JP2014150747A - Mutant microorganisms and methods for producing succinic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a set of technologies to produce citric acid from methanol etc.SOLUTION: Provided is a mutant microorganism prepared by gene modification of a methylotroph host microorganism to enhance 2-oxoglutaric acid dehydrogenase activity and/or to delete or suppress succinic acid dehydrogenase activity, the mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methyl amine, formic acid, formaldehyde and formamide.

Description

  The present invention relates to a mutant microorganism capable of producing succinic acid from methanol or the like, and a method for producing succinic acid using the mutant microorganism.

  Succinic acid is used in resin raw materials, medical raw materials, plating agents, photographic developers, seasonings and the like. Most of succinic acid is produced by hydrogenation of maleic anhydride produced from petroleum, except for food. However, in recent years, there are concerns about the sustainability of chemical processes that depend on petroleum, and it is expected to establish a method for producing succinic acid that does not depend on petroleum.

  On the other hand, succinic acid is one of metabolic intermediates in the TCA (tricarboxylic acid) cycle. Succinic acid production technology using microorganisms has been developed. Here, in order to improve the productivity of succinic acid by microorganisms, improvement of the carbon supply amount of the TCA cycle and accumulation of succinic acid within the TCA cycle are considered important.

  Techniques for promoting carbon supply to the TCA cycle include suppression or deficiency of lactate dehydrogenase activity (Patent Documents 1 and 2), suppression of acetyl CoA hydrolase activity (Patent Document 3), and suppression or deficiency of pyruvate formate lyase activity. (Patent Documents 4 and 5) are known.

Techniques for promoting succinic acid accumulation within the TCA cycle under aerobic conditions include enhancement of 2-oxoglutarate dehydrogenase activity (Patent Document 6), suppression of succinate dehydrogenase activity (Patent Document 7), etc. Techniques are known.
On the other hand, as a technique for promoting the accumulation of succinic acid in a reduced TCA cycle that functions in an anaerobic (reducing) condition in a sugar solution containing bicarbonate ion or carbon dioxide gas, etc., enhancement of fumarate reductase activity (patented) Literature 2), enhancement of pyruvate carboxylase activity (Patent Literatures 1 and 8), enhancement of phosphoenolpyruvate carboxykinase activity (Patent Literature 9), and the like are known.

By the way, among C1 compounds, methanol is inexpensively produced from natural gas and synthesis gas, which is a mixed gas of carbon monoxide, carbon dioxide, and hydrogen obtained by incineration of waste such as biomass and municipal waste. Is done. Natural gas is abundant in fossil resources, and the amount of CO ₂ generated is relatively small. Therefore, natural gas is attracting attention as a next-generation energy source, and the transition from conventional oil to natural gas is progressing. Methanol is easy to handle and store, such as being soluble in water, and is also suitable as a carbon source for microbial culture.

  Methylotroph is a carbon compound that does not have a C—C bond in the molecule, such as methane, methanol, methylamine, dimethylamine, trimethylamine, etc., as the sole carbon source and energy source. It is a generic name for microorganisms. Microorganisms called methanotroph, methane oxidizing bacteria, methanol-assimilating bacteria, methanol-assimilating yeast, methanol-assimilating microorganisms, etc. all belong to methylotrophs. Many bacterial methylotrophs can assimilate methane, and these are often called mesanotrophs.

  A methylotroph uses, as a central metabolism, a reaction in which methanol is converted into formaldehyde and then formaldehyde is converted into an organic substance having a C—C bond. As shown in FIG. 1, serine pathway, ribulose monophosphate pathway (RuMP pathway), and xylulose monophosphate pathway (XuMP pathway) are known as carbon assimilation metabolic pathways via formaldehyde. Methylotrophs classified as bacteria (methylotrophic bacteria) possess the serine pathway or the RuMP pathway. On the other hand, methylotrophs (methylotrophic yeast) classified as yeast possess the XuMP pathway.

  Moreover, methylotrophic bacteria are classified into obligate methylotroph and facultative methylotroph that can use other carbon compounds because of the difference in methanol requirement.

International Publication No. 2005/010182 JP 2005-95169 A International Publication No. 2005/113744 Special table 2012-521190 gazette JP 2010-263911 A International Publication No. 2007/099867 JP 2011-120508 A JP 2007-125030 A International Publication No. 2009/072562

  Regarding the production process from renewable resources, most of the conventional techniques, including the above-described succinic acid production technique, are production methods using microorganisms that depend on organic substances, particularly sugar, glycerol or oil components. However, the amount of sugar, glycerin and oil components that can be used at present from plant resources is insufficient as a carbon source for microorganisms in order to cover the global production of many basic chemicals derived from petroleum. Is essential. That is, the amount of basic chemicals produced by microorganisms that depend on carbohydrates and oil components is limited in the future. In addition, such a process is also concerned about competition with food.

  In view of the above-mentioned present situation, an object of the present invention is to provide a series of techniques for producing succinic acid from methanol or the like.

  One aspect of the present invention for solving the above-mentioned problems is selected from the group consisting of enhancement of 2-oxoglutarate dehydrogenase activity and deficiency or inhibition of succinate dehydrogenase activity against a host microorganism that is a methylotroph. A mutated microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. .

  As described above, in the case of producing succinic acid by a microorganism, in order to promote accumulation of succinic acid within the TCA cycle under aerobic conditions, 2-oxoglutarate dehydrogenase activity is enhanced or succinate dehydrogenase activity is promoted. It is useful to suppress this. In the mutant microorganism of the present invention, the gene is modified so that 2-oxoglutarate dehydrogenase activity is enhanced and / or 2-oxoglutarate dehydrogenase activity is deleted or suppressed with respect to a host microorganism that is a methylotroph. The accumulation of succinic acid in the TCA cycle is improved. According to the mutated microorganism of the present invention, the above-mentioned C1 is based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” inherent in methylotrophs, particularly under aerobic conditions. Succinic acid can be efficiently produced from the compound.

  The host microorganism refers to the original microorganism (wild strain or the like) that is the target of genetic modification.

  The “enhancement” of the enzyme activity means that the enzyme activity is higher than that of an unmodified microorganism (eg, wild strain, host microorganism). In addition, “inhibition” of enzyme activity means that the enzyme activity is lower than that of an unmodified microorganism (eg, wild strain, host microorganism). In addition, “deficiency” of enzyme activity means that the enzyme activity has disappeared.

  “Gene modification” includes at least modification of an existing gene, replacement of an existing gene with another gene, and introduction (addition) of a new gene.

