Method for producing L-lysine through mixed fermentation

Abstract

The present invention relates to a method for producing L-lysine by mixed fermentation using pseudomonas tolerant to and utilizing high-concentration methanol and corynebacterium glutamicum producing L-lysine. According to the method provided by the invention, the L-lysine can be produced with high yield by taking the methanol as the unique carbon source under the condition that the strain does not need to be subjected to genetic engineering modification.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and specifically to a method for producing L-lysine using methanol as the sole carbon source through mixed-culture fermentation. Background Technology

[0002] Carbon dioxide fixation and utilization is a crucial pathway to achieving the "dual carbon" goal. However, due to its stable structure and low energy content, high-value utilization of carbon dioxide presents significant challenges. Selective hydrogenation of carbon dioxide to methanol offers a highly efficient conversion method. In recent years, China has made significant progress in carbon dioxide hydrogenation to methanol projects, having built the world's largest industrial pilot plant for this purpose. Against this backdrop, methanol is not only considered a substitute for fossil fuels but also a highly promising non-carbohydrate carbon source, which can further expand the sources of biorefining feedstocks and meet people's growing needs. The use of methanol as a carbon source in microbial cell factories has already been applied to produce various high-value-added chemicals, including fatty acids and their derivatives.

[0003] With the excessive consumption of petrochemical resources, increasingly serious environmental problems, and global warming, energy conservation and emission reduction have become a global consensus. Studies show that methane (CH4) accounts for as much as 20% of greenhouse gases contributing to global warming, and its global warming potential over a 20-year period is 86 times that of carbon dioxide (CO2). Currently, anthropogenic emissions account for 63% of total methane emissions (fossil fuel and biomass combustion, livestock farming, and landfills), while natural sources account for 37% (wetlands, oceans, rivers, lakes, and permafrost). At present, my country's methane (biogas, natural gas) utilization is still mainly through combustion, a relatively singular method with a need to improve utilization rates, and it also increases greenhouse gas emissions (burning 1 mol of methane releases 1 mol of carbon dioxide). With the continuous increase in biogas production and proven reserves of natural gas (shale gas, combustible ice) in my country, there is an urgent need to supplement and expand green utilization pathways and technologies for methane. Methanol is a product of methane conversion processes. With the increasing depletion of global oil resources and the reduction in the unit cost of methanol, using methanol as a new source of petrochemical raw materials has become a trend.

[0004] Although there is currently a relative overcapacity in global methanol production, and fluctuations in the methanol market cannot be ruled out due to various reasons, my country has abundant coal resources. As a cornerstone product of C1 chemicals, methanol should continue to expand its application areas, vigorously produce and develop its downstream products, thereby promoting the development of the entire methanol industry.

[0005] On the other hand, constructing microbial cell factories to achieve the bioconversion of methanol into value-added chemicals has advantages such as green processes, mild conditions, and diverse product systems. It can expand the methanol-based product chain and is an important means to achieve green biomanufacturing. In recent years, the application progress and challenges of natural and synthetic methyltrophic bacteria in methanol bioconversion have been discussed. Constructing microbial cell factories using synthetic biology techniques to produce valuable chemicals from renewable raw materials is a major goal of green biomanufacturing. Currently, both natural and synthetic methyltrophic bacteria face certain difficulties in achieving the bioconversion of methanol into value-added chemicals: natural methyltrophic bacteria lack effective gene-editing tools and mainly produce single-cell proteins, primarily using yeast-like fungi (such as Pichia pastoris) as the main methanol chassis cells; methanol bacteria are limited by difficulties in modifying metabolic pathways and excessively long doubling times; and synthetic methyltrophic bacteria (modified E. coli to assimilate methanol) is a very hot topic, currently still focused on research and far from industrialization.

[0006] In summary, the following technical challenges still exist in this field:

[0007] (1) As a microbial cell factory, the production strains of amino acids, especially L-lysine, have low tolerance to methanol and can generally only use sugar carbon sources. They are difficult to grow in cultures where methanol is the only carbon source, let alone produce L-lysine.

[0008] (2) Methyltrophic bacteria that can use methanol as the sole carbon source generally do not have the metabolic pathway to produce amino acids, especially L-lysine, which makes it difficult for methyltrophic bacteria to become suitable chassis cells for microbial cell factories.

[0009] (3) If either of the two strains is engineered through genetic processes, whether by introducing the corresponding L-lysine production pathway enzyme or methanol metabolism pathway enzyme, it is not only time-consuming and laborious, but the growth and production effect of the transformed strain are still limited.

[0010] Therefore, there is an urgent need in this field to develop a fermentation method that can rapidly and efficiently produce L-lysine using methanol as the sole carbon source.

[0011] [Reference Documents]

[0012] 1. Chinese patent application CN 104774792A;

[0013] 2. Jiang Jialiang et al., High-density culture of methane-oxidizing bacteria using methanol as carbon source, Chemical Engineer, 2013, No. 2, pp. 5-8 and 14. Summary of the Invention

[0014] This invention develops a method for producing L-lysine using methanol as the sole carbon source by combining a first bacterial strain capable of metabolizing carbohydrate carbon sources to produce L-lysine with a second bacterial strain capable of tolerating high concentrations of methanol through mixed bacterial fermentation, thus comprehensively utilizing the metabolic pathways of the two strains.

[0015] Specifically, the present invention solves the technical problems existing in the prior art through the following technical solutions:

[0016] 1. A method for producing L-lysine via fermentation using methanol as the sole carbon source, comprising:

[0017] (1) A first bacterial strain capable of metabolizing carbohydrate carbon sources to produce L-lysine is mixed with a second bacterial strain capable of tolerating high concentrations of methanol, and fermented in a medium using methanol as the sole carbon source, wherein the first bacterial strain is different from the second bacterial strain; and

[0018] (2) Optionally, L-lysine is isolated from the resulting fermentation broth.

