High-yield strategy of porphyrin compound and application thereof

Abstract

The invention provides a high-yield strategy and application of a porphyrin compound. The strategy comprises: fermentative culturing a porphyrin compound producing strain to produce a porphyrin compound; wherein an additive selected from the group consisting of 5-aminolevulinic acid (5-ALA), glucose, glutamate, succinic acid, or a combination thereof, is added before the start of fermentation or during the fermentation. According to the technical scheme, the yield of the porphyrin compound can be remarkably increased, the cost is low, and operation is easy.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and fermentation engineering, and more specifically, relates to a high-yield strategy for a porphyrin compound and an application thereof. Background Art

[0002] Porphyrins are widely found in nature. They are composed of four pyrrole subunits, and adjacent subunits are bridged by methylene groups and cyclized to form porphyrin macrocyclic molecules. Porphyrin compounds are essential for maintaining the life of plants and mammals. The eight β positions on the pyrrole ring and the four hydrogen atoms inside the porphyrin ring can be replaced by other groups to form porphyrin derivatives.

[0003]

[0004] Most porphyrin compounds exist as metallated porphyrin macrocyclic molecules. When they are combined with apoproteins, they play an important role in light capture, electron transfer and oxygen binding. Porphyrin compounds in nature include heme, chlorophyll (Chlorophyll, Chl), bacterial chlorophyll (Bacteriochlorophyll, Bchl), vitamin B 12 (Vitamin B 12 ,VB 12 ), Siroheme, coenzyme F430 and heme d1, etc., play an important role in the life activities of organisms.

[0005] Porphyrin compounds can be obtained from natural substances containing porphyrins in nature by extraction, separation, purification and other methods. At present, there are two ways to obtain the target porphyrin molecules in the art: structural modification of natural porphyrins and total synthesis of porphyrin compounds. Although the structural modification of natural porphyrins can easily transform the structure, it is limited by the structure itself, and the selection of exocyclic functional groups is also very limited. In addition, the physiological activity of the porphyrin compound itself is also limited. Therefore, people usually need to obtain porphyrin molecules with specific physiological activity and function by total synthesis.

[0006] In the past, some methods for synthesizing porphyrin compounds include the Rothemund method, the Adler method, the Lindesy method, and the microwave excitation method. The Rothemund method uses aldehyde compounds and pyrrole as raw materials, pyridine and methanol as solvents, reacts in a glass tube with an air outlet, reacts in a 90-95°C water bath for 30 minutes, cools the reaction solution and filters it, obtains the filtrate and removes the methanol, vacuum concentrates it, introduces it into ether, filters it, washes it, and uses hydrochloric acid, sodium acetate, sodium hydroxide, and sodium sulfate to concentrate it using the Willstatter method to precipitate the porphyrin. The Adler method is an improvement on the Rothemund method, and is also a method based on multi-step chemical reactions.

[0007] In summary, this field urgently needs to further explore optimized biosynthetic methods for preparing porphyrin compounds. Summary of the invention

[0008] The purpose of the present invention is to provide a high-yield strategy for porphyrin compounds and application thereof.

[0009] In a first aspect of the present invention, a fermentation method for high-yield porphyrin compounds is provided, comprising: fermenting and culturing porphyrin compound-producing bacteria to produce porphyrin compounds; wherein, before the start of fermentation or during fermentation, an additional component selected from the following group is added: 5-aminolevulinic acid (5-ALA), glucose, glutamate, succinic acid, or a combination thereof.

[0010] In one or more preferred embodiments, the added ingredient is 5-aminolevulinic acid; the added amount of 5-aminolevulinic acid is 0.5-5 g / L, preferably 1-4 g / L (such as 2 or 3 g / L), more preferably 1.5-2.5 g / L (optimally 2±0.2 g / L).

[0011] In one or more preferred embodiments, the 5-aminolevulinic acid is added to the initial fermentation medium.

