CN113862315A - Formula suitable for producing L-phenylalanine by fermenting escherichia coli and application thereof - Google Patents
Formula suitable for producing L-phenylalanine by fermenting escherichia coli and application thereof Download PDFInfo
- Publication number
- CN113862315A CN113862315A CN202111163151.5A CN202111163151A CN113862315A CN 113862315 A CN113862315 A CN 113862315A CN 202111163151 A CN202111163151 A CN 202111163151A CN 113862315 A CN113862315 A CN 113862315A
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- fermentation
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- phenylalanine
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- 229960005190 phenylalanine Drugs 0.000 title claims abstract description 57
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 31
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims abstract description 103
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- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims abstract description 25
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/222—Phenylalanine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention provides a formula suitable for producing L-phenylalanine by fermenting escherichia coli and application thereof, wherein the functional component of the formula is CaCl2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O, pyridoxal phosphate, vitamin B2 and biotin, the formula uses vitamin B2 for the fermentation of L-Phe for the first time, beneficial effects are achieved, and CoCl is optimized for the first time2.6H2O, PLP, the adverse effect of biotin is determined for the first time, and FeSO is further optimized4.7H2O、MnSO4.H2The dosage of trace elements such as O and the like finally improves the yield and conversion of L-PheThe fermentation method can achieve the aims of accelerating the growth and the propagation of thalli, improving the biomass, prolonging the biomass stabilization period and the acid production peak period, improving the yield and the conversion rate of L-phenylalanine, saving the cost and the like.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a formula suitable for producing L-phenylalanine by fermenting escherichia coli and application thereof.
Background
L-Phenylalanine (L-Phe), L-tyrosine (L-Tyr) and L-tryptophan (L-Trp) belong to aromatic amino acids, are one of shikimic acid pathway products, can be used as a synthetic precursor of aspartame as a novel sweetening agent, can also be used as a carrier of anticancer drugs, have great application value in the aspects of food and medicine, and are usually obtained by a microbial fermentation method at present.
The metabolic pathway for synthesizing L-Phe by using glucose and the like as carbon sources is long, and the L-Phe needs to pass through glycolysis pathway (EMP), citric acid cycle (TCA), Pentose Phosphate Pathway (PPP) and shikimic acid pathway (shikimic acid pathway), wherein a plurality of key enzymes are closely related to the synthesis of L-Phe. The metal ions are used as important enzyme activators, and the type and the dosage of the metal ions influence the strength of the enzyme activity of the key enzyme so as to influence the yield of L-Phe and the conversion rate of saccharic acid. The mechanism of activation/inhibition of enzymes by metal ions is intricate and complex, and appears as follows: the enzyme cannot be activated without or with too little metal ion, such as 5-10 × 10-3moL/L of Mg2+Can activate NADH+Synthase, but not below or above this concentration; the action of metal ions on the enzyme has a certain selectivity, e.g. Ca2+Activating myosin adenosine triphosphate, and inhibiting decarboxylase; sometimes antagonism between ions, e.g. Ca2+Can inhibit Mg2+The activated enzyme, and also the metal ions, may be substituted for one another, e.g. Mn2+Ions may be substituted for Mg2+An activated kinase. Riboflavin (vitamin B2, VB2) is a synthetic precursor of the important coenzymes Flavin Adenine Dinucleotide (FAD) and Flavin Mononucleotide (FMN) in the organism, and FAD and FMN are involved in metabolism involved in hydrogen transfer as coenzymes of flavoproteins, and play important roles in respiration and biological oxidation. Pyridoxal phosphate (PLP) is a coenzyme for a number of enzymes of cell 140 and plays an important role in metabolic reactions such as amino acid synthesis, interconversion and degradation, and common reactions include: transaminases, which catalyze the conversion of alpha-keto acids to amino acids, and a series of racemization, decarboxylation, and aldol reactions. Vitamin H (V)H) Excessive bacteria can cause vigorous metabolism and too fast growth of the bacteria, and the sugar-acid conversion rate is greatly reducedLow problems; too little or no addition of VHCan cause the problems of slow growth of the thalli, low biomass, low acid production efficiency and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a formula suitable for producing L-phenylalanine by fermenting escherichia coli.
The technical problem to be solved by the invention is to provide a method for producing L-phenylalanine by fermentation by applying the formula.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a formula suitable for producing L-phenylalanine by fermenting Escherichia coli comprises CaCl as effective component2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O, pyridoxal phosphate (PLP), vitamin B2 (V)B2) And biotin (V)H) Wherein, the CaCl2.2H2The dosage of O is 11.00-33.00mg/L, and the CuSO4.5H2The dosage of O is 0.39-1.17mg/L, and the CoCl2.6H2The dosage of O is 2.00-8.00mg/L, and the ZnSO4The dosage of the FeSO is 0.60-6.00mg/L4.7H2The dosage of O is 20.00-50.00mg/L, and the MnSO4.H2The dosage of O is 13.73-27.40mg/L, and the NiCl is used2.6H2The amount of O is 2.02-8.08mg/L, the amount of pyridoxal phosphate (PLP) is 3.00-20.00mg/L, and the vitamin B2 (V)B2) The dosage of the biotin (V) is 2.00-8.00mg/LH) The dosage of the composition is 0-2.00 mg/L.
Preferably, the above formula is suitable for producing L-phenylalanine by fermenting Escherichia coli, and the CaCl is2.2H2The dosage of O is 11 mg/L.
