoA isomerase (Epi), ethylmalonyl-CoA mutase (Ecm), mesaconyl-CoA hydratase (Mcd), and methylsuccinyl-CoA dehydrogenase. The enzymes used include Msd, malyl-CoA / β-methylmalyl-CoA lyase (Mcl), propionyl-CoA carboxylase (Pcc), methylmalonyl-CoA mutase (Mcm), and succinyl CoA synthetase (SucCD).

[0038] In other words, in methanolophilic bacteria, after methanol metabolism produces acetyl-CoA, it can be converted into succinic acid through the action of enzymes such as PhaA, PhaB, CroR, Ccr, Epi, Ecm, Mcd, Msd, Mcl, Pcc, Epi, Mcm, and SucCD.

[0039] Please refer to Figure 2. It can be seen that in the TCA cycle, succinate is converted into fumarate by succinate dehydrogenase (SDH), thus consuming the succinate produced by natural methanolophilic bacteria.

[0040] In several embodiments of this disclosure, genetic engineering methods, such as gene editing technology, can be used to reduce or inactivate the activity of succinate dehydrogenase in the TCA cycle of genetically modified strains, thereby causing the genetically modified strains to lose their ability to utilize succinate and begin to accumulate succinate, and to excrete the produced succinate.

[0041] In some embodiments of this disclosure, strain AM1 of the naturally occurring methanolophile *Methylobacterium extorquens* is used, genetically modified to eliminate succinate dehydrogenase subunits A and B (SdhAB) from the TCA cycle. Such genetically modified strains can be cultured in composite media. However, in mineral media, growth inhibition occurs because the strain cannot convert succinate to malic acid.

[0042] Using mineral-based culture media as the fermentation environment, compared to composite media, can reduce the cost of product purification procedures, which helps realize the industrial vision of converting methanol into succinic acid. Furthermore, if malic acid needs to be added during the cultivation or fermentation process, it will further increase the cultivation cost.

[0043] In several embodiments of this disclosure, genetically engineered strains have been developed to address growth inhibition caused by inactivation or reduced activity of succinate dehydrogenase. Therefore, these genetically engineered strains are able to produce high yields of succinate in mineral culture media using methanol as a single carbon source.

[0044] In some embodiments, the mineral culture medium used comprises water, a carbon source (i.e., methanol), inorganic salts (e.g., potassium, sodium, magnesium, calcium, bicarbonates and carbonates, phosphates, sulfates, and hydrochlorides), a pure nitrogen salt (e.g., ammonium or urea), trace metals (e.g., iron, copper, zinc, manganese, cobalt, molybdenum, and optionally borates), optional glycine betaine (also known as betaine), and optional antifoaming agent. The medium does not contain complex (also known as "rich") nutrient sources such as yeast extract, corn steep liquor, soybean hydrolysate, broth, casein hydrolysate, grains, legumes, or other "undefined" nutrient mixtures. Furthermore, the medium may contain one or more pure chemical components to meet specific growth requirements (e.g., for auxotrophic or trophically retarded strains) or to enhance a biochemical pathway. For example, some strains may require vitamins, or vitamins may promote the growth of some strains; therefore, small concentrations of vitamins may be added to the medium.

[0045] In some embodiments, the mineral culture medium used comprises: (NH4)2SO4, MgSO4·7H2O, KH2PO4, Na2HPO4, CaCl2, NaC6H3O7·2H2O, FeSO4·7H2O, ZnSO4·7H2O, MnCl2·4H2O, CuSO4·5H2O, CoCl2·6H2O, H3BO3, Na2WO4·2H2O, and Na2MoO4·2H2O.

[0046] In some embodiments, the culture medium contains methanol as a single carbon source from about 0.5% v / v (volume percentage) to about 2% v / v, such as 0.5% v / v, 1% v / v, 1.5% v / v, or 2% v / v methanol. Since the methanol in the culture medium is metabolized or volatilized, methanol can be added to the culture medium at regular intervals during the culture period (e.g., from about 8 hours to about 16 hours).