[0012] In one or more preferred embodiments, while the initial culture medium includes glucose, glucose is added when the fermentation is at a stage of 30 to 42 hours, preferably 32 to 40 hours (such as 34, 35, 36, 37, 38, 39 hours); preferably, glucose is also added when the fermentation is at a stage of 55 to 70 hours (more preferably 58 to 68 hours, such as 60, 62, 64, 66 hours).

[0013] In one or more preferred embodiments, the amount of glucose added is 5-20 g / L, preferably 7-15 g / L (such as 8, 9, 10, 11, 12 or 13 g / L), more preferably 10±2 g / L (optimally 10±1 g / L).

[0014] In one or more preferred embodiments, the added ingredient is glutamate; the added amount of glutamate is 5-20 g / L, preferably 7-15 g / L (such as 8, 9, 10, 11, 12 or 13 g / L), more preferably 10±2 g / L (optimally 10±1 g / L).

[0015] In one or more preferred embodiments, the glutamate is added to the initial fermentation medium.

[0016] In one or more preferred embodiments, the glutamate comprises: sodium glutamate, glutamine and L-glutamate hydrochloride.

[0017] In one or more preferred embodiments, the added ingredient is succinic acid; the added amount of succinic acid is 5-20 g / L, preferably 7-15 g / L (such as 8, 9, 10, 11, 12 or 13 g / L), more preferably 10±2 g / L (optimally 10±1 g / L).

[0018] In one or more preferred embodiments, the succinic acid is added to the initial fermentation medium.

[0019] In one or more preferred embodiments, the additional ingredients are 5-aminolevulinic acid and glutamate.

[0020] In one or more preferred embodiments, the added amount of 5-aminolevulinic acid is 0.3-4 g / L, preferably 0.5-3 g / L (such as 1, 1.5, 2 or 2.5 g / L), more preferably 0.6-2 g / L (optimally 1±0.3 g / L or 1±0.2 g / L).

[0021] In one or more preferred embodiments, the amount of glutamate added is 5-20 g / L, preferably 7-15 g / L (such as 8, 9, 10, 11, 12 or 13 g / L), more preferably 10±2 g / L (optimally 10±1 g / L).

[0022] In one or more preferred embodiments, the glutamate is added to the initial fermentation medium.

[0023] In one or more preferred embodiments, the glutamate comprises: sodium glutamate, glutamine and L-glutamate hydrochloride.

[0024] In one or more preferred embodiments, the culture medium for fermentation culture includes: glucose, enzymatic corn steep liquor, monosodium glutamate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate.

[0025] In one or more preferred embodiments, the culture medium comprises:

[0026] Glucose: 20-40 parts by weight, preferably 25-35 parts by weight, such as 30 parts by weight;

[0027] Enzymatic corn syrup: 2-4 parts by weight, preferably 2.5-3.5 parts by weight, such as 3 parts by weight;

[0028] MSG: 2-4 parts by weight, preferably 2.5-3.5 parts by weight, such as 3 parts by weight;

[0029] Dipotassium hydrogen phosphate: 1-2 parts by weight, preferably 1.2-1.8 parts by weight, such as 1.5 parts by weight;

[0030] Magnesium sulfate: 7-14 parts by weight, preferably 8-12 parts by weight, such as 10 parts by weight;

[0031] Sodium chloride: 1.5-2.5 parts by weight, preferably 1.8-2.2 parts by weight, such as 2 parts by weight;

[0032] Calcium carbonate: 7-14 parts by weight, preferably 8-12 parts by weight, such as 10 parts by weight.

[0033] In one or more embodiments, the fermentation method is a large-scale method (including a scale greater than a shake flask, such as a small scale (small test), a medium scale (pilot), or a large scale). For example, the fermentation method is carried out in a bioreactor of ≥100 mL, ≥200 mL, ≥250 mL, ≥300 mL, ≥500 mL, ≥1 L, ≥2 L, ≥3 L, ≥5 L, ≥7 L, ≥10 L, ≥20 L, ≥30 L, ≥50 L, or ≥100 L.