Preferably, the formula suitable for producing the L-phenylalanine by fermenting the escherichia coli is characterized in that the CaCl is used as each component2.2H2O 11mg/L，CuSO4.5H2O 0.47mg/L，CoCl2.6H2O 6mg/L，ZnSO4 0.6mg/L，FeSO4.7H2O 28mg/L，MnSO4.H2O 14mg/L，NiCl2.6H2O6.06 mg/L, pyridoxal phosphate 9.5mg/L, vitamin B2 6mg/L。
Preferably, the formulation for fermentation production of L-phenylalanine from Escherichia coli, which is described above, is biotin (V)H) The amount of (b) used was 0 (the conversion of sugar acid was higher without addition of biotin at any concentration).
Preferably, the method for producing L-phenylalanine by fermentation according to claim 1, wherein: the method adopts a strain of escherichia coli and comprises the following specific steps:
(1) the basic culture medium required by fermentation is as follows: 30g/L glucose, 5g/L yeast powder, 1g/L peptone, 1.2g/L citric acid, MgSO4.7H2O 2g/L，KH2PO4 4.0g/L；
(2) And (3) shake flask culture: the shake flask culture medium is shown in (1), and different kinds and amounts of the effective components in claim 1 are added according to the need to determine the optimal formula. The culture method comprises the following steps: inoculating 10% phenol red as acid-base indicator, adjusting pH (7.0) by injecting ammonia water with injector, and culturing for 36 h;
(3) seed tank culture: the seed culture medium is shown as (1), and no functional component is added. The culture method comprises the following steps: the fermentation temperature of the initial fermentation liquid is 37 ℃, the initial ventilation amount is 1.6L/min, the initial stirring rotating speed is 200r/min, the ventilation amount and the rotating speed are adjusted at the later stage to maintain DO at about 50%, the pH of the fermentation liquid is adjusted to 6.8-7.0 by feeding 25% ammonia water, and the fermentation is carried out when the OD600 nm is more than 25 nm;
(4) fed-batch fermentation culture: the fermentation medium is shown in (1), and the formula in the claims 1 and 2 is added according to the requirement, and is uniformly mixed for use. The culture method comprises the following steps: the inoculation amount is 20-30%, the fermentation temperature is 37 ℃, the initial ventilation amount is 0.65L/min, the initial stirring rotating speed is 200r/min, the ventilation amount and the rotating speed are adjusted in the later period to maintain the DO at 35-50%, the rotating speed and the air amount are alternately increased when the dissolved oxygen is lower than 35%, the pH is controlled to be 7.0 by feeding ammonia water, a glucose solution with the concentration of 80% is supplemented when the residual sugar in the fermentation tank is reduced to 0.5-1.0g/L, and the sub-proper sugar control in the subsequent fermentation stage is kept.
The preparation method of the formula suitable for producing the L-phenylalanine by fermenting the escherichia coli comprises the following specific steps:
(1)CaCl2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2accurately weighing the required medicine by using an analytical balance, dissolving the medicine by using deionized water (dripping a small amount of concentrated sulfuric acid if obvious precipitation exists, and fully stirring the medicine until the medicine is dissolved);
(2) pyridoxal phosphate (PLP), vitamin B2 (V)B2) Accurately weighing the required medicine by using an analytical balance, dissolving by using a dilute sodium hydroxide solution (pH 11), filtering by using a sterile filter membrane of 0.22 mu m, and keeping away from light;
(3) and (3) uniformly mixing the components in the steps (1) and (2).
Preferably, the preparation method of the formula suitable for producing the L-phenylalanine by fermenting the escherichia coli exists in biotin (V)H) When the biotin (V) is presentH) Accurately weighing the required medicine by using an analytical balance, dissolving the medicine by using deionized water, storing the medicine in a dark place, and uniformly mixing the medicine with the components in the steps (1) and (2).
Has the advantages that:
the formula for producing L-phenylalanine by fermenting escherichia coli provided by the invention is used for adjusting trace elements (Mg) used in L-Phe fermentation2+、Fe2+、Mn2+、Cu2+、Zn2+、Co2+、Ca2+、Ni2+、PLP、VB2、VH) The dosage is optimized in a specific proportion, the key enzyme activity, the L-Phe yield, the sugar-acid conversion rate and the like are taken as indexes, the superiority of the optimized trace element dosage is shown, the problem of wild trace element dosage is solved, and the method has guiding significance for producing L-Phe by industrialized microbial fermentation.
The formula uses VB2 in the fermentation of L-Phe for the first time, obtains beneficial effects, and optimizes CoCl for the first time2.6H2O, PLP, the amount of the first time determinesAdverse effects of biotin (VH), further optimizes the dosage of trace elements such as FeSO4.7H2O, MnSO4.H2O and the like, and finally improves the yield and the conversion rate of L-Phe, which has positive significance for the industrial production of L-Phe.
The fermentation method can achieve the purposes of accelerating the growth and the propagation of thalli, improving the biomass, prolonging the biomass stabilization period and the acid production peak period, improving the yield and the conversion rate of L-phenylalanine, saving the cost and the like.
Drawings
FIG. 1 shows the effect of a single trace element on the production of L-Phe.
FIG. 2 shows the effect of a single trace element on biomass.
FIG. 3 shows the effect of trace element deletion on fermentation.
FIG. 4 shows the effect of trace element deletion on enzyme activity.
FIG. 5 is a graph of sugar consumption rate versus biomass process.
