CN114410562B - Klebsiella engineering bacterium and application thereof in ethanol production - Google Patents
Klebsiella engineering bacterium and application thereof in ethanol production Download PDFInfo
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Abstract
The invention discloses a Klebsiella engineering bacterium and application thereof in ethanol production, comprising a host bacterium and a plasmid vector transferred into the host bacterium, wherein an oxidoreductase subunit gene for catalyzing short-chain alcohol synthesis is introduced into the plasmid vector, or an acetaldehyde dehydrogenase large subunit gene is introduced into the plasmid vector, and over-expression of the two genes is realized. The invention also provides a construction method and application of the engineering bacteria for producing ethanol. The obtained engineering strain keeps the fermentation performance of xylose and glucose in the lignocellulose hydrolysate, obtains the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase and the output of alcohol, and realizes the effective utilization of the lignocellulose hydrolysate and the efficient production of bioethanol.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to Klebsiella engineering bacteria for synthesizing bioethanol, a construction method thereof and application thereof in cellulosic ethanol production.
Background
In recent years, the production of ethanol from lignocellulose resources as a raw material has become a research focus in the field of fuel ethanol because of the abundance and reproducibility of lignocellulose resources. How to improve the process efficiency and reduce the production cost is an important point of efficient production of the cellulose ethanol and is also a key for determining industrialization of the cellulose ethanol. Saccharomyces cerevisiae or Zymomonas mobilis are the main microbial strains used for ethanol fermentation, but the strain can not decompose and utilize cellulose, and can not effectively transform and utilize xylose in lignocellulose hydrolysate, so that the strain has no advantage in the production of cellulose fuel ethanol. Klebsiella (Klebsiella sp.) is an important fermentation strain in industrial production, used in the fermentation production of various bio-based chemicals such as 1, 3-propanediol, 2, 3-butanediol, biohydrogen, etc. In addition, klebsiella has a broad matrix utilization characteristic, and particularly, some wild strains can utilize lignocellulose hydrolysate to perform fermentation production of bio-based chemicals, for example, one strain Klebsiella oxytoca is reported to be capable of fermenting corncob acidolysis solution to produce 2, 3-butanediol, and one strain Klebsiella oxytoca is reported to be capable of fermenting leprosy bark acidolysis solution to produce 2, 3-butanediol. Among Klebsiella, ethanol is an important metabolite accompanying the production of some bio-based chemicals, for example, a strain of Klebsiella (Klebsiella sp.wl1316) screened in the laboratory can efficiently synthesize bio-hydrogen by using glucose and xylose in lignocellulose hydrolysate, and can accumulate ethanol with higher concentration in the hydrogen production process of fermentation. However, to date, few studies have involved the use of Klebsiella engineering bacteria for bioethanol production.
Although Klebsiella sp.wl1316 wild bacteria in the laboratory can accumulate ethanol with higher concentration in the process of hydrogen production by fermentation, the metabolic flow distribution in the branch of biohydrogen synthesis is the highest. Therefore, based on the whole genome information of Klebsiella sp.WL1316 (the genome data is submitted to NCBI SRA database and BioProject accession number PRJNA 611005), the enhancement of the ethanol anabolism pathway of the strain is realized by homologous over-expression of the key enzymes in the ethanol synthesis pathway, namely the adh gene of the ethanol dehydrogenase and the aldh gene of the acetaldehyde dehydrogenase, so that the engineering strain capable of effectively utilizing lignocellulose hydrolysis sugar liquor and efficiently synthesizing ethanol is obtained, and the efficient engineering strain and the low-cost production mode are provided for the production of cellulose ethanol.
Disclosure of Invention
The invention aims to solve the technical problems that: how to reconstruct wild Klebsiella sp.WL1316, and strengthen the ethanol anabolic pathway of the strain by homologous over-expression of key enzymes in the ethanol synthesis pathway.
