Method for improving fermentation yield of L-tryptophan
阅读说明：本技术 一种提高l-色氨酸发酵产率的方法 (Method for improving fermentation yield of L-tryptophan ) 是由 周旭波 王健 方培新 王华萱 于 2020-12-28 设计创作，主要内容包括：一种提高L-色氨酸发酵产率的方法,在发酵的起始或/和中间阶段,往发酵培养基中加入发酵辅助因子；以发酵培养基的总体积计,每升所述发酵培养基添加的发酵辅助因子包括：0.1×10～(-4)～10×10～(-4)g/L的硫胺素、0.1×10～(-5)～5×10～(-5)g/L的核黄素、0.1×10～(-3)～10×10～(-3)g/L的烟酸、0.1×10～(-5)～2×10～(-5)g/L的磷酸氨基嘌呤、0.1×10～(-3)～10×10～(-3)g/L的泛酸、0.1×10～(-5)～2×10～(-5)g/L的吡哆素、0.1×10～(-4)～10×10～(-4)g/L的生物素、0.1×10～(-6)～10×10～(-6)g/L的叶酸、0.1×10～(-5)～10×10～(-5)g/L的钴胺素和0.1×10～(-5)～10×10～-～5g/L乳清酸。本发明在不增加额外设备和人力投入的情况下,实现了L-色氨酸发酵生产周期的缩短以及产率和转化率的大幅提高,方法简单易行,适合于工业化生产。(A method for improving fermentation yield of L-tryptophan comprises adding fermentation auxiliary factors into fermentation medium at the initial stage or/and middle stage of fermentation; the fermentation auxiliary factors added in each liter of the fermentation medium comprise the following components in percentage by volume of the total fermentation medium: 0.1X 10 ‑4 ～10×10 ‑4 Thiamine in g/L, 0.1X 10 ‑5 ～5×10 ‑5 Riboflavin in g/L, 0.1X 10 ‑3 ～10×10 ‑3 Nicotinic acid, 0.1X 10 in g/L ‑5 ～2×10 ‑5 g/L phosphoaminopurine, 0.1X 10 ‑3 ～10×10 ‑3 g/L pantothenic acid, 0.1X 10 ‑5 ～2×10 ‑5 Pyridoxine, 0.1X 10 in g/L ‑4 ～10×10 ‑4 Biotin of 0.1X 10 in g/L ‑6 ～10×10 ‑6 Folic acid, 0.1X 10 in g/L ‑5 ～10×10 ‑5 g/L of cobalamin and 0.1X 10 ‑5 ～10×10 ‑ 5 g/L orotic acid. The method realizes the shortening of the fermentation production period of the L-tryptophan and the great improvement of the yield and the conversion rate under the condition of not increasing additional equipment and manpower input, is simple and feasible, and is suitable for industrial production.)
1. A method for improving fermentation yield of L-tryptophan is characterized by comprising the following steps: adding fermentation auxiliary factors into a fermentation medium at the beginning or/and the middle stage of fermentation; the fermentation auxiliary factors added in each liter of the fermentation medium comprise the following components in percentage by volume of the total fermentation medium: 0.1X 10-4～10×10-4Thiamine in g/L, 0.1X 10-5～5×10-5Riboflavin in g/L, 0.1X 10-3～10×10-3Nicotinic acid, 0.1X 10 in g/L-5～2×10-5g/L phosphoaminopurine, 0.1X 10-3～10×10-3g/L pantothenic acid, 0.1X 10-5～2×10-5Pyridoxine, 0.1X 10 in g/L-4～10×10-4Biotin of 0.1X 10 in g/L-6～10×10-6Folic acid, 0.1X 10 in g/L-5～10×10-5g/L of cobalamin and 0.1X 10-5～10×10-5g/L orotic acid.
2. The method for improving fermentation yield of L-tryptophan according to claim 1, wherein: the fermentation medium comprises per liter: 5.0-80.0 g of glucose, 2.0-10.0 g of yeast extract powder, 0.1-10.0 g of polypeptide powder, and 1.0-30.0 g of (NH)4)2S040.5-20 g KH2P0400.1-5 g of K2HP04·3H2And 0.1-5.0 g of MgS04·7H20; the initial pH value is 7.0-7.2.