FIG. 6 is a graph of real-time conversion versus production process variation.
FIG. 7 is a graph comparing the production of byproducts.
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art.
The Escherichia coli-producing bacteria used in the examples described below were purchased from the laboratory of Metabolic engineering of the university of Tianjin technology.
Example 1
This example uses shake flask culture to evaluate the effect on fermentation of a single trace element:
the method takes escherichia coli as a production strain, biomass, L-phenylalanine yield, saccharic acid conversion rate, byproduct yield and enzyme activity as evaluation indexes, and the biomass detection mode is as follows: sampling every 2h, properly diluting to a measurable concentration range (0.2-0.9) of a light absorption value, and measuring the light absorption value of the sample at a wavelength of 600nm by using an ultraviolet visible spectrophotometer; the yields of L-phenylalanine, alanine (L-Ala) and acetic acid are measured by an LC20AT high performance liquid chromatograph, and the glutamic acid is detected by an SBA biosensor analyzer; the enzyme activity is defined in a mode that 1 mu moL L product generated by each g fresh weight of thallus in each mL reaction system is defined as 1 enzyme activity unit (mu moL/(h.g fresh weight)); the conversion rate of the sugar acid is calculated by total acid production/total sugar consumption.
TABLE 1 trace elements and amounts thereof
10 groups of experiments are set according to the trace elements shown in the table 1, each group is added with a single trace element, 5 concentration gradients and three parallel experiments are set, the average value of the three groups of parallel experiments is taken as the experiment result, and the result is shown in the figures 1 and 2.
As shown in FIG. 1, the effects of the 10 trace elements involved in the experiment on L-Phe production are mainly in four forms, as CuSO4.5H2O、CoCl2.2H2O、ZnSO4、FeSO4.7H2O is as an example: 1. CuSO4.5H2When the addition amount of O is less than or equal to 0.47mg/L, the yield is improved to a certain extent compared with that of a control group (11.2g/L), and when the addition amount reaches 1.17mg/L, the yield is reduced by 7.1 percent compared with that of the control group; 2. CoCl2.2H2When the O concentration is less than or equal to 6mg/L, the yield is improved along with the increase of the addition amount, the yield is 14.9g/L at 6mg/L and is improved by 33.0 percent compared with a control group, when the addition amount is improved to 8mg/L, the yield is relatively stable, no obvious rising and falling trend appears, and the NiCl with similar effect is also used2.6H2O、PLP、VB2；3、ZnSO4、CaCl2.2H2The addition of O has no obvious influence on the yield of L-Phe and has no analytical significance; 4. FeSO4.7H2When the addition amount of O is less than or equal to 30mg/L, the yield is increased along with the increase of the addition amount, when 30mg/L is added, the yield is 18.2g/L, which is improved by 62.5 percent compared with a control group, and when the addition amount is more than 30mg/L, the yield slips to a certain degree but is always higher than that of the control groupAlso for the control group, MnSO had similar effects4.H2O、VH。
As shown in FIG. 2, FeSO4.7H2O、VHHas a large influence on biomass, Fe2+Has important effects in biological processes such as electron transfer, oxygen transfer, TCA cycle, gene regulation and DNA biosynthesis, and is not easy to release FeSO4.7H2The positive effect of O on biomass, when excessive addition, iron is not beneficial to the growth of microorganisms under aerobic conditions, oxygen and reduced oxygen can react with iron to have destructive influence on life, so that the biomass is 66.4 when the addition amount is 30mg/L, and the biomass is reduced when the addition is continued; vHCan be used as an enzymatic cofactor in the carboxylation of fatty acid biosynthesis, amino acid metabolism and gluconeogenesis, for example as a coenzyme for acetyl CoA carboxylase, involved in the synthesis of fatty acids and consequently affecting the synthesis of phospholipids which, as a major component of the cell membrane, are the major factors limiting cell growth, so that VHThe biomass was increased as the amount of the compound (1) was increased, but the fluctuation was gradually decreased, and the biomass was 62.0% in the experimental group containing 2mg/L, which was increased by 19% as compared with the control. MnSO4.H2The addition of O also promotes biomass, Mn2+Ions act as cofactors of a plurality of key enzymes in EMP, influence utilization of sugar, further influence energy metabolism and further influence growth metabolism of microorganisms, but Mg2+When present, this promoting effect is not significant. Relative of other trace elements to FeSO4.7H2O、VH、MnSO4.H2The influence of O on the biomass is not obvious, but when the O is added in a proper amount, the O still has a certain promoting effect.