In order to solve the technical problems, the invention provides the following technical scheme:
an engineering bacterium of klebsiella, wherein the engineering bacterium overexpresses an oxidoreductase subunit adh gene and an acetaldehyde dehydrogenase large subunit aldh gene which catalyze short-chain alcohol synthesis, and the amino acid sequence of the oxidoreductase subunit which catalyzes short-chain alcohol synthesis is shown as SEQ ID NO. 1; the amino acid sequence of the large subunit of the acetaldehyde dehydrogenase is shown as SEQ ID NO. 2;
the nucleotide sequence of the adh gene encoding the subunit of the oxidoreductase catalyzing the synthesis of the short-chain alcohol is shown as SEQ ID NO.3, and the nucleotide sequence of the aldh gene encoding the large subunit of the acetaldehyde dehydrogenase is shown as SEQ ID NO. 4.
The preparation method of the Klebsiella engineering bacteria comprises the following specific steps:
(1) Extracted genome DNA of klebsiella;
(2) Using genome DNA as a template, and adopting primer pairs ADF1/ADR1 and ALF1/ALR1 to respectively amplify an oxidoreductase subunit adh gene and an acetaldehyde dehydrogenase large subunit aldh gene for catalyzing the synthesis of short-chain alcohol;
(3) Connecting the purified gene amplification fragment with a pUCm-T vector, transforming escherichia coli DH5 alpha competent cells, screening positive clones, and sequencing and verifying;
(4) Adopting a primer pair ADF11/ADR11 and ALF12/ALR12 to amplify an adh gene and an aldh gene containing enzyme cutting sites respectively, transforming, screening positive clones according to the method, and sequencing and verifying to obtain recombinant plasmids adh-pUCm-T or aldh-pUCm-T;
(5) After double enzyme digestion of recombinant plasmids adh-pUCm-T or aldh-pUCm-T, connecting a target gene fragment with a pET-28a vector, transforming escherichia coli DH5 alpha competent cells, screening resistance (Kan), constructing an over-expression plasmid adh-pET-28a or aldh-pET-28a, and sequencing and verifying;
(6) Amplifying and extracting adh-pET-28a or aldh-pET-28a, and further converting the competent cells of the klebsiella to obtain the klebsiella engineering bacteria AD or AL.
Preferably, the primer sequences used for the adh gene cloning are as follows:
ADF1：5’-TGTTTATCACCGGGGCGACCT-3’
ADR1：5’-GCATCACCTCCACCCGGTTAA-3’
the primer sequences used for the aldh gene cloning are as follows:
ALF1：5’-ATGCGTAAACGTAAAGTCGCC-3’
ALR1：5’-TCATGCTGTCGCTCCCGCC-3’
the primer sequences used for subcloning the adh gene are as follows:
ADF11：5’-TGAATTCGTTTATCACCGGGGCGACCT-3’
ADR11：5’-CTCGAGGCATCACCTCCACCCGGTTAA-3’
the primer sequences used for subcloning the aldh gene are as follows:
ALF11：5’-GCGGCCGCATGCGTAAACGTAAAGTCGCC-3’
ALR11：5’-CTCGAGTCATGCTGTCGCTCCCGCC-3’。
the application of the Klebsiella engineering bacteria in ethanol production comprises the following specific steps:
inoculating 3mL of seed culture medium containing kanamycin resistance to the activated Klebsiella engineering bacteria single colony for overnight culture; inoculating the strain into fresh 30mL seed culture medium for about 2h, adding filtered sterilized IPTG to a final concentration of 1mmol/L, and continuously performing induction culture at 37 ℃ for 6h; further inoculating a fermentation medium according to 10% of inoculum size, wherein the initial sugar concentration of the fermentation medium is 50g/L, the initial pH is 7.5, the IPTG induction concentration is 1mmol/L, performing shake culture at 37 ℃ and 180r/min for 12 hours, transferring to a second-stage anaerobic fermentation, and performing constant-temperature culture at 37 ℃ for 72 hours; the fermentation supernatant was collected by centrifugation and used to detect ethanol, glucose and xylose concentrations.