[0019] 2. The method according to item 1, wherein the first bacterial strain is a Corynebacterium glutamicum sp. strain that metabolizes carbohydrate carbon sources to produce L-lysine.

[0020] 3. The method according to item 1 or 2, wherein the first microbial strain is selected from the group consisting of ATCC 21513, ATCC21514, ATCC 21300 and ATCC 13032.

[0021] 4. The method according to any one of items 1-3, wherein the second bacterial species is a Pseudomonas sp. or Micrococcus luteus sp. species that is resistant to high concentrations of methanol.

[0022] 5. The method according to any one of items 1-4, wherein the second bacterial species is Pseudomonas CGMCC NO:26412 or Micrococcus luteus CGMCC NO:26413.

[0023] 6. The method according to any one of items 1-5, wherein the first strain is pre-cultured in a medium containing methanol before step (1), preferably the pre-culture is carried out in a solid or liquid medium containing 10-20 g / L methanol by subculturing 1-30 times, more preferably the first strain after pre-culture can have an OD value of 4.5 or higher after shaking culture in a medium containing 20 g / L methanol for 24 hours.

[0024] 7. The method according to any one of items 1-6, wherein the second strain is pre-cultured in a medium containing methanol before step (1), preferably in a solid or liquid medium containing 10-20 g / L methanol, and more preferably the second strain after pre-culture can have an OD value of 3.5 or higher after being shaken and cultured in a medium containing 20 g / L methanol for 72 hours.

[0025] 8. The method according to any one of items 1-7, wherein in step (1), the first strain and the second strain are simultaneously inoculated into the production fermentation vessel.

[0026] 9. The method according to item 8, wherein cultures obtained by culturing the first and / or second bacterial strains from their respective glycerol tubes or slants for 24-48 hours are simultaneously inoculated.

[0027] 10. The method according to item 8, wherein the cultures obtained by culturing the first and / or second bacterial strains from their respective glycerol tubes or slant for 24-48 hours are respectively cultured as single bacteria for 24-36 hours, and then mixed inoculated.

[0028] 11. The method according to any one of items 1-7, wherein in step (1), the first strain and the second strain are inoculated into the production fermentation container separately, preferably the second strain is inoculated into the production fermentation container before the first strain.

[0029] 12. The method according to claim 11, wherein the culture obtained by culturing the second strain from the respective glycerol tubes or slant for 24-48 hours is first inoculated into a fermentation vessel and cultured for 24-36 hours, and then the culture obtained by culturing the first strain from the glycerol tubes or slant for 24-48 hours is inoculated into the fermentation vessel.

[0030] 13. The method according to any one of items 1-12, wherein step (1) is performed under the following conditions:

[0031] (a) The experiment was conducted in a culture medium using 15-20 g / L, preferably 20 g / L, methanol as the sole carbon source;

[0032] (b) The process shall be carried out at a temperature of 28-32°C, preferably 30°C;

[0033] (c) Performed under oscillation at 150-250 rpm, preferably 200 rpm;

[0034] (d) The total duration is more than 24 hours, and the selection process is more than 30 hours;

[0035] (e) After 30 hours, reduce the methanol concentration to below 7 g / L, preferably below 0.5 g / L; and / or

[0036] (f) After 30 hours, the concentration of L-lysine is increased to 0.45 g / L, preferably 1.25 g / L or higher.

[0037] 14. The method according to any one of claims 1-13, wherein the culture medium using methanol as the sole carbon source comprises the following components:

[0038] (1) Sodium chloride;

[0039] (2) Ammonium sulfate;

[0040] (3) Dipotassium hydrogen phosphate trihydrate; and

[0041] (4) Methanol.

[0042] 15. The method according to claim 14, wherein the culture medium further comprises the following components: urea, monosodium glutamate, magnesium chloride hexahydrate, manganese sulfate monohydrate, copper sulfate, EDTA, calcium chloride; ferrous sulfate and B vitamins, wherein the B vitamins include VB5, VB1 and VB7.

[0043] To make the technical solution of the present invention clearer, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0044] Figure 1 The first strain capable of metabolizing carbohydrate carbon sources to produce L-lysine was shown in shake-flask fermentation culture in a methanol-containing medium and the production of L-lysine (Comparative Example 1).

[0045] Figure 2 This demonstrates the production of L-lysine by shake-flask fermentation of a second strain capable of tolerating high concentrations of methanol in a methanol-containing medium (Comparative Example 2).

[0046] Figure 3 The experiment demonstrates how seed cultures of the first and second strains were simultaneously inoculated into shake flasks for mixed-culture fermentation and L-lysine production (Example 1).

[0047] Figure 4 The second strain seed culture was cultured for 24-48 hours and then inoculated with the first strain seed culture into a shake flask for mixed fermentation culture and L-lysine production (Example 2).

[0048] Figure 5 The experiment showed that seed cultures of the first and second strains were cultured for 24-48 hours, followed by mixed-culture fermentation and L-lysine production (Example 3); and

[0049] Figure 6This shows the yield of L-lysine produced by different implementation schemes. Invention Details

[0051] 1. The method for producing L-lysine by fermentation according to the present invention

[0052] In one aspect, the present invention provides a method for producing L-lysine by fermentation using methanol as the sole carbon source. In one embodiment, the method comprises: (1) mixing a first bacterial strain capable of metabolizing carbohydrate carbon sources to produce L-lysine with a second bacterial strain capable of tolerating high concentrations of methanol, and fermenting the mixture in a medium using methanol as the sole carbon source. In one embodiment, the first bacterial strain is different from the second bacterial strain. In one embodiment, the method further comprises: (2) optionally separating L-lysine from the resulting fermentation broth.