[0034] In another aspect of the present invention, there is provided an application of any of the above methods for: preparing a porphyrin compound; or increasing the yield of a porphyrin compound by a porphyrin compound producing bacteria; or increasing the conversion rate of a porphyrin compound produced by a porphyrin compound producing bacteria; or shortening the fermentation cycle of a porphyrin compound produced by a porphyrin compound producing bacteria.

[0035] In one or more preferred embodiments, the porphyrin compound-producing bacteria include bacteria selected from the following group: photosynthetic bacteria, Paracoccus denitrifying, Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, Rhizobium centroglobulinum, and Methanobacterium barkleyi.

[0036] In one or more preferred embodiments, the photosynthetic bacteria include bacteria selected from the group consisting of Rhodobacter sphaeroides, Rhodobacter capsulatus, Escherichia coli, Rhodobacter azotobacter or Rhodobacter colloidus.

[0037] In one or more preferred embodiments, the porphyrin compound-producing bacteria is a strain in which fnrL is downregulated (eg, deleted, forming ΔfnrL).

[0038] In one or more preferred embodiments, the porphyrin compound includes (but is not limited to): coproporphyrin III or its chelates, analogs, derivatives or downstream products (such as ferrocoproporphyrin III).

[0039] In another aspect of the present invention, there is provided the use of 5-aminolevulinic acid (5-ALA), glucose, glutamate, succinic acid, or a combination thereof for: increasing the yield of porphyrin compounds produced by porphyrin compound-producing bacteria; or, increasing the conversion rate of porphyrin compounds produced by porphyrin compound-producing bacteria.

[0040] In one or more preferred embodiments, the combination is a combination of 5-aminolevulinic acid and glutamate; more preferably, in terms of mass volume ratio, 5-aminolevulinic acid:glutamate is 1:(2-50).

[0041] In one or more preferred embodiments, the combination is a combination of 5-aminolevulinic acid and glutamate; in terms of mass volume ratio, 5-aminolevulinic acid:glutamate is 1:(3-40), preferably 1:(4-30), more preferably 1:(5-25), more preferably 1:(6-20), more preferably 1:(7-18), more preferably 1:(8-15), more preferably 1:(9-12), specifically 1:10.

[0042] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 , fermentation curve of porphyrin compounds after adding 5-ALA.

[0044] Figure 2 , fermentation curve of porphyrin compounds after adding 5-ALA in batches.

[0045] Figure 3 , fermentation curve of porphyrin compounds after adding glucose.

[0046] Figure 4 , fermentation curve of porphyrin compounds after adding sodium glutamate.

[0047] Figure 5 , fermentation curve of porphyrin compounds after adding glycine.

[0048] Figure 6 , the yield of porphyrin compounds with the addition of sodium glutamate and 5-ALA.

[0049] Figure 7 , the production of ferroporphyrin in the fermentation system with the addition of 5-ALA.

[0050] Figure 8, the effect of succinic acid addition on coproporphyrin production. DETAILED DESCRIPTION

[0051] The inventors are committed to the study of high-yield strategies for porphyrin compounds. Based on extensive and in-depth research, an optimized porphyrin compound production strategy is provided, which includes adding the following group of additives to the fermentation system: 5-aminolevulinic acid (5-ALA), glucose, glutamate, succinic acid, or a combination thereof. The production strategy of the present invention has high yield, fast production speed, high efficiency, low cost, and easy control of porphyrin compounds.

[0052] After in-depth research, screening and process investigation, the inventors considered the effects of various chemical / biological reagents on the production of coproporphyrin III, and based on a large number of analyses and research experiments, it was determined that 5-ALA, glucose, glutamic acid and succinic acid have a positive effect on increasing the yield of porphyrin compounds. Many other attempts, such as adding glycine, are not suitable for increasing the yield of porphyrin compounds, and have no effect or even produce a counter-effect.