  Another aspect of the present invention for solving the same problem is that the host microorganism, which is a methylotroph, is enhanced by enhancing fumarate reductase activity, enhancing pyruvate carboxylase activity, and enhancing phosphoenolpyruvate carboxykinase activity. The gene is modified so that at least one selected from the group consisting of succinic acid is produced, and succinic acid can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A mutant microorganism.

As described above, the reduced TCA cycle functions under anaerobic (reductive) conditions. In the case of producing succinic acid by microorganisms, it is useful to enhance fumarate reductase activity, pyruvate carboxylase activity, or phosphoenolpyruvate carboxykinase activity in order to promote accumulation of succinic acid in the reduced TCA cycle. It is. The mutant microorganism of the present invention has a gene modified so that at least one of these enzyme activities is enhanced with respect to a host microorganism that is a methylotroph, and accumulation of succinic acid in a reduced TCA cycle is improved. doing.
According to the mutant microorganism of the present invention, the above-mentioned C1 compound is based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” inherent in methylotrophs, particularly under anaerobic conditions. Can efficiently produce succinic acid.

  Preferably, the gene is further modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. Has been.

As described above, when succinic acid is produced by a microorganism, the activity of lactate dehydrogenase, acetyl CoA hydrolase, or pyruvate formate lyase is suppressed in order to promote carbon supply to the TCA cycle. Useful. The mutant microorganism of the present invention is further modified by a gene so that at least one of these enzyme activities is deficient or suppressed, and the carbon supply to the TCA cycle is improved. According to microorganisms, succinic acid can be more efficiently produced from the aforementioned C1 compound.

  Another aspect of the present invention for solving the same problem is that a host microorganism that is a methylotroph has a deficiency or inhibition of lactate dehydrogenase activity, a deficiency or inhibition of acetyl CoA hydrolase activity, and a pyruvate formate lyase activity. Succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide is modified so that at least one selected from the group consisting of deficiency or suppression is performed It is a mutant microorganism that can produce

As described above, when succinic acid is produced by a microorganism, the activity of lactate dehydrogenase, acetyl CoA hydrolase, or pyruvate formate lyase is suppressed in order to promote carbon supply to the TCA cycle. Useful. The mutant microorganism of the present invention has a modified gene so that at least one of these enzyme activities is deleted or suppressed with respect to a host microorganism which is a methylotroph, and the carbon supply to the TCA cycle is improved. doing. The mutant microorganism of the present invention can produce succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
According to the mutant microorganism of the present invention, succinic acid is efficiently obtained from the aforementioned C1 compound based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” inherent to methylotrophs. Can be produced.

  Preferably, the formaldehyde immobilization pathway has at least one C1 carbon assimilation pathway selected from the group consisting of serine pathway, ribulose monophosphate pathway, and xylose monophosphate pathway.

  Preferably, a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host microorganism.

  With this configuration, formaldehyde immobilization ability by the ribulose monophosphate pathway is imparted or enhanced.

  Preferably, the methylotroph is methanol-assimilating yeast, a gene encoding an enzyme that converts methanol into formaldehyde by dehydrogenation is further introduced, and the gene is expressed in the host microorganism.

  In general, in yeast, alcohol oxidase is responsible for the conversion reaction from methanol to formaldehyde. For this reason, oxygen is required for the conversion reaction, and specifically, it is necessary to aerate vigorously during the culture. Therefore, in this aspect, a gene encoding “enzyme that converts methanol into formaldehyde by dehydrogenation” is introduced so that the conversion reaction from methanol to formaldehyde can be performed without relying on oxygen.

  In another aspect of the present invention for solving the same problem, a gene for imparting a function of converting methanol and / or formic acid into formaldehyde and a gene for immobilizing formaldehyde are introduced into a host microorganism, And the gene is modified so that at least one selected from the group consisting of enhancement of 2-oxoglutarate dehydrogenase activity and deletion or suppression of succinate dehydrogenase activity is performed in the host microorganism, , A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methanol, methylamine, formic acid, formaldehyde, and formamide.

In the mutant microorganism of the present invention, a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” are introduced into the host microorganism, and further, 2-oxoglutarate dehydrogenase activity is introduced. The gene is modified so that at least one selected from the group consisting of enhancement of succinate dehydrogenase activity and deficiency or suppression of succinate dehydrogenase activity is performed. And succinic acid can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the mutant microorganism of the present invention has the same characteristics as a methylotroph because a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” have been introduced. doing. Based on the “function of converting methanol and / or formic acid to formaldehyde” and “formaldehyde immobilization ability” imparted by these foreign genes, succinic acid is obtained from the above-mentioned C1 compound under aerobic conditions. It can be produced efficiently.

  In another aspect of the present invention for solving the same problem, a gene for imparting a function of converting methanol and / or formic acid into formaldehyde and a gene for immobilizing formaldehyde are introduced into a host microorganism, And the gene is expressed in the host microorganism so that at least one selected from the group consisting of enhanced fumarate reductase activity, enhanced pyruvate carboxylase activity, and enhanced phosphoenolpyruvate carboxykinase activity is performed. Is a modified microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

In the mutant microorganism of the present invention, a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability” are introduced into the host microorganism, and further, the fumarate reductase activity is enhanced. The gene is modified so that at least one selected from the group consisting of enhancement of pyruvate carboxylase activity and enhancement of phosphoenolpyruvate carboxykinase activity is performed. And succinic acid can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the mutant microorganism of the present invention also has the same characteristics as methylotrophs. Based on the “function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” imparted by a foreign gene, succinic acid is efficiently removed from the aforementioned C1 compound, particularly under anaerobic conditions. Can be produced.

  Preferably, the gene is further modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. Has been.

  With this configuration, succinic acid can be more efficiently produced from the above-described C1 compound.

  In another aspect of the present invention for solving the same problem, a gene for imparting a function of converting methanol and / or formic acid into formaldehyde and a gene for immobilizing formaldehyde are introduced into a host microorganism, And the gene is expressed in the host microorganism, and at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. The mutated microorganism is capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

In the mutant microorganism of the present invention, the host microorganism is introduced with a “gene that imparts a function of converting methanol and / or formic acid into formaldehyde” and a “gene that imparts formaldehyde immobilization ability”, and further has a deficiency in lactate dehydrogenase activity or The gene is modified so that at least one selected from the group consisting of suppression, deficiency or suppression of acetyl CoA hydrolase activity, and deficiency or suppression of pyruvate formate lyase activity is performed. And succinic acid can be produced from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.
That is, the mutant microorganism of the present invention also has the same characteristics as methylotrophs. And based on the "function which converts methanol and / or formic acid into formaldehyde" and "formaldehyde immobilization ability" which were provided by the foreign gene, succinic acid can be efficiently produced from the above-mentioned C1 compound.

