Porphyrin compound fermentation process and control strategy

Abstract

The invention provides a porphyrin compound fermentation process and a control strategy. According to the strategy disclosed by the invention, the yield of the porphyrin compound produced by the porphyrin compound producing strain can be increased, the conversion rate is increased, and the fermentation period is shortened. The fermentation process and the control strategy provided by the invention provide support for fermentation production and amplification of the porphyrin compound.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and biosynthesis, and more specifically, relates to a porphyrin compound fermentation process and a control strategy. Background Art

[0002] Porphyrins are closed hexadecanalic compounds based on four pyrrole structures, which 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] Porphyrin compounds are widely found in nature. They are called "pigments of life" because they can show different colors by chelating different metal ions. Hemoglobin, chlorophyll and vitamin B12, which are well known to people, are important porphyrin compounds.

[0005] Early porphyrins were obtained from natural products containing porphyrin compounds, such as heme and chlorophyll, by extraction, separation, and purification. There are two ways to obtain the target porphyrin molecule: structural modification of natural porphyrins and total synthesis of porphyrin compounds. Although structural modification of natural porphyrins can easily transform the structure, it is limited by the structure itself, and the choice of exocyclic functional groups is also very limited. In addition, it also limits the physiological activity of the porphyrin compound itself. Therefore, people usually need to use total synthesis to obtain porphyrin molecules with specific physiological activities and functions.

[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] Although the production mechanism of porphyrin compounds in organisms has been explored by those skilled in the art, there is still no mature and efficient biological fermentation production method for porphyrin compounds. Summary of the invention

[0008] The purpose of the present invention is to provide a porphyrin compound fermentation process and control strategy.

[0009] In a first aspect of the present invention, a method for producing a porphyrin compound is provided, comprising: fermenting and culturing a porphyrin compound-producing bacterium, and controlling the glucose content (residual sugar) and dissolved oxygen in the fermentation broth in stages under the conditions of pH 5 to 8.5 and temperature 28 to 35° C.; wherein the staged control of the glucose content in the fermentation broth comprises: within the range of glucose concentration of 4 to 20 g / L, reducing the dissolved oxygen and increasing the glucose concentration in the early stage of fermentation, and increasing the dissolved oxygen and reducing the glucose concentration in the middle and late stages of fermentation.

[0010] In one or more preferred embodiments, the segmented control of the glucose content in the fermentation broth includes: adjusting the glucose content to 12-18 g / L (preferably 13-17 g / L, more preferably 14-16 g / L) in the early stage of fermentation, and reducing the glucose content in the middle and late stages of fermentation to 25-60% of the early stage of fermentation; preferably, the glucose content in the middle and late stages of fermentation is 4-10 g / L (such as 5-9 g / L, more specifically 6, 7, 8 g / L).

[0011] In one or more preferred embodiments, the segmented control of dissolved oxygen in the fermentation liquid includes: adjusting the rotation speed to 600±20rpm in the early stage of fermentation, and transitioning to 700±20rpm in the middle and late stages of fermentation; preferably, the rotation speed in the middle and late stages of fermentation is first transitioned to 650±20rpm, and then transitioned to 700±20rpm; wherein the air flow rate is 3.5±0.5L / min.

[0012] In one or more preferred embodiments, the temperature is 30-34°C, preferably 31-33°C (such as 32°C).

[0013] In one or more preferred embodiments, the pH is 5.5-7.5, preferably 6-7, more preferably pH 6.2-6.8 (such as pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7); more preferably, the pH value is adjusted using aqueous ammonia.

[0014] In one or more preferred embodiments, with respect to pH, at the start of fermentation, the pH is adjusted to the maximum value within a range that does not affect the growth of the strain, thereby exerting pressure on the initial growth of the bacteria; thereafter, the pH naturally drops to a lower limit (5 to 6.6; such as 5.5, 6, 6.2 or 6.5), and then adjustment is performed to maintain a pH of 6 to 7 (more preferably pH 6.2 to 6.8; such as pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7).

[0015] In one or more preferred embodiments, the maximum value within the range that does not affect the growth of the strain is pH 7.3-8.7; more preferably pH 7.4-8.6 (such as 7.5, 7.6, 7.8, 8, 8.2, 8.4, 8.5).

[0016] In one or more preferred embodiments, the fermentation culture is carried out under conditions of reduced light (such as reducing light by 10-100%, specifically reducing it by more than 20%, more than 40%, more than 60%, more than 80%, more than 90%, more than 95%).

[0017] In one or more preferred embodiments, the early fermentation period is from the start of fermentation to 30 to 45 hours (such as 32, 35, 38, 40, 42, 43 hours); the period thereafter is the middle and late fermentation period; preferably, the fermentation is terminated after 65 to 100 hours (such as 68, 70, 75, 76, 80, 85, 90, 95 hours; more preferably 70 to 80 hours).

[0018] In one or more preferred embodiments, the fermentation culture is carried out in a bioreactor; preferably, the bioreactor comprises a stirring propeller; more preferably, the stirring propeller is a sloped stirring propeller.