[0053] As used in the present invention, the terms "comprising" or "including" include "including", "consisting mainly of (made of)", "consisting essentially of", and "consisting of". The term "consisting essentially of" means that in addition to the essential ingredients or components, a composition (such as a culture medium or an active ingredient that plays a role in a culture medium) may contain minor ingredients and / or impurities that do not affect the effective ingredients in small amounts.

[0054] As used in the present invention, the "porphyrin compound" includes ferroporphyrin III, coproporphyrin III, heme, etc. The heme is a downstream product of ferroporphyrin III or coproporphyrin III. Further, the "porphyrin compound" may also include protoporphyrin IX, hematin, vitamin B12, chlorophyll, etc. The "porphyrin compound" may also be a variation or derivative form based on the compound disclosed in the present invention, for example, the parent core structure of the compound remains unchanged, but a group (such as an aliphatic hydrocarbon group containing 1-4 carbon atoms (preferably 1-2 carbon atoms)) is substituted at individual (such as 1-3, 1-2) positions.

[0055] As used in the present invention, the "porphyrin compound production bacteria" may include strains selected from the following: photosynthetic bacteria, Paracoccus denitrificans, Bacillus subtilis, Agrobacterium tumefaciens, Rhizobium mesoamericanum, Methanosarcina barkeri str. Fusaro, Escherichia coli or C. glutamicum. In a preferred embodiment, the photosynthetic bacteria are selected from purple non-sulfur photosynthetic bacteria. The purple non-sulfur bacteria may be selected from Rhodobacter and Rhodospirillum. In a more preferred embodiment, the photosynthetic bacteria include bacteria selected from the group consisting of Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter azotoformans, or Rubrivivax gelatinosus. Since hemN has homologous genes in fungi such as the above-mentioned strains, it can be expected that the regulatory factor in these bacteria should have the same or similar functions. Preferably, in the present invention, the strain is an engineered bacterium transformed by metabolic engineering; more preferably, the porphyrin compound producing bacterium is a strain in which fnrL is downregulated (such as missing, forming ΔfnrL).

[0056] In other embodiments, the host bacteria is Escherichia coli (E. coli), yeast or Corynebacterium glutamicum (C. glutamicum), and the host bacteria can produce porphyrin compounds through metabolic engineering and / or with the help of naturally occurring synthetic pathways therein.

[0057] The starting strain on which the transformation of the present invention is based can be a wild-type strain, a genetically engineered strain and / or a chemically mutated strain.

[0058] In a preferred embodiment of the present invention, the initial added components are added to the initial culture medium in a suitable amount to promote production. The recommended amounts of 5-aminolevulinic acid, glutamate, succinic acid or a combination thereof are shown in Table 1.

[0059] Table 1

[0060]

[0061] As a preferred embodiment of the present invention, while glucose is included in the initial culture medium, glucose is added at the stage of 30 to 42 hours, preferably 32 to 40 hours of fermentation; preferably, glucose is also added at the stage of 55 to 70 hours of fermentation; the amount of glucose added can be 5 to 20 g / L, preferably 7 to 15 g / L, and more preferably 10 ± 2 g / L. The supplementary addition of glucose greatly increases the amount of bacterial cells, and the increase in the amount of bacterial cells also increases the production of porphyrin compounds. Supplementary addition of glucose can be used as an addition strategy for high-yield porphyrin compounds.

[0062] The inventors also found that the higher the addition of a component, the more beneficial it is. Only the appropriate amount is beneficial. For example, although the addition of glucose is beneficial to high yield, the analysis results show that the tolerance concentration of the bacteria to 5-ALA is limited, and multiple additions of 5-ALA do not effectively increase the yield of coproporphyrin. Therefore, the preferred addition method of 5-ALA is to initially add 2g / L, and subsequent additions may not be conducive to strain production.

