CN115595342A - Method for improving tryptophan production level based on control of specific oxygen consumption rate - Google Patents
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Abstract
A method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate belongs to the technical field of amino acid fermentation. In order to improve the yield and conversion rate of tryptophan and reduce the synthesis of byproducts, the invention provides a method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate, which is characterized in that in the process of producing tryptophan by using escherichia coli fermentation, when the amount of oxygen consumed per dry weight of thalli in unit time is slowly reduced to a target value from the beginning of the peak in the early stage of the fermentation process, the amount of oxygen consumed per dry weight of thalli in unit time is controlled in stages by supplementing compound nitrogen. The method can realize the balance between acid production and conversion rate, realize the aim of improving the acid production and conversion rate, and is suitable for large-scale industrial production.
Description
Technical Field
The invention belongs to the technical field of amino acid fermentation, and particularly relates to a method for improving the production level of tryptophan based on control of specific oxygen consumption rate.
Background
L-tryptophan, also known as α -aminoindolylpropanoic acid, has the formula: c 11 H 12 N 2 O 2 The amino acid is a precursor of important neurotransmitter-5-hydroxytryptamine of human bodies, is one of essential amino acids of human bodies, has good nutritive value and medicinal value, and is widely used in industries of feed, medicine, health care and the like as a large amount of amino acid fermentation products.
The production of L-tryptophan is mainly realized by taking escherichia coli or corynebacterium glutamicum which is modified by genetic engineering technology as a production strain and adopting a microbial fermentation method. The microbial fermentation method has the advantages of low raw material price, simple process control, reliable product quality and the like. In recent years, how to produce tryptophan products with low cost and high efficiency is the key for promoting the continuous progress of the technology of main amino acid manufacturers in China, so in order to reduce the cost and increase the benefit, higher requirements are put forward on not only nutrient components of a culture medium but also the rationality of the control of the fermentation process.
The consumption capacity of the thalli to oxygen in the aerobic fermentation process can reflect the strength of respiratory metabolism. Especially for the high aerobic tryptophan fermentation process, the level of controlling the oxygen consumption rate has important influence on the generation of thalli, products and byproducts by substrates metabolized by escherichia coli, and finally influences the tryptophan product concentration and the conversion rate, so that the reasonable control of the oxygen consumption rate is the key for realizing high yield and high conversion rate. Although some documents report that the expression of tryptophan is improved in the fermentation process by controlling the dissolved oxygen or the oxygen uptake rate, the influence of the cell concentration on the oxygen consumption of unit bacteria in the fermentation process is not fully considered; there is no report of controlling the amount of oxygen substances consumed in unit dry weight and unit time in the fermentation process by continuous feeding to realize optimal control of the tryptophan fermentation process, and improving the tryptophan fermentation yield and conversion rate.
Disclosure of Invention
In order to improve the yield and the conversion rate of tryptophan and reduce the synthesis of byproducts, the invention provides a method for improving the production level of tryptophan on the basis of controlling the specific oxygen consumption rate, which is characterized in that in the process of producing tryptophan by using escherichia coli through fermentation, when the oxygen consumption per dry weight of thalli per unit time is slowly reduced to 5.0mmol/h/g from the peak in the early stage of the fermentation process, the oxygen consumption per dry weight of thalli per unit time is controlled to be 2.5-4.0mmol/h/g by supplementing a compound nitrogen material.
The composite nitrogen material is further limited to have the composition of 0.2g/L of amino acid powder, 0.3g/L of corn steep liquor dry powder, 0.2g/L of yeast powder, 0.03g/L of ammonium sulfate, 0.01g/L of defoaming agent and the balance of water, and the pH value is 8.0.
Further limiting, stirring linkage is adopted in the fermentation process, and dissolved oxygen is controlled to be not less than 20%.
Further limiting, in the fermentation process, when the glucose concentration in the fermentation liquor is lower than 1.0g/L after 2-4h of fermentation, a glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process.
Further, when the amount of oxygen consumed per dry cell weight per unit time was reduced to 5.0mmol/h/g, the amount of oxygen consumed per dry cell weight per unit time was controlled in stages by adding a composite nitrogen feed.
Further limiting, the staged control means controlling the amount of oxygen consumed per unit cell dry weight per unit time to be 3.5-4.0mmol/h/g when fermenting for 8-20 h; controlling the amount of oxygen consumed in unit cell dry weight unit time to be 3.0-3.5mmol/h/g when fermenting for 20-32 h; controlling the amount of oxygen consumed per dry weight of the bacterial cells to be 2.5-3.0mmol/h/g in unit time from 32h to the end of fermentation.