[0053] In one embodiment, the first bacterial strain is *Corynebacterium glutamicum* sp., which metabolizes carbohydrate carbon sources to produce L-lysine. In one embodiment, the first bacterial strain is selected from the group consisting of ATCC 21513, ATCC 21514, ATCC 21300, and ATCC 13032. In one embodiment, the first bacterial strain is *Corynebacterium glutamicum* ATCC 21513. In one embodiment, the second bacterial strain is a *Pseudomonas* sp. or *Micrococcus luteus* sp., which is resistant to high concentrations of methanol. In one embodiment, the second bacterial strain is *Pseudomonas* CGMCC NO:26412 or *Micrococcus luteus* CGMCC NO:26413.

[0054] In one embodiment, the first bacterial strain is pre-cultured in a methanol-containing medium prior to step (1). In one embodiment, the pre-culture of the first bacterial strain is carried out in a solid or liquid medium containing 10-20 g / L methanol by subculturing for 1-30, 5-30, 10-30, 15-30, 20-30, or 25-30 generations. In one embodiment, the first bacterial strain, after said pre-culture, can achieve an OD of 24 hours after shaking culture in a medium containing 20 g / L methanol. 600The values ​​are 2.0 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3.0 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, 3.5 or higher, 3.6 or higher, 3.7 or higher, 3.8 or higher, 3.9 or higher, 4.0 or higher, 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, or 4.5 or higher. In one embodiment, the first strain pre-cultured can achieve an OD value of 2.0 or higher after shaking culture in a medium with 20 g / L methanol as the sole carbon source for 24 hours. 600 Values ​​are 2.0 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3.0 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, 3.5 or higher, 3.6 or higher, 3.7 or higher, 3.8 or higher, 3.9 or higher, 4.0 or higher, 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, or 4.5 or higher.

[0055] In one embodiment, the second bacterial strain is pre-cultured in a methanol-containing medium prior to step (1). In one embodiment, the pre-culture of the second bacterial strain is carried out in a solid or liquid medium containing 10-20 g / L methanol. In one embodiment, the pre-cultured second bacterial strain can achieve an OD of 72 hours after shaking culture in a medium containing 20 g / L methanol. 600 The values ​​are 1.8 or higher, 1.9 or higher, 2.0 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3.0 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, or 3.5 or higher. In one embodiment, the second strain pre-cultured can achieve an OD value of 1.8 or higher after shaking culture in a medium with 20 g / L methanol as the sole carbon source for 72 hours. 600 Values ​​are 1.8 or higher, 1.9 or higher, 2.0 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 2.6 or higher, 2.7 or higher, 2.8 or higher, 2.9 or higher, 3.0 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, or 3.5 or higher.

[0056] In one embodiment, in step (1), the first and second strains are simultaneously inoculated into the production fermentation vessel. In one embodiment, cultures obtained by culturing the first and / or second strains from their respective glycerol tubes or slant for 24-48 hours are simultaneously inoculated into the production fermentation vessel. In one embodiment, cultures obtained by culturing the first and / or second strains from their respective glycerol tubes or slant for 24-48 hours are separately cultured as single strains for 24-36 hours before mixed inoculation.

[0057] In one embodiment, in step (1), the first strain and the second strain are inoculated into the production fermentation vessel separately (i.e., not simultaneously). In one embodiment, the second strain is inoculated into the production fermentation vessel before the first strain. In one embodiment, the culture obtained by culturing the second strain from its respective glycerol tube or slant for 24-48 hours is first inoculated into the fermentation vessel and cultured for 24-36 hours, and then the culture obtained by culturing the first strain from its glycerol tube or slant for 24-48 hours is inoculated into the fermentation vessel.

[0058] In one embodiment, step (1) is carried out under one or more of the following conditions: (a) in a culture medium with 15-20 g / L methanol as the sole carbon source; (b) at a temperature of 28-32°C; (c) under shaking at 150-250 rpm; (d) for a total of more than 24 hours; (e) after 30 hours, reducing the methanol concentration to below 7 g / L; and / or (f) after 30 hours, increasing the L-lysine concentration to above 0.45 g / L. In one embodiment, step (1) is carried out under one or more of the following conditions: (a) in a culture medium with 20 g / L methanol as the sole carbon source; (b) at a temperature of 30°C; (c) under shaking at 200 rpm; (d) for a total of more than 30 hours; (e) after 30 hours, reducing the methanol concentration to below 0.5 g / L; and / or (f) after 30 hours, increasing the L-lysine concentration to above 1.25 g / L.

[0059] In one embodiment, the culture medium comprises the following components: (1) sodium chloride; (2) ammonium sulfate; (3) dipotassium hydrogen phosphate trihydrate; and (4) methanol. In one embodiment, the culture medium further comprises the following components: urea, monosodium glutamate, magnesium chloride hexahydrate, manganese sulfate monohydrate, copper sulfate, EDTA, calcium chloride, ferrous sulfate, and B vitamins. In one embodiment, the B vitamins include VB5, VB1, and VB7.

[0060] 2. Advantages and beneficial effects of the present invention

[0061] Compared with existing methods, the method of the present invention has at least the following advantages:

[0062] (1) It can metabolize L-lysine through the coordinated fermentation of two bacteria with methanol as the sole carbon source, thus realizing the biotransformation of the carbon source from one-carbon methanol to multi-carbon amino acids, and achieving the transformation of methanol as a carbon source into biochemical products.

[0063] (2) By domesticating Corynebacterium glutamicum to tolerate a certain concentration of methanol, the mixed fermentation of two different carbon source bacteria was achieved with methanol as the sole carbon source.

[0064] (3) Find a new method to solve the problems of gene editing difficulties in natural methyltrophic bacteria, complex metabolic pathways, and difficulties in converting biochemical products with methanol as the sole carbon source.

[0065] (4) It was realized that Corynebacterium glutamicum, which uses glucose as a carbon source to ferment and metabolize amino acids, can grow and metabolize L-lysine in a culture medium with methanol as the sole carbon source.