  Preferably, the gene imparting formaldehyde immobilization ability is a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase.

  With this configuration, formaldehyde immobilization ability by the ribulose monophosphate pathway is imparted.

  Preferably, the formaldehyde immobilization pathway has at least one C1 carbon assimilation pathway selected from the group consisting of serine pathway, ribulose monophosphate pathway, and xylose monophosphate pathway.

  Another aspect of the present invention for solving the same problem is that a host microorganism having a ribulose monophosphate pathway is introduced with a gene imparting a function of converting methanol and / or formic acid into formaldehyde, and the gene is introduced into the host. A gene is modified so that at least one selected from the group consisting of enhancement of 2-oxoglutarate dehydrogenase activity and deficiency or suppression of succinate dehydrogenase activity is performed in microorganisms, methane, methanol, methylamine , A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of formic acid, formaldehyde, and formamide.

  Another aspect of the present invention for solving the same problem is that a host microorganism having a ribulose monophosphate pathway is introduced with a gene imparting a function of converting methanol and / or formic acid into formaldehyde, and the gene is introduced into the host. The gene is modified so that at least one selected from the group consisting of enhancement of fumarate reductase activity, enhancement of pyruvate carboxylase activity, and enhancement of phosphoenolpyruvate carboxykinase activity is performed in a microorganism, , A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methanol, methylamine, formic acid, formaldehyde, and formamide.

  Preferably, the gene is further modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. Has been.

  Another aspect of the present invention for solving the same problem is that a host microorganism having a ribulose monophosphate pathway is introduced with a gene imparting a function of converting methanol and / or formic acid into formaldehyde, and the gene is introduced into the host. The gene is expressed in a microorganism so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. A mutant microorganism that is modified and capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.

  These aspects correspond to, for example, an embodiment in which a non-methylotroph having a ribulose monophosphate pathway is a host microorganism.

  Preferably, a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host microorganism.

  Preferably, the gene imparting the function of converting methanol to formaldehyde is a gene encoding methanol dehydrogenase or alcohol oxidase, and the gene imparting the function of converting formic acid to formaldehyde is a gene encoding formaldehyde dehydrogenase.

  Both methanol dehydrogenase and alcohol dehydrogenase have an action of converting methanol into formaldehyde. Formaldehyde dehydrogenase has a function of converting formic acid into formaldehyde. These enzymes are all methane metabolizing enzymes in methylotrophs belonging to bacteria. On the other hand, methylotrophs belonging to yeast do not have methane oxidation activity, but have an action of converting methanol into formaldehyde by the action of alcohol oxidase. Yeast also has an enzymatic activity to convert formic acid into formaldehyde.

  Preferably, a gene imparting a function of converting methane to methanol is further introduced, and the gene is expressed in the host microorganism.

  Preferably, the gene imparting the function of converting methane to methanol is a gene encoding methane monooxygenase.

  Methane monooxygenase has an action of converting methane to methanol. Methane monooxygenase is also one of the methane metabolizing enzymes in methylotrophs.

  Preferably, the gene modification is introduced into the genome.

  Preferably, a gene encoding a transporter protein that promotes extracellular excretion of succinic acid is introduced, and the gene is expressed.

  With this configuration, the produced succinic acid is efficiently discharged out of the cell.

  In another aspect of the present invention, the mutant microorganism is cultured using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source. This is a method for producing succinic acid by causing microorganisms to produce succinic acid.

  The present invention relates to a method for producing succinic acid. In the present invention, succinic acid is introduced into the mutant microorganism by culturing the mutant microorganism described above using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source. Let it be produced. According to the present invention, succinic acid can be produced from methanol or the like.

  In another aspect of the present invention, the mutant microorganism is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, and the mutant microorganism is contacted with the C1 compound. Is a method for producing succinic acid from succinic acid.

  In the present invention, the mutant microorganism described above is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, and succinic acid is produced from the C1 compound. Also according to the present invention, succinic acid can be produced from methanol or the like.

  According to the mutant microorganism of the present invention, succinic acid can be efficiently produced from methane, methanol, methylamine, formic acid, formaldehyde, or formamide.

  The same applies to the method for producing succinic acid of the present invention, and succinic acid can be efficiently produced from methane, methanol, methylamine, formic acid, formaldehyde, or formamide.

It is explanatory drawing showing the carbon assimilation metabolic pathway through formaldehyde.

  Hereinafter, embodiments of the present invention will be described. In the present invention, the term “gene” can be replaced by the term “nucleic acid” or “DNA”.

  The mutant microorganism of the present invention basically has a host microorganism having “a function of converting methanol and / or formic acid into formaldehyde” and “formaldehyde immobilization ability” with a lack of activity of an enzyme involved in succinic acid synthesis. It has been genetically modified to be suppressed or enhanced.

  Host microorganisms employed in the present invention include both host microorganisms that are methylotrophs and a wide range of host microorganisms including non-methylotrophs.

  As described above, methylotroph is a C1 compound that uses a carbon compound having no C—C bond in the molecule, such as methane, methanol, methylamine, dimethylamine, and trimethylamine, as a sole carbon source and energy source. It refers to a chemical microorganism. In general, methylotrophs inherently have a carbon assimilation metabolic pathway through formaldehyde, specifically, a function (route) for converting methanol and / or formic acid to formaldehyde and a formaldehyde immobilization ability (formaldehyde immobilization pathway). I have it.

  Examples of the formaldehyde immobilization pathway include serine pathway, ribulose monophosphate pathway (RuMP pathway), and xylulose monophosphate pathway (XuMP pathway) shown in FIG. In general, methylotrophs possess a serine pathway, a RuMP pathway, or a XuMP pathway as a carbon assimilation metabolic pathway via formaldehyde.

Here, each formaldehyde fixation path | route (FIG. 1) is demonstrated.
An important reaction for formaldehyde fixation by the serine pathway is a serine production reaction from glycine and 5,10-methylene-tetrahydrofolic acid by serine hydroxymethyltransferase. 5,10-methylene-tetrahydrofolic acid is formed by the combination of formaldehyde and tetrahydrofolic acid. In the serine pathway, one molecule of acetyl CoA is directly generated from one molecule of formaldehyde.