[0047] In some embodiments, genetically engineered microorganisms can grow and produce succinic acid without the addition of malic acid during cultivation. That is, malic acid does not need to be added to the culture medium during the initial and subsequent stages of cultivation. In some embodiments, no other organic acids need to be added during the cultivation of succinic acid by genetically engineered microorganisms.

[0048] The following experimental examples provide various genetically modified microorganisms and related experimental test results. The OD600 value of a spectrophotometer was used to quantify the growth of the strains, and high-performance liquid chromatography (HPLC) was used to analyze succinic acid, its derivatives, or other byproducts in the quantitative culture medium.

[0049] Experimental Example 1

[0050] In this experimental example, a methanolophilic bacterium capable of producing succinate was constructed to achieve methanol utilization and direct production of succinate, thus realizing a one-step conversion from methanol to succinate. In this experimental example, strain AM1 of *Methylobacterium extorquens* was selected as the host microorganism.

[0051] In this experimental example, the strain *M. extorquens AM1ΔcelABC* was used as the base strain (TT-1), where ΔcelABC was used to mitigate the inefficient exchange of oxygen and nutrients in cells due to cellulose accumulation. Further, the *sdhAB* gene was removed from the base strain TT-1, resulting in a strain of *M. extorquens AM1ΔcelABCΔsdhAB* (also known as strain AR-1) with succinic acid production potential. This strain loses the ability to utilize succinic acid but can accumulate it.

[0052] Subsequently, the basic strain TT-1 and the sdhAB gene-deficient strain AR-1 were cultured in a compound medium (MC media) containing 60 mM succinic acid. As shown in Figure 3, AR-1 could not grow after 24 hours, indicating that a methanolophilic strain with the potential to produce succinic acid was successfully established.

[0053] Experimental Example 2

[0054] This experiment used the sdhAB gene defect strain: AR-1 (M. extorquens AM1ΔcelABCΔsdhAB), cultured in a compound medium containing 1% (v / v) methanol and a mineral medium (MM media), to test the growth of the strain and the production of succinic acid.

[0055] Figure 4A shows the test results of the culture in a composite medium, the growth status of strain AR-1 and the succinic acid production in 20 mL of composite medium. After 48 hours of culture in the composite medium, strain AR-1 entered the exponential growth phase (log phase) and produced 2.1 ± 0.1 g / L of succinic acid at the 96-hour time point. There was a positive correlation between succinic acid production and strain growth.

[0056] Figures 4B and 4C show the test results of strains TT-1 and AR-1 cultured in mineral medium. Figure 4B shows the growth of strains TT-1 and AR-1 in 20 mL of mineral medium, and Figure 4C shows the succinic acid production of strains TT-1 and AR-1 in 20 mL of mineral medium. The results show that the growth of strain AR-1 was significantly inhibited in mineral medium, requiring 180 hours of growth to reach an OD600 value of 1.65 and produce 502 ± 67 mg / L of succinic acid.

[0057] Experiment Example 3

[0058] This experiment tested the growth rescue of the strain by adding an intermediate metabolite. Differences in growth rate observed after adding a specific intermediate metabolite can serve as a basis for analyzing bottlenecks in the AR-1 metabolic pathway.

[0059] The sdhAB gene-deficient strain AR-1 was cultured in mineral medium containing 1% (v / v) methanol, and 1.18 g / L acetate, 0.375 g / L glycine, 0.37 g / L glyoxylate, 1.76 g / L pyruvate, 2.68 g / L malic acid, 2.66 g / L aspartate (ASP), and 2.92 g / L α-ketoglutarate (α-KG) were added to different culture containers.