3. The method for improving fermentation yield of L-tryptophan according to claim 1, wherein: fermentation cofactors added per liter of the fermentation medium include: 0.5X 10-4～5×10-4g/L thiamine, 0.5X 10-5～5×10-5g/L riboflavin, 0.5X 10-3～5×10-3g/L nicotinic acid, 0.5X 10-5～2×10-5g/L phosphoaminopurine, 0.5X 10-3～5×10- 3g/L pantothenic acid, 0.5X 10-5～2×10-5g/L pyridoxine, 0.5X 10-4～5×10-4g/L biotin, 0.5X 10-6～5×10-6Folic acid, 0.5X 10 g/L-5～5×10-5g/L cobalamin, 0.5X 10-5～5×10-5g/L orotic acid.
4. The method for improving fermentation yield of L-tryptophan according to claim 1, wherein: fermentation cofactors added per liter of the fermentation medium include: 1X 10-4～5×10-4g/L thiamine, 0.5X 10-5～2×10-5g/L riboflavin, 3X 10-3～5×10-3g/L nicotinic acid, 0.5X 10-5～1×10-5g/L phosphoaminopurine, 1X 10-3～5×10-3g/L pantothenic acid, 1X 10-5～2×10-5g/L pyridoxine,0.5×10-4～2×10-4g/L Biotin, 2X 10-6～5×10-6g/L Folic acid, 2X 10-5～5×10-5g/L cobalamin, 2X 10-5～5×10-5g/L orotic acid.
5. The method for improving fermentation yield of L-tryptophan according to claim 1, wherein: and adding the fermentation auxiliary factors in a batch or continuous manner when the fermentation is started for 6-36 h.
6. The method for improving fermentation yield of L-tryptophan according to claim 1, wherein: the strain used for L-tryptophan fermentation is Escherichia coli.
Technical Field
The invention belongs to the technical field of amino acid production by a fermentation method, and relates to a method for improving the fermentation yield of L-tryptophan.
Background
The L-tryptophan has the chemical name of beta-indolylalanine and the chemical name of L-2-amino-3-indolylpropanoic acid, is one of eight essential amino acids in the life activities of human bodies and animals, exists in organisms in a free state or a combined state, plays a very important role in the growth, development and metabolism of the human bodies and the animals, is called as a second essential amino acid, and is widely applied to various aspects such as medicines, foods, feeds and the like.
The production method of L-tryptophan mainly comprises a microbial conversion method, an enzyme method and a direct fermentation method. At present, the main tryptophan manufacturers in the world mainly produce tryptophan by a microbial direct fermentation method, wherein the direct fermentation method uses cheap raw materials such as glucose, cane molasses and the like as carbon sources and utilizes excellent tryptophan production strains to produce tryptophan. However, most fermentation production technologies for producing L-tryptophan by microbial fermentation have low acid production level and high cost, and the production level and yield can not meet the market demands. Therefore, it is of great importance to develop research for improving the technical level of tryptophan fermentation.
In recent years, with the rising of raw material prices and the increasing market competition, in order to improve the tryptophan yield and reduce the production cost, a great deal of research reports on tryptophan fermentation are provided, and mutant strains for tryptophan synthesis are bred and screened mainly by the traditional mutagenesis and genetic engineering methods to improve the L-tryptophan yield. These methods are often directed to a single gene modification or an increase in the activity of a certain reactive enzyme, and have a great gap in increasing the fermentation level of L-tryptophan and in practical production. In recent years, a few studies show that the fermentation of L-tryptophan is promoted to a certain extent by adding a proper amount of amino acid, organic acid, vitamin and the like into a fermentation medium, but the studies only stay on the addition of a single experimental factor, the influence on the improvement of the fermentation level of tryptophan is extremely limited, the fermentation of L-tryptophan in the studies is in a low level, and the gap is large for realizing industrial production.
