CN104878024A - Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method - Google Patents
Recombinant Escherichia coli construction and (S)-2-hydroxy-3-phenylpropionic acid synthesis method Download PDFInfo
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Abstract
The invention relates to an L- lactate dehydrogenase gene, and the nucleotide sequence represents as SEQ ID NO. 1. The invention further provides an expression vector with the gene, a recombinant Escherichia coli, a recombinant Escherichia coli construction method and the application thereof during phenylpyruvic acid conversion and (S)-2-hydroxy-3-phenylpropionic acid synthesis. The method includes after performing codon optimization on (Bacillus megaterium Z2013513) CCTCC NO. M2013244 L- lactate dehydrogenase gene, constructing the expression vector, inducing expression of the Escherichia coli, and obtaining the L-PLA production strain with simple culture condition, convenience for usage and wide application. The yield for the recombinant Escherichia coli converting the L-PLA in whole-cell and single-batch manners is 27.13mmol / L, the converting rate is 77.5%, and the basis is provided for increasing the L-PLA yield and producing rate for subsequent fermentor continuous feeding.
Description
Technical field
The invention belongs to biological technical field, particularly the structure of a strain recombination bacillus coli and the method for synthesis (S)-PLA thereof.
Background technology
Phenyllactic acid (phenyl lactic acid, PLA), i.e. PLA, PLA or β-phenyllactic acid.PLA is pale yellow powder, slightly singularity smell, and molecular formula is C
9h
9o
3, relative molecular weight is 166, fusing point 121 ~ 125 DEG C, to sour and thermally-stabilised.PLA is a kind of new type natural sanitas of discovered in recent years, is extensively present in lactobacillus-fermented product and honey, is by the meta-bolites of phenylalanine in multiple-microorganism (mainly milk-acid bacteria).This aromatic organic acid produced by generally recognized as safe milk-acid bacteria metabolism shows after deliberation to humans and animals cytotoxic.PLA be continue milk-acid bacteria produce first-generation antibacterial substance if lactic acid, acetic acid and s-generation antibacterial substance are as the third generation natural small molecule antibacterial substance beyond the bacteriocins such as nisin (nisin), pediocin (pedioein).Compared with most natural antiseptic agent, phenyllactic acid antimicrobial spectrum is wider, can suppress multiple food-borne pathogens, spoilage organism, particularly can Antifungi pollution, is to make lactobacillus-fermented product antifungal property by force with compared with one of the main active substances of long shelf-life; Solvability is good, is easy to spread in food system; Stability is high, and have broad pH scope and thermostability, these advantages are beneficial to it and have broad application prospects in foodstuffs industry.
PLA second carbon atom is chiral carbon atom, therefore (S)-PLA is had, i.e. L-PLA and (R)-PLA, i.e. D-PLA two kinds of enantiomers, and two kinds of isomer are to the effect of organism and the application in medicine, chemical industry thereof, and there were significant differences, therefore obtaining the high single enantiomer PLA of optical purity is its prerequisite in the widespread use of food and medicine field.
Early-stage Study finds that some bacillus compare stronger PLA synthesis capability as Bacillus megaterium Z2013513 (CCTCC NO:M2013244) has compared with milk-acid bacteria, and because its nutritional requirement is low, growth rapid, security is good and have good potentiality to be exploited.But due to the distinctive sporulation of bacillus and cannibalism effect, inhibit the further raising of PLA synthesis capability, and catalysis can only produce D-PLA, but can not L-PLA be produced.If Microbe synthesis L-PLA production efficiency can be improved further and high-density cells biosynthesizing L-PLA will have very important significance.
Summary of the invention
The first object of the present invention is to provide a kind of LDH gene, solves current bacillus synthesis PLA production efficiency lower, catalysis can only produce D-PLA, but can not produce the problem of L-PLA.
The second object of the present invention is to provide the recombinant expression plasmid carrying above-mentioned LDH gene.
The third object of the present invention is to provide the recombination bacillus coli containing above-mentioned recombinant plasmid.
The fourth object of the present invention is to provide the construction process of above-mentioned recombination bacillus coli.
The fifth object of the present invention is to provide the purposes of above-mentioned recombination bacillus coli.
The sixth object of the present invention is to provide the method that above-mentioned recombination bacillus coli transforms phenyl-pyruvic acid synthesis L-phenyl-lactic acid.
The present invention is achieved through the following technical solutions:
One, a LDH gene, its nucleotide sequence is as shown in SEQ ID NO:1.