[0060] Figure 5 shows the growth recovery test and succinic acid production of the sdhAB gene-deficient strain (AR-1). Here, nd indicates no detected succinic acid production. The results showed that the addition of malic acid effectively restored the growth of strain AR-1. Under malic acid-added conditions, the strain's OD600 value was approximately 2.2 at 48 hours, close to the OD600 value of the control group (strain TT-1) at 2.9, while at 96 hours, the growth was approximately twice that of the control group, producing 100 ± 26 mg / L of succinic acid.

[0061] Experiment Example 4

[0062] In this experiment, the glyoxylate shunt gene was overexpressed in methanolophiles to restore the growth of the sdhAB gene-deficient strain (AR-1) and increase succinic acid production. See Figure 6A, which illustrates the serine cycle, TCA cycle, EMCP metabolic pathways in methanolophiles, as well as the glyoxylate shunt pathway.

[0063] Since methanolophiles lack the natural glyoxylate pathway, in this experimental example, the genes for exogenous glyoxylate pathway enzymes were transferred into methanolophiles. Referring to Figure 6A, the enzymes involved in the conversion of isocitrate to succinate, glyoxylate, and malate include isocitrate lyase (AceA) and malate synthase (AceB).

[0064] Figures 6B and 6C show the constructed expression plasmids. Plastid pAR40 carries the aceA gene from *E. coli*, the sequence of which is shown in SEQ ID:1 of the sequence listing. Plastid pAR31 carries both the aceA and aceB genes from *E. coli*, the sequence of which is shown in SEQ ID:1 of the sequence listing, and the sequence of which is shown in SEQ ID:2 of the sequence listing. The sequence of the PmxaF fragment is the methanol promoter of *Methanolophilus*. The sequence of the oriV-traJ fragment is the conjugation gene transfer gene of AM1 in *Methanolophilus*. The sequence of the ColE1 fragment is a commonly used replicon in *E. coli*. The sequence of the KanR fragment is the gene sequence of the kanamycin resistance protein.

[0065] Figure 6D shows the growth and succinic acid production of strain AR-1 after transfecting it with plasmids pAR40 or pAR31, respectively, and culturing it in mineral medium containing 1% (v / v) methanol for 48 hours. The results showed that aceA alone could restore AR-1 growth to an OD600 value of 0.5 and produce 0.3 g / L succinic acid after 48 hours. Furthermore, compared to the control group, simultaneous expression of aceA and aceB also increased succinic acid production.

[0066] Experimental Example 5

[0067] In this experiment, the sdhAB gene defective strain AR-1 was allowed to evolve naturally in a mineral medium to accelerate the growth rate of this strain and obtain a methanol-loving mutant strain that can grow rapidly in a mineral medium with methanol as the sole carbon source.

[0068] Figure 7A illustrates the workflow of the Adaptive Laboratory Evolution (ALE) strategy. First, strain AR-1 was pre-cultured for 3 days in 2 mL of mixed medium (MC media) containing 1% (v / v) methanol. Then, 1% (v / v) of the strain cells was inoculated into another test tube containing 2 mL of mineral medium (MM media) containing 1% (v / v) methanol, serving as the starting point for evolution. Once the strain reached a plateau, 1% (v / v) of the bacterial culture was transferred to a new mineral medium with added methanol. Through repeated subculturing, the strain's ability to utilize 1% (v / v) methanol was acclimated, allowing for the isolation of a faster-growing strain. Throughout the pre-culture and subculturing process, the strain was cultured at 30°C and 200 rpm. Growth rate was assessed by the change in OD600 value per unit time during the rapid growth phase. During the subculture, the evolved mutant strain mixtures were collected approximately every 1 to 1.5 months. The mixtures were then stored at -80°C with a 1:1 mixture of 40% (v / v) glycerol and water as a protectant. Ultimately, a total of four mutant strain mixtures (A, B, C, and D) were obtained, along with strain Otter-1, which was screened from a single colony in mutant strain mixture D.