Disclosure of Invention
The invention aims to provide a method for improving fermentation yield of L-tryptophan.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for improving fermentation yield of L-tryptophan comprises adding fermentation auxiliary factors into fermentation medium at the initial stage or/and middle stage of fermentation; the fermentation auxiliary factors added in each liter of the fermentation medium comprise the following components in percentage by volume of the total fermentation medium: 0.1X 10-4～10×10-4Thiamine in g/L, 0.1X 10-5～5×10-5Riboflavin in g/L, 0.1X 10-3～10×10-3Nicotinic acid, 0.1X 10 in g/L-5～2×10-5g/L phosphoaminopurine, 0.1X 10-3～10×10-3g/L pantothenic acid, 0.1X 10-5～2×10-5Pyridoxine, 0.1X 10 in g/L-4～10×10-4Biotin of 0.1X 10 in g/L-6～10×10-6Folic acid, 0.1X 10 in g/L-5～10×10-5g/L of cobalamin and 0.1X 10-5～10×10-5g/L orotic acid.
The preferable technical scheme is as follows: the fermentation medium comprises per liter: 5.0-80.0 g of glucose, 2.0-10.0 g of yeast extract powder, 0.1-10.0 g of polypeptide powder, and 1.0-30.0 g of (NH)4)2S040.5-20 g KH2P0400.1-5 g of K2HP04·3H2And 0.1-5.0 g of MgS04·7H20; the initial pH value is 7.0-7.2.
The preferable technical scheme is as follows: fermentation cofactors added per liter of the fermentation medium include: 0.5X 10-4～5×10-4g/L thiamine, 0.5X 10-5～5×10-5g/L riboflavin, 0.5X 10-3～5×10-3g/L nicotinic acid, 0.5X 10-5～2×10-5g/L phosphoaminopurine, 0.5X 10-3～5×10-3g/L pantothenic acid, 0.5X 10-5～2×10-5g/L pyridoxine, 0.5X 10-4～5×10-4g/L biotin, 0.5X 10-6～5×10-6Folic acid, 0.5X 10 g/L-5～5×10-5g/L cobalamin, 0.5X 10-5～5×10-5g/L orotic acid.
The preferable technical scheme is as follows: fermentation cofactors added per liter of the fermentation medium include: 1X 10-4～5×10-4g/L thiamine, 0.5X 10-5～2×10-5g/L riboflavin, 3X 10-3～5×10-3g/L nicotinic acid, 0.5X 10-5～1×10-5g/L phosphoaminopurine, 1X 10-3～5×10-3g/L pantothenic acid, 1X 10-5～2×10-5g/L pyridoxine, 0.5X 10-4～2×10-4g/L Biotin, 2X 10-6～5×10-6g/L Folic acid, 2X 10-5～5×10-5g/L cobalamin, 2X 10-5～5×10-5g/L orotic acid.
The preferable technical scheme is as follows: and adding the fermentation auxiliary factors in a batch or continuous manner when the fermentation is started for 6-36 h.
The preferable technical scheme is as follows: the strain used for L-tryptophan fermentation is Escherichia coli.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the invention adopts a cofactor metabolism regulation strategy and a metabolic flux analysis method, quantitatively researches the influence of fermentation auxiliary factors such as thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin, orotic acid and the like on the metabolic flux distribution change of L-tryptophan producing bacteria and the synergistic effect of the fermentation auxiliary factors in a tryptophan metabolic system, and systematically adds thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid in a specific ratio when the L-tryptophan is fermented for 6-36 hours to improve the yield of the L-tryptophan producing bacteria in the L-tryptophan fermentation process. The method realizes the shortening of the fermentation production period of the L-tryptophan and the great improvement of the yield and the conversion rate under the condition of not increasing additional equipment and manpower input, is simple and easy to implement, and is suitable for industrial production.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Comparative example:
test number one: fermentation medium: 42.5g/L glucose, 6g/L yeast extract powder, 5.05g/L polypeptide powder, (NH)4)2S04 15g/L，KH2P04 10.25g/L，K2HP04·3H20 2.55g/L，MgS04·7H202.55 g/L, initial pH 7.1.
Test number two: 80.0g/L glucose, 10.0g/L yeast extract powder, 0.1g/L polypeptide powder, (NH)4)2S04 1.0g/L，KH2P04 20g/L，K2HP04·3H25 g/L，MgS04·7H200.1 g/L, initial pH 7.0.