[0066] This invention has at least the following beneficial technical effects:

[0067] 1. A breakthrough has been achieved in the one-carbon conversion of biochemical products through mixed bacterial fermentation.

[0068] 2. It does not require modification of the metabolic pathway of Pseudomonas methanolis, thus avoiding the difficulties of gene editing and the uncertainty of bacterial growth.

[0069] 3. With the increasing maturity of the technology for producing methanol by adding hydrogen to carbon dioxide, a new method has been found for utilizing carbon dioxide and thus reducing greenhouse gas emissions. Detailed Implementation

[0070] The present invention will be further described below with reference to specific embodiments, but these specific embodiments should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make various changes or modifications to these specific embodiments without departing from the scope of the technical solution of the present invention, and the changed and modified implementation schemes still fall within the protection scope of the present invention.

[0071] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0072] Solid culture medium F1

[0073] Sucrose: 10 g / L; Beef extract: 10 g / L; Yeast powder: 10 g / L; Urea: 2.5 g / L; Sodium chloride: 2 g / L; Agar powder: 20 g / L; pH: adjusted to 7.0 with KOH solution.

[0074] Rejuvenating medium F2

[0075] Sucrose: 20 g / L; Polypeptone: 10 g / L; Yeast extract: 5 g / L; Urea: 3.5 g / L; Cysteine: 0.5 g / L; Ammonium sulfate: 5 g / L; Dipotassium hydrogen phosphate: 10.5 g / L; Magnesium sulfate: 0.5 g / L; Potassium dihydrogen phosphate: 4 g / L; Biotin: 0.2 mg / L; Vitamin B1: 1.5 mg / L; Nicotinamide (Vitamin B3): 3 mg / L; Calcium dextropantoate (Vitamin B5): 2 mg / L

[0076] Inorganic salt complex culture medium F3

[0077] Sodium chloride: 2 g / L, urea: 2 g / L, ammonium sulfate: 6 g / L, dipotassium hydrogen phosphate trihydrate: 8 g / L, monosodium glutamate: 2 g / L, magnesium chloride hexahydrate: 0.5 g / L, manganese sulfate monohydrate: 0.1 g / L, copper sulfate: 0.01 g / L, EDTA: 0.05 g / L, calcium chloride: 0.1 g / L, ferrous sulfate: 0.15 g / L, vitamin B5: 0.1 g / L, vitamin B1: 0.1 g / L, vitamin B7: 0.05 g / L.

[0078] Inorganic salt solid composite culture medium F3g

[0079] Sodium chloride: 2 g / L, agar powder: 20 g / L, urea: 2 g / L, ammonium sulfate: 6 g / L, dipotassium hydrogen phosphate trihydrate: 8 g / L, monosodium glutamate: 2 g / L, magnesium chloride hexahydrate: 0.5 g / L, manganese sulfate monohydrate: 0.1 g / L, copper sulfate: 0.01 g / L, EDTA: 0.05 g / L, calcium chloride: 0.1 g / L, ferrous sulfate: 0.15 g / L, VB5: 0.1 g / L, VB1: 0.1 g / L, VB7: 0.05 g / L.

[0080] Example 1. Pre-culture of the first bacterial strain in methanol-containing medium

[0081] 1.1 Strain rejuvenation:

[0082] The commercially available L-lysine-glutamic acid-producing Corynebacterium ATCC 21513 glycerol cryovial was removed from the -80℃ freezer and streaked onto solid medium F1. After incubation at 30℃ for 48 hours, larger single colonies were picked and inoculated into shake flasks (200 / 500 mL) containing rejuvenation medium F2. The flasks were then incubated at 30℃ with shaking at 200 rpm for 24 hours. After fermentation, the rejuvenated bacterial culture was obtained. The OD of the culture was... 600 The value is around 6.0, indicating that the bacterial solution is awaiting acclimatization and tolerance to methanol.

[0083] 1.2 Methanol pre-culture of bacterial strains

[0084] 1.2.1 Tolerance Process: 2 mL of the activated ATCC 21513 bacterial suspension from 1.1 was inoculated into a 200 / 500 mL shake flask containing 10 g / L methanol in rejuvenation medium F2. The flask was incubated at 30°C with shaking at 200 rpm for 24 hours. The OD of the bacterial suspension was measured. 600 The OD value was around 2.0, indicating that methanol had a certain inhibitory effect on the growth of ATCC 21513. A bacterial suspension with an OD of 2.0 was streaked onto solid medium F1 containing 10 g / L methanol and incubated at 30°C for 48 hours. Larger single colonies were then picked and inoculated into shake flasks (200 / 500 mL) containing 10 g / L methanol rejuvenation medium F2. The flasks were incubated at 30°C with shaking at 200 rpm for 24 hours. The OD value of the bacterial suspension was then... 600 The OD value was approximately 3.0. A bacterial suspension with an OD of 3.0 was streaked onto a solid agar plate (F1) containing 20 g / L methanol. After incubating at 30°C for 48 hours, a single colony with a larger size was picked, diluted in 200 μl of sterile water, and evenly spread onto another solid agar plate (F1) containing 20 g / L methanol. This process was repeated, and single colonies from generations 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 were inoculated into shake flasks and incubated at 30°C with shaking at 200 rpm for 24 hours. The OD value was then measured. 600 The changes are shown in Table 1. After the 30th generation of plate subculturing, a single colony was picked and inoculated into a shake-flask (200 / 500 mL) containing 20 g / L methanol-containing rejuvenation medium F2. The culture was incubated at 30℃ and 200 rpm for 24 hours. The OD of the bacterial culture was... 600 It is around 4.5, OD 600 The viability of the strain was improved by increasing the growth factor from 2.5 in the first generation to 4.5, resulting in strain ATCC 21513, which can grow in F2 rejuvenation medium containing 20 g / L methanol. This strain was then stored in glycerol tubes.