Two, the recombinant expression plasmid pET-28a-ldhL of above-mentioned LDH gene is carried.
Three, the recombination bacillus coli containing above-mentioned carrier.
The construction process of four, above-mentioned recombination bacillus coli, the method comprises the following steps:
(1) according to colibacillary codon preference to bacillus megaterium (
bacillus megateriumz2013513) CCTCC NO:M 2013244 LDH gene carries out codon optimized;
(2) codon optimized LDH channel genes pET-28a (+) carrier is obtained recombinant expression vector pET-28a-ldhL;
(3) the recombinant expression vector pET-28a-ldhL obtained is transformed
e. colrecombination bacillus coli is obtained after i BL21 (DE3).
Five, above-mentioned recombination bacillus coli is transforming the application in phenyl-pyruvic acid synthesis (S)-PLA.
Six, above-mentioned recombination bacillus coli transforms the method for phenyl-pyruvic acid synthesis (S)-PLA, and the method comprises the following steps:
(1) full cell collecting cells, activate in recombination bacillus coli list colony inoculation to seed culture medium, this seed liquor be seeded in enlarged culturing base and be cultured to mid-log phase, add and produce enzyme inducer IPTG, low temperature induction also collects thalline to obtain full cell bacteria suspension;
(2) current adding substrate phenyl-pyruvic acid and glucose, Whole Cell Bioconversion synthesis (S)-PLA.
Adopt the positively effect of technique scheme: the present invention to bacillus megaterium (
bacillus megateriumz2013513) CCTCC NO:M2013244 LDH gene carry out codon optimized after, be built into expression vector, abduction delivering in intestinal bacteria, obtain simple, easy to use, the widely used L-PLA of a kind of culture condition and produce bacterial strain; Described recombination bacillus coli, through conversion test in batches, batch feeding conversion test and the conversion test of cell repeatability, all obtains higher PPA transformation efficiency and L-PLA output, and this improves L-PLA output for subsequent fermentation tank continuous feeding and productive rate is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is recombinant plasmid pET-28a-ldhL schematic diagram.
Fig. 2 is engineering bacteria SDS-polyacrylamide gel electrophoresis figure.Swimming lane M is protein standard; Swimming lane 1 is
e. colibL21 (DE3)/pET-28a-ldhL, swimming lane 2 is
e. colibL21 (DE3)/pET-28a, swimming lane 3 is
e. colibL21 (DE3).
Fig. 3 is the collection of illustrative plates that high performance liquid chromatography chiral column detects recombination bacillus coli full cell conversion fluid L-PLA in batches.Solid line represents the detected result of PLA in conversion fluid, and represented by dotted arrows contains the detected result of standard substance D-PLA and L-PLA solution.
Fig. 4 is the time curve of substrate batch feeding Synthesis L-PLA.
Fig. 5 is high performance liquid chromatography chiral analysis bacillus megaterium full cell synthesis PLA.Solid line represents the detected result of PLA in conversion fluid, and represented by dotted arrows is containing the detected result of the solution of standard substance D-PLA and L-PLA.
Embodiment
The source of biomaterial in the present invention:
1, bacillus megaterium (
bacillus megateriumz2013513) CCTCC NO:M2013244 is disclosed in Chinese Patent Application No. 201410000615.4, applying date 2014-01-02, publication date 2014-04-09, in publication number CN103710291A.
2, prokaryotic expression carrier pET-28a (+) is purchased from Novagen.
3, the LDH gene of synthetic and PCR primer all entrust the raw work biosynthesizing in Shanghai.
Below in conjunction with embodiment and comparative example, the technical scheme to this explanation is described further, but should not be construed as limitation of the present invention:
embodiment 1
The present embodiment illustrates recombination bacillus coli
e. colithe structure of BL21 (DE3)/pET-28a-ldhL.
1.L-serum lactic dehydrogenase derives from bacillus megaterium
bacillus megateriumz2013513 (CCTCC NO:M2013244), carries out codon optimized according to e. coli codon Preference to L-LDH gene, and its nucleotide sequence is as shown in SEQ ID NO:1.