[0019] In one or more preferred embodiments, the culture medium (fermentation medium) used for fermentation culture is a culture medium suitable for the growth and production of the porphyrin compound producing bacteria, and contains sufficient nutrients required for production and growth; preferably, the culture medium includes: glucose, enzymatic corn syrup, monosodium glutamate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate.

[0020] In one or more preferred embodiments, the culture medium comprises:

[0021] Glucose: 20-40 parts by weight, preferably 25-35 parts by weight, such as 30 parts by weight;

[0022] Enzymatic corn syrup: 2-4 parts by weight, preferably 2.5-3.5 parts by weight, such as 3 parts by weight;

[0023] MSG: 2-4 parts by weight, preferably 2.5-3.5 parts by weight, such as 3 parts by weight;

[0024] Dipotassium hydrogen phosphate: 1-2 parts by weight, preferably 1.2-1.8 parts by weight, such as 1.5 parts by weight;

[0025] Magnesium sulfate: 7-14 parts by weight, preferably 8-12 parts by weight, such as 10 parts by weight;

[0026] Sodium chloride: 1.5-2.5 parts by weight, preferably 1.8-2.2 parts by weight, such as 2 parts by weight;

[0027] Calcium carbonate: 7-14 parts by weight, preferably 8-12 parts by weight, such as 10 parts by weight.

[0028] In one or more preferred embodiments, the fermentation method is 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.

[0029] 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 bacterium; or increasing the conversion rate of a substrate (such as glucose) for producing a porphyrin compound by a porphyrin compound producing bacterium; or shortening the fermentation cycle for producing a porphyrin compound by a porphyrin compound producing bacterium.

[0030] In one or more preferred embodiments, the porphyrin compound-producing bacteria include bacteria selected from the following group: photosynthetic bacteria, denitrifying Paracoccus, Bacillus subtilis, Agrobacterium tumefaciens, Sinorhizobium or Methanobacterium buckleyi; preferably, the photosynthetic bacteria include bacteria selected from the following group: spherical red bacteria, capsule red bacteria, nitrogen-fixing red bacteria or gelatinous red longevity bacteria.

[0031] In one or more preferred embodiments, the porphyrin compound includes (but is not limited to): coproporphyrin III or its chelate, analog, derivative, precursor or downstream product (such as ferrocoproporphyrin III).

[0032] 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

[0033] Figure 1 , 7 .Some parameters of fermentation control in 5L fermenter (New Brunswick).

[0034] Figure 2 , 7 .5L fermentation tank fermentation site photo.

[0035] Figure 3 , 5 Some parameters of fermentation control in L fermenter (Bai Lun Company).

[0036] Figure 4 , 5 L fermentation Actual photos at different time points during the fermentation process.

[0037] Figure 5 , Example 4 Compared with the process of Example 3, the output of coproporphyrin III changes.

[0038] Figure 6 , 7.5L fermentation tank with Rhodobacter capsulatus fermentation results.

[0039] Figure 7 , 7 .E. coli fermentation results in a 5L fermenter. DETAILED DESCRIPTION

[0040] The present invention discloses a high-yield porphyrin compound strain fermentation process and control strategy. The strategy described in the present invention can enable porphyrin compound producing bacteria to produce high-yield porphyrin compounds, and has universality in various strains. The fermentation process and control strategy described in the present invention provide support for the fermentation production and amplification of porphyrin compounds.

[0041] Porphyrin compounds are a class of macromolecular heterocyclic compounds formed by four α-carbon atoms of pyrrole subunits connected by a methine bridge (=CH-). The parent compound is porphine, and porphine with a substituent is called porphyrin. Porphyrin exists in nature in the form of coordination with metal ions, such as heme can be produced when coordinated with iron.

[0042] As used herein, the "porphyrin compound" includes: coproporphyrin III or its chelate, analog, derivative, precursor or downstream product (such as ferrocoproporphyrin III). It can include: coproporphyrin III, ferrocoproporphyrin III, etc. Further, the process of the present invention or in combination with further process / addition of reaction components, the "porphyrin compound" may also include protoporphyrin IX, hemoglobin, vitamin B12, chlorophyll, etc.

[0043] As used herein, the "porphyrin compound" producing bacteria include bacteria selected from the following groups: photosynthetic bacteria, Paracoccus denitrificans, Bacillus subtilis, Agrobacterium tumefaciens, Rhizobium mesoamericanum, Methanosarcina barkeri str. Fusaro. Preferably, the photosynthetic bacteria are selected from purple non-sulfur photosynthetic bacteria. The purple non-sulfur bacteria can be selected from the genus Rhodobacter and Rhodospirillum. More preferably, the photosynthetic bacteria include bacteria selected from the following groups: Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter azotoformans, or Rubrivivax gelatinosus.

[0044] As used herein, the "bioreactor" is a container or equipment used for biological cell culture or involving biochemical reaction processes and having appropriate environmental conditions, such as but not limited to culture bottles, constant temperature shaking culture shake flasks and fermenters.