Example 2
The part is cultured by using a shake flask, escherichia coli is taken as a production strain, and the influence on fermentation when single trace elements are deleted is researched:
in combination with the results of the experiment in example 1, CaCl is considered to be2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O、PLP、VB2、VHThe optimum addition amounts are respectively 11.0mg/L, 0.47mg/L, 6mg/L, 0.6mg/L, 30mg/L, 15mg/L, 6.06mg/L, 10mg/L, 6mg/L and 1mg/L, under the premise of adding optimum concentrations, all 10 trace elements involved in the experiment are taken as a control group, the influence of single deletion of the trace elements on the fermentation is researched, the result is taken as the average value of three groups of parallel experiments, and the experimental method is the same as that in example 1, as shown in FIG. 3:
as shown in FIG. 3, from the biomass difference, FeSO4.7H2O、VHThe influence of the deletion on the biomass is the largest, the deletion is respectively reduced by 10.0 percent and 10.6 percent compared with the control group, and the influence of the residual 8 trace elements on the biomass is not obvious, so that the FeSO can be seen4.7H2O、VHThe necessity in the growth process of the thallus can not be compensated by the addition of other trace elements4.7H2O、VH(ii) deletion of (a); from the viewpoint of L-Phe production, MnSO4.H2O、FeSO4.7H2The influence of O deletion on yield is the most remarkable, and is respectively reduced by 15.0 percent and 10.1 percent compared with a control group, and CoCl is used as a secondary factor2.6H2O, PLP, when the two are missing, the yield is reduced by 7.0%, 8.5%, and Mn is reduced compared with the control group2+、Fe2+And Co2+Ions as an enzymatic activator of NADH enzyme, can greatly increase the production of L-Phe, even though Mn is reported2+Capable of preferentially binding NADH enzyme, but this experiment demonstrates Mn2+、Fe2+、Co2+The individual deletion of (A) affects the production of L-Phe. The loss of PLP also affects the final yield, presumably by acting as a coenzyme for other key enzymes, and thus L-Phe. The absence of other trace elements did not cause a significant yield loss, with CuSO4.5H2The loss of O instead increases the yield by 2.1% compared with the control group; from the viewpoint of conversion rate of sugar and acid, CoCl2.6H2O、FeSO4.7H2O、MnSO4.H2O、PLP、VB2The deletion respectively reduces the conversion rate of the saccharic acid by 2.8 percent and 4.6 percent compared with the control group％、3.5％、6.3％、2.8％，CoCl2.6H2O、FeSO4.7H2O、MnSO4.H2O, PLP the effect on the conversion rate was the same as the tendency of the yield to change, and therefore it was considered that the reduction in the conversion rate was caused by the reduction in the yield, VB2Is a prosthetic group of some oxidoreductases in organisms, has catalytic action on reactions such as electron transfer, dehydrogenation and the like, and can be considered as VB2The energy metabolism of the thalli is promoted, and the saccharic acid conversion rate is further improved, but the specific action mechanism of the thalli is still to be researched. VHThe deletion of (A) improves the sugar-acid conversion rate to a great extent, improves the sugar-acid conversion rate by 8.5 percent compared with a control group, obviously reduces the corresponding bacterium amount, and has VHThe presence of (2) results in an increase in the amount of bacteria, a shift of the sugar excess to the growth and metabolism direction of the bacteria, and a decrease in the conversion rate of sugar to acid, even though V is demonstrated in the result of 2.1HThe single existence can improve the yield of the L-Phe to a certain degree, but the improvement can be completely replaced by other trace elements, so that the V is added from an external sourceHIs not suitable for producing L-Phe by fermentation of Escherichia coli.
Example 3
The part is cultured by using a shake flask, and escherichia coli is taken as a production strain, so that the influence of trace elements on the enzyme activity is researched.
According to the experimental results in example 2, CoCl can be obtained2.6H2O、FeSO4.7H2O、MnSO4.H2O、PLP、VB2The influence on the fermentation production of L-phe is large, and the 5 substances are guessed to be coenzymes or cofactors of key enzymes in the synthesis path of the L-phe, so that the experiment of the part takes the optimal addition amount of the 5 substances as a control group, and CoCl is singly deleted2.6H2O、FeSO4.7H2O、MnSO4.H2O、PLP、VB2For the experimental group, samples were taken to detect the enzymatic activities of DAHP enzyme, CM enzyme, PDT enzyme, and aromatic amino acid Transaminase (Transaminase), the experimental procedure was the same as in example 1, and the experimental results are shown in fig. 4:
control group DAHP enzyme activity was 4347.3. mu. moL. (h.g)-1，FeSO4.7H2The enzyme activity is reduced by 21.5% compared with the control group due to the deletion of O, and the MnSO4.H2The enzyme activity is reduced by 23.4 percent due to the deletion of O, which proves that FeSO4.7H2O、MnSO4.H2The indispensable performance of O, and the deficiency of other trace elements does not cause the obvious reduction of enzyme activity; CM enzyme activity of control group was 3292.5. mu. moL. (h.g)-1，FeSO4.7H2The enzyme activity is reduced by 35.2% compared with that of a control group due to the deletion of O, and the enzyme activity is not obviously reduced due to the remaining trace elements, so that the CM enzyme is considered to be Fe2+(ii) dependent; PDT enzyme activity of control group was 3321.4 μmoL (h.g)-1，FeSO4.7H2Deletion of O decreased the enzyme activity by 33.6%, similar to the case of CM enzyme, and therefore Fe was considered2+Cofactors critical for CM-PDT enzymes; control group aromatic amino acid transaminase activity was 3813.8 μmoL (h.g)-1Deletion of PLP reduced the enzyme activity by 39.3%, thus it is believed that PLP is a critical cofactor for aromatic amino acid transaminases. VB2The deletion of (A) causes each enzyme to be reduced to a certain degree relative to a control group, but does not cause the enzyme activity of a specific key enzyme to be obviously reduced, so that VB2Coenzyme as oxidative dehydrogenation enzyme does not directly affect the production of aromatic amino acid, but indirectly improves the yield and conversion rate of L-Phe by accelerating energy metabolism in pathways such as EMP and HMP. CoCl2.6H2The deletion of O slightly reduces the enzyme activities of DAHP and CM, does not affect the activities of other three enzymes, but combines the conclusion in 2.2.1, CoCl2.6H2The loss of O obviously reduces the yield and the conversion rate, and the substance is supposed to be used as a cofactor of shikimic acid pathway related enzymes, thereby influencing the generation of L-Phe.