The important reaction for formaldehyde fixation by the RuMP pathway is ribulose 5-phosphate (Ru5P) and formaldehyde by 3-hexulose-6-phosphate synthase (hereinafter sometimes abbreviated as “HPS”). Of D-arabino 3 hexulose 6-phosphate from lysine and D-arabino 3 hexulose by 6-phospho-3-hexuloisomerase (hereinafter sometimes abbreviated as “PHI”) It is a production reaction of fructose 6-phosphate (F6P) from 6-phosphate.
F6P and the like produced by this pathway are also used for glycolysis, and then produce acetyl CoA, glyceraldehyde triphosphate (G3P), and pyruvic acid. In the case of F6P, it is converted into two molecules of G3P per molecule, and then two molecules of acetyl CoA are generated via two molecules of pyruvic acid.

  An important reaction for formaldehyde fixation by the XuMP pathway is the formation reaction of dihydroxyacetone (Du) and glyceraldehyde triphosphate (G3P) from xylulose pentaphosphate (Xu5P) and formaldehyde by dihydroxyacetone synthase. . G3P produced by this pathway is also used for glycolysis and converted to pyruvic acid and acetyl CoA. Dihydroxyacetone can also be subjected to glycolysis by phosphorylation and converted to G3P, pyruvate, and acetyl CoA.

  The mutant microorganism of the present invention is capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. For example, in the case of a mutant microorganism having methanol dehydrogenase or alcohol oxidase, methanol can be converted to formaldehyde.

  In the case of a mutant microorganism having methane monooxygenase in addition to methanol dehydrogenase or alcohol oxidase, methane can be converted to methanol, and subsequently methanol can be converted to formaldehyde.

  Furthermore, in the case of a mutant microorganism having formaldehyde dehydrogenase, formic acid can be converted to formaldehyde.

  In general, methylotrophs (methylotrophic bacteria) classified as bacteria have methane monooxygenase and methanol dehydrogenase, so that formaldehyde can be synthesized from methane or methanol. In addition, methylotrophs (methylotrophic yeasts) classified as yeast have alcohol oxidase, so that formaldehyde can be synthesized from methanol. Moreover, methylotroph has formaldehyde dehydrogenase and can convert formic acid into formaldehyde.

  The methanol dehydrogenase includes pyrroloquinoline quinone (PQQ) -dependent methanol dehydrogenase found in methylotrophs of gram-negative bacteria, NAD (P) -dependent methanol dehydrogenase and alcohol dehydrogenase found in methylotrophs of gram-positive bacteria, gram-positive Contains DMNA (N, N'-dimethyl-4-nitrosoaniline) -dependent methanol oxidoreductase (Park H. et al., Microbiology 2010, 156, 463-471) found in bacterial methylotrophs, from methanol in yeast Conversion to formaldehyde is usually catalyzed by alcohol oxidase, which is oxygen dependent.

In the case of a mutant microorganism having amine oxidase or methylamine dehydrogenase, methylamine can be converted to formaldehyde. These enzymes are known to be possessed by some methylotrophs and bacteria belonging to the genus Arthrobacter (Anthony C., The Biochemistry of Methylotroph, 1982, Academic Press Inc.).
An enzyme that converts formamide to formaldehyde has been found in some microorganisms (Anthony C., The Biochemistry of Methylotroph, 1982, Academic Press Inc.).

  And succinic acid can be produced via formaldehyde.

  The type of methylotroph used as the host microorganism is not particularly limited, and for example, those classified into bacteria and yeast can be employed.

  Examples of methylotrophic bacteria include, for example, Methylacidphilum, Methylosinus, Methylocystis, Methylobacterium, Methylocella, Methylococcus, Methylomonas, Methylobacter, Methylobacillus, Methylophilus, Methylotenera, Methylovorus, Genus, Methyloversatilis, Mycobacterium, Arthrobacter, Bacillus, Beggiatoa, Burkholderia, Granulibacter, Hyphomicrobium, Pseudomonas, Achromobactor, Paracoccus, Crenothrix, Clonothrix, Rhodohobacter, Rhodobacter Examples include bacteria belonging to the genus Thiomicrospira, Verrucomicrobia, and the like.

  Examples of methylotrophic yeasts include yeasts belonging to the genus Pichia, Candida, Saccharomyces, Hansenula, Torulopsis, Kloeckera, and the like. Examples of Pichia genus yeast include P. haplophila, P. pastoris, P. trehalophila, P. lindnerii, and the like. Examples of Candida yeast include C. parapsilosis, C. methanolica, C. boidinii, C. alcomigas, and the like. Examples of Saccharomyces yeast include Saccharomyces metha-nonfoams. Examples of Hansenula yeast include H. wickerhamii, H. capsulata, H. glucozyma, H. henricii, H. minuta, H. nonfermentans, H. philodendra, H. polymorpha, and the like. Examples of Torulopsis yeast include T. methanolovescens, T. glabrata, T. nemodendra, T. pinus, T. methanofloat, T. enokii, T. menthanophiles, T. methanosorbosa, T. methanodomercqii, and the like.

  When the host microorganism is a non-methylotroph, it does not necessarily have a pathway for converting methanol or the like to formaldehyde, so it is necessary to provide at least a “function for converting methanol and / or formic acid to formaldehyde”. . Furthermore, it is preferable to provide “a function of converting methane to methanol”. These functional additions can be realized by introducing a gene encoding the above-described enzyme into a host microorganism.

  For example, as a gene imparting a function of converting methanol into formaldehyde, a gene encoding methanol dehydrogenase (for example, EC1.1.1.244, EC1.1.2.7) or a gene encoding alcohol oxidase (for example, EC1.13.13) Can be used. Moreover, a gene encoding formaldehyde dehydrogenase (for example, EC1.2.1.46) can be used as a gene imparting a function of converting formic acid into formaldehyde. Furthermore, a gene encoding methane monooxygenase can be used as a gene imparting the function of converting methane to methanol.

  Moreover, a plasmid that imparts methanol assimilation is known. For example, the assimilation ability of Bacillus methanolicus depends on a plasmid encoding an enzyme group involved in methanol metabolism (Brautaset T. et al., J. Bacteriology 2004, 186 (5), 1229-1238). By introducing such a plasmid into a related non-methylotroph, it is possible to confer methanol assimilation ability. Furthermore, by modifying such a plasmid, it is possible to impart methanol-assimilating properties to various non-methylotrophs.