[0045] As used herein, the term "fermentation", "fermentation culture" or "culture" can be used interchangeably and refers to the process in which microbial cells grow under aerobic conditions using exogenously provided nutrients (such as carbon sources, nitrogen sources, etc.).

[0046] As used herein, the "early fermentation period" is from the start of fermentation to the 30th to 45th hour, such as 32nd, 35th, 38th, 40th, 42nd, and 43rd hour. Sometimes, the "early fermentation period" may also refer to a period when the bacterial cells grow relatively fast.

[0047] As used herein, the "mid-to-late fermentation" refers to the time after the early fermentation. Sometimes, the "mid-to-late fermentation" may also refer to the period when the bacteria grow to a certain extent and the growth rate slows down, at which time the production of products often increases.

[0048] As used herein, the term "bioreactor" is used interchangeably with "fermenter."

[0049] There have been no related reports on the production of porphyrin compounds by small-scale fermentation of microorganisms represented by Rhodobacter sphaeroides. At present, the production potential of such microbial fermentation to produce porphyrin compounds is unknown in the art, and there is no mature large-scale (larger than shake flask) fermentation process. The present invention explores and optimizes the production process of porphyrin compounds, so as to enable the industrialization of porphyrin compounds such as coproporphyrin III produced by microbial fermentation as soon as possible, and provides a new way to establish a fermentation production process.

[0050] In the research and test of the inventor, 5L tank and 7.5L tank were used for fed-batch fermentation production, and many factors affecting fermentation were analyzed, and parameters such as fermentation temperature, fermentation pH, dissolved oxygen (DO) in the fermentation tank and sugar concentration were determined. The inventor also fermented according to the optimized fed-batch fermentation conditions, and took the fermentation of coproporphyrin III by spherical red bacteria as an example, and established a suitable fed-batch fermentation process for producing porphyrin compounds by bacterial strain fermentation. On this basis, non-critical factors such as light conditions, tank size, and stirring paddle style were optimized, and it was found that the three also had a certain impact on fermentation, and the shading environment could accelerate the growth and production of bacterial strains, and the inclined stirring paddle had a certain lifting effect on dissolved oxygen. The inventor also found that the initial pH pressure promoted the growth and production of bacterial strains in the later stage. After a series of explorations and optimizations, the inventor adopted the mode of segmented control of dissolved oxygen and residual sugar to further improve the conversion efficiency of coproporphyrin III and further shorten the fermentation cycle.

[0051] Based on the in-depth research and experiments of the inventors, the present invention provides a method for producing a porphyrin compound, comprising: fermenting and culturing porphyrin compound-producing bacteria, and controlling the glucose content (residual sugar) and dissolved oxygen in the fermentation broth in stages under the conditions of pH 5-8.5 and temperature 28-35°C; wherein the staged control of the glucose content in the fermentation broth comprises: within the range of glucose concentration of 4-20 g / L, reducing the dissolved oxygen and increasing the glucose concentration in the early stage of fermentation, and increasing the dissolved oxygen and reducing the glucose concentration in the middle and late stages of fermentation.

[0052] It should be understood that although the fermentation culture described in the present invention is carried out in a specific bioreactor, a variety of bioreactors can be applied to the present invention as long as they can provide appropriate dissolved oxygen, temperature, ventilation and stirring conditions.

[0053] 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 ≥1 L, ≥2 L, ≥3 L, ≥5 L, ≥7 L, ≥10 L, ≥20 L, ≥30 L, ≥50 L, or ≥100 L.

[0054] In one or more embodiments, the bioreactor includes a stirring blade, which is beneficial for effectively dissolving oxygen in the fermentation liquid. As a preferred embodiment of the present invention, the stirring blade can be an inclined stirring blade, but is not limited thereto.

[0055] In the present invention, the culture medium (fermentation medium) used for fermentation culture is a culture medium suitable for the growth and production of the porphyrin compound producing bacteria, containing sufficient nutrients required for production and growth; preferably, the culture medium includes: glucose, enzymatic corn syrup, monosodium glutamate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate.

[0056] It is understood that the medium components used to cultivate the strains in the present invention can be replaced by other components with similar functions, and other components can be appropriately added or removed according to the characteristics of specific strains in different situations. The porphyrin compound producing bacteria of the present invention can be wild-type strains, genetically engineered strains and / or chemically mutated / modified / screened strains. The porphyrin compound producing bacteria of the present invention can be a derivative strain with higher yield or more optimized enzyme system obtained by further improvement through laboratory domestication, genetic breeding, molecular genetic manipulation and the like.

[0057] The culture system or fermentation system of the present invention can be scaled up for industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on their general knowledge to facilitate the growth or production of the strain.

[0058] The present invention also provides an application of the method, which is used for: preparing a porphyrin compound; or increasing the yield of a porphyrin compound produced by a porphyrin compound producing bacterium; or increasing the conversion rate of a substrate (such as glucose) produced by a porphyrin compound producing bacterium; or shortening the fermentation cycle of a porphyrin compound produced by a porphyrin compound producing bacterium. The increase in yield, the increase in conversion rate, and the shortening of the fermentation cycle are all shown in the specific embodiments of the present invention.