Example 4
In this example, shake flask culture was used, escherichia coli was used as a production strain, and orthogonal experiments were used to further optimize the trace element usage.
According to the results of examples 1, 2 and 3, the influence sequence of 10 trace elements involved in the experiment on the improvement of the L-Phe yield is as follows: MnSO4.H2O＞FeSO4.7H2O＞CoCl2.6H2O＞PLP＞VB2＞VH＞NiCl2.6H2O＞＞CaCl2.2H2O、CuSO4.5H2O、ZnSO4(the last 3 positions are not consecutive and are much smaller than the first 7 positions), wherein VB2、VHCan be replaced by other trace elements; the influence sequence on the improvement of the conversion rate of the L-Phe saccharic acid is as follows: PLP > FeSO4.7H2O＞MnSO4.H2O＞VB2、CoCl2.6H2O＞＞VH、NiCl2.6H2O、CaCl2.2H2O、CuSO4.5H2O、ZnSO4(the last 5 positions are not in sequence and are far smaller than the first 5 positions), so the experiment of the part is carried out on PLP and FeSO through orthogonal experiments as shown in Table 2 on the basis of the results of single-factor experiments4.7H2O、MnSO4.H2O、VB2、CoCl2.6H2The amounts of O5 trace elements were further investigated.
TABLE 2 orthogonal experiment factor horizon
TABLE 3L-Phe production orthogonal experimental results
According to the table 3, the factor FeSO4.7H2Maximum O mean 1, i.e. optimum FeSO4.7H2The O concentration is 28 mg/L; factor MnSO4.H2The O mean value 1 is maximum, namely the optimal concentration is 14.0 mg/L; factor CoCl2.6H2The O mean value 2 is maximum, namely the optimal concentration is 5.5 mg/L; the factor PLP average value 3 is maximum, namely the optimal concentration is 10 mg/L; factor VB2Mean 4 max, i.e. optimum concentration of 65 mg/L. Factor FeSO4.7H2O、MnSO4.H2The extreme difference of O reaches 1.850 and 2.857 respectively, which are main factors influencing the yield of L-Phe, wherein MnSO4.H2O is a key factor, so the dosage of the two trace elements should be strictly controlled. Factor CoCl2.6H2O、PLP、VB2The range is small, the influence on the yield is not obvious, and the range is a secondary factor, so that the use amount optimization significance is not large.
TABLE 4L-Phe sugar acid conversion orthogonal experimental results
As can be seen from Table 4, the factor FeSO4.7H2Maximum O mean 1, i.e. optimum FeSO4.7H2The O concentration is 28 mg/L; factor MnSO4.H2The O mean value 2 is maximum, namely the optimal concentration is 14.5 mg/L; the factor CoCl2.6H2O has the maximum mean value 2, namely the optimal concentration is 5.5 mg/L; the factor PLP average value 2 is maximum, namely the optimal concentration is 10 mg/L; factor VB2The mean value 2 is maximum, namely the optimal concentration is 5.5 mg/L. Factor FeSO4.7H2O、MnSO4.H2O, PLP, the range of the standard deviation reaches 1.500, 0.825 and 0.650 respectively, and the standard deviation is a main factor influencing the conversion rate of saccharic acid, wherein FeSO4.7H2O is a key factor, so the dosage of the 3 trace elements should be strictly controlled. Factor CoCl2.6H2O、VB2The range is small, the influence on the conversion rate is not obvious, and the range is a secondary factor, so the use amount optimization significance is not large.
From the viewpoint of the comprehensive yield of L-Phe and the conversion rate of saccharic acid, FeSO4.7H2O、MnSO4.H2O, PLP, the yield and the conversion rate are greatly influenced by further optimizing the dosage, and the factor FeSO4.7H2The two mean values are maximum when O is 28mg/L, becauseElemental MnSO4.H2The average of the yield is the maximum when O is 14mg/L, and the average of the conversion rate is the maximum when O is 14.5mg/L, so that MnSO can be selectively selected4.H2The average value of the yield is the largest when the PLP is 10mg/L, and the average value of the conversion rate is the largest when the PLP is 9.5mg/L, but the influence of the PLP on the conversion rate is far greater than the influence on the yield, so that 9.5mg/L is selected as the optimal addition amount.
CoCl2.6H2O、VB2The concentration change of (A) does not significantly affect the conversion rate and yield in this part of the experiment, so it can be considered that the optimum amount of both has been reached, and it is meaningless to optimize again.
Example 5
This example uses a 5L fermentor culture with E.coli as the production strain to perform fermentor validation experiments including a seed tank culture and a fed-batch fermentation culture.
Examples 1, 2, 3 and 4 determine the adverse effect of biotin on L-phe fermentation, determine the addition of the other 9 trace elements through a single-factor experiment, analyze the key trace elements affecting the key enzyme activity, and further optimize the key 5 trace elements through an orthogonal experiment, but on the basis of the previous experiment, escherichia coli is taken as a production strain, CaCl is taken as a CaCl production strain2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O、PLP、VB211.0mg/L, 0.47mg/L, 6mg/L, 0.6mg/L, 28mg/L, 14mg/L, 6.06mg/L, 10mg/L and 6mg/L are respectively added to serve as experimental groups, no trace element is added to serve as a control experiment, a fermentation experiment is carried out by using a 5L fermentation tank, and the optimization result of the trace element dosage at the early stage is checked.