  As described above, non-methylotrophs are treated in the same way as methylotrophs by providing them with the function of converting methanol and / or formic acid into formaldehyde, and further by providing the ability to immobilize formaldehyde. Is possible. The formaldehyde-immobilizing ability can be imparted, for example, by introducing a gene encoding an enzyme that acts in the serine pathway, RuMP pathway, or XuMP pathway described above into a non-methylotroph.

  In one preferred embodiment, the host cell is methanol-assimilating yeast, and a gene encoding an enzyme that converts methanol into formaldehyde by dehydrogenation is further introduced, and the gene is expressed in the host microorganism. For example, a target mutant microorganism can be obtained by using a Pichia genus yeast having methanol assimilation ability as a host cell and further introducing a methanol dehydrogenase gene. According to this embodiment, a mutant microorganism capable of producing succinic acid capable of converting methanol into formaldehyde without depending on oxygen can be obtained.

  The case where a RuMP route is assigned will be further described as an example. The provision of the RuMP pathway is, for example, the above-described 3-hexulose 6-phosphate synthase (HPS; eg EC4.1.2.43) gene and 6-phospho-3-hexoisomerase (PHI; eg EC5.3.1.27). This can be realized by introducing a gene. That is, ribulose 5-phosphate (Ru5P) and fructose 6-phosphate (F6P), which are substrates or products of formaldehyde immobilization reaction by HPS / PHI, are all biological organisms as metabolic intermediates of the pentose phosphate pathway and the calvin cycle. Exists universally. Therefore, by introducing HPS / PHI, it is possible to impart formaldehyde fixing ability to all organisms including Escherichia coli, Bacillus subtilis, yeast and the like.

  You may introduce | transduce a HPS gene and a PHI gene into the host microorganism which originally has RuMP pathway. Thereby, the ability to fix formaldehyde by the RuMP pathway can be enhanced. For example, an alcohol dehydrogenase such as methanol dehydrogenase (for example, EC1.1.1.244, EC1.1.2.7), 3-hexulose, or the like, which has a RuMP pathway or a pathogen of the same quality as Bacillus subtilis. By introducing a gene encoding an enzyme such as 6-phosphate synthase (HPS; eg EC4.1.2.43), 6-phospho-3-hexoisomerase (PHI; eg EC5.3.1.27), methanol In addition to imparting a function of converting aldehyde into formaldehyde (that is, methanol assimilation), the ability to fix formaldehyde can be enhanced.

  The HPS gene and the PHI gene may be introduced into a host microorganism that is a methylotroph. That is, by introducing HPS / PHI into a methylotroph having a serine pathway, RuMP pathway, or XuMP pathway, the ability to immobilize formaldehyde by the RuMP pathway can be enhanced. As a result, the formaldehyde resistance of the mutant microorganism can be increased, and as a result, the resistance to methanol and formic acid and the ability to assimilate can be increased. This makes it possible to improve the culture efficiency of mutant microorganisms and the production efficiency of succinic acid.

  On the other hand, when conferring formaldehyde immobilization ability by the serine pathway, the above-mentioned serine hydroxymethyltransferase (for example, EC2.1.2.1) gene can be used. For example, non-methylotrophs include alcohol dehydrogenase genes such as methanol dehydrogenase, 5,10-methylenetetrahydrofolate (CH2 = H4F) synthase gene, and serine hydroxymethyltransferase (eg EC2.1.2. 1) By introducing a gene or the like, it is possible to impart methanol utilization and formaldehyde immobilization ability by serine pathway.

  In the mutant microorganism of the present invention, one or more genetic modifications shown in the following (a) to (c) are applied to the host microorganism. Specifically, "at least one of (a)", "at least one of (b)", "at least one of (c)", "at least one of (a) and at least one of (c) Or a genetic modification consisting of “at least one of (b) and at least one of (c)”.

(A) Genetic modifications that improve succinic acid accumulation within the TCA cycle under aerobic conditions:
(A-1) enhancement of 2-oxoglutarate dehydrogenase activity,
(A-2) Loss or inhibition of succinate dehydrogenase activity

(B) Genetic modification that improves succinic acid accumulation within the reduced TCA cycle under anaerobic conditions:
(B-1) enhancement of fumarate reductase activity,
(B-2) enhancement of pyruvate carboxylase activity,
(B-3) Enhancement of phosphoenolpyruvate carboxykinase activity

(C) Genetic modification that promotes carbon supply to the TCA cycle:
(C-1) deficiency or inhibition of lactate dehydrogenase activity,
(C-2) deficiency or inhibition of acetyl CoA hydrolase activity,
(C-3) Deficiency or inhibition of pyruvate formate lyase activity

  2-oxoglutarate dehydrogenase (2-oxoglutarate dehydrogenase) is an enzyme that converts 2-oxoglutarate to succinyl CoA. Succinate dehydrogenase is an enzyme that converts succinic acid to fumaric acid. By enhancing 2-oxoglutarate dehydrogenase activity or by deleting or suppressing succinate dehydrogenase, accumulation of succinate in the TCA cycle can be promoted. Only one or both of the enhancement of 2-oxoglutarate dehydrogenase activity and the deficiency or suppression of succinate dehydrogenase may be performed.

Fumarate reductase is an enzyme that converts fumarate to succinic acid. Pyruvate carboxylase is an enzyme that converts pyruvate into oxaloacetate. Phosphoenolpyruvate carboxykinase is an enzyme that catalyzes the reaction of decarboxylating oxaloacetate to synthesize phosphoenolpyruvate and its reverse reaction.
As described above, the reduced TCA cycle functions under anaerobic (reductive) conditions. Therefore, in order to promote the accumulation of succinic acid in the reduced TCA cycle under anaerobic conditions, the intermediate metabolism of the TCA cycle is enhanced by enhancing the fumarate reductase activity, pyruvate carboxylase activity, or phosphoenolpyruvate carboxykinase activity. A large amount of succinic acid as a product can be accumulated.
These three enzyme activity enhancements may be performed for only one enzyme, or may be performed for two or more enzymes.

Lactate dehydrogenase is an enzyme that converts pyruvate into lactic acid. Acetyl-CoA hydrolase is an enzyme that produces acetic acid from acetyl-CoA and water. Pyruvate formate lyase is an enzyme that generates acetyl CoA and formic acid from pyruvate and coenzyme CoA. By deficient or inhibiting the activity of these enzymes, it is possible to maintain a high level of carbon inflow into the TCA cycle, which is important for mass production of succinic acid. In addition, since the production of formic acid and acetic acid strongly inhibits cell growth, inhibition of cell growth can be avoided by deleting or suppressing acetyl CoA hydrolase activity and pyruvate formate lyase activity.
The deficiency or suppression of these three types of enzyme activities may be performed for only one enzyme, or may be performed for two or more enzymes.