[0059] Compared with traditional natural separation methods or chemical synthesis methods, microbial fermentation has the advantages of fast speed and less influence by external factors; the yield of some compounds synthesized by microorganisms is much higher than that of plant extraction, and it has become an important means of obtaining natural products. Porphyrin compounds have low natural abundance, and chemical synthesis methods are cumbersome and complicated. In the present invention, microbial fermentation is used to efficiently and directionally synthesize porphyrin compounds, which effectively reduces the cost of separating and purifying such compounds.

[0060] 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.

[0061] Materials and Methods

[0062] 1. Bioreactor and related equipment

[0063] 7.5L fermentation tank ( 115, Eppendof);

[0064] 5L fermentation tank (5L four-tank, Bailun Bio);

[0065] SBA-40D Biosensor Analyzer.

[0066] 2. Strains and main basic culture medium

[0067] Bacterial strain: Rhodobacter sphaeroides ATH2.4.1.

[0068] The formula of the primary seed plate medium is shown in Table 1.

[0069] Table 1

[0070]

[0071] The formula of the secondary seed culture medium is shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075] The formula of the tertiary fermentation medium is shown in Table 3.

[0076] Table 3

[0077]

[0078] 3. Fermentation method

[0079] In the subsequent examples, a 7.5 L tank was used in the fermentation in Examples 1, 2, and 4, and a 5 L tank was used in the fermentation in Example 3.

[0080] Basic fermentation process of 7.5L fermenter:

[0081] (1) Prepare 2.4 L of tertiary fermentation medium in a 7.5 L tank, tighten the screws, and seal the fermentation tank.

[0082] (2) Calibrate the pH electrode and insert the pH electrode and dissolved oxygen electrode into the fermentation tank.

[0083] (3) Connect the feed bottle and related pipelines, clamp the pipelines that may spray liquid with water stop clamps, and wrap the electrodes with aluminum foil.

[0084] (4) After sterilization at 121°C for 30 minutes, use a cold trap to cool the tank while ventilating it to prevent contamination.

[0085] (5) Wait until the tank temperature drops to 32°C and all parameters stabilize, then connect the motor and start stirring.

[0086] (6) Use ammonia water to adjust the pH to 7 and calibrate the maximum value of the dissolved oxygen electrode according to the maximum speed of 700 rpm.

[0087] (7) Cultivate the secondary seed solution until the OD is 10 and then inoculate it, controlling the inoculation ratio to 18%-20%.

[0088] (8) The feeding and aeration process during the fermentation process is as follows: after inoculation, the rotation speed is maintained at 600 rpm, the ventilation (air flow rate) is maintained at 2 L / min, and the temperature is maintained at 32°C; after 9 hours, the ventilation volume is changed to 2.2 L / min; after 30 hours, the rotation speed is maintained at 650 rpm, and the ventilation volume is maintained at 2.4 L / min; after 24 hours, 1 / 3 of 10-fold fermentation medium is added; after 30 hours, the ventilation volume is changed to 2.6 L / min; after 36 hours, 1 / 3 of 10-fold fermentation medium is added; after 48 hours, the rotation speed is maintained at 700 rpm, the ventilation is maintained at 3 L / min, and 1 / 3 of 10-fold fermentation medium is added at the same time.

[0089] In the above steps (1)-(8), the specific conditions in the corresponding embodiments are described or optimized in the subsequent embodiments.

[0090] The basic fermentation process of 5L fermenter is:

[0091] The operation of the 5L fermenter was the same as that of the 7.5L fermenter, and 1.8L of the tertiary fermentation medium was prepared so that the initial liquid volume after inoculation was 2L. The vertical paddle was replaced with an inclined paddle, and the height of the stirring paddle was reduced so that it could fully stir the medium. The specific conditions in the corresponding embodiments are described separately in the subsequent embodiments.

[0092] 4. Multi-factor analysis in fermentation

[0093] (a) pH analysis

[0094] The inventors observed that the difference in initial pH is closely related to the length of the lag phase of the strain, the state of the bacteria, etc., so they considered optimizing the initial pH and process pH of the fermentation:

[0095] (1) The pH at the time of initial inoculation in the fermentation of Example 1, Example 2, and Example 3 was controlled at 6.7, 7.0, and 8.5, respectively.

[0096] (2) During the fermentation process of Example 1, control experiments were set up to stabilize the pH at 6.5 by pH-related alkali supplementation, to rapidly increase the pH and then fall back, and to rapidly decrease the pH and then rebound, to explore the effects of different pH fluctuations on fermentation production.

[0097] (b) Glucose analysis

[0098] In Example 1 and Example 2-3, the strategies of one-time addition of glucose and flow addition of glucose were adopted respectively. Meanwhile, the residual sugar content of Example 2-3 was controlled at about 20 g / L and 15 g / L respectively to determine the effects of different sugar supplementation strategies on strain growth.