As can be seen from FIG. 5, the biomass and sugar consumption rate of the experimental group are always higher than those of the control group in the fermentation process, the maximum biomass 122.8 is far greater than that of the control group by 101.3, and the time for reaching the maximum sugar consumption rate is 4 hours earlier than that of the control group, but is similar in value; when the fermentation is carried out for 34h, the biomass of the control group begins to show a descending trend, the sugar consumption rate also shows a large downward slide, and the biomass and the sugar consumption rate of the experimental group are both in a stable period; when the fermentation is carried out for 40h, sugar uptake of the control group can hardly continue, the biomass continuously decreases, and the fermentation can not continue, at the moment, the biomass of the experimental group is in a stable period, but the sugar consumption rate is reduced; after fermentation for 50h, the biomass of the experimental group still has no obvious reduction trend, but the sugar consumption rate is reduced seriously at the moment, so the fermentation period is prolonged by 10h compared with the control group after the fermentation is finished.
As can be seen from FIG. 6, when the trace element is deficient, the yield of L-Phe is generally low and steadily increased, and the final yield is 56.4g/L, which is about the same as the conversion curve trend, and the overall trend is upward, and the final yield is decreased in 22-26h, because the L-Phe forms soft pseudo-crystal state near the crystallization concentration, and along with a large amount of fermentation foam, the L-Phe is difficult to continue to be generated, but the thallus continuously consumes sugar, which results in the decrease of the conversion rate; after the yield of the experimental group is slowly accumulated for 0-20h, the peak period of acid production is reached within 20-35h, the increase rate is reduced, but the total amount still increases, the final yield is 85.4g/L, and is increased by 51.4% compared with the control group. The real-time conversion rate curve fluctuation degree of the experimental group is larger than that of the control group, the curve fluctuation degree is increased rapidly in 0-6h, the culture time of seed liquid is longer, so that the thallus starts to produce acid immediately after fermentation, the thallus amplification speed is slower, the conversion rate is increased rapidly, the biomass starts to increase rapidly after 6h, although the acid production is increased, most of glucose is used for thallus growth, the sugar acid conversion rate is in a descending trend, the yield is close to the crystallization concentration around 24h and is the same as that of the control group, the conversion rate is further reduced, after L-Phe is crystallized, the thallus enters an acid production peak period, the conversion rate and the yield are both increased rapidly until the acid production rate is slowed down again in the later fermentation period (after 40 h), but the sugar consumption rate is not obviously reduced, the conversion rate is reduced again, the final conversion rate is 26.3%, and is far larger than 21.4% of the control group, even if the curve fluctuation of the conversion rate of the experimental group is larger, the real-time conversion rate is higher than that of the control group in the whole fermentation process, so that the experimental group has more advantages.
As shown in FIG. 7, the experimental group produced 0.8g/L acetic acid, 1.2g/L glutamic acid (L-Glu) and 0.2g/L tryptophan (L-Trp), which did not affect the growth metabolism of the cells and the subsequent extraction. The acetic acid content of the control group is 6.7g/L, and the acetic acid content can seriously influence the growth and metabolism of thalli at the moment and is a main reason for the rapid reduction of biomass at the later fermentation stage of the control group; the content of glutamic acid and tryptophan reaches 3.3g/L and 1.4g/L, which are respectively increased by 175 percent and 600 percent compared with a control group, thereby not only influencing the conversion rate, but also improving the difficulty of subsequent separation and extraction; alanine (1.8g/L) is detected in the fermentation liquor of the control group, which is not detected in the experimental group, and the reason is presumed that a large amount of pyruvic acid is accumulated in the glycolysis pathway due to insufficient enzyme activity of key enzyme of the shikimic acid pathway, and the pyruvic acid is used as a direct precursor of the alanine and can generate the alanine through one-step reaction.
The results show that the optimized types and the optimized dosage of the trace elements improve the sugar-acid conversion rate of the yield of the L-phenylalanine and improve the product competitiveness.
Example 6
This example uses a 5L fermentor culture with E.coli as the production strain to perform fermentor validation experiments including a seed tank culture and a fed-batch fermentation culture.
A formula suitable for producing L-phenylalanine by fermenting Escherichia coli comprises CaCl as effective component2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O, pyridoxal phosphate (PLP), vitamin B2 (V)B2) And biotin (V)H) Wherein, the CaCl2.2H2The dosage of O is 11.00mg/L, and the CuSO4.5H2The dosage of O is 0.47mg/L, and the CoCl2.6H2The dosage of O is 6.00mg/L, and the ZnSO4The dosage of the FeSO is 0.6mg/L4.7H2The dosage of O is 28mg/L, and the MnSO4.H2The dosage of O is 14mg/L, and the NiCl2.6H2The dosage of O is 6.06mg/L, the dosage of pyridoxal phosphate (PLP) is 9.5mg/L, and the vitaminB2(VB2) The dosage of the biotin (V) is 6.00mg/LH) The amount of (B) was 0mg/L (no biotin was added).
The preparation method of the components comprises the following steps:
accurately weighing CaCl by using analytical balances respectively2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2Dissolving O33 mg, 1.41mg, 18mg, 1.8mg, 84mg, 42mg and 18.18mg in deionized water, and sterilizing in a jar; accurate weighing of pyridoxal phosphate (PLP), vitamin B2 (V) using an analytical balanceB2)28.5mg and 18mg, dissolving with dilute sodium hydroxide solution (pH 11), filtering with 0.22 μm sterile filter membrane, sterilizing, and adding into the tank; mixing the above components uniformly.