[0063] The inventors unexpectedly found that the combined use of 5-aminolevulinic acid and glutamate in appropriate amounts can greatly increase the yield of porphyrin compounds. The increase in yield is surprising compared to the use of either alone, indicating that the two have a synergistic effect of stimulating the increase in yield.

[0064] In the present invention, in addition to the specific chemical components listed in the embodiments of the present invention, chemical components known in the art that have the same or similar functions as them can also be applied to the present invention. Analogs or compounds with the same functions of the specifically listed components, equivalent compounds, analogs, derivatives and / or their salts, hydrates or precursors that induce the same target can also be used to replace the above-mentioned specifically listed components to achieve the same technical effects. These analogs and compounds with the same functions should also be included in the present invention.

[0065] Analogs of compounds include, but are not limited to, isomers and racemates of compounds. Compounds have one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.

[0066] The "precursor of a compound" refers to a compound that can be converted into a compound of any of the above compounds, or a salt or solution composed of a compound of any of the above compounds in the culture medium after being added or treated by an appropriate method.

[0067] As a preferred embodiment of the present invention, the culture medium is further added with ingredients for preventing bacterial contamination of cell culture, especially Gram-positive and Gram-negative bacterial contamination, such as some antibiotics, defoaming bases or pH regulators.

[0068] The high-yield porphyrin compound strain fermentation strategy of the present invention can enable the porphyrin compound production strain to produce high-yield porphyrin compounds, and has universality for porphyrin compounds. The fermentation strategy of the present invention provides support for the efficient production of porphyrin compounds.

[0069] In the present invention, the fermentation method can be a large-scale method (including a scale larger than a shake flask, such as a small scale (small test), a medium scale (pilot), or a large scale). For example, the fermentation method is carried out in a bioreactor of ≥1 L, ≥2 L, ≥3 L, ≥5 L, ≥7 L, ≥10 L, ≥20 L, ≥30 L, ≥50 L, or ≥100 L.

[0070] In the process of fermentation to produce porphyrin compounds, adding cheap additives such as 5-aminolevulinic acid, succinyl coenzyme A, glycine, glutamic acid, etc. can greatly increase the porphyrin yield, improve the fermentation benefits, and have low cost and controllable cost, which is conducive to the large-scale biosynthesis of porphyrin compounds. And this is a general strategy for porphyrin synthesis.

[0071] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0072] Materials and Methods

[0073] Strain: Rhodobacter sphaeroides RSI-ΔfnrL strain (CN112760275A).

[0074] The formula of the plate culture medium is shown in Table 2.

[0075] Table 2

[0076]

[0077] The formula of the seed culture medium is shown in Table 3.

[0078] Table 3

[0079]

[0080] The formula of the fermentation medium is shown in Table 4.

[0081] Table 4

[0082]

[0083] The shake flask fermentation conditions were 32°C and 220 rpm.

[0084] Example 1: Analysis of product conversion after initial addition of 5-ALA

[0085] The wild type of Rhodobacter sphaeroides was used as a control group, and the porphyrin-producing bacteria obtained by previous transformation were used as test strains (RSI-ΔfnrL strain, CN112760275A; hereinafter referred to as mut), and shake flask (shake flask volume was 250 mL, liquid volume was 45 mL) fermentation was carried out, and 0 g / L, 1 g / L and 2 g / L of 5-ALA were added to the culture medium before inoculation for the modified porphyrin-producing bacteria.

[0086] When 5-ALA was exogenously added to the wild-type strain, the signal peak of coproporphyrin III could not be detected in its fermentation broth.

[0087] The modified strains were able to produce coproporphyrin III, and their production was analyzed, such as Figure 1 The measurement results are as follows:

[0088] The modified strain (mut) to which 5-ALA was not added in the initial fermentation medium and the subsequent culture stages entered a plateau phase after 46 h of growth, and its coproporphyrin III production was 1.1 g / L.

