CN117305144A - Recombinant pichia pastoris, construction method thereof and application thereof in production of L-lactic acid - Google Patents

Recombinant pichia pastoris, construction method thereof and application thereof in production of L-lactic acid Download PDF

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CN117305144A
CN117305144A CN202311049949.6A CN202311049949A CN117305144A CN 117305144 A CN117305144 A CN 117305144A CN 202311049949 A CN202311049949 A CN 202311049949A CN 117305144 A CN117305144 A CN 117305144A
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pichia pastoris
lactic acid
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李�灿
王旺银
张亚静
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Dalian Institute of Chemical Physics of CAS
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Abstract

The application discloses recombinant pichia pastoris, a construction method thereof and application thereof in producing L-lactic acid, and belongs to the technical field of microbial fermentation. The recombinant pichia pastoris provided by the invention contains an L-lactate dehydrogenase gene; the nucleotide sequence of the L-lactate dehydrogenase gene is shown as SEQ ID No. 1. The recombinant pichia pastoris provided by the invention can utilize small molecules such as 'liquid sunlight' methanol, dihydroxyacetone or glycerol as substrates to produce L-lactic acid. The yield of the obtained L-lactic acid can reach 30g/L, the conversion rate is close to 100%, and the optical purity is more than 99.5%, which indicates that the recombinant pichia pastoris constructed and obtained by the invention can be applied to the field of biological fermentation to efficiently produce high-quality L-lactic acid.

Description

Recombinant pichia pastoris, construction method thereof and application thereof in production of L-lactic acid
Technical Field
The application belongs to the technical field of microbial fermentation, and particularly relates to recombinant pichia pastoris, a construction method thereof and application thereof in L-lactic acid production.
Background
With the increasing problem of global plastic pollution, biodegradable materials such as polylactic acid are widely focused and studied. L-lactic acid is an important industrial chemical for synthesizing polylactic acid, and the demand thereof is also increasing. The current industrial lactic acid production methods are mainly divided into chemical synthesis methods and microbial fermentation methods. Among these, the chemical synthesis methods are mainly the glyoxylic acid method and the acrylonitrile method, and racemic lactic acid, namely DL-lactic acid, is usually produced, and the optical purity is too low to meet the application requirements. The microbial fermentation method generally adopts starch such as corn, wheat and the like as raw materials, saccharifies the starch by amylase and saccharifying enzyme, and converts sugar into L-lactic acid with high optical purity by microbial fermentation, so as to be applied to industrial synthesis of poly-L-lactic acid. However, as the demand of L-lactic acid increases, microbial fermentation using a glycosyl carbon source as a substrate may form a situation of 'competing with people for food' in the future, thereby threatening food safety. Therefore, it is important to find a new green sustainable L-lactic acid production mode.
The development of green low-carbon artificial photosynthesis technology is becoming an important research direction for domestic scholars. The artificial photosynthesis is a process of converting water and carbon dioxide into liquid sunlight hydrocarbon by utilizing renewable energy sources such as solar energy and the like through chemical catalysis and biological catalysis, and can effectively solve the problems of energy sources, environment and sustainable development.
Therefore, by referring to the artificial light synthesis concept, a sustainable production mode for efficiently producing L-lactic acid by using solar energy and taking carbon dioxide and water as substrates is developed, and has important significance. According to the production mode, the traditional glycosyl raw materials are replaced by carbon dioxide, so that the grain safety problem can be relieved, and the upgrading conversion from solar-driven carbon dioxide to high-added-value bio-based chemicals can be realized, so that the problems of energy and environment and sustainable development can be effectively solved. However, studies on how to produce L-lactic acid using "liquid sunlight" such as small molecules of methanol, dihydroxyacetone, and glycerol as substrates have been recently reported.
Disclosure of Invention
In order to solve the technical problems, the invention provides recombinant pichia pastoris, a construction method thereof and application thereof in producing L-lactic acid, wherein the recombinant pichia pastoris can utilize liquid sunlight small organic molecules such as methanol, dihydroxyacetone, glycerol and the like as substrates for fermentation, so that the L-lactic acid with high optical purity is obtained.