The method for producing the L-phenylalanine by using the formula comprises the following specific steps:
(1) taking out the strain from a refrigerator at minus 80 ℃, evenly scratching the liquid strain on an LB solid culture medium by using an inoculating loop in a super clean bench, carrying out inverted culture at 37 ℃ for 12h, and carrying out passage twice.
(2)5L of seed tank culture: washing off bacteria from a solid inclined plane by using 0.9% sterile normal saline, inoculating the bacteria into a seeding tank for culture, wherein the volume of initial fermentation liquor is 3L, the fermentation temperature is 37 ℃, the initial ventilation volume is 1.6L/min, the initial stirring rotating speed is 200r/min, the ventilation volume and the rotating speed are adjusted at the later stage to keep DO at about 50%, the pH of the fermentation liquor is adjusted to 6.8-7.0 by adding 25% ammonia water, and the fermentation is carried out when the OD600 nm is more than 25; the seed culture medium is as follows: 30g/L glucose, 5g/L yeast powder, 1g/L peptone, 1.2g/L citric acid, MgSO4.7H2O 2g/L，KH2PO4 4.0g/L；
(3)5L tank fed-batch fermentation culture: the inoculation amount is 25%, the fermentation temperature is 37 ℃, the initial aeration amount is 0.65L/min, the initial stirring rotation speed is 200r/min, the aeration amount and the rotation speed are adjusted at the later stage to maintain DO at 35-50%, the rotation speed and the air volume are alternately increased when the dissolved oxygen is lower than 35%, the pH is controlled to be 7.0 by feeding ammonia water, and when the residual sugar in the fermentation tank is reduced to 0.5-1.0g/LAnd (4) beginning to supplement the 80% glucose solution, and keeping a sub-proper amount of sugar control in the subsequent fermentation stage until the fermentation is finished. The fermentation medium is as follows: 30g/L glucose, 5g/L yeast powder, 1g/L peptone, 1.2g/L citric acid, MgSO4.7H2O 2g/L，KH2PO44.0g/L, and adding the components with the above dosage, and mixing uniformly for use.
The final experimental results are: the yield of the L-phenylalanine is 85.4g/L, the saccharic acid conversion rate is 26.3 percent, the biomass is 122.8, and the byproducts of the alanine, the glutamic acid, the tryptophan and the acetic acid are respectively 0, 1.2, 0.2 and 0.8 g/L.
Example 7
This example uses a 5L fermentor culture with E.coli as the production strain to perform comparative fermentor experiments including a seed tank culture and a fed-batch fermentation culture.
A formula suitable for producing L-phenylalanine by fermenting Escherichia coli comprises CaCl as effective component2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O, pyridoxal phosphate (PLP), vitamin B2 (V)B2) And biotin (V)H) Wherein, the CaCl2.2H2The dosage of O is 33.00mg/L, and the CuSO4.5H2The dosage of O is 0.39mg/L, and the CoCl2.6H2The dosage of O is 2.00mg/L, and the ZnSO4The dosage of the FeSO is 0.60mg/L4.7H2The dosage of O is 50.00mg/L, and the MnSO4.H2The dosage of O is 22mg/L of NiCl2.6H2The dosage of O is 8.08mg/L, the dosage of pyridoxal phosphate (PLP) is 3.00mg/L, and the vitamin B2 (V)B2) The dosage of the biotin (V) is 2.00mg/LH) The dosage of the compound is 2.00 mg/L.
Coli as a production bacterium, the fermentation step was the same as in example 6, the detection and analysis method was the same as in example 1, the preparation method of the above-mentioned components was the same as in example 6, and biotin (V) was precisely weighed using an analytical balanceH) Sterilizing in a jar, mixing the componentsMixing uniformly to obtain the final product.
The final experimental results are: the yield of the L-phenylalanine is 78.6g/L, the saccharic acid conversion rate is 24.8 percent, the biomass is 117.6, and the byproducts of the alanine, the glutamic acid, the tryptophan and the acetic acid are respectively 0, 2.4, 2.1 and 1.3 g/L.
Example 8
This example uses a 5L fermentor culture with E.coli as the production strain to perform comparative fermentor experiments including a seed tank culture and a fed-batch fermentation culture.
A formula suitable for producing L-phenylalanine by fermenting Escherichia coli comprises CaCl as effective component2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O, pyridoxal phosphate (PLP), vitamin B2 (V)B2) And biotin (V)H) Wherein, the CaCl2.2H2The dosage of O is 11.00mg/L, and the CuSO4.5H2The dosage of O is 1.17mg/L, and the CoCl2.6H2The dosage of O is 8.00mg/L, and the ZnSO4The dosage of the FeSO is 6.00mg/L4.7H2The dosage of O is 20.00mg/L, and the MnSO4.H2The dosage of O is 18mg/L of NiCl2.6H2The dosage of O is 2.02mg/L, the dosage of pyridoxal phosphate (PLP) is 20.00mg/L, and the dosage of vitamin B2 (V)B2) The dosage of the biotin (V) is 8.00mg/LH) The dosage of the compound is 0.50 mg/L.
Coli as a production bacterium, the fermentation step was the same as in example 6, the detection and analysis method was the same as in example 1, the preparation method of the above-mentioned components was the same as in example 6, and biotin (V) was precisely weighed using an analytical balanceH) Sterilizing in a jar, and mixing the above components.