[0099] (c) Light analysis

[0100] The inventors also analyzed the effect of the light regulation system on the porphyrin synthesis pathway. In Example 3, the tank body was wrapped with aluminum foil after 20 hours to keep it in a light-proof state. The day and night time periods in Example 1 and Example 2 were compared to explore whether the shading conditions had an improvement effect on the yield. In Example 4, according to the optimized parameters of Examples 1, 2, and 3, a comprehensive evaluation and fermentation were performed. Therefore, shading was also used in Example 4. The specific operation was to wrap the tank body with aluminum foil on the transparent glass tank body of the fermenter to avoid light.

[0101] (d) Dissolved oxygen analysis

[0102] (1) In Example 1, dissolved oxygen-associated stirring is set to maintain the dissolved oxygen at 20%; in Examples 2 and 3, a constant stirring speed is set at 600 rpm in the early stage according to the process, and gradually transitions to 700 rpm in the middle and late stages.

[0103] (2) In the fermentation of Example 1, the air flow rate was maintained according to the process control for 60 h; in the fermentation of Examples 2 and 3, the air flow rate was increased to 3.5 L / min.

[0104] (3) In Example 3, a smaller 5 L tank was used, and multi-hole ventilation and inclined paddle vortex stirring were used to increase the dissolved oxygen in the fermenter.

[0105] (e) Temperature analysis

[0106] In the tank fermentation experiments of Examples 1 and 4, the tank temperature was maintained at 33°C, and in the tank fermentation experiments of Examples 2 and 3, the tank temperature was maintained at 32°C.

[0107] (f) Adversity Analysis

[0108] In the tank fermentation experiments of Example 3 and Example 4, the initial pH was controlled at a maximum value within a range that did not affect the growth of the strain, thereby exerting pressure on the initial growth of the bacteria.

[0109] (g) Phased analysis

[0110] In the tank fermentation of Example 4, based on the experience of the tank fermentation of Examples 1-3, different strategies were optimized for dissolved oxygen and residual sugar concentration in the early stage and the middle and late stages. Reducing dissolved oxygen in the early stage increased the residual sugar concentration, while increasing dissolved oxygen in the middle and late stages reduced the residual sugar concentration.

[0111] Example 1, 7.5L fermenter fermentation process 1

[0112] A New Brunswick 7.5L fermenter was used. The fermentation process referred to the basic fermentation process, and the following adjustments were made: After calibrating the dissolved oxygen electrode, the stirring was linked to the dissolved oxygen to maintain the dissolved oxygen at 20%, and the speed change range was limited to 200-700rpm. The temperature after inoculation was 33°C to maintain the entire fermentation process. The initial pH was adjusted to 6.7 before inoculation, and ammonia water was added to the pH after 20 hours to maintain the pH at 6.5. At the same time, in order to explore the effect of the acidic environment on the fermentation production of the bacteria, the pH-linked ammonia water addition was turned off at 24-36h, 42-46h and 48-56h to allow the pH to drop naturally. Fermentation medium without glucose was added at 24h, 36h and 48h (50-70mL each time). The initial concentration of glucose was 20g / L, and the time points of glucose supplementation are shown in Table 1. Figure 1 As shown in (Fig. The symbol indicates the time point of adding glucose. Each time, 30-40 mL of 80% glucose solution was added to maintain the glucose concentration at 10 g / L. At the same time, 48 g of glucose was added in the two time periods of 39-45 h and 48-60 h (48 g of glucose was added in 18 hours at an average flow rate of 2.67 g / h). The main control parameters of the fermentation process in the 7.5 L fermenter are as follows: Figure 1 .

[0113] The inventor observed that in the initial stage of fermentation, the pH of the fermentation liquid first rose to 7.9 and then naturally decreased, reaching a preset value of 6.5 at 12 hours. The rotation speed was gradually increased from 200 rpm to 700 rpm, maintaining the dissolved oxygen at 20%. The residual sugar dropped to 3.6 g / L. From the above parameters, it can be seen that the overall growth of the strain was good at this stage, the OD value rose to 20, and the coproporphyrin III product also accumulated 0.1 g / L.

[0114] Subsequently, the inventors observed that the growth rate of the strain slowed down due to the low residual sugar, and the pH increased at the same time, and the two had a certain correlation. In the 24-36h time period (turning off the pH-associated ammonia supplement), the pH of the fermentation broth dropped sharply to 4.9, causing the death of the strain, and then the pH naturally rose to 7. During this period of time, the overall growth and production rates of the strain slowed down, which preliminarily indicated that when the pH was below 5, it would have a more serious impact on the growth and production of the strain. The phenomenon in the 42-46h and 48-56h time periods (turning off the pH-associated ammonia supplement) further illustrates this problem.

[0115] During the 36-42h period, the pH of the fermentation broth was stably controlled at 6.5, and the sugar concentration was maintained at a sufficient level of 10g / L. During this period, the stirring speed reached the maximum, and the dissolved oxygen was close to zero, indicating that the strain was in a relatively ideal state and grew faster. At the same time, the production of coproporphyrin III (CP III) also increased to 1.4g / L, reaching a peak.