2. obtain codon optimized LDH gene through synthetic, design upstream and downstream primer, primer sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3.After pcr amplification, nucleic acid agarose gel electrophoresis glue reclaims, and is connected to pMD19-T carrier, is transformed into by cloning vector
e. colijM109 obtains
e. colijM109-pMD-ldhL, extracts recombinant plasmid and carries out double digestion checking, by positive colony after checking
e. colijM109-pMD-ldhL carries out nucleotide sequence order-checking.After order-checking is correct, the product of cloning vector pMD-ldhL double digestion is carried out the recovery of nucleic acid electrophoresis glue, obtain goal gene
l-ldh, connect expression plasmid pET-28a, be converted into
e. coliin BL21 (DE3), verify through plasmid enzyme restriction checking and bacterium colony PCR, obtain
e. colibL21 (DE3)/pET-28a-ldhL, and through the expression amount (Fig. 2) of SDS-Polyacrylamide Gel Electrophoresis L-LDH.
embodiment 2
The present embodiment illustrates the preparation of the full cell of recombination bacillus coli.
(1) actication of culture: picking
e. coliin BL21 (DE3)/pET-28a-ldhL mono-colony inoculation to 10 mL LB liquid nutrient medium (containing 50 mg/L kantlex), 37 DEG C, 200 r/min overnight incubation.
(2) seed culture: by above-mentioned steps 1) gained nutrient solution accesses in 100 mL LB substratum (containing 50 mg/L kantlex) according to 1% inoculum size (volume fraction), and 37 DEG C, 200 r/min culturing cell OD
600be 0.4 ~ 1.0.
(3) abduction delivering: to above-mentioned 4) add IPTG in the bacterium liquid that obtains, in 25 DEG C, 200 r/min cultivate 4 ~ 6 h to Growth of Cells mid-log phase, by gained nutrient solution in 4 DEG C, centrifugal 5 min of 6000 r/min, wash 2 times with sterilizing sodium phosphate buffer (0.1 mol/L, pH=7.0) and obtain resting cell thalline.
embodiment 3
The present embodiment illustrates that full cell catalyzes and synthesizes L-PLA in batches.
(1) resting cell: to suspend above-described embodiment 2 gained thalline with 5 ml conversion fluids, and pour in 100 ml triangular flasks.Containing 0.1 mol/L sodium phosphate buffer and 100 mmol/L glucose in conversion fluid.Be suspended in after in conversion fluid until bacterium mud is full and uniform, add 5 ml 70 mmol/L PPA solution and final concentration is 35 mmol/L, on shaking table, carry out conversion reaction after mixing, temperature of reaction is 37 DEG C, 200 r/min oscillatory reaction 60 min.
(2) by above-mentioned steps 1) gained reaction conversion fluid through 4 DEG C, supernatant liquor is got after centrifugal 2 min of 10000 r/min, high performance liquid chromatograph is adopted to measure the content of L-PLA, testing conditions is: chiral column OJ-RH, moving phase (volume ratio): acetonitrile: methyl alcohol: trifluoroacetic acid: deionized water=50:50:1.5:900, flow velocity 0.4 mL/min, column temperature 25 DEG C, determined wavelength 210 nm, sample size 5 μ L.As shown in Figure 3, there is obvious crest in L-PLA to concrete outcome.
embodiment 4
The present embodiment illustrates that full cell batch feeding catalyzes and synthesizes L-PLA.
With 50 ml sodium phosphate buffers (0.1 mol/L, pH=7.0, containing 100 mmol/L glucose) suspension above-described embodiment 2 gained thalline, be placed in 250 ml triangular flasks.After the full and uniform suspension of bacterium mud, add 0.36 g PPA, namely in 50 ml conversion fluids, PPA final concentration is 35 mmol/L, on shaking table, conversion reaction is carried out after mixing, temperature of reaction is 37 DEG C, 200 r/min oscillatory reactions, per hourly gets 0.5 ml conversion fluid, centrifugal 2 min of 10000 r/min, supernatant liquor adopts HPLC to detect.PPA and glucose is supplemented immediately after every sub-sampling.Concrete test-results as shown in Figure 4.Stream through 4 h adds conversion reaction, and obtaining L-PLA ultimate density is 128 mmol/L, and molar yield reaches 63.96 %.
embodiment 5
The present embodiment illustrates that cell recycling repeatedly transforms preparation L-PLA.
(1) resting cell: to suspend above-described embodiment 2 gained thalline with 5 ml conversion fluids, and pour in 100 ml triangular flasks.Conversion fluid is 0.1 mol/L sodium phosphate buffer (containing 100 mmol/L glucose).Be suspended in after in conversion fluid until bacterium mud is full and uniform, add 5 ml 70 mmol/L PPA solution and final concentration is 35 mmol/L, on shaking table, conversion reaction is carried out after mixing, temperature of reaction is 37 DEG C, after 200 r/min oscillatory reaction 60 min, centrifugal 5 min separating thallus and the reaction solutions of 6000 r/min, the thalline obtained is centrifugal after sodium phosphate buffer cleans twice repeats conversion reaction for next batch, and reaction supernatant liquor is used for HPLC and measures the concentration of PPA and L-PLA and calculate L-PLA productive rate.