The beneficial effects obtained by the invention are as follows: according to the invention, through over-expressing the key enzyme genes of 2 ethanol anabolism branches in klebsiella, the engineering strain capable of synthesizing ethanol by utilizing lignocellulose hydrolysate is obtained, and the ethanol yield and key enzyme activity of the engineering strain in the process of fermenting lignocellulose hydrolysate to produce ethanol can reach higher level. The engineering bacteria constructed by the invention have better application prospect.
Drawings
FIG. 1 is a map of the adh-pET-28a (a) and aldh-pET-28a (b) overexpression plasmids of the invention.
FIG. 2 shows the double cleavage of the adh-pET-28a (a) and aldh-pET-28a (b) overexpressing plasmids of the invention (bands of interest indicated by arrows).
FIG. 3 shows SDS-PAGE patterns (bands indicated by arrows) of expressed proteins of the AD engineering bacteria (a) and the AL engineering bacteria (b) of the present invention.
FIG. 4 shows ethanol yield and reducing sugar utilization of the AD engineering bacteria of the invention.
FIG. 5 shows the ethanol yield and the reducing sugar utilization rate of the AL engineering bacteria of the invention.
FIG. 6 shows the alcohol dehydrogenase activity and the bacterial growth OD of the AD-engineered bacterium of the present invention 600 A curve.
FIG. 7 shows the alcohol dehydrogenase activity and the bacterial growth OD of the AL-engineering bacteria of the present invention 600 A curve.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate a more complete, accurate and thorough understanding of the present invention's inventive concepts and technical solutions by those skilled in the art.
The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents, materials, kits and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
The following examples relate to reagents used in molecular biology procedures, bacterial genomic DNA extraction, T-vector PCR product cloning, plasmid extraction, PCR product purification kits, and DNA fragment recovery kits, all of which are manufactured by Shanghai, inc.; the restriction enzymes and T4 DNA ligase involved in the examples are Takara products of Takara Shuzo Co., ltd; the DNA synthesis and sequencing work involved in the examples was done by the biological engineering (Shanghai) Co., ltd.
Example 1: construction of overexpression strains adh-pET-28a-Klebsiella sp.WL1316 (AD) and aldh-pET-28a-Klebsiella sp.WL1316 (AL):
the host bacterium is Klebsiella capable of producing hydrogen by utilizing lignocellulose hydrolysate for fermentation, and screening of hydrogen-producing bacteria Klebsiella sp.WL1316 and metabolic regulation of synthesizing biological hydrogen by fermenting cotton stalk hydrolysate (2018) of a doctor paper of Li Yanbin are used for elaborating screening sources, fermentation matrix utilization, hydrogen production characteristics and genome information of the strain. Genomic data of this strain has been submitted to the NCBI SRA database and obtained with bioprject accession number PRJNA611005. The metabolic pathway reconstructed based on genome information shows that the Klebsiella ethanol synthesis and the biological hydrogen synthesis are both derived from a mixed acid fermentation pathway, and therefore, the key enzyme genes which overexpress the ethanol synthesis branch are expected to enhance the metabolism branch of ethanol synthesis, thereby improving the ethanol yield.
The functional genes of the strain are predicted and analyzed based on genome information, and the key enzyme genes of the ethanol synthesis branch are selected to be the oxidoreductase subunit (adh) gene and the acetaldehyde dehydrogenase large subunit (aldh) gene for catalyzing the synthesis of short-chain alcohols, so that the two genes are over-expressed in klebsiella.