[0116] Then, at 60h, as the residual sugar dropped below 1g / L, the sugar consumption rate of the bacteria decreased. At the same time, the amount of bacteria and the amount of products began to drop sharply, and the pH increased significantly, indicating that the environment at this time could no longer maintain the stability of the bacteria. A large number of bacteria lysed and died. At this time, the amount of bacteria (including dead bacteria) reached a peak, and the OD value was 86.

[0117] Example 2, 7.5L fermenter fermentation process 2

[0118] In this embodiment, the strategy of adding glucose by flow is adopted to maintain the residual sugar at a level above 5g / L at all times. During most of the fermentation time, the residual sugar is maintained at a sufficient level of 20g / L to ensure that the strain does not stagnate or decay due to insufficient glucose. The temperature is kept constant at 32°C, hoping to slightly slow down the level of protein synthesis and reduce the probability of protein misfolding: during the fermentation process, the temperature rises, the reaction rate increases, the growth metabolism accelerates, and the product is generated in advance. However, the higher the temperature, the faster the protein synthesis rate, the easier it is to form misfolding, and the catalytic function of the enzyme is reduced. This can be achieved by slightly lowering the temperature. At the same time, it can also make more carbon flow in the bacteria shift from growth to production, thereby increasing the yield. In terms of pH control, the pH of the fermentation liquid is adjusted to 7 before inoculation, and the pH is always controlled during the fermentation process after 12h of ammonia supplementation to maintain the pH above 6.2. The fermentation site diagram of the 7.5L fermenter is as follows Figure 2 .

[0119] The inventors observed that the strain maintained a substantially constant growth rate from 0 to 96 h, and entered a growth plateau after 96 h.

[0120] In the 0-18h period, the pH gradually decreased from the initial 7 to 6.5 after being maintained for about 12h and the associated ammonia supplement was started, and the dissolved oxygen rapidly decreased from the initial 95% to a hypoxic state of 3%. During this stage, the glucose was maintained at a relatively high concentration of about 20g / L, the strain grew rapidly, the OD value reached the level of 30.5, and there was no growth lag period after inoculation, while the production rate of porphyrin compounds such as coproporphyrin III was relatively low, only 0.17g / L, which was basically the same as the output in Example 1 during the same period, but significantly lower than about 0.5g / L of the two shake flask fermentations.

[0121] After entering the hypoxic environment for 18 hours, the production of coproporphyrin III began to increase significantly.

[0122] In the following 18-96h period, the strain stabilized at a higher growth production rate and reached a peak value of 135 at 96h with a coproporphyrin III production of 10g / L and a ferroporphyrin III production of 0.5g / L.

[0123] After 96 hours, the strain basically stopped growing and producing, but the sugar consumption continued. It is speculated that the growth of the strain and the accumulation of coproporphyrin III reached a stable state. The pH of this stage was basically stable at 6.5, and the residual sugar content was also maintained above 12g / L, indicating that the strain reached a stable state of proliferation and decay in a relatively suitable and nutritious environment, and entered the fermentation platform period. During the fermentation process, the glucose conversion rate was 0.0416g / g coproporphyrin III.

[0124] Example 3, 5L fermentation tank fermentation process

[0125] In this embodiment, the experience of Examples 1-2 is combined to continue the analysis and optimization. The inventors conducted a small-scale fermentation on a 5L tank produced by Bailun Company. In the fermentation of this embodiment, the middle stirring paddle was replaced by a vertical paddle with an inclined paddle to increase the dissolved oxygen by stirring and mixing the fermented liquid more fully up and down. According to the process setting, the constant stirring speed is 600rpm in the early stage, and gradually transitions to 700rpm in the middle and late stages (connect to the fermentation for 0-18h, use a rotation speed of 600rpm, and then adjust the rotation speed to 650rpm. After fermentation to the middle and late stages for about 48-52h, adjust the rotation speed to 700rpm and continue until the end of the fermentation). At the same time, the residual sugar level is controlled at a level of about 15g / L by the method of adding glucose, which is in contrast to 20g / L in Example 2. In addition, the initial pH before inoculation was controlled at 8.5 to explore the effect of higher pH on the initial growth of the strain. At the 20th hour, the tank body was wrapped with aluminum foil to explore the effect of shading on fermentation production. The main fermentation control parameters of the 5L fermenter are as follows Figure 3 , the actual pictures at different time points during the fermentation process are as follows Figure 4 .

[0126] During the 0-12h period, the strain grew and the pH fluctuated downward. At the 19th hour, the associated ammonia supplement was started to maintain the pH above 6.2.

[0127] During the 12-30h period, the strain gradually grew faster and the dissolved oxygen also decreased rapidly, but there was no obvious accumulation of coproporphyrin III. Compared with the accumulation of coproporphyrin III in the fermenter of Example 2 after 18h, there was a production lag period of 12h.