Further limiting, before the fermentation, slant culture, primary seed culture and secondary seed culture are carried out on the escherichia coli;
the composition of the slant culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of beef extract, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 15g/L of agar powder, 0.05g/L of tetracycline and the balance of water;
the seed culture medium comprises 10g/L of glucose, 4g/L of yeast powder, 0.5g/L of citric acid, 1g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1mg/L of VB, 0.35mg/L of biotin, 1.5g/L of magnesium sulfate heptahydrate, 2.8mg/L of ferrous sulfate heptahydrate, 2mL/L of trace element mixed solution and the balance of water;
the culture medium selected for fermentation consists of 30g/L of glucose, 10g/L of yeast powder, 2.5g/L of citric acid, 4g/L of ammonium sulfate, 5g/L of monopotassium phosphate, 2.8g/L of magnesium sulfate heptahydrate, 90mg/L of ferrous sulfate heptahydrate, 15mg/L of VB, 2mg/L of biotin and 4mL/L of trace element mixed solution.
Further limited, the first-stage seed culture is to inoculate the thalli after slant culture into a seed culture medium, and shake-flask culture is carried out for 10-12h at 37 ℃ and 220 rpm.
Further limiting, the secondary seed culture is to inoculate the primary seed liquid into a seed culture medium according to the inoculation amount of 1%, and culture for 10-12h under the conditions of 36-37 ℃, the aeration ratio of 0.5VVM,200rpm, the tank pressure of 0.03-0.05MPa, the pH value of 7.0-7.2 and the dissolved oxygen content of more than or equal to 30%.
Further limiting, the fermentation is to inoculate the secondary seed liquid into a fermentation medium according to the inoculation amount of 10%, and to culture for 36-40h under the conditions of 36-37 ℃, the aeration ratio of 1.0VVM, the dissolved oxygen ratio of more than or equal to 20%, the tank pressure of 0.03-0.05MPa and the pH value of 6.8-7.0.
The specific oxygen consumption rate in the present invention means the amount of oxygen species consumed per dry cell weight per unit time.
The invention has the beneficial effects that:
the invention realizes the regulation of the metabolic activity of the thalli by controlling the rate of material supplement, and achieves the purpose of controlling the specific oxygen consumption rate, compared with the prior art, the invention has the following beneficial effects:
1. compared with the conventional feeding mode of supplementing sugar and ammonia according to dissolved oxygen and pH, the invention is characterized in that continuous feeding is carried out after the specific oxygen consumption rate in the fermentation process reaches different specific oxygen consumption rates with peak drop from initial culture utilization through continuous feeding, the metabolic activity of thalli is maintained, effective carbon and nitrogen nutrition is continuously fed after the tryptophan synthesis stage, and the stable synthesis of products, the continuous increase of high-speed acid production and the good metabolic stability of thalli can be realized by maintaining the proper specific oxygen consumption rate. After the automatic control technology is adopted, the method can be directly amplified to the industrial production scale according to the specific oxygen consumption rate control strategy.
2. The continuous feeding and staged control of the specific oxygen consumption rate can realize the adjustment of improving the tryptophan conversion rate, reduce byproducts and be beneficial to the control of the product quality. Particularly, according to the actual industrialized production requirement, the conversion of carbon atoms among products, thalli and maintenance can be controlled by controlling the specific oxygen consumption rate of different stages, the growth of the thalli and the maintenance of the metabolic activity of the thalli are facilitated under the condition of maintaining the specific oxygen consumption rate at 3.5-4.0mmol/h/g, and more byproducts are synthesized; under the condition of maintaining specific oxygen consumption rate above 2.5-3.0mmol/h/g, tryptophan synthesis is favored, but the substrate is converted into CO 2 The conversion rate is reduced due to the release, and the thalli are easy to age and die subsequently, so that the thalli are put in a tank in advance; and the balance between acid production and conversion rate can be realized under the condition of maintaining the specific oxygen consumption rate of 3.0-3.5mmol/h/g, and the aims of improving the acid production and the conversion rate are fulfilled.
Drawings
FIG. 1 is a graph showing the change in specific oxygen consumption rate during fermentation in each example;
FIG. 2 is a graph showing the change of the content of glutamic acid produced as a by-product in the fermentation process in each example.
Detailed Description
The present invention will be further described with reference to the following examples and accompanying drawings, which are not intended to limit the invention, but rather, within the spirit and principle of the invention, any modifications, equivalents, improvements and the like made are intended to be included within the scope of the present invention.