  As described above, “enhancement” of enzyme activity means that the enzyme activity is higher than that of an unmodified microorganism (eg, wild strain, host microorganism). Preferably, the enzyme activity is 5 to 10 times or more compared to that of the unmodified microorganism.

  As described above, “inhibition” of enzyme activity means that the enzyme activity is lower than that of an unmodified microorganism (eg, wild strain, host microorganism). Preferably, the enzyme activity is 20% or less as compared with the unmodified microorganism. In addition, “deficiency” of enzyme activity means that the enzyme activity has disappeared.

  Specific examples of the enzyme are listed below.

2-oxoglutarate dehydrogenase (for example, EC 1.2.4.2, EC 1.2.1.52)
Examples of genes (indicated by Uni-Prot / Swiss-Prot No.): P20967 (Saccharomyces cerevisiae); P23129 (Bacillus subtilis); P0AFG3 (E.coli); Q8NRC3 (Corynebacterium glutamicum);

-Succinate dehydrogenase (eg EC 1.3.5.1)
Examples of genes (indicated by Uni-Prot / Swiss-Prot No.): P69054 (E. coli); P0AC41 (E. cli); P07014 (E. coli); P33421 (Saccharomyces cerevisiae); P37298 (Saccharmomyces cerevisiae); Q08230 (Saccharmomyces cerevisiae) etc.

・ Fumarate reductase (eg EC 1.3.1.6, EC 1.3.5.4, etc.)
Examples of genes (indicated by Uni-Prot / Swiss-Prot No.): P0A8Q3 (E. coli); P0A8Q0 (E. coli); P0A8Q4 (E. coli); P00363 (E. coli); P0AC47 (E. coli) Q4QM68 (Haemophilus influenza); A5UDP2 (Haemophilus influenza); A5UHY2 (Haemophilus influenza), etc.

・ Pyruvate carboxylase (eg EC 6.4.4.1)
Examples of genes (indicated by Uni-Prot / Swiss-Prot No.): P9KWU4 (Bacillus subtilis); P32327 (Saccharomyces cerevisiae); P11154 (Saccharomyces cerevisiae); P78992 (Pichia pastoris), etc.

・ Phosphoenolpyruvate carboxykinase (eg EC 4.1.1.32)
Examples of genes (indicated by Uni-Prot / Swiss-Prot No.): P22259 (E. coli); P54418 (Bacillus subtilis); P10963 (Saccharomyces cerevisiae); Q4QM91 (Haemophilus influenza); Q3KJP4 (Pseudomonas fluorescens); A4QHQ4 Corynebacterium glutamicum) etc.

Lactate dehydrogenase (eg, EC 1.1.2.3, 1.1.2.4, 1.1.1.27, 1.1.1.28)
Examples of genes (indicated by UniProt KB / Swiss-Prot No.): P06149 (E. coli); P33232 (E. coli); P52643 (E. coli); P13714 (Bacillus subtilis); P00175 (Saccharomyces cerevisiae), etc.

Acetyl CoA hydrolase (eg EC 3.1.2.1)
Examples of genes (indicated by UniProt KB / Swiss-Prot No.): Q81S17 (Bacillus anthracis); P32316 (Saccharomyces cerevisiae); C4R404 (Pichia pastoris) etc.

Pyruvate formate lyase (eg EC 2.3.1.54)
Examples of genes (indicated by UniProt KB / Swiss-Prot No.): P09373 (E. coli); P32674 (E. coli); Q7A7X6 (Staphylrococcus aureus); P43753 (Haemophilus influenzae), etc.

  As described above, “gene modification” includes at least three of modification of an existing gene, replacement of an existing gene with another gene, and introduction (addition) of a new gene.

There are two major methods of genetic modification for deleting, suppressing, or enhancing the activity of the enzyme: a method for increasing or decreasing the number of molecules of the expressed enzyme and a method for increasing or decreasing the activity of the expressed enzyme itself. As an example of the former, there is a method of increasing or decreasing the expression level of the target enzyme gene at the transcription level or translation level. Specifically, modification of expression control sequences such as promoter sequences and Shine-Dalgarno (SD) sequences can be mentioned.
An example of the latter is introducing a mutation into a target enzyme gene on the genome.

When the enzyme activity is deficient or suppressed, an embodiment in which an existing enzyme gene of the host microorganism is replaced with another enzyme gene is preferably employed. For example, by replacing an existing enzyme gene on the genome with a sequence that causes a frame shift, a sequence that causes a point mutation, a partial fragment gene of a target enzyme, etc., deletion or suppression of enzyme activity is realized. can do.
When it is desired to enhance the enzyme activity, the existing enzyme gene may be replaced with a gene whose enzyme activity has been enhanced by, for example, codon modification.

  For example, gene replacement can be performed by homologous recombination. The homology of the base sequence required for homologous recombination is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more. In addition, gene manipulation methods by homologous recombination methods have already been established in many bacteria and the like, and there are a method using linear DNA, a method using a plasmid, etc. (US Pat. No. 6,303,383; No. 007491).

  The target gene modification can also be performed by subjecting the host microorganism to a mutation treatment with a mutagen or the like. For example, mutations in which the host microorganism is treated with radiation or a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, and the target enzyme activity is deficient, suppressed, or enhanced Microorganisms can be selected.

  The target mutant microorganism can also be obtained by introducing (adding) a new gene to the host microorganism. This embodiment is useful mainly when it is desired to enhance the activity of the target enzyme. For example, the same gene as the enzyme gene originally possessed by the host microorganism can be further introduced from the outside to increase the copy number of the gene. In addition, an enzyme gene modified to a codon suitable for the host microorganism can be introduced. The enzyme gene to be introduced is preferably of the same origin as the host microorganism, but may be of a different origin. Any of prokaryotic origin and eukaryotic origin may be sufficient. Information on the types of enzymes that can be used, gene sequences, amino acid sequences, activities and the like can be obtained from, for example, the enzyme information system “Enzyme Database-BRENDA” (http://www.brenda-enzymes.info/).

  When the succinic acid concentration is increased in the cell, succinic acid production is suppressed. In order to improve this, it is preferable to have a function of promoting extracellular discharge of excessively generated succinic acid. Therefore, in a preferred embodiment, a gene encoding a transporter protein that promotes extracellular succinate excretion is introduced, and the gene is expressed in the mutant microorganism.

