CN102250822A - Reconstruction method for producing Vitamin C precursor 2-keto-L-gulonic acid (2-KLG) with gluconobacter oxydans - Google Patents

Abstract

The invention discloses a method for transforming gluconobacterium oxydans to produce vitamin C precursor 2-keto-L-gulonic acid, which belongs to the field of genetic engineering. The present invention expresses the genes of sorbose dehydrogenase (SDH) and sorbone dehydrogenase (SNDH) derived from Ketogulonigenium vulgare in Gluconobacter oxydans through genetic engineering technology In this study, a strain of G.oxydans engineered strain that utilizes sorbitol to produce 2-KLG was obtained. G.oxydans is a commonly used strain in the first step of the fermentation process in the production of 2-KLG by the two-step fermentation method. Expressing the sdh and sndh genes in G.oxydans can relieve the problem of the dependence of the small bacteria on the accompanying bacteria and realize the transformation from D- The direct conversion of sorbitol to 2-KLG simplifies the production process of vitamin C, and the output of 2-KLG can reach 83g/L, which has a good application prospect.

Description

A method for transforming Gluconobacter oxidans to produce vitamin C precursor 2-keto-L-gulonic acid

technical field

The invention relates to a high-yielding 2-KLG gluconobacter oxidase engineering bacterium and a construction method thereof, which adopts molecular means to introduce SDH and SNDH to realize the production of 2-KLG by metabolizing sorbitol, and belongs to the field of genetic engineering.

Background technique

Vitamin C (Vitamin C, VC), also known as Ascorbic acid (Ascorbic acid), is an essential vitamin and antioxidant, widely used in industries such as medicine, food, feed and cosmetics. At present, the industrial production of vitamin C in China adopts a two-step fermentation method. In the second-step mixed-bacteria fermentation system, only small bacteria perform sugar-acid conversion. It is difficult for small bacteria to grow alone, and they need to be co-cultured with large bacteria to grow normally. The fermentation control of the two kinds of bacteria has added great difficulties to the production process, and the acid-producing properties of the small bacteria are unstable, resulting in unstable production, and the tanks are often inverted due to the degradation of the bacteria, resulting in repeated heavy losses in production. The fermentation process of my country's vitamin C fermentation technology from sorbitol to 2-KLG involves three kinds of bacteria, which will inevitably cause a lot of waste of substrate and medium in the process of microbial metabolism. The fermentation process is divided into two steps, which not only prolongs the production cycle but also causes A huge waste of energy and manpower. Therefore, the existing vitamin C two-step fermentation process still has great potential for technological progress. How to simplify the fermentation process is an urgent problem to be solved.

Contents of the invention

The purpose of the present invention is to provide a high-yielding 2-KLG G.oxydans engineering bacteria.

The engineering bacteria can express sdh and sndh genes.

The nucleotide sequences of the sdh and sndh genes are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

The sdh and sndh genes are cloned on the G.oxydans-E.coli shuttle plasmid vector pGUC1.

Another technical problem to be solved by the present invention is to provide a method for constructing a high-yield 2-KLG G.oxydans genetically engineered bacterium.

In order to solve the problems of the technologies described above, the specific solutions of the present invention are:

1) Design primers to clone sdh and sndh genes according to the sdh and sndh gene sequences annotated in the whole genome sequencing results of K.vulgare DSM 4205 in our laboratory;

2) connecting the sdh and sndh genes with the vector to obtain a recombinant expression vector;

3) Transform the obtained recombinant expression vector into Gluconobacter oxydans (Gluconobacter oxydans, ATCC 621H) to obtain a recombinant strain.

Another technical problem to be solved by the present invention is to provide a method for fermenting and producing 2-KLG. After the engineered bacteria seeds are cultivated, a 5L fermenter is inoculated at a ratio of 15%, and after 16 hours of fermentation, the feed-in carbon source is 2.5g/L.h. Fermentation parameters: speed 500rpm, pH 5.1~5.4, ventilation 1.5vvm, control tank temperature 34°C, tank pressure 0.05MPa.

Seed and slant medium (g/L): sorbitol 15, yeast extract 0.6, calcium carbonate 0.4, pH 4.8-5.1, agar 20 (slant medium), pH 7.0, sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg /mL.

Fermentation medium (g/L): sorbitol 25, yeast extract 0.2, calcium carbonate 0.2, initial pH 5.1-5.4, sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg/mL.

Determination of sorbitol and 2-KLG content: liquid chromatography (LC)

Fermentation samples were diluted tenfold with mobile phase and filtered through a 0.45 μm filter membrane. Agilent 1100system, RioRad Aminex HPX-87H chromatographic column; mobile phase: 2.75 μmol/L concentrated sulfuric acid; column temperature: 35°C; flow rate: 0.6mL/min; injection volume: 5 μL; detector: differential refractive index detector.