[0128] Comparing before and after 20h shading, it can be seen that the growth of the bacteria is accelerated after shading, and the rate of decline of dissolved oxygen becomes faster, until the dissolved oxygen drops to a low oxygen state around 30h. The production rate of coproporphyrin III is significantly improved. At 96h, the OD reaches 150, the coproporphyrin III production reaches 16.5g / L, and the iron coproporphyrin III reaches 0.75g / L.

[0129] After 96 hours, the bacteria entered a growth plateau, the sugar consumption rate was low, the OD value increased slightly, and the coproporphyrin III production fluctuated slightly. The fermentation cycle of this example is similar to the fermentation in the tank of Example 2, and both entered a growth and production plateau at around 96 hours.

[0130] Finally, the yield of the main product coproporphyrin III in the tank fermentation of this embodiment increased by 65% ​​and the iron coproporphyrin III increased by 150% compared with that in Example 2. During the fermentation process, the conversion rate of coproporphyrin III was 0.0737 g / g, which was 77% higher than that in the tank fermentation of Example 2.

[0131] At the same time, the higher initial pH provided a stress effect, which significantly promoted the later growth and production of the strain.

[0132] Example 4, 7.5L fermenter fermentation process 3

[0133] In this embodiment, on the basis of the aforementioned embodiment 3, a New Brunswick 7.5L tank was used, and a straight-faced paddle was used to accelerate the rate of decline of dissolved oxygen in the early stage, enter hypoxic conditions in advance, and increase the ventilation flow rate in the middle and late stages (according to the process setting, the constant stirring speed was 600rpm in the early stage, gradually transitioned to about 650rpm in the middle and late stages, and then transitioned to about 700rpm. After adjusting to about 700rpm, the corresponding air flow rate was 3.5L / min). In terms of residual sugar, the early stage was the same as in embodiment 3, and the residual sugar was maintained at a residual sugar level of 15g / L by the method of adding glucose, but the sugar supplement rate was reduced in the middle stage of fermentation to maintain the residual sugar at a level of 5-10g / L. In terms of pH, the initial pH before inoculation was controlled at 8.5 (the pH fluctuated downward after the start of fermentation, and then stabilized at 6.5). That is, the comprehensive fermentation results of the previous batches were adopted, that is, the pH also adopted the control method in embodiment 3, the air flow rate adopted the segmented control method in embodiment 1, and the temperature of the whole process was 32°C.

[0134] During the 0-4h period, the strain grew stably.

[0135] During the 12-20h period, the strain began to grow faster and the dissolved oxygen quickly dropped to below 10%.

[0136] At 18h, the stirring speed was increased from 600rpm to 650rpm, and the dissolved oxygen increased slightly and then continued to decrease to the micro-limited oxygen state. In the subsequent 20-40h period, the residual sugar was stabilized at 15g / L, the pH was around 6.5, and the dissolved oxygen in the fermentation system was close to zero. The strain was in a relatively ideal growth and production state during this period, and the bacteria grew rapidly to an OD value of 95, and the production rate of coproporphyrin III was also significantly accelerated.

[0137] In the 40-70h time period, a hypoglycemic strategy was adopted to reduce the residual sugar content and maintain about 5-10g / L, and at 52h, the stirring speed was increased to 700rpm, and compared with the previous time period, the bacterial cell volume growth was slower during this period. During this period, coproporphyrin III accumulation was significantly accelerated, and in the 70-74h time period, coproporphyrin III production increased, and bacterial cell growth rate had slowed down. In order to make the sugar consumption consistent with Example 3 in the present embodiment, sugar supplementation was stopped at the 74h time point, and fermentation was terminated at the 76h time point when the bacterial strain growth production tended to be stable, pH and dissolved oxygen all significantly increased.

[0138] The final strain OD value reached 137.5, the coproporphyrin III yield was 19.6 g / L, and the iron coproporphyrin III yield was 0.62 g / L. During the fermentation process, the glucose conversion rate was 0.0893 g / g coproporphyrin III, which was 21% higher than that in Example 3.

[0139] Compared with the process of Example 3, the process of this embodiment has a significant improvement in the output of coproporphyrin III. Figure 5 As shown, the coproporphyrin III production reached about 20 g / L.

[0140] Example 5, Rhodobacter capsulatus fermentation

[0141] The same fermentation process as in Example 4 was used, except that the strain was replaced with R. capsulatus SB1003. The results were as follows: Figure 6 As shown, it can be seen that Rhodobacter capsulatus can also effectively produce coproporphyrin III with a high yield.

[0142] Fermentation with E. coli DH5a showed the following results: Figure 7 As shown, it can be seen that Escherichia coli can also effectively produce coproporphyrin III with a high yield.

[0143] Summarize

[0144] There are many fermentation process parameters, including many basic parameters and personalized parameters, so that the fermentation process varies greatly according to the different fermentation strains and target products, requiring a lot of exploration and optimization work. The inventors analyzed many fermentation factors / parameters, and established and optimized the coproporphyrin III fermentation process based on comprehensive analysis and repeated exploration.