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the present invention provides a recombinant pichia pastoris strain, the recombinant pichia pastoris containing an L-lactate dehydrogenase gene;
the nucleotide sequence of the L-lactate dehydrogenase gene is shown as SEQ ID No. 1.
In a second aspect, the invention provides a construction method of the recombinant pichia pastoris, which comprises the following steps:
step 1), connecting the L-lactate dehydrogenase gene with an initial expression vector, a promoter and a terminator to obtain a recombinant expression vector;
and 2) transferring the recombinant expression vector into pichia pastoris to obtain the recombinant pichia pastoris.
Optionally, the initial expression vector in step 1) comprises pPICZA;
the promoter in the step 1) comprises any one of GAPp, PGI1p and TEF1 p;
the terminator in step 1) includes FBP1tt.
Optionally, the pPICZA contains a bleomycin resistance gene.
Optionally, the nucleotide sequence of the GAPp is shown as SEQ ID No. 2;
the nucleotide sequence of the PGI1p is shown as SEQ ID No. 3;
the nucleotide sequence of the TEF1p is shown as SEQ ID No. 4.
Alternatively, the nucleotide sequence of FBP1tt is shown as SEQ ID No. 5.
Optionally, step 1) after the ligation of the L-lactate dehydrogenase gene with the initial expression vector, the promoter and the terminator is completed, a step of performing cleavage with restriction enzyme BlpI is further included.
Optionally, the temperature of the enzyme digestion is 35-40 ℃;
the enzyme digestion time is 25-35 min.
In a third aspect, the invention provides an application of the recombinant pichia pastoris in L-lactic acid production by organic small molecule fermentation.
In a fourth aspect, the present invention provides a method for producing L-lactic acid by fermentation using small organic molecules, comprising the steps of:
inoculating the recombinant pichia pastoris into a culture medium for fermentation culture, and collecting fermentation products to obtain the L-lactic acid.
Optionally, the initial inoculum size of the recombinant pichia pastoris is 1-20 OD 600 。
Alternatively, the medium has small organic molecules as fermentation substrates.
Alternatively, the small organic molecule includes any one of methanol, dihydroxyacetone, and glycerin.
Optionally, the initial addition amount of the small organic molecules is 10-30 g/L.
Alternatively, the initial amount of organic small molecule added is independently selected from any of 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, or a range of values between any two.
Optionally, the pH of the culture medium is 5-6.
Optionally, the temperature of the fermentation culture is 28-30 ℃.
Alternatively, the fermentation culture mode comprises shake flask fermentation culture or semi-continuous fermentation culture.
Optionally, the time of shake flask fermentation culture is 36-72 h;
the time of the semi-continuous fermentation culture is 48-96 hours.
Optionally, the aeration amount in the semi-continuous fermentation culture is 36-48 sL/h.
Alternatively, the aeration amount in the semi-continuous fermentation culture is independently selected from any value or range of values between any two of 36sL/h, 38sL/h, 40sL/h, 42sL/h, 44sL/h, 46sL/h, 48sL/h.
Optionally, the initial dissolved oxygen content in the semi-continuous fermentation culture is not less than 30%.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention introduces the expression vector with the L-lactate dehydrogenase gene into pichia pastoris, and inserts the target gene into the genome of the pichia pastoris in a homologous recombination mode to obtain the recombinant pichia pastoris.
(2) The recombinant pichia pastoris strain provided by the invention can utilize small molecules such as methanol, dihydroxyacetone, glycerol and the like as substrates to produce L-lactic acid. The yield of the obtained L-lactic acid can reach 30g/L, the conversion rate is close to 100%, and the optical purity can reach more than 99.5%. In the production mode, the traditional glycosyl raw materials are replaced by carbon dioxide, so that the grain safety problem can be relieved, and the upgrading conversion from solar-driven carbon dioxide to high-added-value bio-based chemicals can be realized, so that the problems of energy and environment and sustainable development can be effectively solved.
Drawings
FIG. 1 shows the growth of Pichia pastoris GS115 of the present invention in methanol, dihydroxyacetone and glycerol medium;
FIG. 2 shows the shake flask fermentation of recombinant Pichia pastoris GS115-G, GS-P, GS115-T in Delft minimum medium using dihydroxyacetone as a substrate to produce L-lactic acid;
FIG. 3 shows the production of L-lactic acid by semi-continuous fermentation of recombinant Pichia pastoris GS115-G in a Delft minimum medium using dihydroxyacetone as a substrate in a fermenter;
FIG. 4 shows the production of L-lactic acid by semi-continuous fermentation of recombinant Pichia pastoris GS115-G of the present invention in YPHASA medium using dihydroxyacetone as a substrate in a fermenter.