The tryptophan producing escherichia coli used by the invention is disclosed in Chinese patent application with the application number of 2016111887431, the invention name of which is a clear liquid fermentation culture medium and a method for improving L-tryptophan, and the preservation number of the strain is CGMCC NO.11073.
The culture medium of the invention is as follows:
the slant culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of beef extract, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 15g/L of agar powder, 0.05g/L of tetracycline and the balance of water;
the seed culture medium comprises 10g/L of glucose, 4g/L of yeast powder, 0.5g/L of citric acid, 1g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1mg/L of VB, 0.35mg/L of biotin, 1.5g/L of magnesium sulfate heptahydrate, 2.8mg/L of ferrous sulfate heptahydrate, 2mL/L of trace element mixed solution and the balance of water;
the fermentation medium consists of 30g/L of glucose, 10g/L of yeast powder, 2.5g/L of citric acid, 4g/L of ammonium sulfate, 5g/L of monopotassium phosphate, 2.8g/L of magnesium sulfate heptahydrate, 90mg/L of ferrous sulfate heptahydrate, 15mg/L of VB, 2mg/L of biotin and 4mL/L of trace element mixed solution;
the composite nitrogen material consists of amino acid powder 0.2g/L, corn steep liquor dry powder 0.3g/L, yeast powder 0.2g/L, ammonium sulfate 0.03g/L, defoaming agent 0.01g/L, and water in balance, and has a pH value of 8.0.
Example 1:
the embodiment provides a method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate, which comprises four stages of slant culture, primary seed culture, secondary seed culture and fermentation, and comprises the following specific steps:
slant culture: inoculating the Escherichia coli from the culture tube onto sterile slant culture medium at 37 deg.C for 2 days.
First-order seed culture: scraping 1cm 2 The slant-cultured E.coli cells were inoculated into a flask (500 mL) containing sterilized 100mL of seed medium and packed with 8-layer gauze. The culture conditions are as follows: incubated at 37 ℃ and 220rpm for 10h.
Secondary seed culture: the method comprises the steps of filling 10L of seed culture medium into a 15L seed tank, sterilizing, keeping the pressure at 0.05MPa by using sterile air, inoculating 100mL (1% of inoculum size) of cultured primary seed liquid into the seed tank by adopting a pressure difference method for culturing, wherein the culture condition is 36 ℃, the aeration ratio is 0.5VVM, the initial stirring rotation speed is 200rpm, the tank pressure is 0.03-0.05MPa, the pH value is 7.0, the dissolved oxygen is controlled to be not less than 30% in the whole process by adjusting the air flow and the rotation speed, and the culture period is 10h.
Fermentation culture: adding 20L fermentation medium into 50L fermentation tank, sterilizing the fermentation medium, cooling, transferring the secondary seed solution into the fermentation tank for culture under conditions of inoculation amount of 10%, culture temperature of 36 deg.C and aeration ratio of 1.0VVM, the initial rotation speed is 200rpm, the dissolved oxygen is controlled to be not less than 20% by adjusting the air quantity and the whole rotation speed, the tank pressure is 0.03-0.05MPa, the pH is controlled to be 6.8 by supplementing composite nitrogen and introducing ammonia water, and the culture period is 36h.
And (3) a feeding control strategy: in the fermentation process, when the glucose concentration in the fermentation liquor is lower than 1.0g/L after 2-4h of fermentation, a glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process. When the oxygen consumption per dry weight of the thallus in unit time is slowly reduced to 5.0mmol/h/g from the peak in the early stage of the fermentation process, the oxygen consumption per dry weight of the thallus in unit time is controlled to be 3.5-4.0mmol/h/g by supplementing the composite nitrogen.
During fermentation, CO in fermentation tail gas was measured by using a process mass spectrometer PASS2000 from Shunhui Yu Heng Ltd 2 、O 2 、N 2 And Ar gas components, capacitance value data measured on line by using an on-line living cell analyzer of Hamilton company, and the specific oxygen consumption rate of the fermentation process is continuously calculated by utilizing a known fermentation process analysis software package developed by Shanghai fast spectrum intelligent company Limited.
After the fermentation process is finished, the related indexes of the fermentation liquor are measured and analyzed, and the result is that the fermentation titer is 52.3g/L and the conversion rate is 18.3%.