The present invention expresses the sdh and sndh genes derived from Ketogulonigenium vulgare in Gluconobacter oxydans through genetic engineering transformation, and obtains a strain that utilizes sorbitol to produce 2-KLG G.oxydans engineering bacteria. G.oxydans engineering bacteria are used to produce 2-KLG by one-step fermentation of sorbitol, which solves the problem of the dependence of small bacteria on accompanying bacteria and simplifies the production process of vitamin C. The output of 2-KLG can reach 83g/L, which has a good application prospect. The construction method provided by the invention is simple and suitable for standardization.

Detailed ways

The construction of embodiment 1 expression vector

Design primers P1: 5'GGAATTCCATATGATGAAACCGACTTCGCTGCTTTGGGC3'; P2: 5'CGCGGATCCTTATTGCGGCAGGGCGAAGACGTAGA3', clone the sdh gene sequence of K.vulgareDSM4205 after amplification and clone it into the G.oxydans-E.coli shuttle plasmid vector pGUC1, and construct the expression vector pGUC1-sdh. After the constructed expression vector was transformed into Escherichia coli JM109, the transformants were selected, the plasmid was extracted and digested with NdeI and BamHI, a 1740bp band appeared, which proved that the expression vector pGUC1-sdh had been successfully constructed. Primers P3: 5'CGCGGATCCATGTTACCCAAATCATTGAAACATAAGA3'; P4: 5'CGAGCTCTCAGCGGG TGGCAGCGG3' were designed, and the sndh gene sequence annotated in the whole genome sequencing results of K. vulgare DSM4205 in our laboratory was amplified and cloned into the shuttle plasmid vector pGUC1 to construct The expression vector pGUC1-sdh-sndh, after transforming the constructed expression vector into Escherichia coli JM109, selecting the transformant, extracting the plasmid and digesting it with BamHI and SacI, a 1830bp band appeared, proving that the expression vector pGUC1-sdh has been successfully constructed -sldh.

The construction of embodiment 2G.oxydans engineering bacteria

Transform the constructed expression vector into E.coli JM109. Since the recombinant plasmid carries the ampicillin resistance gene, transform E.coli JM109 competent, apply to LB containing ampicillin (yeast extract 5g/L, peptone 10g/L, NaCl 10g/L, solid medium plus 20g /L agar, adjust the pH to 7.0, and sterilize at 121°C for 15 minutes), pick the transformants that grow normally on the plate after transformation, and extract the plasmids for PCR verification. Bands of 1740bp and 1830bp appear respectively, and the same bands cannot be obtained by PCR in the control. It is proved that it has been successfully transformed into E.coli, and then the extracted plasmid is transformed into G.oxydans to obtain G.oxydans-pGUC1-sdh-sndh engineering bacteria.

Embodiment 3 fermentation produces 2-KLG

Seed and slant medium (g/L): sorbitol 20, yeast extract 2, calcium carbonate 0.4, pH 4.8-5.1, agar 20 (slant medium), pH 7.0, sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg /mL.

Fermentation medium (g/L): sorbitol 80, yeast extract 5, calcium carbonate 0.2, initial pH 5.1-5.4, sterilized at 121°C for 15 minutes, final concentration of ampicillin 100 μg/mL.

Culture conditions: inoculate the recombinant bacteria from the slant into 20mL seed medium, culture at 30°C and 200rpm for 32h, then add 15% to the fermentation medium, and carry out shake flask fermentation at 34°C and 220rpm, and the fermentation period is 48h. The yield of 2-KLG was 52g/L.

Embodiment 4 fermentation produces 2-KLG

After culturing the engineered bacteria seeds, inoculate a 5L fermenter at a ratio of 15%. After 16 hours of fermentation, the amount of carbon source is 2.5g/L.h. The tank pressure is 0.05MPa, and the 2-KLG yield reaches 83g/L after fermentation.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

sequence listing

<110> Jiangnan University

the

<110> Jiangnan University

the

<120> A method for transforming Gluconobacter oxidans to produce vitamin C precursor 2-keto-L-gulonic acid