Detailed Description
The present application is further illustrated below in conjunction with specific examples. The following description is given of several embodiments of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art may make various changes or modifications using the technical contents disclosed above without departing from the scope of the technical solutions of the present application, which are equivalent to the equivalent embodiments.
Unless otherwise indicated, all starting materials in the examples of the present application were purchased commercially and used without any particular treatment.
Unless otherwise indicated, the analytical methods in the examples all employed conventional arrangements of instruments or equipment and conventional analytical methods.
Pichia used in the following examples was Pichia pastoris Pichia pastoris GS glycerol, purchased from Biyun Biotechnology Co., ltd, product number D0412.
Example 1
Construction of recombinant Pichia pastoris for L-lactic acid production:
1. constructing an expression vector containing an L-lactate dehydrogenase gene:
(1) The pPICZA expression vector skeleton gene containing bleomycin resistance gene is obtained by amplification using pPICZA plasmid DNA as a template and using a primer pPICZA-S/pPICZA-A, and is called fragment pPICZA.
The amplification system is as follows: high-fidelity PCR enzymeMax DNA Polymerase (2X) 25. Mu.l, 1. Mu.l of DNA template (50 ng/. Mu.l), 1. Mu.l of each primer (10. Mu.M), 22. Mu.l of distilled water, and a total volume of 50. Mu.l.
The amplification conditions were: pre-denaturation at 94 ℃ for 5min (1 cycle); denaturation at 94℃for 15 seconds, annealing at 55℃for 15 seconds, extension at 72℃for 35 seconds (30 cycles); extension at 72℃for 7 min (1 cycle).
(2) The GAPp, PGI1p and TEF1p promoter genes, designated as fragments GAPp, PGI1p and TEF1p, were PCR amplified using genomic DNA of Pichia pastoris Pichia pastoris GS strain as a template and primers GAPp-S/GAPp-A, PGI p-S/PGI1p-A and TEF1p-S/TEF1p-A, respectively. The amplification system and conditions are the same as in step (1).
(3) The PCR amplification of the FBP1tt terminator gene, called fragment FBP1tt, was performed using the genomic DNA of Pichia pastoris Pichia pastoris GS strain as template and the primer FBP1tt-S/FBP 1tt-A. The amplification system and the amplification conditions are the same as in the step (1).
(4) The L-lactate dehydrogenase gene (LDH), designated as fragments LDH-G, LDH-P and LDH-T, was PCR amplified using genomic DNA of the lactococcus lactis Lactococcus lactis strain (lactococcus lactis NZ9000, purchased from Barceichhorns) as a template, and the primers LDH-GAP-S, LDH-PGI1-S and LDH-TEF1-S, respectively, with LDH-A. The amplification system and the amplification conditions are the same as in the step (1).
(5) And (3) taking the fragments GAPp, PGI1p and TEF1p obtained in the step (2), the fragment FBP1tt obtained in the step (3) and the fragment LDH-G, LDH-P, LDH-T obtained in the step (4) as templates, respectively carrying out overlapping PCR amplification by using primers GAPp-S, PGI1p-S, TEF1p-S and FBP1tt-A, and respectively connecting the three fragments into a complete gene fragment, thereby obtaining three gene fragments GAPp-LDH-FBP1tt, PGI1p-LDH-FBP1tt and TEF1p-LDH-FBP1tt.
(6) The plasmid gene fragment pPICZA obtained in the step (1) and the three target gene (LDH) fragments GAPp-LDH-FBP1tt, PGI1p-LDH-FBP1tt and TEF1p-LDH-FBP1tt obtained in the step (5) were taken at 110ng and 55ng, respectively, and 10. Mu.l were addedHiFi DNA Assembly Master Mix buffer, adding distilled water to 20 μl, reacting for 20min at 50deg.C to obtain three complete expression vectors containing target genes, namely pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh.