Example 2:
the embodiment provides a method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate, which comprises four stages of slant culture, primary seed culture, secondary seed culture and fermentation, and comprises the following specific steps:
slant culture: inoculating the colibacillus from the culture tube to the aseptic slant culture medium at 37 deg.C for 2 days.
First-order seed culture: scrape 1cm 2 The slant-cultured E.coli cells were inoculated into a flask (500 mL) containing sterilized 100mL of seed medium and packed with 8-layer gauze. The culture conditions are as follows: incubated at 37 ℃ and 220rpm for 12h.
Secondary seed culture: the method comprises the steps of filling 10L of seed culture medium into a 15L seed tank, sterilizing, keeping the pressure at 0.05MPa by using sterile air, inoculating 100mL (1% of inoculum size) of cultured primary seed liquid into the seed tank by adopting a pressure difference method for culturing, wherein the culture condition is 37 ℃, the aeration ratio is 0.5VVM, the initial stirring rotation speed is 200rpm, the tank pressure is 0.03-0.05MPa, the pH value is 7.2, the dissolved oxygen is controlled to be not less than 30% in the whole process by adjusting the air flow and the rotation speed, and the culture period is 12h.
Fermentation culture: putting 20L of fermentation medium into a 50L fermentation tank, sterilizing and cooling the fermentation medium, transferring the secondary seed liquid into the fermentation tank for culture, wherein the inoculation amount is 10%, the culture condition is that the temperature is 37 ℃, the aeration ratio is 1.0VVM, the initial rotation speed is 200rpm, the dissolved oxygen in the fermentation process is controlled to be not less than 20% through adjusting the air volume and the rotation speed in the whole process, the tank pressure is 0.03-0.05MPa, the pH is controlled to be 7.0 through supplementing composite nitrogen and introducing ammonia water, and the culture period is 40h.
And (3) a feeding control strategy: when the glucose concentration in the fermentation liquor is lower than 1.0g/L after fermentation for 2-4h, the glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process. When the oxygen consumption per dry weight of the thallus in unit time is slowly reduced to 6.0mmol/h/g from the peak in the early stage of the fermentation process, the oxygen consumption per dry weight of the thallus in unit time is controlled to be 3.0-3.5mmol/h/g by supplementing the composite nitrogen.
During fermentation, CO in fermentation tail gas was measured by using a process mass spectrometer PASS2000 from Shunhui Yu Heng Ltd 2 、O 2 、N 2 Ar gas component, capacitance value data measured on line by using an on-line living cell analyzer of Hamilton company, and the continuous calculation of the specific oxygen in the fermentation process is realized on a well-known fermentation process analysis software package developed by Shanghai fast spectrum Intelligent Limited companyThe rate of consumption.
After the fermentation process is finished, the related indexes of the fermentation liquor are measured and analyzed, the fermentation titer is 58.3g/L, and the conversion rate is 19.3%.
Example 3:
the embodiment provides a method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate, which comprises four stages of slant culture, primary seed culture, secondary seed culture and fermentation, and comprises the following specific steps:
slant culture: inoculating the colibacillus from the culture tube to the aseptic slant culture medium at 37 deg.C for 2 days.
First-order seed culture: scraping 1cm 2 The slant-cultured E.coli cells were inoculated into a flask (500 mL) containing sterilized 100mL of seed medium and packed with 8-layer gauze. The culture conditions are as follows: incubated at 37 ℃ and 220rpm for 11h.
Secondary seed culture: the method comprises the steps of filling 10L of seed culture medium into a 15L seed tank, sterilizing, keeping the pressure at 0.05MPa by using sterile air, inoculating 100mL (1% of inoculum size) of cultured primary seed liquid into the seed tank by adopting a pressure difference method for culturing, wherein the culture condition is 37 ℃, the aeration ratio is 0.5VVM, the initial stirring rotation speed is 200rpm, the tank pressure is 0.03-0.05MPa, the pH value is 7.1, the dissolved oxygen is controlled to be not less than 30% in the whole process by adjusting the air flow and the rotation speed, and the culture period is 11h.
Fermentation culture: putting 20L of fermentation medium into a 50L fermentation tank, sterilizing and cooling the fermentation medium, transferring the secondary seed liquid into the fermentation tank for culture, wherein the inoculation amount is 10%, the culture condition is that the temperature is 37 ℃, the aeration ratio is 1.0VVM, the initial rotation speed is 200rpm, the dissolved oxygen in the fermentation process is controlled to be not less than 20% through adjusting the air volume and the rotation speed in the whole process, the tank pressure is 0.03-0.05MPa, the pH is controlled to be 6.9 through supplementing composite nitrogen and introducing ammonia water, and the culture period is 38h.