[0069] Figure 7B shows a comparison of the growth of strain AR-1, mutants A, B, C, D, and Otter-1 in mineral medium containing 1% (v / v) methanol. It can be seen that adopting an adaptive laboratory evolution strategy can effectively accelerate the growth rate of strains in mineral medium, which is beneficial for shortening the time for succinic acid production. As described in Experimental Example 2, strain growth may be positively correlated with product formation, and using mineral medium as the fermentation environment can reduce the cost of product purification procedures compared to composite media. Therefore, the fastest-growing strain, Otter-1, is of great significance for realizing the industrialization vision of converting methanol to succinic acid.

[0070] Experimental Example 6

[0071] This experiment used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to measure the differences in gene expression in the mutant strain Otter-1 after evolution, using this as a basis for analyzing the metabolic pathway of the mutant strain Otter-1 and increasing succinic acid production. This experiment selected several target genes in the central metabolic pathway of methanolophiles (see Figure 2, showing the metabolic steps associated with these target genes): methanol dehydrogenase (mxaF), serine hydroxymethyl transferase (glyA), malate dehydrogenase (mdh), malyl-CoA lyase (mcl), acetoacetyl-CoA reductase (phaB), crotonyl-CoA carboxylase (ccr), and citrate synthase (gltA).

[0072] See Figure 8, which shows the relative gene expression results of RT-qPCR, where gene expression levels are compared with the corresponding gene expression levels in strain AR-1. The results show that the expression of the mxaF gene was increased by approximately 2.4-fold and the glyA gene by approximately 1.3-fold, indicating enhanced methanol metabolism in the mutant strain Otter-1. Furthermore, the expression of the phaB gene was increased by 1.7-fold, while the expression of the gltA gene was significantly decreased. This suggests that the succinic acid produced by the mutant strain Otter-1 using methanol may primarily originate from the ethylmalonido-CoA pathway (EMCP).

[0073] These results indicate that the Otter-1 strain, obtained through adaptive evolution, overcame the problem of growth inhibition by evolving enhanced methanol metabolism, inhibition of the TCA pathway, and increased carbon flux of EMCP.

[0074] Experimental Example 7

[0075] In this experiment, succinic acid was fed-batch fermented in conical flasks. 1% (v / v) methanol was added at hour 0, and 1% (v / v) methanol was added every 12 hours. The pH of the culture medium was also adjusted to restore it to pH 7.

[0076] As shown in Figure 9, 4.3 g / L of succinic acid can be produced after 108 hours of cultivation, demonstrating the high potential of methanolophilic bacteria in succinic acid production.

[0077] Experimental Example 8

[0078] In this experimental example, a nitrogen source was added during fermentation, and continuous batch fermentation was carried out to produce succinic acid.

[0079] Adding a nitrogen source to the succinic acid feed batch fermentation program extends the growth of the strain and accumulates higher concentrations of succinic acid.

[0080] During the test, 1% (v / v) methanol was added to the mineral medium on day 0, and methanol was replenished every half day (12 hours), along with 1 g / L of ammonium nitrate (NH4)2NO3, and the pH of the medium was adjusted back to 7. As shown in Figure 10, the addition of a nitrogen source increased the yield of succinic acid to 5.9 g / L.

[0081] Figure 10 also illustrates the subsequent replacement of the culture medium with fresh mineral medium, using a continuous batch fermentation method to revive the strain and produce succinic acid. The results showed that the strain achieved a maximum succinic acid yield of 7.3 g / L after the second culture medium replacement. This result can serve as a strategy for modifying culture conditions to increase succinic acid yield. In other words, the strain can be cultured continuously for several days; this fermentation method can effectively enhance succinic acid production capacity.