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<213> Ketogulonigenium vulgare DSM4205

the

<400> 1

atgaaaccga cttcgctgct ttgggccagt gctggcgcac ttgcattgct tgccgcaccc 60

gcctttgctc aagtgacccc cgtcaccgat gaattgctgg cgaacccgcc cgctggtgaa 120

tggatcagct acggtcagaa ccaagaaaac taccgtcact cgcccctgac gcagatcacg 180

actgagaacg tcggccaact gcaactggtc tgggcgcgcg gcatgcagcc gggcaaagtc 240

caagtcacgc ccctgatcca tgacggcgtc atgtatctgg caaacccggg cgacgtgatc 300

caggccatcg acgccaaaac tggcgatctg atctgggaac accgccgcca actgccgaac 360

atcgccacgc tgaacagctt tggcgagccg acccgcggca tggcgctgta cggcaccaac 420

gtttactttg tttcgtggga caaccacctg gtcgccctcg acaccgcaac tggccaagtg 480

acgttcgacg tcgaccgcgg ccaaggcgaa gacatggttt cgaactcgtc gggcccgatc 540

gtggcaaacg gcgtgatcgt tgccggttcg acctgccaat actcgccgtt cggctgcttt 600

gtctcgggcc acgactcggc caccggtgaa gagctgtggc gcaactactt catcccgcgc 660

gctggcgaag agggtgatga gacttggggc aacgattacg aagcccgttg gatgaccggt 720

gcctggggcc agatcaccta tgaccccgtc accaaccttg tccactacgg ctcgaccgct 780

gtgggtccgg cgtcggaaac ccaacgcggc accccgggcg gcacgctgta cggcacgaac 840

acccgtttcg ccgtgcgtcc tgacacgggc gagattgtct ggcgtcacca gaccctgccc 900

cgcgacaact gggaccagga atgcacgttc gagatgatgg tcaccaatgt ggatgtccaa 960

ccctcgaccg agatggaagg tctgcagtcg atcaacccga acgccgcaac tggcgagcgt 1020

cgcgtgctga ccggcgttcc gtgcaaaacc ggcaccatgt ggcagttcga cgccgaaacc 1080

ggcgaattcc tgtgggcccg tgataccaac taccagaaca tgatcgaatc catcgacgaa 1140

aacggcatcg tgaccgtgaa cgaagatgcg atcctgaagg aactggatgt tgaatatgac 1200

gtctgcccga ccttcttggg cggccgcgac tggccgtcgg ccgcactgaa ccccgacagc 1260

ggcatctact tcatcccgct gaacaacgtc tgctatgaca tgatggccgt cgatcaggaa 1320

ttcacctcga tggacgtcta taacaccagc aacgtgacca agctgccgcc cggcaaggat 1380

atgatcggtc gtattgacgc gatcgacatc agcacgggtc gtacgctgtg gtcggtcgaa 1440

cgtgctgcgg cgaactattc gcccgtcttg tcgaccggcg gcggcgttct gttcaacggt 1500

ggtacggatc gttacttccg cgccctcagc caagaaaccg gcgagaccct gtggcagacc 1560

cgccttgcaa ccgtcgcgtc gggccaggcc atctcttacg aggttgacgg catgcaatat 1620

gtcgccatcg caggtggtgg tgtcagctat ggctcgggcc tgaactcggc actggctggc 1680

gagcgagtcg actcgaccgc catcggtaac gccgtctacg tcttcgccct gccgcaataa 1740

the

the

<210> 2

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<212> DNA

<213> Ketogulonigenium vulgare DSM4205

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<400> 2

atgttaccca aatcattgaa acataagaat ggcgccatgc gccttgtcgc agcctcgacc 60

cttgcgctga tgatcggcgc gggtgcccat gcgcaggtaa acccggtcga agtgccggtg 120

ggcgcgaacg agacctttac ctcgcgcgtg ctgaccaccg gcctgtcgaa cccttgggaa 180

atcacctggg gccccgacaa tatgctgtgg gtgaccgagc gatcttccgg cgaagtgacg 240

cgcgtcgacc ccaataccgg cgagcagcag gtcctgctga ccctgaccga tttcagcgtc 300

gatgtgcaac accagggcct acttggcctc gcgctgcatc ctgagtttat gcaagagagc 360

ggcaacgact acgtctatat cgtctacact tataacaccg gcaccgaaga agcgcccgat 420

ccgcatcaaa agctggtgcg ttatgcctat gacgctgccg cgcagcagct ggtcgatccg 480

gttgatctgg tcgcaggcat tcccgcaggc aacgaccaca atggcggtcg catcaaattc 540

gcccccgatg gccaacacat cttttacacg ctgggcgagc aaggcgcgaa ctttggcggt 600

aacttccgcc gtccgaacca cgcgcaactg ctgccgacgc aagagcaggt cgacgcgggc 660

gattgggtcg cctattcggg caagatcctg cgcgtgaacc ttgacggcac gatccccgaa 720

gacaaccccg agatcgaggg cgtgcgtagc catatcttta cctatggcca ccgtaacccg 780

cagggcatca cctttggccc cgacggcacc atttatgcca ccgaacacgg ccccgatacg 840

gatgacgagc tgaacatcat cgccggcggt ggcaactatg ggtggccgaa tgtggccggc 900

tatcgcgatg gcaaatccta tgtctacgct gattggagcc aagcgcccgc tgaccagcgt 960

tacaccggtc gcgccggtat ccccgacacc gtgccgcaat tccccgagct ggaattcgcg 1020

cccgagatgg tcgatccgct gacaacctat tggacggtgg ataatgatta cgatttcacc 1080