And (3) a feeding control strategy: when the glucose concentration in the fermentation liquor is lower than 1.0g/L after 2-4h of fermentation, a glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process. When the oxygen consumption per dry weight of the thallus in unit time is slowly reduced to 5.0mmol/h/g from the peak in the early stage of the fermentation process, the oxygen consumption per dry weight of the thallus in unit time is controlled to be 2.5-3.0mmol/h/g by supplementing the composite nitrogen.
During fermentation, CO in fermentation tail gas was measured by using a process mass spectrometer PASS2000 from Shunhui Yu Heng Ltd 2 、O 2 、N 2 And Ar gas components, capacitance value data measured on line by using an on-line living cell analyzer of Hamilton company is used, and the specific oxygen consumption rate of the fermentation process is continuously calculated on a 'well-known' fermentation process analysis software package developed by Shanghai fast spectrum intelligent company Limited.
After the fermentation process is finished, the related indexes of the fermentation liquor are measured and analyzed, and the result is that the fermentation titer is 48.2g/L and the conversion rate is 16.8%.
Example 4:
the embodiment provides a method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate, which comprises four stages of slant culture, primary seed culture, secondary seed culture and fermentation, and comprises the following specific steps:
slant culture: inoculating the colibacillus from the culture tube to the aseptic slant culture medium at 37 deg.C for 2 days.
First-order seed culture: scraping 1cm 2 The slant-cultured E.coli cells were inoculated into a flask (500 mL) containing sterilized 100mL of seed medium and packed with 8-layer gauze. The culture conditions are as follows: incubated at 37 ℃ and 220rpm for 11h.
Secondary seed culture: the method comprises the steps of filling 10L of seed culture medium into a 15L seed tank, sterilizing, keeping the pressure at 0.05MPa by using sterile air, inoculating 100mL (1% of inoculum size) of cultured primary seed liquid into the seed tank by adopting a pressure difference method for culturing, wherein the culture condition is 37 ℃, the aeration ratio is 0.5VVM, the initial stirring rotation speed is 200rpm, the tank pressure is 0.03-0.05MPa, the pH value is 7.1, the dissolved oxygen is controlled to be not less than 30% in the whole process by adjusting the air flow and the rotation speed, and the culture period is 11h.
Fermentation culture: putting 20L of fermentation medium into a 50L fermentation tank, sterilizing and cooling the fermentation medium, transferring the secondary seed liquid into the fermentation tank for culture, wherein the inoculation amount is 10%, the culture condition is that the temperature is 37 ℃, the aeration ratio is 1.0VVM, the initial rotation speed is 200rpm, the dissolved oxygen in the fermentation process is controlled to be not less than 20% through adjusting the air volume and the rotation speed in the whole process, the tank pressure is 0.03-0.05MPa, the pH is controlled to be 6.9 through supplementing composite nitrogen and introducing ammonia water, and the culture period is 38h.
And (3) feeding control strategy: when the glucose concentration in the fermentation liquor is lower than 1.0g/L after fermentation for 2-4h, the glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process. When the oxygen consumption per dry weight of the thallus in unit time is slowly reduced to 5.0mmol/h/g from the peak in the early stage of the fermentation process, the oxygen consumption per dry weight of the thallus in unit time is controlled by adding composite nitrogen: controlling the amount of oxygen consumed in unit cell dry weight unit time to be 3.5-4.0mmol/h/g when fermenting for 8-28 h; controlling the oxygen consumption per unit dry weight of the thallus at 3.0-3.5mmol/h/g during fermentation for 28-38 h.
During fermentation, CO in fermentation tail gas was measured by using a process mass spectrometer PASS2000 from Shunhui Yu Heng Ltd 2 、O 2 、N 2 And Ar gas components, capacitance value data measured on line by using an on-line living cell analyzer of Hamilton company is used, and the specific oxygen consumption rate of the fermentation process is continuously calculated on a 'well-known' fermentation process analysis software package developed by Shanghai fast spectrum intelligent company Limited.
After the fermentation process is finished, relevant indexes of the fermentation liquor are measured and analyzed, and the result is that the fermentation titer is 60.8g/L and the conversion rate is 20.5%.
Example 5:
the embodiment provides a method for improving the production level of tryptophan based on controlling the specific oxygen consumption rate, which comprises four stages of slant culture, primary seed culture, secondary seed culture and fermentation, and comprises the following specific steps:
slant culture: inoculating the colibacillus from the culture tube to the aseptic slant culture medium at 37 deg.C for 2 days.