The transporter protein is not particularly limited as long as it has physical properties and functions capable of discharging succinic acid and other dicarboxylic acids to the outside of cells in prokaryotic cells. Preferably, those that also function in prokaryotes without undergoing post-translational modifications such as glycosylation, such as those derived from bacteria, more preferably derived from gram-positive bacteria.
Examples of transporter proteins suitable for extracellular export of succinic acid include those derived from prokaryotes such as Drug / metabolite transporter (DMT), C4-dicarboxylate ABC transporter (ABC), MFS family major facilitator transporter (MFS), C4- dicarboxylate transporter A (DctA), C4-dicarboxylate uptake B (DcuB), L-tartrate / succinate antiporter (TtdT, YgjE), Dicarboxylate amino acid-cation symporter (DAACS), Divalent anion sodium symporter (DASS), Tripartite ATP-independent Examples include transporter proteins belonging to families such as periplasmic (TRAP), CitMHS, and MaeN, and variants thereof.
For eukaryotes, transporter proteins belonging to families such as Mitochondrial succinate / fumarate carrier (SFC), Sodium / dicarboxylate co-transporter, Tonoplast dicarboxylate transporter (TDT), Aluminum-activated malate transporter (ALMT), and their mutations The body is mentioned.
In addition, since various transporter functions depend not only on the intracellular and extracellular succinic acid concentration but also on the redox potential, pH, cation concentration, anion concentration, phosphoric acid concentration, etc., it is efficient by combining with the culture condition study. In some cases, extracellular succinate can be achieved.

The method for introducing a gene into the host microorganism is not particularly limited, and may be appropriately selected depending on the type of the host microorganism. For example, a vector that can be introduced into a host microorganism and can express an integrated gene can be used.
For example, when the host microorganism is a prokaryotic organism such as a bacterium, the vector may contain a promoter at a position where the host microorganism can replicate autonomously in the host microorganism or can be integrated into a chromosome and can transcribe the inserted gene. Can be used. For example, it is preferable to construct a series of components comprising a promoter, a ribosome binding sequence, the above gene, and a transcription termination sequence in the host microorganism using the vector.

  For example, examples of methods for integrating methylotrophic bacteria into the chromosome include Methylobacillus flagellatus having a ribulose monophosphate pathway and Methylobacterium extorquencs having a serine pathway by disrupting the target gene (Chistoserdova L. et al. , Microbiology 2000, 146, 233-238; Chistoserdov AY., Et al., J. Bacteriol 1994, 176, 4052-4065). These are gene introduction methods into the genome using circular DNA, but in the genus Methylophilus and the like, gene introduction methods into the genome using linear DNA have also been developed (Japanese Patent Application Laid-Open No. 2004-229626). . In general, when recombination by a host microorganism is difficult, genomic recombination with linear DNA is more efficient than with circular DNA. In general, the homologous recombination method preferably targets a gene present in multiple copies on the genome, such as an inverted-repeat sequence. In addition to homologous recombination, methods for introducing multiple copies into the genome include a method for loading in a transposon. As a method for introducing a gene into a methylotrophic bacterium, for example, a broad host range vector pAYC32 (Chistoserdov AY., Et al., Plasmid 1986, 16, 161-167), pRP301 (Lane M., et al., Arch. Microbiol. 1986, 144 (1), 29-34), pBBR1, pBHR1 (Antoine R. et al., Molecular Microbiology 1992, 6, 1785-1799), pCM80 (Marx CJ. Et al., Microbiology 2001, 147, 2065-2075), etc.

  The gene introduction method in methylotrophic yeast is established mainly by Pichia pastoris, and vectors such as pPIC3.5K, pPIC6, pGAPZ, and pFLD (Invitrogen) are commercially available.

As plasmids that can be used for gene transfer into Bacillus bacteria, Bacillus subtilis includes pMTLBS72 (Nquyen HD. Et al., Plasmid 2005, 54 (3), 241-248), pHT01 (Funakoshi), pHT43 (Funakoshi). Bacillus megaterium includes p3STOP1623hp (Funakoshi), pSP _YocH hp (Funakoshi), and Bacillus brevis includes pNI DNA (Takara Bio).

When a plurality of types of genes are introduced into a host microorganism using a vector, each gene may be incorporated into one vector or may be incorporated into separate vectors. Further, when a plurality of genes are incorporated into one vector, each gene may be expressed under a common promoter or may be expressed under different promoters.
Examples of introducing multiple types of genes include two or more genes selected from the group consisting of fumarate reductase gene, pyruvate carboxylase gene, and phosphoenolpyruvate carboxykinase gene.
As another example, there is an embodiment in which at least one gene selected from the group consisting of a fumarate reductase gene, a pyruvate carboxylase gene, and a phosphoenolpyruvate carboxykinase gene and a succinate transporter protein gene are introduced. It is done.
As another example, a mode in which a 2-oxoglutarate dehydrogenase gene and a succinate transporter protein gene are introduced can be mentioned.

  As described above, known vectors that can be used in methylotrophs and the like have been shown. However, regions related to transcriptional control such as promoters and terminators, replication regions, and the like can be modified according to the purpose. As a modification method, it may be changed to another natural gene sequence in each host microorganism or its related species, or may be changed to an artificial gene sequence.

  Moreover, in addition to the above-mentioned modification by gene transfer, combining mutation methods such as mutation and genome shuffling, etc., the expression level of the transgene in the host microorganism, the citrate excretion function of the host microorganism, citrate resistance, etc. By improving, it becomes possible to further improve the productivity of citric acid.

  In the mutant microorganism of the present invention, genes other than those described above may be further introduced. Examples of the gene to be introduced include the above-mentioned methanol dehydrogenase gene, alcohol dehydrogenase gene, methane monooxidase gene, HPS / PHI gene, serine hydroxymethyltransferase gene, 5,10-methylenetetrahydrofolate synthase gene, and serine hydroxymethyltransferase Genes, etc.

  In one aspect of the method for producing citric acid of the present invention, at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide is used as the carbon source. And cultivate the mutant microorganism to produce citric acid. About these C1 compounds used as a carbon source, only one may be used and it may be used in combination of 2 or more. Moreover, it is preferable to use these C1 compounds as a main carbon source, and it is more preferable that it is the only carbon source.