gccaattgcg gctggatctg taatccgacg atcgcgcctt cgtctgccta ttactatgcg 1140

gcgggcgaga gcggtatcgc ggcttgggat aattcgatcc tgatcccgac gctgaaacat 1200

ggcggcatct atgtgcagca cctcagcgat gatggccaat ctgtcgacgg cctgcccgag 1260

ctgtggttca gcacccagaa ccgctatcgc gatatcgaga tcagccccga taaccatgtt 1320

tttgtggcga ccgacaactt tggcacctcg gcgcagaaat atggcgagac cggctttacc 1380

aacgtgctgc ataaccccgg cgcgatcctt gtctttagct atgtcggcga ggatgctgcg 1440

ggtcagaccg gaatgatgac cgcgcccgca ccgcagacgc aatacacgca agtgcccgcc 1500

gagggtgcag gcgcgggcgc gactgaggtt gcggatgtcg attacgacac gctgttcacc 1560

gaaggccaga ccctttatgg cagcgcatgt gccgcgtgcc atggtgccgc tggccaaggt 1620

gcgcagggcc cgacctttgt gggcgtgccg gatgtgacgg gtgacaagga ctaccttgcc 1680

cgcaccatca tccacggttt tggctatatg ccgtcgtttg cgactcggct ggatgacgag 1740

gaggttgccg ccatcgcgac ctttatccgc aacagctggg gcaatgacga aggcatcctg 1800

accccggccg aggccgctgc cacccgctga 1830

the

<210> 3

<211> 39

<212> DNA

<213> Synthetic sequences

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<220>

<223> Designed according to gene sequence for gene amplification.

the

<400> 3

ggaattccat atgatgaaac cgacttcgct gctttgggc 39

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<210> 4

<211> 35

<212> DNA

<213> Synthetic sequences

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<220>

<223> Designed according to gene sequence for gene amplification.

the

<400> 4

cgcggatcct tattgcggca gggcgaagac gtaga 35

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<210> 5

<211> 37

<212> DNA

<213> Synthetic sequences

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<220>

<223> Designed according to gene sequence for gene amplification.

the

<400> 5

cgcggatcca tgttacccaa atcattgaaa cataaga 37

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<210> 6

<211> 24

<212> DNA

<213> Synthetic sequences

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<220>

<223> Designed according to gene sequence for gene amplification.

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cgagctctca gcgggtggca gcgg 24

the

Claims (7)

1. the oxidizing glucose acidfast bacilli of a high yield 2-KLG (Gluconobacter oxydans) genetic engineering bacterium is characterized in that expressing external source sdh, sndh gene.

2. the described genetic engineering bacterium of claim 1 is characterized in that described sdh gene nucleotide series is shown in SEQ IDNO.1.

3. the described genetic engineering bacterium of claim 1 is characterized in that described sndh gene nucleotide series is shown in SEQID NO.2.

4. the described genetic engineering bacterium of claim 1 is characterized in that described sdh, sndh gene clone are on G.oxydans-E.coli shuttle vector pGUC1.

5. make up the method for the described genetic engineering bacterium of claim 1, it is characterized in that comprising the steps:

1) design primer clone sdh, sndh gene nucleotide series;

2) recombinant expression vector is arrived in sdh, sndh gene clone;

3) will obtain recombinant bacterial strain behind the recombinant expression vector conversion oxidizing glucose acidfast bacilli (Gluconobacter oxydans) that obtain.

6. application rights requires the method that 1 described genetic engineering bacterium is produced 2-KLG, after it is characterized in that the engineering bacteria seed is cultivated, in 15% ratio inoculation 5L fermentor tank, stream dosage carbon source 2.5g/L.h behind the fermentation 16h, fermentation parameter: rotating speed 500rpm, pH 5.1～5.4, air flow 1.5vvm, 34 ℃ of controlling tank temperature, tank pressure 0.05MPa.

7. the described method of claim 6 is characterized in that described carbon source is a sorbyl alcohol.