[0082] Experiment Example 9

[0083] This experiment further characterized the mutant strain Otter-1, using Next Generation Sequencing (NGS) to explore the differences in gene sequence between the mutant strain Otter-1 and the basal strain TT-1 after evolution. Tables 1 and 2 below show the results of the NGS analysis of the mutant strain Otter-1, including the mutation types occurring in the chromosome and endogenous megaplastic body of the mutant strain Otter-1 after adaptive evolution. Table 1 shows the sites of base insertion or deletion, and Table 2 shows the sites of base substitution. NC_012808.1 is the sequence number of the chromosome of the methanolophile AM1, and NC_012811.1 is the sequence number of the endogenous megaplastic body.

[0084] Table 1 chromosome Location Encoded products Type (INDEL) describe NC_012808.1 (chromosome) 482893 FliK (Flagellar hook-length control protein) insert G -> GC 2012134 Hypothetical protein Missing GCCGTC -> G 2329710 NTE family protein rssA Missing AG -> A 3037768 Maltose alpha-D-glucosyltransferase Missing TC -> T 3159070 ATP-dependent helicase Missing CG -> C 4001526 Glutathione S-transferase Missing GCGTGC -> G 4001533 Missing GT -> G 4422401 TonB-dependent receptor insert G -> GA 4422405 Missing GC -> G 4423386 TonB-dependent receptor insert G -> GA 4423393 insert C -> CTG 4755174 Enoyl-CoA hydratase / isomerase family protein Missing GC -> G 4808561 Hypothetical protein Missing ACCACGCGCC -> A 5018430 MBL fold metallo-hydrolase insert C -> CG NC_012811.1 (Megaplastid) 580985 Hypothetical protein insert G -> GC 770862 Hypothetical protein Missing TG -> T

[0085] Table 2 chromosome Location Encoded products Type (SNP) describe NC_012808.1 (chromosome) 501077 Proteins containing the DUF4239 domain. replace G -> A 1724528 ABC transporter ATP-binding protein T -> G 2012145 Hypothetical protein C -> G 2050899 TonB-dependent siderophore receptor C -> G 2511236 Proteins containing the LEPR-XLL domain. T -> C 2936238 Hypothetical protein Hypothetical protein NC_012811.1 (Megaplastid) 170244 IS110 family transposase ISMex12 T -> C

[0086] Achieving higher levels of succinic acid production in the fermentation process is desirable. The following experimental examples tested different culture conditions to further improve the succinic acid biosynthesis yield of strain Otter-1.

[0087] Experimental Example 10

[0088] In the EMCP pathway, both mutases (see Figure 1), Ecm and Mcm, utilize adenosylcobalamin (AdoB12) as a cofactor for reversible carbon skeleton recombination. AdoB12 is a type of vitamin B12, its molecular structure consisting of a corrin ring centered on a cobalt ion (Co2+) and a nucleotide with 5,6-dimethylbenzoic acid as the base. This experiment investigated the effect of cobalt ion (Co2+) concentration on strain growth and succinic acid production. CoCl2 at concentrations of 0, 0.4, 4, 12, 24, and 48 µM were added to a cobalt chloride (CoCl2)-free mineral medium, with a 4 µM CoCl2 concentration serving as the control group. Additional B vitamins were also tested for comparison. The B vitamins include thiamine (also known as vitamin B1), riboflavin (also known as vitamin B2), nicotinamide (also known as vitamin B3), pantothenic acid (also known as vitamin B5), pyridoxine (also known as vitamin B6), biotin (also known as vitamin B7), folic acid (also known as vitamin B9), and cyanocobalamin (also known as vitamin B12).

[0089] Figures 11A through 11F illustrate the effects of different cobalt [Co2+] concentrations on the growth and succinic acid production of strain Otter-1. Figures 11A and 11D compare growth and succinic acid production at cobalt [Co2+] concentrations of 0 and 0.4 µM, based on a 4 µM concentration. Figures 11B and 11E show growth and succinic acid production at cobalt [Co2+] concentrations of 12, 24, and 48 µM. Figures 11C and 11F show growth and succinic acid production after the addition of additional B vitamins. In this experimental example, the initial cell density was OD600 of 0.1.