In the case of obligate methylotrophs, it is fundamental to use a synthetic medium containing C1 compound as the only carbon source, but a small amount of natural medium such as yeast extract, corn steep liquor, meat extract and vitamins are added to this. This also promotes the growth of bacteria. In the case of facultative methylotrophs, substances other than C1 compounds such as carbohydrates and lipids may be used as the carbon source in the cell growth stage, and the carbon source may be changed to the C1 compound in the succinic acid production stage. The microorganism can be cultured under aerobic, microaerobic or anaerobic conditions depending on the purpose. Any of batch culture, fed-batch culture, and continuous culture may be used.
For example, when methanol is used as the carbon source, it is usually used at a concentration of 1.0% (v / v) for bacteria and at a concentration of 3.0% (v / v) for yeast. When the resistance to is improved, it can be cultured at higher concentrations.

In another aspect of the succinic acid production method of the present invention, the mutant microorganism is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, A mutant microorganism is caused to produce succinic acid from the C1 compound. That is, regardless of whether cell division (cell proliferation) is involved, succinic acid can be produced by contacting the above-mentioned C1 compound with a mutant microorganism. For example, the above-mentioned C1 compound can be continuously supplied to the immobilized mutant microorganism, and succinic acid can be continuously produced.
Also in this aspect, about these C1 compounds, only one may be used and it may be used in combination of 2 or more.

  Produced succinic acid is accumulated intracellularly or released extracellularly. For example, purified succinic acid can be obtained by recovering succinic acid released outside the cell and isolating and purifying it.

Claims (25)

The gene is modified so that at least one selected from the group consisting of enhancement of 2-oxoglutarate dehydrogenase activity and deletion or suppression of succinate dehydrogenase activity is performed on a host microorganism that is a methylotroph,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. The gene is modified so that at least one selected from the group consisting of enhancement of fumarate reductase activity, enhancement of pyruvate carboxylase activity, and enhancement of phosphoenolpyruvate carboxykinase activity is performed on a host microorganism that is a methylotroph And
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   Furthermore, the gene has been modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. Item 3. The mutant microorganism according to Item 1 or 2. A host microorganism that is a methylotroph is subjected to at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity. The gene was modified
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   The mutant microorganism according to any one of claims 1 to 4, which has at least one C1 carbon assimilation pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylose monophosphate pathway as a formaldehyde immobilization pathway.   The gene encoding 3-hexulose 6-phosphate synthase and the gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host microorganism. Mutant microorganisms described in 1.   The methylotroph is a methanol-assimilating yeast, a gene encoding an enzyme that converts methanol into formaldehyde by a dehydrogenation reaction is further introduced, and the gene is expressed in a host microorganism. Mutant microorganism. A gene that imparts a function of converting methanol and / or formic acid into formaldehyde and a gene that imparts formaldehyde immobilization ability into the host microorganism, and the gene is expressed in the host microorganism;
The gene is modified so that at least one selected from the group consisting of enhancement of 2-oxoglutarate dehydrogenase activity and deletion or suppression of succinate dehydrogenase activity is performed,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A gene that imparts a function of converting methanol and / or formic acid into formaldehyde and a gene that imparts formaldehyde immobilization ability into the host microorganism, and the gene is expressed in the host microorganism;
The gene is modified so that at least one selected from the group consisting of enhancement of fumarate reductase activity, enhancement of pyruvate carboxylase activity, and enhancement of phosphoenolpyruvate carboxykinase activity is performed,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   Furthermore, the gene has been modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. Item 10. The mutant microorganism according to Item 8 or 9. A gene that imparts a function of converting methanol and / or formic acid into formaldehyde and a gene that imparts formaldehyde immobilization ability into the host microorganism, and the gene is expressed in the host microorganism;
The gene is modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   The mutation according to any one of claims 8 to 11, wherein the gene imparting formaldehyde immobilization ability is a gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase. Microorganisms.   The mutant microorganism according to any one of claims 8 to 11, which has at least one C1 carbon assimilation pathway selected from the group consisting of a serine pathway, a ribulose monophosphate pathway, and a xylose monophosphate pathway as a formaldehyde immobilization pathway. A gene that gives a function of converting methanol and / or formic acid into formaldehyde is introduced into a host microorganism having a ribulose monophosphate pathway, and the gene is expressed in the host microorganism.
The gene is modified so that at least one selected from the group consisting of enhancement of 2-oxoglutarate dehydrogenase activity and deletion or suppression of succinate dehydrogenase activity is performed,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide. A gene that gives a function of converting methanol and / or formic acid into formaldehyde is introduced into a host microorganism having a ribulose monophosphate pathway, and the gene is expressed in the host microorganism.
The gene is modified so that at least one selected from the group consisting of enhancement of fumarate reductase activity, enhancement of pyruvate carboxylase activity, and enhancement of phosphoenolpyruvate carboxykinase activity is performed,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   Furthermore, the gene has been modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed. Item 16. The mutant microorganism according to Item 14 or 15. A gene that gives a function of converting methanol and / or formic acid into formaldehyde is introduced into a host microorganism having a ribulose monophosphate pathway, and the gene is expressed in the host microorganism.
The gene is modified so that at least one selected from the group consisting of deficiency or inhibition of lactate dehydrogenase activity, deficiency or inhibition of acetyl CoA hydrolase activity, and deficiency or inhibition of pyruvate formate lyase activity is performed,
A mutant microorganism capable of producing succinic acid from at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide.   A gene encoding 3-hexulose 6-phosphate synthase and a gene encoding 6-phospho-3-hexoisomerase are further introduced, and the gene is expressed in the host microorganism. The mutant microorganism according to 11, 13, 14, 15, 16 or 17.   The gene imparting the function of converting methanol to formaldehyde is a gene encoding methanol dehydrogenase or alcohol oxidase, and the gene imparting the function of converting formic acid to formaldehyde is a gene encoding formaldehyde dehydrogenase. 18. The mutant microorganism according to any one of 18.   The mutant microorganism according to any one of claims 8 to 19, wherein a gene imparting a function of converting methane to methanol is further introduced, and the gene is expressed in the host microorganism.   The mutant microorganism according to claim 20, wherein the gene imparting a function of converting methane to methanol is a gene encoding methane monooxygenase.   The mutant microorganism according to any one of claims 1 to 21, wherein the gene modification is introduced into the genome.   The mutant microorganism according to any one of claims 1 to 22, wherein a gene encoding a transporter protein that promotes extracellular discharge of succinic acid is introduced, and the gene is expressed.   The mutant microorganism according to any one of claims 1 to 23 is cultured using at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a carbon source, A method for producing succinic acid by causing a mutant microorganism to produce succinic acid.   24. The mutant microorganism according to any one of claims 1 to 23 is contacted with at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde, and formamide, and the mutant microorganism is contacted with the C1. A method for producing succinic acid, comprising producing succinic acid from a compound.

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