[0085] Since the first bacterial strain can be any Corynebacterium glutamicum sp. that produces L-lysine from metabolized carbohydrate carbon sources, based on the above operation on ATCC 21513, ATCC21514, ATCC 21300, and ATCC 13032 were also pre-cultured in methanol, and bacterial strains ATCC 21514, ATCC 21300, and ATCC 13032 that can grow in F2 rejuvenation medium containing 20 g / L methanol were obtained, respectively.

[0086] 1.2.2 Verify the tolerance of the first pre-cultured bacterial strain to methanol-inorganic salt medium:

[0087] Glyceryl sludge from the pre-cultured ATCC 21513 strain was streaked onto solid medium F1 containing 20 g / L methanol. After incubation at 30°C for 48 hours, larger single colonies were picked and inoculated into shake flasks (200 / 500 mL) containing 20 g / L methanol rejuvenation medium F2. The flasks were then incubated at 30°C with shaking at 200 rpm for 24 hours. The OD of the bacterial culture was measured. 600 The OD value was approximately 4.5. The fermentation broth was centrifuged at 3500 rpm for 3 minutes in a 50 mL sterile centrifuge tube, then resuspended in 150 mL of sterile culture medium F3. After resuspending, 20 g / L methanol was added, and the OD value was measured. 600 The concentration was 3.0, and the sample was transferred to a shake flask (200 / 500 mL). The flask was incubated at 30°C and 200 rpm for 24 hours, and the OD value was then measured. 600 The OD value was 2.75, and the methanol content was 19.7 g / L, indicating that strain ATCC21513 could maintain its biomass in compound inorganic salt medium F3 containing 20 g / L methanol, but did not degrade methanol. The obtained culture was inoculated at a 10% inoculation rate into baffled shake flasks (200 / 500 mL) containing 20 g / L methanol rejuvenation medium F2, and cultured at 30℃ with shaking at 200 rpm for 24 hours. 600 The value was around 4.7, indicating that the bacterial viability had not declined. This also verified the tolerance of pre-cultured ATCC 13032 in methanol-inorganic salt medium (OD value of the bacterial culture). 600 (Approximately 4.3).

[0088] Table 1. OD of fermentation broth after methanol plate subculturing of the first strain 600 change

[0089]

[0090] Example 2. Methanol pre-culture of the second bacterial strain

[0091] Take the methanol-resistant Pseudomonas MJMm-2 cryopreserved in our laboratory from a -80℃ freezer, streak it onto a solid medium F3g containing 20 g / L methanol, and incubate at 30℃ for 72 hours. Then, pick larger single colonies and inoculate them into shake flasks (200 / 500 mL) containing 20 g / L methanol medium F3. Incubate at 30℃ and 200 rpm for 72 hours. After fermentation, obtain a first-generation revitalized bacterial suspension. The OD of the bacterial suspension... 600 The concentration is 3.2, the methanol residue is 2.5 g / L, and the OD value is... 600The fermentation broth from step 3.2 was further streaked onto solid medium F3 containing 20 g / L methanol and incubated at 30°C for 72 hours. Larger single colonies were then picked and inoculated into shake flasks (200 / 500 mL) containing 20 g / L methanol of medium F3. The flasks were incubated at 30°C and 200 rpm for 72 hours with shaking. After fermentation, the second-generation revitalized bacterial culture was obtained. The OD of the bacterial culture was... 600 The concentration was 3.55, and it was stored in a glycerol tube for subsequent experiments.

[0092] Since the second bacterial species can be a Pseudomonas sp. or Micrococcus luteus sp. that can tolerate high concentrations of methanol, based on the above operation on MJMm-2, MJMm-1 was also pre-cultured in methanol to obtain the second-generation revitalized and activated bacterial solution of MJMm-1.

[0093] Comparative Example 1. Shake-flask fermentation and L-lysine production experiment of the first strain

[0094] The ATCC 21513 glycerol strain, pre-cultured in methanol, was streaked onto a solid culture medium F1 containing 20 g / L methanol. After incubation at 30°C for 48 hours, bacterial colonies were picked and inoculated onto culture medium F2 containing 20 g / L methanol in shake flasks (50 / 250 mL). The culture was then incubated at 30°C with shaking at 200 rpm for 24 hours. The OD of the bacterial culture was measured. 600 The OD value was approximately 4.8. 30 mL of fermentation broth was centrifuged at 3500 rpm for 3 minutes in a 50 mL sterile centrifuge tube, then resuspended in 50 mL of sterile culture medium F3. After resuspending, 20 g / L methanol was added, and the OD value was measured. 600 The concentration was 2.5, and the mixture was transferred to a baffled shake flask (50 / 250 mL). The flask was incubated at 30°C and 200 rpm for 30 hours, with samples taken every 2 hours to measure the OD value of the fermentation broth. 600 Methanol concentration and L-lysine metabolism, such as Figure 1 As shown.

[0095] OD in fermentation broth 600 Changes: During the 30-hour fermentation process, OD 600 Slightly decreased;

[0096] Changes in methanol concentration: During the 30-hour fermentation process, the methanol concentration remained essentially unchanged.

[0097] L-Lysine production: During the 30-hour fermentation process, the L-lysine production remained at 0.

[0098] Results analysis: The rejuvenated bacterium ATCC 21513 was able to maintain its biomass (OD) essentially in F3 medium containing 20 g / L methanol. 600 (There was no significant increase or decrease, it only existed in the form of live bacteria), but it could not degrade methanol. L-lysine was not detected throughout the fermentation process, indicating that the Corynebacterium glutamicum strain ATCC 21513, which was precultured in methanol, could not produce L-lysine in F3 medium containing 20 g / L methanol.