[0090] As shown in Figures 11A and 11D, the absence of cobalt ions in the environment severely affects the growth and yield of the strain. However, cobalt ions in the concentration range of 0.4 µM to 4 µM showed no significant effect.

[0091] Figures 11B and 11E show that when the cobalt ion concentration is increased to 12 µM, the yield of succinic acid is effectively increased to 5.7 g / L. However, further increases in the cobalt ion concentration to 24 µM and 48 µM inhibit growth and yield.

[0092] Figures 11C and 11F show the results of the additional B vitamins, which were not significantly different from the control group. This may be because the vitamin B12 used was cyanocobalamin instead of adenosylcobalamin, while Ecm and Mcm enzymes prefer adenosylcobalamin.

[0093] In summary, based on the test results of this experimental example, in some subsequent embodiments, the succinic acid quantification experiment uses a mineral culture medium containing 12 µM of cobalt ions. In some embodiments, the cobalt ion content in the mineral culture medium is less than 20 µM, for example, ranging from 0.4 µM to 20 µM, preferably from 1 µM to 18 µM, or from 2 µM to 16 µM.

[0094] Experimental Example 11

[0095] In the above-mentioned experimental examples of succinic acid production by fermentation with Otter-1 strain, we found that a total of about 6 to 7 g / L of NaOH was required to maintain the pH of the culture environment at 7 for 120 hours; however, this may lead to excessive accumulation of sodium ions (Na+), thereby inhibiting the growth of the strain and the production of succinic acid.

[0096] In this experimental example, 0, 2, 5, 10, 15, 30, and 60 g / L NaCl were added to the culture medium of strain Otter-1 at 0 hours to test the effect of different sodium ion concentrations on the growth of the strain. Strain growth was measured at 48 hours.

[0097] As shown in Figure 12, a NaCl concentration of 5 g / L inhibited approximately 50% of the strain's growth. This suggests that one reason for the stagnation of Otter-1's growth in the later stages may be the toxicity caused by the accumulation of excessive sodium ions.

[0098] To address the issue of sodium ion accumulation, KOH and NH4OH, common neutralizing agents in other culture media, were selected for testing in this experimental example. During the culture period, 0.3 to 0.4 g / L of KOH or 0.5 to 0.6 g / L of NH4OH were added on average every 12 hours. The initial cell density was OD600 of 0.1. Growth and succinic acid production of the Otter-1 strain were measured every 12 hours for a total culture period of 108 hours.

[0099] As shown in Figures 13A and 13B, the results of adjusting the pH using KOH were similar to those of using NaOH, both in terms of strain growth and succinic acid production. This may also be due to excessive metal ions (K+). The test group using NH4OH showed an approximately 2-fold increase in OD600 value compared to the other two groups after 108 hours, with a succinic acid production reaching 7.2 g / L, accumulating at a rate of approximately 0.12 g / L / hour. This may be because at suitable concentrations, NH4+ can also be utilized by the strain as a nitrogen source, and there is less risk of excessive metal ion accumulation.

[0100] In some embodiments, based on the tests in this experimental example, the concentration of NaCl in the culture medium is less than 5 g / L. In some embodiments, NH4OH can be added as a neutralizing agent for the culture medium; for example, the concentration of NH4OH in the culture medium can be 6 to 7 g / L.

[0101] Experimental Example 12

[0102] Since the choice of fermentation vessel affects the amount of methanol volatilization, which is detrimental to the analysis of the succinic acid yield of the strain, this experimental example combines the above-mentioned optimal culture conditions (mineral medium containing 12 µM cobalt and pH adjusted with NH4OH), and the fermentation shake flasks used have angular bodies that can create turbulence. Two different caps were used for testing. The control group used a common white polypropylene (PP) plastic cap, also known as a "white PP cap"; the test group used a sponge cap, also known as a "sponge cap". No additional NH4NO3 was added in this experimental example, as NH4OH was already added to supplement the nitrogen source.