Test number three: 5.0g/L glucose, 2.0g/L yeast extract powder, 10.0g/L polypeptide powder, (NH)4)2S04 30.0g/L，KH2P04 0.5g/L，K2HP04·3H20 0.1g/L，MgS04·7H25.0g/L, initial pH 7.2.
Inoculating the escherichia coli liquid in the logarithmic phase into a 30L automatic control fermentation tank containing a fermentation culture medium according to the inoculation amount of 10%, initially fixing the volume to 12L, controlling the temperature to be 37 ℃, introducing appropriate air, adjusting the stirring speed, controlling the dissolved oxygen to be 20-50%, controlling the pH to be 7.0-7.2 by automatically feeding ammonia water in a flowing manner, defoaming by feeding appropriate amount of foam killer, and controlling the residual sugar to be 0.1% by feeding glucose solution with the concentration of 600 g/L. When the fermentation is stopped after 48 hours, and the fermentation tank is placed, the acid production and sugar acid conversion rate of the L-tryptophan are shown in the table 1.
Test number
Acid production by L-tryptophan
Conversion rate of sugar and acid
A
36.87g/L
16.13％
II
37.26g/L
15.88％
III
37.51g/L
16.09％
Average
37.21g/L
16.03％
Example 1: method for improving fermentation yield of L-tryptophan
The fermentation medium is as follows: fermentation medium: 42.5g/L glucose, 6g/L yeast extract powder, 5.05g/L polypeptide powder, (NH)4)2S04 15g/L，KH2P04 10.25g/L，K2HP04·3H20 2.55g/L，MgS04·7H202.55 g/L, initial pH 7.1; any one of the following fermentation cofactors was added to the fermentation medium, respectively, to conduct an experiment. The cofactors are added at the beginning of the fermentation.
Cofactor 1: 0.1X 10-4g/L thiamine, 0.1X 10-5g/L riboflavin, 0.1X 10-3g/L nicotinic acid, 0.1X 10-5g/L phosphoaminopurine, 0.1X 10-3g/L pantothenic acid, 0.1X 10-5g/L pyridoxine, 0.1X 10-4g/L biotin, 0.1×10-6Folic acid, 0.1X 10 g/L-5g/L cobalamin, 0.1X 10-5g/L orotic acid.
Cofactor 2: 0.5X 10-4g/L thiamine, 0.5X 10-5g/L riboflavin, 0.5X 10-3g/L nicotinic acid, 0.5X 10-5g/L phosphoaminopurine, 0.5X 10-3Pantothenic acid, 0.5X 10-5g/L pyridoxine, 0.5X 10-4g/L biotin, 0.5X 10-6Folic acid, 0.5X 10 g/L-5g/L cobalamin, 0.5X 10-5g/L orotic acid.
Cofactor 3: 1X 10-4g/L thiamine, 2X 10-5g/L riboflavin, 3X 10-3g/L nicotinic acid, 1X 10-5g/L phosphoaminopurine, 1X 10-3g/L pantothenic acid, 1X 10-5g/L pyridoxine, 2X 10-4g/L Biotin, 2X 10-6g/L Folic acid, 2X 10-5g/L cobalamin, 2X 10-5g/L orotic acid.
Inoculating the escherichia coli liquid in the logarithmic phase into a 30L automatic control fermentation tank containing a fermentation culture medium according to the inoculation amount of 10%, initially fixing the volume to 12L, controlling the temperature to be 37 ℃, introducing appropriate air, adjusting the appropriate stirring speed, controlling the dissolved oxygen to be 20-50%, controlling the pH to be 7.0-7.2 by automatically feeding ammonia water in a flowing manner, defoaming by feeding appropriate amount of natural enemy, controlling the residual sugar to be 0.1% by feeding glucose solution with the concentration of 600g/L, and stopping fermentation until 48 h. The L-tryptophan yield and the sugar acid conversion rate at the time of tank discharge are shown in Table 2, and the improvement rates in the tables are calculated based on the comparative examples.