[0099] Comparative Example 2. Shake-flask fermentation and L-lysine production experiment of the second strain

[0100] 200 μl of the second-generation revitalized strain MJMm-2 was inoculated into 10 / 30 mL test tubes of F3 medium containing 20 g / L methanol. After culturing at 30 °C and 200 RPM for 48 hours, 5 mL of the culture was inoculated into 50 / 250 mL shake flasks with baffles of F3 medium containing 20 g / L methanol. The culture was then incubated at 30 °C and 200 RPM for 30 hours. Odion displacement (OD) of the fermentation broth was measured every 2 hours. 600 Methanol concentration and L-lysine metabolism, such as Figure 2 As shown.

[0101] Fermentation broth OD 600 Changes: During the 30-hour fermentation process, OD 600 It continued to grow to 2.7;

[0102] Changes in methanol concentration: During the 30-hour fermentation process, the methanol concentration decreased from 20 g / L to 0.3 g / L;

[0103] L-Lysine production: During the 30-hour fermentation process, the L-lysine production remained at 0.

[0104] Results analysis: The revitalized bacterium MJMm-2 grew normally in F3 medium containing 20 g / L methanol, consuming most of the methanol. OD 600 The growth rate reached 2.7, and no L-lysine was detected during the entire fermentation process, indicating that strain MJMm-2 could grow normally in F3 medium containing 20 g / L methanol, but could not produce L-lysine.

[0105] Example 3. Mixed-culture shake-flask fermentation experiment I (simultaneous inoculation)

[0106] 200 μl of glycerol-containing Pseudomonas mJMm-2 was inoculated into a 10 / 30 mL test tube of F3 medium containing 20 g / L methanol and cultured at 30 °C and 200 RPM for 48 hours. OD was then measured. 600 =2.4.

[0107] ATCC 21513 glycerol bacteria, pre-cultured in methanol, were streaked onto solid medium F1 containing 20 g / L methanol. After incubation at 30°C for 48 hours, bacterial colonies were picked and inoculated into shake flasks (50 / 250 mL) containing medium F2 containing 20 g / L methanol. The culturing was carried out at 30°C with shaking at 200 rpm for 24 hours. The OD of the bacterial culture was measured. 600 The concentration was approximately 4.8. 30 mL of fermentation broth was centrifuged in a 50 mL sterile centrifuge tube at 3500 rpm for 3 minutes and then resuspended in 50 mL of sterile culture medium F3.

[0108] Take 1 mL (OD) 600 =2.4) Pseudomonas MJMm-2 culture medium in test tubes containing culture medium F3 and 5 mL (OD 600 The resuspension of Corynebacterium glutamicum ATCC 21513 (4.8%) in F3 medium was simultaneously inoculated into a shake flask (50 / 250 mL) containing F3 medium with 20 g / L methanol. The mixture was cultured at 30°C and 200 rpm for 30 hours. The OD value of the fermentation broth was measured during the fermentation process. 600 Methanol content and L-lysine content, fermentation process as follows Figure 3 As shown.

[0109] Fermentation broth OD 600 Changes: During the 30-hour fermentation process, OD 600 Increased from the initial 1.0 to 2.8;

[0110] Changes in methanol concentration: During the 30-hour fermentation process, methanol concentration decreased from an initial 20 g / L to 6.4 g / L;

[0111] L-Lysine yield: During the 30-hour fermentation process, the L-Lysine yield increased from the initial 0 g / L to 0.47 g / L after 30 hours.

[0112] Results analysis: During the mixed fermentation of *Pseudomonas* MJMm-2 and *Corynebacterium glutamicum* ATCC 21513 simultaneously inoculated into F3 medium containing 20 g / L methanol, the methanol concentration decreased from 20 g / L to 6.4 g / L, and the OD... 600 The yield of L-lysine increased from an initial 1.0 to 2.8, rising from 0 g / L initially to 0.47 g / L after 30 hours. Compared to Comparative Examples 1-2, methanol was degraded by *Pseudomonas* MJMm-2, and the carbon source from the methanol degradation flowed towards L-lysine. Although MJMm-2 could not utilize methanol to produce L-lysine, the intermediate product from MJMm-2's methanol degradation was used as a substrate by *Corynebacterium glutamicum* ATCC 21513 to produce L-lysine. The mixed-culture fermentation results demonstrated the production of L-lysine using methanol as a substrate.

[0113] For verification purposes, a similar experiment was also performed on a mixed culture of *Micrococcus luteus* MJMm-1 and *Corynebacterium glutamicum* ATCC 13032. The experimental results were consistent with... Figure 3 resemblance:

[0114] Fermentation broth OD 600 Changes: During the 30-hour fermentation process, OD 600 Increased from the initial 1.0 to 2.6;

[0115] Changes in methanol concentration: During the 30-hour fermentation process, methanol concentration decreased from an initial 20 g / L to 9.8 g / L;

[0116] L-Lysine yield: During the 30-hour fermentation process, the L-Lysine yield increased from the initial 0 g / L to 0.42 g / L after 30 hours.

[0117] Example 4. Mixed-culture shake-flask fermentation experiment II (staggered inoculation, inoculating the second strain first, then the first strain)

[0118] Analysis of the results of the mixed-culture fermentation scheme after simultaneous inoculation in Example 3 shows that L-lysine was successfully produced from methanol using mixed-culture fermentation, achieving a breakthrough in amino acid production. However, the OD of the mixed-culture fermentation in Example 3... 600 The growth was relatively slow, and the methanol degradation rate was also slower than that of single-strain fermentation of MJMm-2. Based on the experimental data, it is speculated that there may be a difference in the growth and metabolic rates of the two strains after simultaneous inoculation. In particular, the growth of the second strain (such as MJMm-2) limited the subsequent production of the first strain (such as ATCC21513). This example verifies whether fermenting the second strain (such as MJMm-2) first and accumulating a certain biomass before adding the first strain (such as ATCC 21513) can more efficiently and quickly utilize methanol to produce L-lysine through mixed culture.