[0103] In this embodiment, both the white PP cap and the sponge-top cap are oxygen-permeable. Using a cap with a sponge not only reduces methanol evaporation but also decreases water vapor evaporation, possibly due to the porous material of the sponge head. Therefore, the gas exchange rate of the shake flask with the sponge-top cap is lower than that of the shake flask with the white PP cap.

[0104] Figure 14 is a bar chart showing the amount of methanol remaining in the methanol-containing aqueous solution in the shake flasks of the control and experimental groups after 12 hours.

[0105] The effects of the two cap types on the fermentation of strain Otter-1 were then tested. For the control group (white PP cap) and the test group (sponge cap), methanol was added every 12 hours and the pH was adjusted to 7. However, methanol was no longer added to the test group (sponge cap) after 108 hours. The initial cell density was OD600 of 0.5. The initial methanol concentration was 7.9 g / L. Furthermore, changes in methanol concentration, succinic acid production, and strain growth were monitored every 12 hours.

[0106] As shown in Figures 15A to 15C, there was no significant difference in methanol concentration changes between the control group (with a white PP cap) and the test group (with a sponge cap) in the early stage of fermentation (before 84 hours), approximately 6 to 7 g / L per 12 hours. This data includes both the amount of methanol volatilized and the amount actually metabolized by the strain. This may be because the strain metabolizes methanol relatively quickly in the early stage to meet its growth needs, thus the impact of methanol volatilization is small and difficult to observe. However, in the later stage of fermentation, the methanol change in the test group with the sponge cap gradually decreased, with a change of 2 to 3 g / L per 12 hours. This indicates that the rate at which the strain metabolizes methanol also slows down as growth stagnates (see Figure 15C). In contrast, the control group with the white PP cap continued to show a methanol change of approximately 6 to 7 g / L per 12 hours, but the strain growth and succinic acid production, like those in the test group with the sponge cap, ceased to increase, indicating the severity of methanol volatilization.

[0107] It is worth noting that, under the condition that the methanol concentration is not less than 2 g / L, simply changing the bottle cap caused a difference in the succinic acid yield between the two test groups (see Figure 15B). The yields of the white PP cap and the sponge cap groups were 7.7 g / L and 9.5 g / L, respectively.

[0108] In some embodiments, fermenters are used to cultivate the strains on a large scale and produce succinic acid. In some embodiments, the fermenters incorporate means to reduce methanol volatilization. In some embodiments, condensation devices, such as cooling coils or condensation towers, are installed at the vents of the fermenters to condense and recover volatilized methanol, thereby reducing methanol emissions. In some embodiments, the fermentation temperature can be lowered (e.g., controlled at 20 to 30°C) to reduce volatilization. In some embodiments, the pressure inside the tank can be increased within an appropriate range to reduce the methanol escape rate. In some embodiments, methanol molecules can also be captured through physical adsorption and filtration.

[0109] The genetically engineered strains disclosed herein can overcome growth inhibition caused by succinate dehydrogenase inactivation and methanol as the sole carbon source in mineral culture media. In some embodiments, gene editing technology or adaptive evolution is used to further mutate the genes of methanol-eating bacteria deficient in succinate dehydrogenase, resulting in a strain that produces succinate using methanol as the sole carbon source.

[0110] Since the adaptively evolved strain Otter-1 has overcome the problem of growth inhibition by evolving enhanced methanol metabolism, inhibition of the TCA pathway, and increased carbon flux of EMCP, these changes can be used as a strategy for preparing genetically engineered microorganisms.

[0111] In some implementations, succinic acid-producing strains can be made to have increased methanol metabolism, reduced citric acid pathway, and increased carbon flux in the ethylmalonidase A pathway via gene editing or adaptive evolution.