[0089] The modified strains to which 1g / L and 2g / L of 5-ALA were added to the initial fermentation medium, respectively, had a lower growth rate than the modified strains without 5-ALA in the 0-26h period, and the coproporphyrin III production rate was also low during this period. However, after 38h, there was a significant difference in the production rate of coproporphyrin III in the experimental groups with different concentrations of 5-ALA. The experimental group with 2g / L 5-ALA continued to maintain a high production rate until 46h, the production rate of the experimental group with 1g / L 5-ALA slowed down, and the production of the experimental group without 5-ALA stagnated. In the late fermentation period, the coproporphyrin III production of the experimental group with the initial addition of 1g / L 5-ALA was 1.9g / L, and the coproporphyrin III production of the experimental group with the initial addition of 2g / L 5-ALA was 2.5g / L.

[0090] The inventors also analyzed the content of ferrocoprophyrin in the fermentation broth of the experimental group with the initial addition of 1 g / L 5-ALA. Figure 7 As shown, the fermentation product contained ferrocoprophyrin with a yield of about 17 mg / L, which was increased by about 21.4% compared with the group without adding 5-ALA (about 14 mg / L).

[0091] Example 2: Effect of adding 5-ALA at different concentrations in batches on product production

[0092] Fermentation was performed in a shake flask (shake flask volume of 250 mL, liquid volume of 45 mL), with wild-type Rhodobacter sphaeroides as the control group and the porphyrin-producing bacteria obtained by previous transformation as the test strain. Initially, a sugar-containing fermentation medium (Table 4) was used, with no addition or addition of 2 g / L or 4 g / L of 5-ALA. At different stages of culture, the wild-type and the modified strain (RSI-ΔfnrL strain) were subjected to experimental treatments of no addition of 5-ALA, addition of 2 g / L and 4 g / L 5-ALA. The addition of exogenous 5-ALA includes: adding to the fermentation medium before inoculation, adding during the fermentation process, and adding a certain amount each time.

[0093] like Figure 2 As shown in the figure, in the initial stage of 0-24h, the strain produces porphyrin compounds slowly overall. After adding different concentrations of 5-ALA at 25h, it can be seen that adding 2g / L 5-ALA will promote the synthesis of porphyrin compounds, while adding 4g / L 5-ALA will reduce the synthesis of porphyrin compounds, indicating that the more 5-ALA, the better it is at this stage. The porphyrin production of the experimental group to which 4g / L 5-ALA was added at 37h was greatly improved. At 43-115h, the porphyrin production of the control group to which 5-ALA concentration was further added (43h, 69h, 94h) at this stage was not significantly improved compared with that of the control group to which 5-ALA was not added. 5-ALA was used as a carbon source to significantly improve the OD of the strain.

[0094] Therefore, the fermentation reaction at the shake flask level showed that the bacteria had a limited tolerance to 5-ALA concentration, and multiple additions of 5-ALA did not effectively increase the production of coproporphyrin. Therefore, the best way to add 5-ALA is to add 2g / L initially, and subsequent additions may not be conducive to strain production.

[0095] Example 3: Effect of batch addition of glucose on product production

[0096] On the basis of basic shake flask fermentation (fermentation medium as shown in Table 4), 10 g / L glucose was fed at 37 h and 62 h, respectively.

[0097] like Figure 3 As shown, it can be seen that in the wild-type strain and the modified strain, the addition of glucose can greatly increase the OD of the strain. Adding glucose can make the OD of the wild-type strain reach 85, and the OD of the modified strain reach 52, which is the highest level of shake flask fermentation.