[0119] 200 μl of glycerol bacterium MJMm-2 was inoculated into a 10 / 30 mL test tube of F3 medium containing 20 g / L methanol. After culturing at 30 °C and 200 RPM for 48 hours, 5 mL of the culture was inoculated into a 50 / 250 mL shake flask with baffles of F3 medium containing 20 g / L methanol. The culture was then incubated at 30 °C and 200 RPM for 30 hours. The OD of the fermentation broth was then measured. 600 =2.6, methanol content =0.5g / L, L-lysine content is 0.

[0120] ATCC 21513 glycerol bacteria, pre-cultured in methanol, were streaked onto solid medium F1 containing 20 g / L methanol. After incubation at 30°C for 48 hours, bacterial colonies were picked and inoculated into shake flasks (50 / 250 mL) containing medium F2 containing 20 g / L methanol. The flasks were then incubated at 30°C with shaking at 200 rpm for 24 hours. The OD of the bacterial culture was measured. 600 To obtain a concentration of 5.0, centrifuge 30 mL of fermentation broth in a 50 mL sterile centrifuge tube at 3500 rpm for 3 minutes, and then resuspend it in 5 mL of sterile culture medium F3.

[0121] Inoculate 5 mL of ATCC 21513 F3 resuspension into OD. 600 =2.6, MJMm-2 fermentation broth with methanol content of 0.5 g / L and L-lysine content of 0, 20 g / L of methanol was added, and the initial OD was measured. 600 =5.1, L-lysine content was 0.03 g / L, cultured at 30℃ and 200 RPM, and then cultured for another 30 hours before termination. The OD of the fermentation broth was measured during the fermentation process. 600 The content of methanol and L-lysine, and the fermentation process as follows Figure 4 As shown.

[0122] Changes in OD600 of fermentation broth: After 30 hours of fermentation with a single strain of MJMm-2, OD600... 600 The OD increased from an initial 0.11 to 2.5, and after adding ATCC 21513 F3 resuspension and 20 g / L methanol, the OD... 600 After the mixed culture was cultured for 30 hours, the OD value was reduced to 5.1. 600 It increased to 6.5.

[0123] Changes in methanol concentration: After 30 hours of fermentation with a single strain MJMm-2, the methanol concentration decreased from an initial 20 g / L to 0.5 g / L. After adding ATCC 21513 F3 resuspension and 20 g / L methanol, the methanol concentration decreased to 5.9 g / L after 30 hours of mixed culture.

[0124] L-Lysine metabolism: During the 30-hour fermentation of single-strain MJMm-2, the amount of L-lysine remained at 0. After adding ATCC 21513 F3 resuspension and 20 g / L methanol, the initial L-lysine content was 0.03 g / L. After 30 hours of mixed culture, the L-lysine content increased to 0.84 g / L.

[0125] Results Analysis: Compared with Example 3, which involved co-inoculation of Pseudomonas MJMm-2 and Corynebacterium glutamicum ATCC 21513 for mixed fermentation, the final L-lysine content in this example was increased to 0.84 g / L. Because the second strain (e.g., MJMm-2) was used for single-strain fermentation for 30 hours to obtain sufficient biomass before inoculation with the first strain (e.g., ATCC21513) of approximately the same biomass, the growth rates of both strains were improved. However, the methanol degradation rate after mixed fermentation in this example was still not as fast as that of the second strain (e.g., MJMm-2) alone. The reason for this may be that the culture medium became nutrient-deficient after the single-strain culture of the second strain (e.g., MJMm-2) and the mixed culture.

[0126] Example 5. Mixed-culture shake-flask fermentation experiment III (the second and first strains were cultured separately as single cultures and then mixed for fermentation)

[0127] A comprehensive analysis of the results of the mixed-culture fermentation schemes in Examples 3 and 4 shows that both successfully produced L-lysine from methanol using mixed-culture fermentation, achieving a breakthrough in amino acid production. However, the methanol degradation rate was still not ideal. This may be due to differences in the growth / production rates of the two strains and the potential lack of nutrients in the culture medium. This example verifies the following scheme: the second strain (e.g., MJMm-2) and the first strain (e.g., ATCC 21513) are cultured as single strains. After both strains have obtained sufficient biomass, they are then mixed for fermentation. This can shorten the growth time of the two strains and allow them to quickly enter the amino acid production pathway.

[0128] 200 μl of glycerol bacterium MJMm-2 was inoculated into a 10 / 30 mL test tube of F3 medium containing 20 g / L methanol. After culturing at 30 °C and 200 RPM for 48 hours, 5 mL of the culture was inoculated into a 50 / 250 mL shake flask with baffles of F3 medium containing 20 g / L methanol. The culture was then incubated at 30 °C and 200 RPM for 30 hours. The OD of the fermentation broth was then measured. 600 =2.4, centrifuge 50 mL of fermentation broth in a 50 mL sterile centrifuge tube at 3500 rpm for 3 minutes, and then resuspend it in 5 mL of sterile culture medium F3.

[0129] ATCC 21513 glycerol bacteria, pre-cultured in methanol, were streaked onto solid medium F1 containing 20 g / L methanol and incubated at 30°C for 48 hours. The bacterial colony was then picked and inoculated into shake flasks (50 / 250 mL) containing medium F2 containing 20 g / L methanol. The culturing was continued at 30°C with shaking at 200 rpm for 24 hours. The OD of the bacterial culture was measured. 600 The concentration was 4.5. 30 mL of fermentation broth was centrifuged in a 50 mL sterile centrifuge tube at 3500 rpm for 3 minutes and then resuspended in 5 mL of sterile culture medium F3.