Group of
Production of L-tryptophan
Rate of increase
Cofactor 1
41.16g/L
10.62％
Cofactor 2
42.47g/L
14.14％
Cofactor 3
44.13g/L
18.60％
Example 2: method for improving fermentation yield of L-tryptophan
Fermentation medium: 54g/L glucose, 6.5g/L yeast extract powder, 5.6g/L polypeptide powder, (NH)4)2S04 23g/L，KH2P04 11g/L，K2HP04·3H20 4.3g/L，MgS04·7H24.3 g/L, initial pH 7.1.
Inoculating the escherichia coli liquid in the logarithmic phase into a 30L automatic control fermentation tank containing a fermentation culture medium according to the inoculation amount of 10%, initially fixing the volume to 12L, controlling the temperature to be 37 ℃, introducing appropriate air, adjusting the stirring speed, controlling the dissolved oxygen to be 20-50%, controlling the pH to be 7.0-7.2 by automatically feeding ammonia water in a flowing manner, defoaming by feeding appropriate amount of foam killer, and controlling the residual sugar to be 0.1% by feeding glucose solution with the concentration of 600 g/L. And feeding fermentation auxiliary factors when the fermentation time reaches 6h, and stopping feeding when the fermentation time reaches 36 h. The flow acceleration of each auxiliary factor adopts the following schemes:
addition amount of fermentation auxiliary factor: based on the total volume of the fermentation medium, adding per liter of the fermentation medium: 5X 10- 4Thiamine in g/L, 3X 10-5g/L riboflavin, 5X 10-3Nicotinic acid, 1X 10 in g/L-5g/L phosphoaminopurine, 5X 10-3g/L pantothenic acid, 1X 10-5Pyridoxine, 5X 10 in g/L-4g/L of crudeSubstance, 5X 10-6Folic acid, 5X 10 in g/L-5g/L of cobalamin and 5X 10-5g/L orotic acid. Continuously feeding the fermentation auxiliary factors at the speed of:
scheme 1: thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin, orotic acid at a feeding rate of 4.0. mu.g/h, 0.4. mu.g/h, 40.0. mu.g/h, 0.4. mu.g/h, 4.0. mu.g/h, 0.04. mu.g/h, 0.4. mu.g/h, respectively.
Scheme 2: thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin, orotic acid at a feeding rate of 20.0. mu.g/h, 2.0. mu.g/h, 200.0. mu.g/h, 2.0. mu.g/h, 20.0. mu.g/h, 0.2. mu.g/h, 2.0. mu.g/h, respectively.
Scheme 3: thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin, orotic acid at a feeding rate of 40.0. mu.g/h, 8.0. mu.g/h, 1200.0. mu.g/h, 4.0. mu.g/h, 400.0. mu.g/h, 4.0. mu.g/h, 80.0. mu.g/h, 0.8. mu.g/h, 8.0. mu.g/h, respectively.
The fermentation cofactors are fermented from 6h to 48h, and the yield and saccharic acid conversion rate of L-tryptophan are shown in Table 3 when the fermentation cofactors are placed in the tank, wherein the improvement rates are calculated based on the comparative example.
Group of
Production of L-tryptophan
Increase ratio (%)
Conversion rate of sugar and acid
Increase ratio (%)
Flow acceleration 1
41.51g/L
11.56％
16.83％
4.99％
Flow acceleration 2
43.32g/L
16.42％
17.21％
7.36％
Flow acceleration 3
45.18g/L
21.42％
17.59％
9.73％
Example 3: method for improving fermentation yield of L-tryptophan
Fermentation medium: 80.0g/L glucose, 10.0g/L yeast extract powder, 10.0g/L polypeptide powder, (NH)4)2S0430.0g/L，KH2P04 20g/L，K2HP04·3H20 5g/L，MgS04·7H205.0 g/L, initial pH 7.2.