[0098] according to Figure 3The addition of 10g / L glucose has no direct effect on the production of porphyrin compounds. This is because the production of the strain, especially the synthesis of 5-ALA from glucose, is strictly regulated, resulting in more carbon flow to growth rather than to the porphyrin synthesis pathway. However, the supplementary addition of glucose significantly increased the bacterial mass, and the increase in bacterial mass also increased the production of porphyrin compounds. Supplementary addition of glucose can be used as an addition strategy for high-yield porphyrin compounds.

[0099] Example 4: Effect of adding sodium glutamate on product production

[0100] In the in-depth analysis experiment, the inventors further found that the addition of glutamate has the potential to increase production.

[0101] On the basis of basic shake flask fermentation, the inventors added 10 g / L sodium glutamate into the culture medium before inoculation.

[0102] like Figure 4 As shown, it can be seen that the addition of sodium glutamate has no significant effect on strain growth, but directly has a significant effect on the production of porphyrin compounds. In the time period of 0-54h, whether sodium glutamate is added has no obvious effect on strain growth and production, and the burden on growth caused by 5-ALA is relatively small. After the glucose in the culture medium is completely consumed at about 54h, the strains of the group with added sodium glutamate do not enter primary metabolism, significantly increase the amount of bacteria, and efficient conversion occurs.

[0103] Therefore, glutamate is a good promoter of porphyrin synthesis, has a certain sustained-release effect, and has a lower cost than 5-ALA.

[0104] Furthermore, the inventors analyzed the yield of the combined use of glutamate and 5-ALA. Shake flask fermentation was carried out as described above, and 10 g / L sodium glutamate and 1 g / L 5-ALA were added to the culture medium before inoculation. Figure 6 As shown in the figure, the addition of sodium glutamate and ALA significantly increased the yield of porphyrin compounds. Only about 1 g / L of 5-ALA is required in this reaction, and the cost is lower than the case of adding 5-ALA alone.

[0105] Example 5: Effect of adding glycine and succinic acid on product production

[0106] The inventors also tested the effects of other chemical additives on product production, including glycine and succinyl-CoA.

[0107] On the basis of basic shake flask fermentation, the present inventors added 5 g / L glycine to the culture medium before inoculation.

[0108] like Figure 5As shown, it can be seen that the addition of glycine has a significant inhibitory effect on strain growth and also has a significant reduction in the yield of porphyrin compounds. The inventors observed that this is because the addition of glycine affects the synthesis of bacterial cell walls, making cells fragile and easier to lyse. Therefore, glycine is not an ideal promoter for porphyrin synthesis.

[0109] Accordingly, in the test group with the addition of succinic acid (1 g / L was added to the culture medium before inoculation, and other operating conditions were the same as the addition of sodium glutamate in Example 4), an increase in the intracellular succinyl-CoA content was observed, producing an effect similar to that of the addition of sodium glutamate. Figure 8 The addition of succinic acid significantly increased the yield.

[0110] Summarize

[0111] In summary, based on extensive analysis and research by the inventors, it has been determined that the best strategy for fermentation production of porphyrin compounds is to add appropriate amounts of 5-ALA, glucose, glutamate and / or succinic acid before inoculation / during fermentation. Surprisingly, the yield can be significantly increased by combining 5-ALA and glutamate and adding them together.

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A fermentation method for high-yield porphyrin compounds, include: The porphyrin compound producing bacteria are fermented and cultured to produce the porphyrin compound; wherein, before the start of fermentation or during fermentation, an additional component selected from the following group is added: 5-aminolevulinic acid, glucose, glutamate, succinic acid, or a combination thereof.

2. The method according to claim 1, It is characterized in that The added component is 5-aminolevulinic acid; the added amount of the 5-aminolevulinic acid is 0.5 to 5 g / L, preferably 1 to 4 g / L, more preferably 1.5 to 2.5 g / L; Preferably, the 5-aminolevulinic acid is added to the initial fermentation medium.