(2) with 5 ml conversion fluids suspension above-mentioned (1) gained thalline, and pour in 100 ml triangular flasks.The step repeated in (1) is carried out thalline recycling and is transformed PPA, and thalline transforms 7 times repeatedly to PPA, and the result of each conversion reaction is as shown in the table.Transform through 7 times, obtain 143.04 mmol/L L-PLA, average molar conversion yield 64.54 %.Along with multiplicity increases, cell viability and enzyme are lived and are declined, and the reason such as aqtocytolysis causes transformation efficiency obviously to decline.
Table 1 cell replica test result
| Multiplicity | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| PPA(mmol/L) | 35 | 35 | 30 | 30 | 30 | 30 | 30 |
| L-PLA(mmol/L) | 27.13 | 25.34 | 21.60 | 20.78 | 18.16 | 17.21 | 12.82 |
| Transformation efficiency (%) | 77.51 | 72.40 | 72.00 | 69.26 | 60.53 | 57.37 | 42.73 |
comparative example 1
With without codon optimized bacillus megaterium in contrast, carry out resting cell, analyze the synthesis result of L-PLA, concrete steps are as follows:
(1) actication of culture: in picking bacillus megaterium single colony inoculation to 10 mL LB liquid nutrient medium, 37 DEG C, 200 r/min overnight incubation.
(2) seed culture: by above-mentioned steps 1) gained nutrient solution accesses in 100 mL LB substratum according to 1% inoculum size (volume fraction), 37 DEG C, 200 r/min cultivate 4 ~ 6 h to Growth of Cells mid-log phase, by gained nutrient solution in 4 DEG C, centrifugal 5 min of 6000 r/min, wash 2 times with sterile phosphate buffer (0.1 mol/L, pH=7.0) and obtain thalline.
(3) resting cell: suspend above-mentioned 2 with 5 ml conversion fluids) middle gained thalline, and pour in 100 ml triangular flasks.Conversion fluid is 0.1 mol/L phosphoric acid buffer (containing 100 mmol/L glucose).Be suspended in after in conversion fluid until bacterium mud is full and uniform, add 5 ml 70 mmol/L PPA solution and final concentration is 35 mmol/L, on shaking table, conversion reaction is carried out after mixing, temperature of reaction is 37 DEG C, after 200 r/min oscillatory reaction 2 h, centrifugal 5 min separating thallus and the reaction solutions of 10000 r/min, reaction supernatant liquor is used for HPLC chiral analysis PLA.As shown in Figure 5, it is D-PLA that result shows that bacillus megaterium resting cell PPA synthesizes PLA to result, the synthesis of L-PLA do not detected.
Therefore, can find out, can only D-PLA be synthesized without codon optimized bacillus megaterium, and can not L-PLA be synthesized.And the present invention to bacillus megaterium (
bacillus megateriumz2013513) CCTCC:M2013244 LDH gene carry out codon optimized after, be built into expression vector, abduction delivering in intestinal bacteria, obtain simple, easy to use, the widely used L-PLA of a kind of culture condition and produce bacterial strain; It is 27.13 mmol/L that single batch, the full cell of described recombination bacillus coli transforms L-PLA output, and transformation efficiency reaches 77.5 %, and this improves L-PLA output for subsequent fermentation tank continuous feeding and productive rate is laid a good foundation.