Since Klebsiella and Escherichia coli belong to the classified gram-negative bacteria of the Enterobacteriaceae facultative anaerobism, a prokaryotic expression vector construction scheme similar in genetic manipulation technology to Escherichia coli but different in screening strategy is adopted, specifically as follows:
the genome DNA of klebsiella extracted by using Ezup column type bacterial genome DNA extraction kit is used as a template, and a primer pair ADF1/ADR1 and ALF1/ALR1 are used for respectively amplifying an oxidoreductase subunit adh gene and an acetaldehyde dehydrogenase large subunit aldh gene which catalyze the synthesis of short-chain alcohol, and the PCR reaction conditions are as follows: 2 XPCR Master 10. Mu.L, primer 1 (ADF 1 or ALF 1) 0.8. Mu.L, primer 1 (ADR 1 or ALR 1) 0.8. Mu.L, template DNA0.8μL，ddH 2 O7.6 μl; adh gene PCR procedure: (1) 95 ℃ for 5min; (2) 94℃1min,58℃ 30S,72℃50S,30 cycles; (3) 72 ℃ for 7min; aldh gene PCR procedure: (1) 95 ℃ for 5min; (2) 94℃1min,60℃ 50S,72℃1min,30 cycles; (3) the purified gene was ligated with pUCm-T vector at 72℃for 10min, E.coli DH 5. Alpha. Competent cells were transformed, positive clones were selected and submitted to Bio-company sequencing verification. Further adopting primer pairs ADF11/ADR11 and ALF12/ALR12 to respectively amplify adh gene and aldh gene containing enzyme cutting site, and adopting the above-mentioned method to make conversion, screening positive clone and sequencing verification. Cloning and subcloning both yielded an adh gene of about 700bp and an aldh gene of about 1000bp, consistent with the expected target band size, indicating that both cloning and subcloning products of these genes were obtained.
The primer sequences used for adh gene cloning were as follows:
ADF1：5’-TGTTTATCACCGGGGCGACCT-3’
ADR1：5’-GCATCACCTCCACCCGGTTAA-3’
the primer sequences used for the aldh gene cloning are as follows:
ALF1：5’-ATGCGTAAACGTAAAGTCGCC-3’
ALR1：5’-TCATGCTGTCGCTCCCGCC-3’
the primer sequences used for subcloning the adh gene are as follows:
ADF11：5’-TGAATTCGTTTATCACCGGGGCGACCT-3’
ADR11：5’-CTCGAGGCATCACCTCCACCCGGTTAA-3’
the primer sequences used for subcloning the aldh gene are as follows:
ALF11：5’-GCGGCCGCATGCGTAAACGTAAAGTCGCC-3’
ALR11：5’-CTCGAGTCATGCTGTCGCTCCCGCC-3’
the cloning plasmids adh-pUCm-T and aldh-pUCm-T constructed above were digested with EcoR I/Xho I and Not I/Xho I, respectively, and ligated with the pET-28a vector digested with the same double digestion to transform competent cells of E.coli DH 5. Alpha. And selected for resistance (Kan), thereby constructing overexpression plasmids adh-pET-28a (a) and aldh-pET-28a, as shown in FIG. 1. The constructed over-expression plasmid is subjected to bacterial liquid PCR, plasmid PCR detection and double enzyme digestion verification, and an electrophoresis chart of double enzyme digestion detection of the plasmid adh-pET-28a (a) and the aldh-pET-28a is shown in the attached figure 2, which shows that the enzyme digestion obtains a target strip with the expected size, and the target strip is further delivered to a biological company for sequencing verification. The successfully constructed expression plasmids adh-pET-28a and aldh-pET-28a are further transformed into Klebsiella competent cells to realize homologous over-expression of adh genes and aldh genes, and the successfully constructed strains are the adh-pET28a-Klebsiella sp.WL1316 (AD) and aldh-pET28a-Klebsiella sp.WL1316 (AL) engineering bacteria obtained through double digestion and sequencing verification. The AD engineering bacteria overexpress an oxidoreductase subunit adh gene for catalyzing the synthesis of short-chain alcohol, the AL engineering bacteria overexpress an acetaldehyde dehydrogenase large subunit aldh gene, and the amino acid sequence of the oxidoreductase subunit for catalyzing the synthesis of short-chain alcohol is shown as SEQ ID NO. 1; the amino acid sequence of the large subunit of the acetaldehyde dehydrogenase is shown as SEQ ID NO. 2;
the nucleotide sequence of the adh gene encoding the subunit of the oxidoreductase catalyzing the synthesis of the short-chain alcohol is shown as SEQ ID NO.3, and the nucleotide sequence of the aldh gene encoding the large subunit of the acetaldehyde dehydrogenase is shown as SEQ ID NO. 4.