[0145] In the preferred process of the present invention, a staged control method of dissolved oxygen and residual sugar is adopted, that is, the dissolved oxygen is rapidly reduced and the residual sugar is maintained high in the early stage, and the ventilation and stirring speeds are increased and the residual sugar is maintained low in the middle and late stages, so that the conversion efficiency of coproporphyrin III can be significantly improved and the fermentation cycle is further shortened.

[0146] In the present invention, the temperature is also analyzed, and it is determined that the temperature at which the strain can grow normally is 28-35°C, and the suitable temperature is 31-33°C.

[0147] In the present invention, the pH is also analyzed, and it is determined that the pH range for normal growth of the strain is 5-8.5, and the suitable pH range is 6-7. The inventors also found that the initial high pH pressure promotes the later growth and production of the strain.

[0148] In the present invention, the residual sugar concentration at which the strain can grow normally is also analyzed, and it is determined to be 4-28 g / L, and the appropriate residual sugar concentration is 5-20 g / L.

[0149] In the present invention, the dissolved oxygen is also analyzed, and a dissolved oxygen strategy is determined to maintain the stirring speed and ventilation volume at each stage and gradually increase them.

[0150] Analysis of factors such as lighting conditions and stirring paddle style revealed that they have a certain impact on fermentation. A shading environment can accelerate the growth and production of strains, and an inclined stirring paddle can increase dissolved oxygen to a certain extent.

[0151] The optimized process of the present invention increases the yield of coproporphyrin III from the initial 1.4 g / L to 19.6 g / L, which is the highest yield reported so far. The fermentation cycle is shortened from 96 h to 74 h, and the glucose conversion rate of the substrate is increased to 0.0893 g / g.

[0152] 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 the present invention, just as each document is cited as reference separately.

Claims

1. A method for producing a porphyrin compound, comprising: The porphyrin compound production bacteria are fermented and cultured, and the glucose content and dissolved oxygen in the fermentation liquid are controlled in stages under the conditions of pH 5-8.5 and temperature 28-35°C; wherein the staged control of the glucose content in the fermentation liquid includes: in the range of glucose concentration of 4-20 g / L, reducing the dissolved oxygen and increasing the glucose concentration in the early stage of fermentation, and increasing the dissolved oxygen and reducing the glucose concentration in the middle and late stages of fermentation.

2. The method according to claim 1, characterized in that The stepwise control of the glucose content in the fermentation liquid includes: adjusting the glucose content to 12-18 g / L in the early stage of fermentation, and reducing the glucose content to 25-60% of the early stage of fermentation in the middle and late stages of fermentation; preferably, the glucose content in the middle and late stages of fermentation is 4-10 g / L.

3. The method according to claim 1, characterized in that The segmented control of dissolved oxygen in the fermentation liquid includes: adjusting the rotation speed to 600±20rpm in the early stage of fermentation, and transitioning to 700±20rpm in the middle and late stages of fermentation; preferably, the rotation speed in the middle and late stages of fermentation is first transitioned to 650±20rpm, and then transitioned to 700±20rpm; wherein, the air flow rate is 3.5±0.5L / min.

4. The method according to claim 1, characterized in that The temperature is 30-34°C, preferably 31-33°C; and / or The pH is 5.5 to 7.5, preferably 6 to 7, more preferably pH 6.2 to 6.8; more preferably, the pH value is adjusted using ammonia water.

5. The method according to claim 1, characterized in that Regarding pH, at the beginning of fermentation, the pH is adjusted to the maximum value within the range that does not affect the growth of the strain, which puts pressure on the initial growth of the bacteria; after that, the pH naturally drops to the lower limit and is adjusted to maintain pH 6-7; Preferably, the maximum value within the range that does not affect the growth of the strain is pH 7.3-8.7; more preferably pH 7.4-8.

6.

6. The method according to claim 1, characterized in that The fermentation culture is carried out under reduced light conditions.

7. The method according to claim 1, characterized in that The early fermentation period is from the start of fermentation to the 30th to 45th hour; the period thereafter is the middle and late fermentation period; preferably, the fermentation is terminated at 65 to 100 hours.

8. The method according to claim 1, characterized in that The fermentation culture is carried out in a bioreactor; preferably, the bioreactor comprises a stirring propeller; more preferably, the stirring propeller is an inclined stirring propeller; and / or The culture medium used for fermentation culture is a culture medium suitable for the growth and production of the porphyrin compound producing bacteria, and contains sufficient nutrients required for production and growth; Preferably, the culture medium comprises: glucose, enzymatic corn steep liquor, monosodium glutamate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride and calcium carbonate.

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

10. The method according to any one of claims 1 to 8 or the use according to claim 9, characterized in that: The porphyrin compound producing bacteria include bacteria selected from the following group: photosynthetic bacteria, Paracoccus denitrificans, Bacillus subtilis, Agrobacterium tumefaciens, Rhizobium centromedianum or Methanobacterium barkleyi; preferably, the photosynthetic bacteria include bacteria selected from the following group: Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter nitrogen-fixing or Rhodobacter colloidus; or The porphyrin compound includes: coproporphyrin III or its chelate, analog, derivative, precursor or downstream product.

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