(7) Calcium chloride conversion process: 10 μl of the reaction solution containing the expression vectors pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh obtained in step (6) was added to 100 μl of TOP10 competent cells (available from Tiangen Biochemical Co., ltd.) respectively, and the mixture was subjected to ice bath for 30 minutes, and then heat shock was performed in a water bath at 42℃for 90 seconds, and immediately placed on ice for 2 minutes. 1ml of low-salt LB (5 g/l yeast extract, 10g/l tryptone, 5g/l sodium chloride) medium was added and incubated at 200rpm for 1 hour at 37 ℃. 1ml of the supernatant was centrifuged and the remaining 100. Mu.l of the bacterial liquid was spread on a low-salt LB plate containing bleomycin (30. Mu.g/ml final concentration), and after overnight culture, 5 positive single colonies were selected and colony PCR was verified, with primers GAPp-S/FBP1tt-A, PGI1p-S/FBP1tt-A, TEF p-S/FBP1tt-A and pPICZA-S/pPICZA-A, respectively. And (3) carrying out sample feeding and sequencing analysis on the PCR product, wherein the result is positive cloning, and plasmids pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh are obtained.
2. Introducing the plasmids pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh obtained in the step (7) into the genome of Pichia pastoris by a homologous recombination method to obtain recombinant Pichia pastoris containing the L-lactate dehydrogenase gene, comprising the following steps:
1) The plasmids pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh obtained in the above step (7) were digested with restriction enzyme BlpI (Thermo Fisher) at 37℃for 30 minutes, and washed with PCR purification kit (SanPrep column PCR product purification kit, available from Shanghai Co., ltd.) to obtain linearized plasmids pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh, respectively.
2) Competent cells of pichia pastoris Pichia pastoris GS were prepared. The preparation process comprises the following steps: firstly, single colony of yeast receptor bacteria is selected and inoculated into 10ml of YPD (1% yeast extract powder, 2% peptone and 2% glucose) culture medium, and shake culture is carried out at 30 ℃ for overnight; the seed solution was transferred to 100ml of YPD liquid medium with an initial OD 600 Shake culture at 30 ℃ for 4 hours to OD =0.2 600 When the bacterial liquid is=0.8-1.0, placing the bacterial liquid in a centrifugal machine at 4 ℃ and centrifuging at 5000rpm for 5 minutes, and discarding the supernatant; the cells were then washed twice successively with 100ml and 50ml of sterile water pre-chilled on ice, respectively, and centrifuged at 5000rpm for 10 minutes at 4℃and the supernatant discarded. Finally, washing 1 time with 20ml of 1mol/L sorbitol, and dissolving in 200 mu L of 1mol/L ice-precooled sorbitol to obtain competent pichia pastoris cells.
3) The linearized plasmids pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh obtained in step 1) are introduced into the competent cells obtained in step 2) by means of electrotransformation. The electric conversion conditions are as follows: firstly, cleaning and blow-drying a Bio-Rad electric shock cup with the length of 0.2cm, and pre-cooling the cup on ice; mu.l of competent cells obtained in step 2) were placed on ice, and 30ng of linearized plasmids pPICZA-GAPp-ldh, pPICZA-PGI1p-ldh and pPICZA-TEF1p-ldh, respectively, were added, and placed on ice for 5 minutes and rapidly transferred to a cuvette. An electroporation apparatus of MicroPulser (BioRad Co.) was used, and the electric shock parameter was 1.5kV. 1ml of ice-precooled 1mol/L sorbitol was rapidly transferred to a cuvette after electric shock, 5 times of blowing and transferred to a 2ml sterile centrifuge tube, sealed and incubated at 200rpm for 1 hour at 30 ℃. 1ml of the supernatant was centrifuged and the remaining 100. Mu.l of the bacterial liquid was plated on YPDS (1% yeast extract powder, 2% peptone, 2% glucose, 1mol/L sorbitol) plates containing bleomycin (final concentration of 100. Mu.g/ml), and after two days of incubation at 30℃5 positive single colonies were selected, the colony genomes were roughly extracted and PCR verified, the primers were GAPp-S/FBP1tt-A, PGI p-S/FBP1tt-A and TEF1p-S/FBP1tt-A, respectively. And (3) carrying out sample feeding and sequencing analysis on the PCR result, and obtaining three recombinant pichia pastoris strains GS115-G, GS-P and GS115-T containing different promoters and producing L-lactic acid, wherein the positive cloning result is correct.