Example 2: AD and AL engineering bacteria induce enzyme production, and SDS-PAGE electrophoresis is adopted to detect the expression condition of two enzyme genes:
culturing wild fungus and AD and AL engineering bacteria by streaking respectively, picking single colony, inoculating in 10mL LB culture medium (containing kanamycin) at 37deg.C, and culturing at 180r/min overnight; inoculating 5mL of the bacterial liquid into fresh 100mL of LB culture medium, culturing for 2h, further adding 1mol/L IPTG to make the final concentration of the IPTG be 1mmol/L, continuously performing induction culture at 37 ℃ for 6h, taking 20mL of bacterial liquid, and centrifugally collecting bacterial bodies for later use; extracting total proteins of engineering bacteria and wild bacteria by using a bacterial total protein extraction kit according to the operation steps of a kit instruction, and freezing for later use; preparing a PAGE gel (the concentration of a prepared separation gel is 12 percent according to the expected molecular weight of the target protein) by adopting an SDS-PAGE denatured acrylamide gel rapid preparation kit; mixing the protein collection liquid with 5×protein loading buffer, boiling for 3min, loading sample, and electrophoresis; further staining with coomassie brilliant blue staining solution, decolorizing, and photographing. The SDS-PAGE electrophoresis is shown in figure 3, and the result shows that the AD and AL engineering bacteria have a certain deepening in the bands with the sizes close to 25 kDa and 35kDa compared with the wild bacteria, and the preliminary indication shows that the adh and aldh genes realize homologous over-expression in the Klebsiella.
Example 3: AD engineering bacteria and AL engineering bacteria fermented straw hydrolysate for producing ethanol and reducing sugar utilization change
Inoculating 3mL of seed culture medium (containing Kan) to the activated single colony of the wild bacteria and the engineering bacteria for overnight culture; the next day was inoculated into fresh 30mL of seed medium, cultured for about 2 hours, and the filtration sterilized IPTG was added to a final concentration of 1mmol/L, and the induction culture was continued at 37℃for 6 hours. Further inoculating fermentation medium (initial sugar concentration 50g/L, initial pH 7.5, IPTG induction concentration 1 mmol/L) according to 10% inoculum size, shake culturing at 37deg.C and 180r/min for 12 hr, and transferring into second stage anaerobic fermentation (37deg.C) to 72 hr; taking fermentation liquor every 12 hours, centrifuging and collecting fermentation supernatant, and detecting the concentration of ethanol, glucose and xylose. The ethanol concentration was measured using a K-ETOH ethanol assay kit (Megazyme, ireland), and the glucose and xylose concentrations were quantitatively measured using HPLC in combination with the DNS method and the lichenin colorimetric method. The ethanol concentration of the AD engineering bacteria fermented for 72 hours is monitored, the result is shown in figure 4, and from the ethanol concentration change curve, the ethanol concentration of the AD engineering bacteria is almost higher than that of wild bacteria (except for 24 hours of fermentation) in the fermentation period of 72 hours, especially 48 hours of fermentation reaches the highest peak value, and the ethanol yield can reach 7.54+/-0.55 g/L, which is improved by about 111% compared with the treatment of the wild bacteria at the same time; and the glucose and xylose utilization rate is reduced to a certain extent compared with the wild fungus treatment. The glucose and xylose utilization rate of the AL engineering bacteria is close to that of the wild bacteria, the ethanol yield is higher than that of the control bacteria in 36-60 h of fermentation, and the ethanol with the highest concentration is obtained in 60h of fermentation, and the ethanol yield reaches 4.92+/-0.03 g/L, which is improved by 18% compared with the wild bacteria treatment (figure 5).