First-order seed culture: scraping 1cm 2 The slant-cultured E.coli cells were inoculated into a flask (500 mL) containing sterilized 100mL of seed medium and packed with 8-layer gauze. The culture conditions are as follows: incubated at 37 ℃ and 220rpm for 11h.
Secondary seed culture: the method comprises the steps of filling 10L of seed culture medium into a 15L seed tank, sterilizing, keeping the pressure at 0.05MPa by using sterile air, inoculating 100mL (1% of inoculum size) of cultured primary seed liquid into the seed tank by adopting a pressure difference method for culturing, wherein the culture condition is 37 ℃, the aeration ratio is 0.5VVM, the initial stirring rotation speed is 200rpm, the tank pressure is 0.03-0.05MPa, the pH value is 7.1, the dissolved oxygen is controlled to be not less than 30% in the whole process by adjusting the air flow and the rotation speed, and the culture period is 11h.
Fermentation culture: putting 20L of fermentation medium into a 50L fermentation tank, sterilizing and cooling the fermentation medium, transferring the secondary seed liquid into the fermentation tank for culture, wherein the inoculation amount is 10%, the culture condition is that the temperature is 37 ℃, the aeration ratio is 1.0VVM, the initial rotation speed is 200rpm, the dissolved oxygen in the fermentation process is controlled to be not less than 20% through adjusting the air volume and the rotation speed in the whole process, the tank pressure is 0.03-0.05MPa, the pH is controlled to be 6.9 through supplementing composite nitrogen and introducing ammonia water, and the culture period is 38h.
And (3) feeding control strategy: when the glucose concentration in the fermentation liquor is lower than 1.0g/L after fermentation for 2-4h, the glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process. When the amount of oxygen substances consumed by the dry weight of the thallus in unit time is slowly reduced to 5.0mmol/h/g from the peak in the early stage of the fermentation process, the amount of oxygen consumed by the dry weight of the thallus in unit time is controlled in stages by adding a composite nitrogen material: controlling the amount of oxygen consumed in unit cell dry weight unit time to be 3.5-4.0mmol/h/g when fermenting for 8-20 h; controlling the amount of oxygen consumed in unit cell dry weight unit time to be 3.0-3.5mmol/h/g when fermenting for 20-32 h; controlling the amount of oxygen consumed per dry weight of the bacterial cells to be 2.5-3.0mmol/h/g in unit time from 32h to the end of fermentation.
In the course of fermentation, makeDetermination of CO in fermentation off-gas by Process Mass spectrometer PASS2000 of Shunhyu Heng Ltd 2 、O 2 、N 2 And Ar gas components, capacitance value data measured on line by using an on-line living cell analyzer of Hamilton company is used, and the specific oxygen consumption rate of the fermentation process is continuously calculated on a 'well-known' fermentation process analysis software package developed by Shanghai fast spectrum intelligent company Limited.
After the fermentation process is finished, relevant indexes of the fermentation liquor are measured and analyzed, and the result is that the fermentation titer is 62.8g/L and the conversion rate is 21.5%.
Comparative example:
the present comparative example provides an initial fermentative tryptophan production process comprising four stages, namely slant culture, primary seed culture, secondary seed culture and fermentation, as follows:
slant culture: inoculating the Escherichia coli from the culture tube onto sterile slant culture medium at 37 deg.C for 2 days.
First-stage seed culture: scraping 1cm 2 The slant-cultured E.coli cells were inoculated into a flask (500 mL) containing sterilized 100mL of seed medium and packed with 8-layer gauze. The culture conditions are as follows: incubated at 37 ℃ and 220rpm for 11h.
Secondary seed culture: the method comprises the steps of filling 10L of seed culture medium into a 15L seed tank, sterilizing, keeping the pressure at 0.05MPa by using sterile air, then inoculating 100mL (1 percent of inoculum size) of cultured primary seed liquid into the seed tank by adopting a pressure difference method for culture, wherein the culture condition is 37 ℃, the aeration ratio is 0.5VVM, the initial stirring rotation speed is 200rpm, the tank pressure is 0.03-0.05MPa, the pH is 7.1, the dissolved oxygen is controlled to be not less than 30 percent in the whole process by adjusting the air flow and the rotation speed, and the culture period is 11h.