The flow of the crude extraction genome is as follows: firstly, preparing cell lysate (0.2 mol/L1% SDS solution of lithium acetate) and preparing analytically pure ethanol and 70% ethanol solution; single colony is selected and dispersed in 100 mu l of cell lysate, and incubated in a metal bath at 70 ℃ for 5 minutes; 300 μl of ethanol was added thereto, centrifuged at 15000g for 3 minutes after vortex shaking, and the supernatant was discarded; then washing the precipitate with 70% ethanol solution, centrifuging at 15000g for 3 min, and discarding the supernatant; after the pellet was dried in the air, it was resuspended in 100. Mu.l of water and centrifuged at 15000g for 15 seconds to pellet cell debris, and the obtained supernatant, i.e., the supernatant contained the genomic DNA fragments.
The information on the strains and plasmids constructed according to the invention are shown in Table 1.
TABLE 1 Strain and plasmid information
The primers used in the present invention are shown in Table 2.
TABLE 2 primer information
Example 2
Wild-type pichia pastoris GS115 grows with methanol, dihydroxyacetone and glycerol as substrates:
(1) Culturing seed liquid:
firstly scribing the pichia pastoris on a YPD plate, culturing for two days at 30 ℃, picking up a monoclonal into 10ml of YPD culture medium, and culturing for 24 hours at 200rpm and 30 ℃ to obtain pichia pastoris seed liquid.
YPD medium: 10g/L yeast extract powder, 20g/L peptone and 20g/L glucose.
(2) Transferring the seed liquid into a growth medium:
pichia pastoris in the seed solution is centrifuged at 4000rpm for 5min and washed once with ultrapure water and then transferred to a growth medium containing methanol, dihydroxyacetone and glycerol, respectively, with pH=5.6, initial OD=0.2, 200rpm, and incubation at 30 ℃. The growth is shown in FIG. 1. As can be seen from fig. 1, the wild-type pichia pastoris GS115 of the present invention can continuously accumulate biomass using three small organic molecules, wherein the growth rate is: glycerol > dihydroxyacetone > methanol.
The composition of the growth medium was as follows:
methanol medium: 10g/L yeast extract powder, 20g/L peptone and 10g/L methanol.
Dihydroxyacetone medium: 10g/L yeast extract powder, 20g/L peptone and 10g/L dihydroxyacetone.
Glycerol medium: 10g/L yeast extract powder, 20g/L peptone and 10g/L glycerol.
Example 3
Enzyme Activity assay of recombinant Pichia pastoris GS115-G, GS115-P and GS 115-T:
the enzyme activity of wild-type Pichia pastoris GS115 and recombinant Pichia pastoris GS115-G, GS115-P, GS-T was determined, and the method comprises the following steps:
(1) Seed liquid culture: the seed liquid culture of wild-type Pichia pastoris GS115 and recombinant Pichia pastoris GS115-G, GS115-P, GS-T was the same as in example 2.
(2) Preparation of crude cell enzyme solution: seed solutions of GS115, GS115-G, GS115-P and GS115-T were centrifuged at 4000rpm for 5min and washed once with ultrapure water, and then transferred to Delft minimum medium containing dihydroxyacetone, pH=5.6, initial OD=0.2, 200rpm, and cultured at 30 ℃. Then 20ml of the bacterial liquid in the logarithmic growth phase was collected by centrifugation at 4℃and washed 1 time with pre-chilled ultrapure water, and the bacterial liquid was resuspended in 3ml of Tris-HCl (pH=7.5). The cells were broken with an ultrasonic cell disrupter (SCIENTZ-II 0, ningbo Xinzhi Biotech Co., ltd.) at an ultrasonic intensity of 300W for 20min for 5sec and stopped for 10sec. Finally, the disrupted cells were centrifuged at 9500g for 15min at 4℃to remove cell debris.
Measurement of crude enzyme solution total protein concentration: the crude enzyme solution total protein concentration was measured using BCA protein concentration measuring kit (beijing solebao technologies limited) and the procedure was performed according to the instructions provided by the kit.