Inoculating the escherichia coli liquid in the logarithmic phase into a 30L automatic control fermentation tank containing a fermentation culture medium according to the inoculation amount of 10%, initially fixing the volume to 12L, controlling the temperature to be 37 ℃, introducing appropriate air, adjusting the stirring speed, controlling the dissolved oxygen to be 20-50%, controlling the pH to be 7.0-7.2 by automatically feeding ammonia water in a flowing manner, defoaming by feeding appropriate amount of foam killer, and controlling the residual sugar to be 0.1% by feeding glucose solution with the concentration of 600 g/L. During the fermentation, the fermentation cofactors are fed in one of the following ways.
Thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin, orotic acid at a feeding rate of 4.0. mu.g/h, 0.4. mu.g/h, 40.0. mu.g/h, 0.4. mu.g/h, 4.0. mu.g/h, 0.04. mu.g/h, 0.4. mu.g/h, respectively.
Fed-batch method 1: feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for the first time after fermentation for 6-12 h, wherein the feeding amounts are 48 mu g, 4.8 mu g, 480 mu g, 4.8 mu g, 48 mu g, 0.48 mu g, 4.8 mu g and 4.8 mu g respectively; feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for 18-24 h after fermentation, wherein the content of the pantothenic acid, the pyridoxine, the biotin, the folic acid, the cobalamin and the orotic acid is respectively 48 mu g, 4.8 mu g, 480 mu g, 4.8 mu g, 48 mu g, 0.48 mu g, 4.8 mu g and 4.8 mu g; and feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for 30-36 h, wherein the feed is respectively 24 mu g, 2.4 mu g, 240 mu g, 2.4 mu g, 24 mu g, 0.24 mu g, 2.4 mu g and 2.4 mu g.
Fed-batch mode 2: feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for the first time after fermentation for 6-12 h, wherein the feeding amounts are 240 mu g, 24 mu g, 2400 mu g, 24 mu g, 240 mu g, 2.4 mu g, 24 mu g and 24 mu g respectively; fermenting for 18-24 h, feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid which are 240 mu g, 24 mu g, 2400 mu g, 24 mu g, 240 mu g, 2.4 mu g, 24 mu g and 24 mu g respectively; and feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for 30-36 h after fermentation, wherein the content of the pantothenic acid, the pyridoxine, the biotin, the folic acid, the cobalamin and the orotic acid is respectively 120 mu g, 12 mu g, 1200 mu g, 12 mu g, 120 mu g, 1.2 mu g, 12 mu g and 12 mu g.
Scheme 3: thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin, orotic acid at a feeding rate of 40.0. mu.g/h, 8.0. mu.g/h, 1200.0. mu.g/h, 4.0. mu.g/h, 400.0. mu.g/h, 4.0. mu.g/h, 80.0. mu.g/h, 0.8. mu.g/h, 8.0. mu.g/h, respectively.
Fed-batch mode 3: feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for the first time after fermentation for 6-12 h, wherein the feeding amount is respectively 480 mu g, 96 mu g, 14400 mu g, 48 mu g, 4800 mu g, 48 mu g, 960 mu g, 9.6 mu g, 96 mu g and 96 mu g; feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for 18-24 h in a fermentation manner, wherein the feeding time is 480 mu g, 96 mu g, 14400 mu g, 48 mu g, 4800 mu g, 48 mu g, 960 mu g, 9.6 mu g, 96 mu g and 96 mu g respectively; and feeding thiamine, riboflavin, nicotinic acid, phosphoaminopurine, pantothenic acid, pyridoxine, biotin, folic acid, cobalamin and orotic acid for 30-36 h, wherein the feed is 240 mu g, 48 mu g, 7200 mu g, 24 mu g, 2400 mu g, 24 mu g, 480 mu g, 4.8 mu g, 48 mu g and 48 mu g respectively.
When the fermentation was stopped at 48 hours and the tank was placed, the yield of L-tryptophan and the sugar-acid conversion rate are shown in Table 4, and the increase rates in the tables are calculated based on the comparative examples.
Group of
Production of L-tryptophan
Increase ratio (%)
Conversion rate of sugar and acid
Increase ratio (%)
Fed-batch method 1
41.21g/L
10.75％
16.74％
4.43％
Fed-batch method 2
43.39g/L
16.61％
17.22％
7.42％
Fed-batch mode 3
45.14g/L
21.31％
17.48％
9.05％
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.