The final experimental results are: the yield of the L-phenylalanine is 81.4g/L, the saccharic acid conversion rate is 25.4%, the biomass is 103.2, and the byproducts of the alanine, the glutamic acid, the tryptophan and the acetic acid are respectively 0.6 g/L, 1.3g/L, 0.8g/L and 1.5 g/L.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several modifications and amendments without departing from the principle of the present invention, the construction steps of the strain of the present invention are not sequential, and those skilled in the art should consider the scope of the present invention as the modifications and amendments of the strain modification based on the method of the present invention or the method of the present invention.
Claims (4)
1. A formula suitable for producing L-phenylalanine by fermenting escherichia coli is characterized in that: the functional components are CaCl2.2H2O、CuSO4.5H2O、CoCl2.6H2O、ZnSO4、FeSO4.7H2O、MnSO4.H2O、NiCl2.6H2O, pyridoxal phosphate, vitamin B2And biotin, wherein the dosage of each component is CaCl2.2H2O 11.00-33.00mg/L，CuSO4.5H2O 0.39-1.17mg/L，CoCl2.6H2O 2.00-8.00mg/L，ZnSO4 0.60-6.00mg/L，FeSO4.7H2O 20.00-50.00mg/L，MnSO4.H2O 13.73-27.40mg/L，NiCl2.6H2O2.02-8.08 mg/L, pyridoxal phosphate 3.00-20.00mg/L, vitamin B22.00-8.00mg/L and biotin 0-2.00 mg/L.
2. The formulation of claim 1 suitable for use in the fermentative production of L-phenylalanine from escherichia coli, wherein: the dosage of each component is CaCl2.2H2O 11mg/L，CuSO4.5H2O 0.47mg/L，CoCl2.6H2O 6mg/L，ZnSO4 0.6mg/L，FeSO4.7H2O 28mg/L，MnSO4.H2O 14mg/L，NiCl2.6H2O6.06 mg/L, pyridoxal phosphate 9.5mg/L, vitamin B2 6mg/L。
3. The formulation of claim 1 suitable for use in the fermentative production of L-phenylalanine with escherichia coli, wherein: the dosage of the biotin is 0.
4. The method for producing L-phenylalanine by fermentation using the formulation of claim 1, which is characterized in that: the method adopts a strain of escherichia coli and comprises the following specific steps:
(1) the basic culture medium required by fermentation is as follows: 30g/L glucose, 5g/L yeast powder, 1g/L peptone, 1.2g/L citric acid, MgSO4.7H2O 2g/L，KH2PO4 4.0g/L；
(2) And (3) shake flask culture: the shake flask culture medium is shown as (1), and different kinds and dosage of the effective components in the claim 1 are added according to the requirement to determine the optimal formula; the culture method comprises the following steps: inoculating 10% phenol red as acid-base indicator, adjusting pH (7.0) by injecting ammonia water with injector, and culturing for 36 h;
(3) seed tank culture: the seed culture medium is shown as (1), and no functional component is added; the culture method comprises the following steps: the fermentation temperature of the initial fermentation liquid is 37 ℃, the initial ventilation amount is 1.6L/min, the initial stirring rotating speed is 200r/min, the ventilation amount and the rotating speed are adjusted at the later stage to maintain DO at about 50%, the pH of the fermentation liquid is adjusted to 6.8-7.0 by feeding 25% ammonia water, and the fermentation is carried out when the OD600 nm is more than 25 nm;
(4) fed-batch fermentation culture: the fermentation medium is shown as (1), and the formula in the claims 1 and 2 is added according to the requirement and is used after being uniformly mixed; the culture method comprises the following steps: the inoculation amount is 20-30%, the fermentation temperature is 37 ℃, the initial ventilation amount is 0.65L/min, the initial stirring rotating speed is 200r/min, the ventilation amount and the rotating speed are adjusted in the later period to maintain the DO at 35-50%, the rotating speed and the air amount are alternately increased when the dissolved oxygen is lower than 35%, the pH is controlled to be 7.0 by feeding ammonia water, a glucose solution with the concentration of 80% is supplemented when the residual sugar in the fermentation tank is reduced to 0.5-1.0g/L, and the sub-proper sugar control in the subsequent fermentation stage is kept.
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| CN102181503A (en) * | 2011-04-15 | 2011-09-14 | 江苏汉光生物工程有限公司 | Method for producing L-phenylalanine (L-Phe) through fermentation |
| CN111187794A (en) * | 2020-04-03 | 2020-05-22 | 杭州巴洛特生物科技有限公司 | Method for preparing L-phenylalanine by using escherichia coli fermentation |
| CN112251476A (en) * | 2020-09-25 | 2021-01-22 | 天津科技大学 | Production method of L-phenylalanine |
| CN112251477A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving fermentation yield and sugar-acid conversion rate of L-phenylalanine |
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| CN102181503A (en) * | 2011-04-15 | 2011-09-14 | 江苏汉光生物工程有限公司 | Method for producing L-phenylalanine (L-Phe) through fermentation |
| CN111187794A (en) * | 2020-04-03 | 2020-05-22 | 杭州巴洛特生物科技有限公司 | Method for preparing L-phenylalanine by using escherichia coli fermentation |
| CN112251476A (en) * | 2020-09-25 | 2021-01-22 | 天津科技大学 | Production method of L-phenylalanine |
| CN112251477A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving fermentation yield and sugar-acid conversion rate of L-phenylalanine |
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