[0112] In some embodiments, through gene editing technology or adaptive evolution, genetically engineered microorganisms are endowed with: enhanced expression of at least one gene for methanol metabolism; enhanced expression of at least one gene for the serine cycle; reduced expression of at least one gene for the citrate cycle pathway; and enhanced expression of at least one gene for the ethylmalondiamide-CoA pathway. In some embodiments, the at least one gene for methanol metabolism comprises a methanol dehydrogenase gene (mxaF). In some embodiments, the at least one gene for the serine cycle comprises a serine hydroxymethyltransferase gene (glyA). In some embodiments, the at least one gene for the citrate cycle pathway comprises citrate synthase (gltA). In some embodiments, the at least one gene for the ethylmalondiamide-CoA pathway comprises: acetylacetyl-CoA reductase (phaB) or crotonyl-CoA carboxylase / reductase (ccr), or a combination thereof.

[0113] In some embodiments, the host of the genetically engineered microorganism is a methanolophilic bacterium containing endogenous or exogenous genes, located within the host cell. In some embodiments, recombinant technology can be used to prepare plasmids carrying and expressing genes for methanol metabolism, serine cycling, and / or the ethylmalonidate-CoA pathway. In some embodiments, gene editing technology can be used to reduce the expression of TCA pathway genes in the genetically engineered bacteria.

[0114] The genetically engineered microorganisms provided in various embodiments of this disclosure, by modifying the metabolic pathway of methanolophilic bacteria, can efficiently produce succinic acid using a mineral culture medium with methanol as a single carbon source. Therefore, the biosynthesis of succinic acid can be easily mass-produced industrially, reducing culture costs and offering an environmentally friendly process.

[0115] Although the present disclosure has been disclosed above with multiple implementation methods and embodiments, it is not intended to limit the present disclosure. Anyone skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be determined by the appended claims.

[0116] none

[0117] Domestic storage information (please note in order of storage institution, date, and number) Methylbacterium extorquens Otter-1 strain was deposited on December 11, 2024, at the Food Industry Development Institute Foundation, with the serial number BCRC 911257, located in Hsinchu City, Taiwan. Overseas storage information (please note in the order of storage country, institution, date, and number) none A0101_OR_PSEQ.xml

Claims

1. A genetically engineered microorganism for producing succinic acid, comprising: a host cell, wherein the host cell is derived from a methanolophilic bacterium, the host cell possessing an ethylmalonido-CoA pathway, the activity of succinate dehydrogenase (SDH) in the methanolophilic bacterium being reduced or inactivated, and the genetically engineered microorganism being able to grow and produce succinic acid using methanol as a single carbon source, wherein the genetically engineered microorganism is strain Otter-1, with accession number BCRC 911257.

2. The genetically engineered microorganism for producing succinic acid as described in claim 1, wherein the genetically engineered microorganism has: enhanced expression of the methanol dehydrogenase (mxaF) gene for methanol metabolism; enhanced expression of the serine hydroxymethyltransferase (glyA) gene for the serine cycle; reduced expression of the citrate synthase (gltA) gene for the citrate cycle pathway; and enhanced expression of the acetyl-acetyl-CoA reductase (phaB) gene or the crotonyl-CoA carboxylase / reductase (ccr) gene for the ethylmalonido-CoA pathway.

3. A method for producing succinic acid, comprising: culturing a strain for producing succinic acid, comprising: culturing the genetically engineered microorganism for producing succinic acid as described in any one of claims 1 to 2 in a mineral culture medium, wherein a single carbon source in the mineral culture medium is methanol.

4. The method for producing succinic acid as described in claim 3, wherein the strain for producing succinic acid is cultured by single-batch fermentation or continuous batch fermentation.

5. The method for producing succinic acid as described in claim 4 further comprises: supplementing a nitrogen source to the mineral culture medium.

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