3. The method according to claim 1, It is characterized in that When the initial culture medium includes glucose, glucose is added at the stage of 30 to 42 hours, preferably 32 to 40 hours of fermentation; preferably, glucose is also added at the stage of 55 to 70 hours of fermentation; The added amount of glucose is 5-20 g / L, preferably 7-15 g / L, and more preferably 10±2 g / L.

4. The method according to claim 1, It is characterized in that The added ingredient is glutamate; the added amount of glutamate is 5-20 g / L, preferably 7-15 g / L, more preferably 10±2 g / L; Preferably, the glutamate is added to the initial fermentation medium; Preferably, the glutamate comprises: sodium glutamate, glutamine and L-glutamate hydrochloride.

5. The method according to claim 1, It is characterized in that The added component is succinic acid; the added amount of succinic acid is 5 to 20 g / L, preferably 7 to 15 g / L, more preferably 10±2 g / L; Preferably, the succinic acid is added to the initial fermentation medium.

6. The method according to claim 1, It is characterized in that The added ingredients are 5-aminolevulinic acid and glutamate; The addition amount of 5-aminolevulinic acid is 0.3-4 g / L, preferably 0.5-3 g / L, more preferably 0.6-2 g / L; The amount of glutamate added is 5-20 g / L, preferably 7-15 g / L, more preferably 10±2 g / L; Preferably, the glutamate is added to the initial fermentation medium; Preferably, the glutamate comprises: sodium glutamate, glutamine and L-glutamate hydrochloride.

7. The method according to claim 1, It is characterized in that The culture medium for fermentation culture includes: glucose, enzymatic corn syrup, monosodium glutamate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate; Preferably, the culture medium comprises: Glucose: 20-40 parts by weight, preferably 25-35 parts by weight, such as 30 parts by weight; Enzymatic corn syrup: 2-4 parts by weight, preferably 2.5-3.5 parts by weight, such as 3 parts by weight; MSG: 2-4 parts by weight, preferably 2.5-3.5 parts by weight, such as 3 parts by weight; Dipotassium hydrogen phosphate: 1-2 parts by weight, preferably 1.2-1.8 parts by weight, such as 1.5 parts by weight; Magnesium sulfate: 7-14 parts by weight, preferably 8-12 parts by weight, such as 10 parts by weight; Sodium chloride: 1.5-2.5 parts by weight, preferably 1.8-2.2 parts by weight, such as 2 parts by weight; Calcium carbonate: 7-14 parts by weight, preferably 8-12 parts by weight, such as 10 parts by weight.

8. Use of the method according to any one of claims 1 to 7 for: preparing a porphyrin compound; or Improving the yield of porphyrin compounds produced by porphyrin compound producing bacteria; or Improving the conversion rate of porphyrin compound produced by porphyrin compound producing bacteria; or Shorten the fermentation cycle of porphyrin compound production by porphyrin compound producing bacteria.

9. The method according to any one of claims 1 to 7 or the use according to claim 8, It is characterized in that The porphyrin compound producing bacteria include bacteria selected from the following group: photosynthetic bacteria, Paracoccus denitrificans, Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, Sinorhizobium, and Methanobacterium barkleyi; preferably, the photosynthetic bacteria include bacteria selected from the following group: Rhodobacter sphaeroides, Rhodobacter capsulatum, Escherichia coli, Rhodobacter azotobacter or Rhodobacter colloidus; preferably, the porphyrin compound producing bacteria is a strain in which fnrL is downregulated; or The porphyrin compound includes: coproporphyrin III or its chelate, analog, derivative or downstream product.

10. Application of 5-aminolevulinic acid, glucose, glutamate, succinic acid, or a combination thereof for: increasing the yield of porphyrin compounds produced by porphyrin compound producing bacteria; or, increasing the conversion rate of porphyrin compounds produced by porphyrin compound producing bacteria; Preferably, the combination is a combination of 5-aminolevulinic acid and glutamate; more preferably, in terms of mass volume ratio, 5-aminolevulinic acid:glutamate is 1:(2-50).

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