Sequence table
<110> Changshu Institute of Technology
The structure of <120> mono-strain recombination bacillus coli and the method for synthesis (S)-PLA thereof
<130> xb15052701
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 957
<212> DNA
<213> Artificial
<220>
LDH gene after <223> is codon optimized
<400> 1
atgaaaaccc aatttacccc aaaaacccgt aaagttgctg ttatcggtac tggttttgtt 60
ggctctagct acgctttttc tatggtgaac caaggtattg ccaacgaact ggtgctgatc 120
gatatgaaca aagaaaaagc ggaaggtgaa gcacgcgata tcaaccacgg tatgccattt 180
tccaccccga tgaaaatctg ggctggtgat tataaagact gtgctgacgc tgatctggta 240
gttattaccg cgggcgctaa ccaagctcca ggtgaaaccc gcctggatct ggttgaaaaa 300
aacgttaaaa tttttgaatg cattgtaaaa gatattatga acagcggttt tgacggcatc 360
attctggttg caaccaaccc agttgatatt ctggcacacg ttacccaaaa agtatctggt 420
ctgccaaacg aacgtgtaat tggttctggt accattctgg acaccgctcg cttccgctac 480
ctgctgagcg actatttcga agtagattct cgcaacgttc acgcttatat tatgggtgaa 540
catggtgata ccgaatttcc ggtttggagc cacgcgcaaa ttggcggcgt gaagctggaa 600
cattttatca acactgctgc tattgaaaaa gaaccggata tgaagcatct gttcgaacaa 660
acccgcgatg cggcttacca tattattaac cgtaaaggtg cgacttatta cggtattgca 720
atgggtctgg tacgtattac caaggctatt ctggatgatg agaactctat tctgaccgta 780
tctgctctgc tggaaggtca atacggtatt tctgacgtgt atatcggcgt accagctatc 840
attaacaaaa acggcgtgcg tcaaattatt gaactgaacc tgactccgca cgaacagcag 900
cagctggagc actctgctag cattctgaag caaactcgcg accgtgcttt tgtgtaa 957
<210> 2
<211> 28
<212> DNA
<213> Artificial
<220>
The upstream primer of the LDH gene after <223> amplification is codon optimized
<400> 2
gcgcccatat gatgaaaacc caatttac 28
<210> 3
<211> 25
<212> DNA
<213> Artificial
<220>
The downstream primer of the LDH gene after <223> amplification is codon optimized
<400> 3
ccgggatcct tacacaaaag cacgg 25
Claims (6)
1. a LDH gene, its nucleotide sequence is as shown in SEQ ID NO:1.
2. carry the recombinant expression plasmid pET-28a-ldhL of LDH gene according to claim 1.
3. the recombination bacillus coli containing carrier according to claim 2.
4. the construction process of recombination bacillus coli according to claim 3, the method comprises the following steps:
(1) according to colibacillary codon preference to bacillus megaterium (
bacillus megateriumz2013513) CCTCC NO:M2013244 LDH gene carries out codon optimized;
(2) codon optimized LDH channel genes pET-28a (+) carrier is obtained recombinant expression vector pET-28a-ldhL;
(3) the recombinant expression vector pET-28a-ldhL obtained is transformed
e. colirecombination bacillus coli is obtained after BL21 (DE3).
5. recombination bacillus coli according to claim 3 is transforming the application in phenyl-pyruvic acid synthesis (S)-PLA.
6. recombination bacillus coli according to claim 3 transforms the method for phenyl-pyruvic acid synthesis (S)-PLA, and the method comprises the following steps:
(1) full cell collecting cells, activate in recombination bacillus coli list colony inoculation to seed culture medium, this seed liquor be seeded in enlarged culturing base and be cultured to mid-log phase, add and produce enzyme inducer IPTG, low temperature induction also collects thalline to obtain full cell bacteria suspension;
(2) current adding substrate phenyl-pyruvic acid and glucose, Whole Cell Bioconversion synthesis (S)-PLA.
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| CN105567747A (en) * | 2016-01-26 | 2016-05-11 | 常熟理工学院 | Method for promoting microbial synthesis of 2-hydroxy-3-phenylpropionic acid with bi-phase system |
| CN105543291A (en) * | 2016-02-15 | 2016-05-04 | 江南大学 | Microbial transformation method |
| CN105603006A (en) * | 2016-02-15 | 2016-05-25 | 江南大学 | Microorganism converting method |
| CN105603008A (en) * | 2016-02-15 | 2016-05-25 | 江南大学 | Microorganism converting method |
| CN105624217A (en) * | 2016-02-15 | 2016-06-01 | 江南大学 | Microbial transformation method |
| CN105624217B (en) * | 2016-02-15 | 2019-03-15 | 江南大学 | A kind of method of microorganism conversion |
| CN105603008B (en) * | 2016-02-15 | 2019-03-15 | 江南大学 | A kind of method of microorganism conversion |
| CN105603006B (en) * | 2016-02-15 | 2019-03-15 | 江南大学 | A kind of method of microorganism conversion |
| CN105543291B (en) * | 2016-02-15 | 2019-07-23 | 江南大学 | A kind of method of microorganism conversion |
| CN107177563A (en) * | 2017-06-21 | 2017-09-19 | 江南大学 | A kind of D lactic dehydrogenases in Lactobacillus casei source and its application |
| CN107177564A (en) * | 2017-06-21 | 2017-09-19 | 江南大学 | A kind of L lactic dehydrogenases in Lactobacillus casei source and its application |
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