[0130] The F3 resuspensions of MJMm-2 and ATCC 21513 obtained above were inoculated into 50 / 250 mL shake flasks with baffles containing F3 medium containing 20 g / L methanol. The flasks were incubated at 30 °C and 200 RPM for 30 hours. The OD value of the fermentation broth was measured during the fermentation process. 600 The content of methanol and L-lysine, and the fermentation process as follows Figure 5 As shown.

[0131] Results analysis:

[0132] Fermentation broth OD 600 Changes: After single-cell fermentation of MJMm-2 and ATCC 21513, the cultures were resuspended in sterile medium F3, then mixed and inoculated into medium F3 with the addition of 20 g / L methanol. OD 600 The initial value was 4.0. After 30 hours of mixed-culture fermentation, the OD... 600 Increased to 7.1;

[0133] Changes in methanol concentration: After 30 hours of mixed-culture fermentation, methanol concentration decreased from an initial 20 g / L to 0.3 g / L;

[0134] L-Lysine metabolism: After 30 hours of mixed-culture fermentation, the L-lysine content increased from the initial 0 g / L to 1.25 g / L.

[0135] Analysis of the embodiments of the present invention. Comparison of L-lysine production in different embodiments.

[0136] like Figure 6 As shown, comparing the L-lysine yields of Examples 3-6 and Comparative Examples 1-2, it is evident that this invention achieves a breakthrough in converting one carbon atom (methanol) into biochemical products (amino acids) through mixed-culture fermentation. The method of this invention does not require modification of the metabolic pathways / routes of any microbial strains, significantly reducing the technical barriers and high costs associated with genetic modification. Through optimized technology, this invention ultimately achieves the production of up to 1.25 g / L of L-lysine using a culture medium with 20 g / L methanol as the sole carbon source, providing a new method for utilizing carbon dioxide to reduce greenhouse gas emissions.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to the above-disclosed technical content without departing from the scope of the present invention, and all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for producing L-lysine via fermentation using methanol as the sole carbon source, comprising: (1) A first strain capable of metabolizing carbohydrate carbon sources to produce L-lysine is mixed with a second strain capable of tolerating high concentrations of methanol and fermented in a medium with methanol as the sole carbon source, wherein the first strain is different from the second strain. and (2) Optionally, L-lysine is isolated from the resulting fermentation broth.

2. The method according to claim 1, wherein the first bacterial strain is a Corynebacterium glutamicum sp. strain that metabolizes carbohydrate carbon sources to produce L-lysine.

3. The method according to claim 1 or 2, wherein the first bacterial strain is selected from the group consisting of ATCC 21513, ATCC21514, ATCC 21300 and ATCC 13032.

4. The method according to any one of claims 1-3, wherein the second bacterial species is a Pseudomonas sp. or Micrococcus luteus sp. that is resistant to high concentrations of methanol.

5. The method according to any one of claims 1-4, wherein the second bacterial species is Pseudomonas CGMCC NO:26412 or Micrococcus luteus CGMCC NO:26413.

6. The method according to any one of claims 1-5, wherein the first strain is pre-cultured in a medium containing methanol before step (1), preferably the pre-culture is carried out in a solid or liquid medium containing 10-20 g / L methanol by subculturing for 1-30 generations, more preferably the first strain after pre-culture can have an OD value of 4.5 or higher after shaking culture in a medium containing 20 g / L methanol for 24 hours.

7. The method according to any one of claims 1-6, wherein the second strain is pre-cultured in a medium containing methanol before step (1), preferably in a solid or liquid medium containing 10-20 g / L methanol, and more preferably the second strain after pre-culture can have an OD value of 3.5 or higher after being shaken and cultured in a medium containing 20 g / L methanol for 72 hours.

8. The method according to any one of claims 1-7, wherein in step (1), the first strain and the second strain are simultaneously inoculated into the production fermentation vessel.

9. The method according to claim 8, wherein cultures obtained by culturing the first and / or second bacterial strains from their respective glycerol tubes or slants for 24-48 hours are simultaneously inoculated.

10. The method according to claim 8, wherein the cultures obtained by culturing the first and / or second bacterial strains from their respective glycerol tubes or slant for 24-48 hours are then subjected to single-cell culture for 24-36 hours, and then mixed inoculation is performed.

11. The method according to any one of claims 1-7, wherein in step (1), the first strain and the second strain are inoculated into the production fermentation container separately, preferably the second strain is inoculated into the production fermentation container before the first strain.

12. The method according to claim 11, wherein the culture obtained by culturing the second strain from the respective glycerol tubes or slant for 24-48 hours is first inoculated into a fermentation vessel and cultured for 24-36 hours, and then the culture obtained by culturing the first strain from the glycerol tubes or slant for 24-48 hours is inoculated into the fermentation vessel.

13. The method according to any one of claims 1-12, wherein step (1) is performed under the following conditions: (a) The experiment was conducted in a culture medium using 15-20 g / L, preferably 20 g / L, methanol as the sole carbon source; (b) The process shall be carried out at a temperature of 28-32°C, preferably 30°C; (c) Performed under oscillation at 150-250 rpm, preferably 200 rpm; (d) The total duration is more than 24 hours, and the selection process is more than 30 hours; (e) After 30 hours, reduce the methanol concentration to below 7 g / L, preferably below 0.5 g / L; and / or (f) After 30 hours, the concentration of L-lysine is increased to 0.45 g / L, preferably 1.25 g / L or higher.

14. The method according to any one of claims 1-13, wherein the culture medium using methanol as the sole carbon source comprises the following components: (1) Sodium chloride; (2) Ammonium sulfate; (3) Dipotassium hydrogen phosphate trihydrate; and (4) Methanol.

15. The method according to claim 14, wherein the culture medium further comprises the following components: urea, monosodium glutamate, magnesium chloride hexahydrate, manganese sulfate monohydrate, copper sulfate, EDTA, calcium chloride; ferrous sulfate and B vitamins, wherein the B vitamins include VB5, VB1 and VB7.

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