Fermentation culture: putting 20L of fermentation medium into a 50L fermentation tank, sterilizing and cooling the fermentation medium, transferring the secondary seed liquid into the fermentation tank for culture, wherein the inoculation amount is 10%, the culture condition is that the temperature is 37 ℃, the aeration ratio is 1.0VVM, the initial rotating speed is 200rpm, the dissolved oxygen in the fermentation process is controlled to be not less than 20% in the whole process by adjusting the air volume and the rotating speed, the tank pressure is 0.03-0.05MPa, the pH is controlled to be 6.9 by ammonia water, and the culture period is 38h.
And (3) a feeding control strategy: and the specific oxygen consumption rate is controlled without adopting composite nitrogen supplement, and when the glucose concentration in the fermentation liquor is lower than 1.0g/L after fermentation for 2-4h, a glucose solution with the concentration of 650g/L is supplemented, so that the glucose concentration in the fermentation liquor is maintained at 0.01-0.2g/L in the fermentation process. Ammonia water is fed according to pH self-control, and the pH is controlled to be not less than 6.9 and not more than 7.0.
During fermentation, CO in fermentation tail gas was measured by using a process mass spectrometer PASS2000 from Shunhui Yu Heng Ltd 2 、O 2 、N 2 And Ar gas components, capacitance value data measured on line by using an on-line living cell analyzer of Hamilton company is used, and the specific oxygen consumption rate of the fermentation process is continuously calculated on a 'well-known' fermentation process analysis software package developed by Shanghai fast spectrum intelligent company Limited.
After the fermentation process is finished, the related indexes of the fermentation liquor are measured and analyzed, and the result is that the fermentation titer is 43.5g/L and the conversion rate is 15.9%.
And (4) analyzing results: to investigate the effect of specific oxygen consumption rate on tryptophan fermentation, different specific oxygen consumption rates were controlled by examples 1-3 (example 1 at 3.5-4.0mmol/h/g, example 2 at 3.0-3.5mmol/h/g, example 3 at 2.5-3.0 mmol/h/g), respectively. Corresponding to the combination of examples 1 to 3 the fermentation titer and conversion rate are known as follows: there is an optimum control range for the specific oxygen consumption rate. As can be seen from the comparative analysis of tryptophan concentration and conversion rate of examples 1-3, the specific oxygen consumption rate of 2.5-3.0mmol/h/g is maintained in example 3, and the acid production rate and conversion rate are both low, which indicates that the supplemented glucose is mainly used for the growth of thalli, while the final electron acceptor of reducing power and energy generated by TCA cycle is oxygen, so that under the condition of low specific oxygen consumption rate, the intermediate metabolite and tryptophan synthesis precursor substances obviously generated by glucose metabolism are pumped away for the synthesis of thalli, but are not beneficial to the synthesis of tryptophan, and the byproducts of lactic acid, acetic acid and glutamic acid are not excessively accumulated too much (see FIG. 2); while the specific oxygen consumption rate of 3.5-4.0mmol/h/g is maintained in the example 1, and the corresponding fermentation titer and the conversion rate show that the TCA cycle speed is higher and the acid yield ratio is obviously improved under the condition of maintaining the high specific oxygen consumption rate, but the accumulation of the byproduct glutamic acid is obviously higher under the condition of the fast circulating speed rate of the TCA cycle, which indicates that the intermediate alpha-ketoglutaric acid in the TCA cycle is converted into the glutamic acid; while the results of example 2 maintaining the specific oxygen consumption rate of 3.0-3.5mmol/h/g are unexpected, the acid production and conversion rate are obviously better than those of example 1 and example 3, which shows that maintaining the proper specific oxygen consumption rate in the fermentation process has an important role in intracellular metabolic balance, namely, the thallus growth, tryptophan synthesis and TCA cycle balance have the optimal control ratio. In addition, glutamic acid in example 2 tended to be lower than that in example 1. In view of this, examples 4 and 5 were designed to control the specific oxygen consumption rate in stages, taking into account the different requirements of the different fermentation stages versus the oxygen rate on the basis of examples 1-3, in combination with the fermentation results of examples 1-3. Example 4 controls the specific oxygen consumption rate for two stages and example 5 controls the specific oxygen consumption rate for three stages. As is clear from the fermentation results, the tryptophan concentration and the conversion rate were higher than those in examples 1 to 3 by controlling the specific oxygen consumption rate in stages, and the conversion rate was improved without significant accumulation of glutamic acid as a by-product. The specific oxygen consumption rate of each example during fermentation is shown in figure 1. The results of the content of glutamic acid as a main by-product are shown in FIG. 2. Example 6 is an initial process control in which no control of the specific oxygen consumption rate by the composite nitrogen feed addition is used. It is evident that the specific oxygen consumption after 28h remains significantly lower than that of examples 1-5 only at a level of 1.5-1.8 mmol/h/g. The results show that the metabolic capacity of the thalli is poor in the later period, more glucose consumed by the thalli is consumed as energy, and the synthesis rate of the reverse tryptophan is low. Without significant accumulation of glutamic acid as a by-product.