(3) Determination of L-lactate dehydrogenase Activity: 1ml of the reaction mixture contained 100mM phosphate buffer (pH=7.5), 0.25mM NADH,50mM pyruvic acid, and 100. Mu.l of crude enzyme solution. Since pyruvic acid is reduced to L-lactic acid by NADH under the action of L-lactate dehydrogenase. Thus the decrease in absorbance of NADH at 340nm can be measured to determine the enzymatic activity of lactate dehydrogenase; the extinction coefficient was 6.22mM-1cm-1. The enzyme activity units are defined as: the amount of enzyme required to oxidize 1. Mu. Mol NADH per minute is one unit of enzyme activity under standard conditions (25 ℃).
The results showed that wild-type Pichia pastoris GS115 had little enzymatic activity, whereas the enzymatic activities of recombinant Pichia pastoris GS115-G, GS115-P and GS115-T were 48.4, 35.8 and 11.2mU/mg protein, respectively.
Example 4
Recombinant pichia pastoris GS115-G, GS115-P and GS115-T are fermented by using dihydroxyacetone as a substrate in a shake flask to produce L-lactic acid:
(1) Culturing the seed liquid once:
the recombinant pichia pastoris GS115-G, GS-P and GS115-T glycerinum are firstly streaked on YPD plates containing bleomycin (the final concentration is 100 mug/ml), and after two days of culture at 30 ℃, monoclonal is selected and cultured in 10ml YPD culture medium at 200rpm and 30 ℃ for 24 hours, and recombinant pichia pastoris primary seed liquid is obtained.
(2) Culturing secondary seed liquid:
the primary seed solution of recombinant Pichia pastoris GS115-G, GS-P and GS115-T is washed by a Delft minimum medium, then transferred to the Delft minimum medium containing 10g/L dihydroxyacetone, pH=5.6, initial OD=0.2, and cultured for 24 hours at 200rpm and 30 ℃ to obtain the secondary seed solution of recombinant Pichia pastoris.
Delft minimum Medium: 2.5g/L (NH) 4 ) 2 SO 4 ，14.4g/L KH 2 PO 4 ，0.5g/L MgSO 4 ·7H 2 O,10g/L dihydroxyacetone, 40mg/L histidine, 2mL/L metal ion and 1mL/L vitamin, 100. Mu.g/mL bleomycin. The metal ion mother liquor composition is shown in table 3.
TABLE 3 Metal ion mother liquor composition
FeSO 4 ·7H 2 O 3.0g/L
ZnSO 4 ·7H 2 O 4.5g/L
CaCl 2 ·2H 2 O 4.5g/L
MnCl 2 ·4H 2 O 1g/L
CoCl 2 ·6H 2 O 300mg/L
CuSO 4 ·5H 2 O 300mg/L
Na 2 MoO 4 ·2H 2 O 400mg/L
H 3 BO 3 1g/L
KI 100mg/L
Na 2 EDTA·2H 2 O 19g/L
The vitamin mother liquor composition is shown in table 4.
TABLE 4 vitamin mother liquor composition
D-biotin 50mg/L
D-pantothenic acid calcium salt 1.0g/L
Thiamine hydrochloride 1.0g/L
Pyridoxine hydrochloride 1.0g/L
Nicotinic acid 1.0g/L
Para aminobenzoic acid 0.2g/L
Inositol (inositol) 25g/L
(3) Shaking flask fermentation
The recombinant Pichia pastoris GS115-G, GS-P and GS115-T secondary seed solution were centrifuged at 4000rpm for 5min and then transferred directly to fresh (NH) 4 ) 2 SO 4 In Delft minimum medium, 80rpm,30℃for 48 hours, samples were taken every 12 hours, and the pH was adjusted to 5.6 with 4M aqueous sodium hydroxide. The initial OD of inoculation was approximately 18 and after 48 hours the growth OD was approximately 22. The fermentation is shown in FIG. 2. As can be seen from FIG. 2, the recombinant Pichia pastoris GS115-G, GS-P and GS115-T continuously consume dihydroxyacetone and produce L-lactic acid, the yield of the L-lactic acid reaches 30G/L after 48 hours of fermentation, the conversion rate of the recombinant Pichia pastoris GS115-G is close to 100%, and the optical purity is more than 99.5%.
Delft minimum Medium: 14.4g/L KH 2 PO 4 ，0.5g/L MgSO 4 ·7H 2 O,30g/L dihydroxyacetone, 40mg/L histidine, 2mL/L metal ion and 1mL/L vitamin, 100. Mu.g/mL bleomycin.
The analysis method comprises the following steps: the components in the medium were determined using an Agilent-1260 high performance liquid chromatograph. The concentration of lactic acid and dihydroxyacetone was determined using primaryAminex HPX-87H organic acid analytical column, available from Biorad, had a mobile phase of 5mM sulfuric acid. Lactic acid optical purity analysis Using Daxiaoqing pharmaceutical chiral technology (Shanghai Co., ltd.)MA (+) chiral column analysis.
Example 5
Recombinant Pichia pastoris GS115-G is used for producing L-lactic acid by semi-continuous fermentation by taking dihydroxyacetone as a substrate:
(1) Culturing the seed liquid once:
the seed solution was cultured once in the same manner as in example 4.
(2) Culturing secondary seed liquid:
the secondary seed liquid was cultured in the same manner as in example 4.
(3) Semi-continuous fermentation:
the apparatus used for the semi-continuous fermentation was a DASSIP parallel bioreactor system (Eppendorf, germany, manufacturer) with a fermenter volume of 1L and a fermentation medium of 300ml. Before inoculation, inoculating the fermentation medium into a fermentation tank, sterilizing at 121 ℃ for 2 hours, and assembling and debugging after the fermentation tank is cooled.
Recombinant pichia pastoris GS115-G secondary seed solution is inoculated to a Delft minimum fermentation medium according to the inoculum size of initial concentration OD=1.0, and fermentation broth is obtained. The fermentation temperature was 30deg.C, and pH was adjusted to 5.6 with 4M sodium hydroxide and 2M hydrochloric acid aqueous solution. The initial stirring was set at 400rpm and increased to a maximum of 1200rpm depending on the level of dissolved oxygen. Aeration was initially set at 36sL/h and increased to a maximum of 48sL/h depending on DO level. In the early stage of the fermentation process, the dissolved oxygen level is maintained above 30%; after the cell density reaches 20, the dissolved oxygen level is reduced to below 10%. The fermentation medium is the same as the shake flask fermentation, the initial concentration of dihydroxyacetone is 20g/L, and the dihydroxyacetone is kept at 10-30 g/L in the fermentation process. Fermenting for 3 days. The results are shown in FIG. 3. As can be seen from FIG. 3, recombinant Pichia pastoris GS115-G continuously consumes dihydroxyacetone, continuously accumulates biomass and produces L-lactic acid, and OD after 60 hours of fermentation 600 The yield of the L-lactic acid reaches 25g/L, and the optical purity reaches more than 99.5 percent.
The analytical method was the same as in example 4.
Example 6
Recombinant Pichia pastoris GS115-G is used for producing L-lactic acid by semi-continuous fermentation by taking dihydroxyacetone as a substrate:
(1) Culturing the seed liquid once:
the seed solution was cultured once in the same manner as in example 4.
(2) Culturing secondary seed liquid:
transferring the primary seed solution of the recombinant pichia pastoris GS115-G into YPHASH culture medium containing 10G/L dihydroxyacetone, wherein the pH value is=5.6, the initial OD is=0.2, the speed is 200rpm, and the culture is carried out at 30 ℃ for 24 hours, so as to obtain the secondary seed solution of the recombinant pichia pastoris.
YPDHA medium: 10g/L yeast extract powder, 20g/L peptone, 10g/L dihydroxyacetone and 100 mu g/ml bleomycin.
(3) Semi-continuous fermentation:
the apparatus used for the semi-continuous fermentation was a DASSIP parallel bioreactor system (Eppendorf, germany, manufacturer) with a fermenter volume of 1L and a fermentation medium of 300ml. Before inoculation, the YPHASA fermentation medium is inoculated into a fermentation tank, sterilized at a high temperature of 121 ℃ for 2 hours, and assembled and debugged after the fermentation tank is cooled.