The invention is not limited to 5 examples, and the improvement of the components or the improvement of the fermentation titer by the similar process strategies of the invention is within the protection scope of the invention.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for improving the production level of tryptophan based on control of specific oxygen consumption rate is characterized in that in the process of producing tryptophan by fermentation of Escherichia coli, when the amount of oxygen consumed per unit cell dry weight unit time is reduced to 5.0mmol/h/g, the amount of oxygen consumed per unit cell dry weight unit time is controlled to be 2.5-4.0mmol/h/g by supplementing composite nitrogen.
2. The method as claimed in claim 1, wherein the composite nitrogen material has the composition of 0.2g/L of amino acid powder, 0.3g/L of corn steep liquor dry powder, 0.2g/L of yeast powder, 0.03g/L of ammonium sulfate, 0.01g/L of defoaming agent and the balance of water, and the pH value is 8.0.
3. The method according to claim 1, wherein stirring linkage is adopted in the fermentation process, and the dissolved oxygen is controlled to be not less than 20%.
4. The method according to claim 1, wherein during the fermentation, when the glucose concentration in the fermentation broth after 2-4h fermentation is lower than 1.0g/L, the glucose solution with the concentration of 650g/L is added, so that the glucose concentration in the fermentation broth during the fermentation is maintained at 0.01-0.2g/L.
5. The method of claim 1, wherein the amount of oxygen consumed per dry cell weight per unit time is controlled in stages by adding the composite nitrogen material when the amount of oxygen consumed per dry cell weight per unit time is up to 5.0 mmol/h/g.
6. The method according to claim 5, wherein the staging control is to control the amount of oxygen consumed per dry weight of the cells to be 3.5 to 4.0mmol/h/g per time unit, during the fermentation for 8 to 20 hours; controlling the oxygen consumption of unit thallus dry weight in unit time to be 3.0-3.5mmol/h/g when fermenting for 20-32 h; controlling the amount of oxygen consumed per dry weight of the bacterial cells to be 2.5-3.0mmol/h/g in unit time from 32h to the end of fermentation.
7. The method of claim 1, wherein prior to fermentation, further comprising subjecting the E.coli to slant culture, primary seed culture and secondary seed culture;
the slant culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of beef extract, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 15g/L of agar powder, 0.05g/L of tetracycline and the balance of water;
the seed culture medium comprises 10g/L of glucose, 4g/L of yeast powder, 0.5g/L of citric acid, 1g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 11 mg/L of VB, 0.35mg/L of biotin, 1.5g/L of magnesium sulfate heptahydrate, 2.8mg/L of ferrous sulfate heptahydrate, 2mL/L of trace element mixed solution and the balance of water;
the culture medium selected for fermentation consists of 30g/L of glucose, 10g/L of yeast powder, 2.5g/L of citric acid, 4g/L of ammonium sulfate, 5g/L of monopotassium phosphate, 2.8g/L of magnesium sulfate heptahydrate, 90mg/L of ferrous sulfate heptahydrate, 15mg/L of VB, 2mg/L of biotin and 4mL/L of trace element mixed solution.
8. The method of claim 7, wherein the primary seed culture is performed by inoculating the slant cultured thalli into a seed culture medium, and performing shake flask culture at 37 ℃ and 220rpm for 10-12h.
9. The method as claimed in claim 7, wherein the secondary seed culture is carried out by inoculating the primary seed solution into the seed culture medium according to the inoculation amount of 1%, and culturing at 36-37 deg.C, aeration ratio of 0.5VVM,200rpm, tank pressure of 0.03-0.05MPa, pH of 7.0-7.2, and dissolved oxygen content of 30% or more for 10-12h.
10. The method according to claim 7, wherein the fermentation is to inoculate the secondary seed liquid into a fermentation medium according to the inoculation amount of 10%, and to culture for 36-40h under the conditions of 36-37 ℃, the aeration ratio of 1.0VVM, the dissolved oxygen ratio of more than or equal to 20%, the tank pressure of 0.03-0.05MPa and the pH of 6.8-7.0.
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