Recombinant pichia pastoris GS115-G secondary seed solution is inoculated to YPHASH fermentation medium according to the inoculum size of initial concentration OD=1.0, and fermentation liquid is obtained. The fermentation temperature was 30deg.C, and pH was adjusted to 5.6 with 4M sodium hydroxide and 2M hydrochloric acid aqueous solution. The initial stirring was set at 400rpm and increased to a maximum of 1200rpm depending on the level of dissolved oxygen. Aeration was initially set at 36sL/h and increased to a maximum of 48sL/h depending on DO level. The dissolved oxygen level was maintained above 30% throughout the fermentation. The fermentation medium is the same as the shake flask fermentation, the initial concentration of dihydroxyacetone is 20g/L, and the dihydroxyacetone is kept at 10-30 g/L in the fermentation process. Fermenting for 3 days. The results are shown in FIG. 4. As can be seen from FIG. 4, recombinant Pichia pastoris GS115-G continuously consumes dihydroxyacetone, continuously accumulates biomass and produces L-lactic acid, and OD after 60 hours of fermentation 600 The yield of the L-lactic acid reaches 23, the yield of the L-lactic acid reaches 16g/L, and the optical purity reaches more than 99.5 percent.
The analytical method was the same as in example 4.
The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.

Claims (10)

1. A recombinant pichia pastoris, wherein the recombinant pichia pastoris contains an L-lactate dehydrogenase gene;
the nucleotide sequence of the L-lactate dehydrogenase gene is shown as SEQ ID No. 1.
2. The method for constructing recombinant pichia pastoris according to claim 1, comprising the steps of:
step 1), connecting the L-lactate dehydrogenase gene with an initial expression vector, a promoter and a terminator to obtain a recombinant expression vector;
and 2) transferring the recombinant expression vector into pichia pastoris to obtain the recombinant pichia pastoris.
3. The method of constructing recombinant pichia pastoris according to claim 2, wherein the initial expression vector of step 1) comprises pPICZA;
the promoter in the step 1) comprises any one of GAPp, PGI1p and TEF1 p;
the terminator in step 1) includes FBP1tt.
4. The method for constructing recombinant pichia pastoris according to claim 3, wherein the pPICZA contains a bleomycin resistance gene;
preferably, the nucleotide sequence of the GAPp is shown as SEQ ID No. 2;
the nucleotide sequence of the PGI1p is shown as SEQ ID No. 3;
the nucleotide sequence of the TEF1p is shown as SEQ ID No. 4;
preferably, the nucleotide sequence of FBP1tt is shown in SEQ ID No. 5.
5. The use of the recombinant pichia pastoris according to claim 1 for the fermentative production of L-lactic acid using small organic molecules.
6. A method for producing L-lactic acid by using organic micromolecule fermentation, which is characterized by comprising the following steps:
inoculating the recombinant pichia pastoris in the claim 1 into a culture medium for fermentation culture, and collecting a fermentation product to obtain the L-lactic acid.
7. The method for producing L-lactic acid by fermentation of small organic molecules according to claim 6, wherein the initial inoculum size of the recombinant Pichia pastoris is 1-20 OD 600 。
8. The method for producing L-lactic acid by fermentation using small organic molecules according to claim 6, wherein the medium has small organic molecules as fermentation substrates;
preferably, the small organic molecule includes any one of methanol, dihydroxyacetone, and glycerol;
preferably, the initial addition amount of the small organic molecules is 10-30 g/L.
9. The method for producing L-lactic acid by fermentation using small organic molecules according to claim 6, wherein the pH of the medium is 5 to 6;
preferably, the temperature of the fermentation culture is 28-30 ℃.
10. The method for producing L-lactic acid by fermentation using small organic molecules according to claim 6, wherein the fermentation culture mode comprises shaking fermentation culture or semi-continuous fermentation culture;
preferably, the time of shake flask fermentation culture is 36-72 h;
the time of the semi-continuous fermentation culture is 48-96 hours;
preferably, the aeration amount in the semi-continuous fermentation culture is 36-48 sL/h;
preferably, the initial dissolved oxygen content in the semi-continuous fermentation culture is not less than 30%.
CN202311049949.6A 2023-08-18 2023-08-18 Recombinant pichia pastoris, construction method thereof and application thereof in production of L-lactic acid Pending CN117305144A (en)

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