Example 4: changes in key enzyme activity and thallus growth of AD engineering bacteria and AL engineering bacteria fermented straw hydrolysate
AD engineering bacteria and AL engineering bacteria were fermented in the manner of example 3, and the fermentation liquid was obtained every 12 hours, and a part of the fermentation liquid was used for the cell OD 600 Detecting, centrifuging the other part, collecting bacterial cells, washing, crushing cells with an ultrasonic cell disrupter, and removing with ethanolExtracting buffer solution in a hydrogenase activity detection kit and an acetaldehyde dehydrogenase activity detection kit (Suzhou Ming Biotechnology Co., ltd.) and detecting ethanol dehydrogenase activity and acetaldehyde dehydrogenase activity according to the operation steps of the kit instruction; thallus OD 600 Detection was performed at 600nm using a spectrophotometer. As can be seen from FIG. 6, the growth OD of the AD engineering bacteria 600 The values were significantly lower than that of wild bacteria, with a reduction of about 2-fold, similar to the behavior of some other microorganisms in the industry, such as Saccharomyces cerevisiae, pseudomonas mobaraensis, expressing genes related to alcohol dehydrogenase, causing a significant decrease in the growth of the cells, which may also be responsible for the decrease in glucose and xylose utilization of the AD engineering bacteria in example 3; from the change of the enzyme activity curve, the activity of the alcohol dehydrogenase of the AD engineering bacteria in the initial fermentation period (12 h) is obviously higher than that of the wild bacteria, the enzyme activity value of the AD engineering bacteria is improved by about 41 times compared with that of the wild bacteria, and the enzyme activity with high fermentation time point can promote the accumulation of the ethanol, so that the accumulated ethanol can reach a peak value in 48h in spite of the fact that the activity of the alcohol dehydrogenase of the AD engineering bacteria is similar to or slightly higher than that of the wild bacteria; in addition, there are different kinds of alcohol dehydrogenases in klebsiella, one of which can cause decomposition of alcohol, which is also the reason why the accumulation of alcohol is reduced after reaching the peak value, which is similar to the activity change and the rule of alcohol dehydrogenase of other microorganisms in industry such as saccharomyces cerevisiae and anaerobic thermophilic bacillus. As can be seen from FIG. 7, the growth of the AL engineering bacteria is slightly lower than that of the wild bacteria in the whole fermentation period; from the change of the enzyme activity curve, the activity of the acetaldehyde dehydrogenase of the AL engineering bacteria is closer to that of the wild bacteria in the whole fermentation period, and is only slightly higher than that of the wild bacteria in the later fermentation period (60-72 h), which is probably the reason for the improvement of the ethanol yield of the engineering strain in the fermentation of about 60h in the embodiment 3. As a facultative anaerobic bacterium, klebsiella synthesizes ethanol through a mixed acid fermentation pathway, while acetaldehyde dehydrogenase is mainly a key enzyme of acetic acid and ethanol synthesis branches, the expression of the large subunit gene of acetaldehyde dehydrogenase in this study only causes a slight increase in the activity of acetaldehyde dehydrogenase in the late stage of fermentation, so that the overexpression of this gene may mainly cause an increase in the metabolic flux distribution of this metabolic branch, thereby achievingTo promote ethanol synthesis.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by the above embodiments, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
Sequence listing
<110> university of Anhui engineering
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Arg Val Ala Lys Val His Tyr Ala Glu Ile Val Ala Ser Ile Ala Ser
145 150 155 160
Gln Ser Ala Gly Pro Gly Thr Arg Ala Asn Ile Asp Glu Phe Thr Glu
165 170 175
Thr Thr Ser Gln Ala Ile Glu Lys Val Gly Gly Ala Gly Lys Gly Lys
180 185 190
Ala Ile Ile Val Leu Asn Pro Ala Glu Pro Pro Leu Met Met Arg Asp
195 200 205
Thr Val Tyr Val Leu Ser Glu Leu Ala Ser Gln Glu Ala Ile Ala Ala
210 215 220
Ser Ile Ala Glu Met Ala Ala Ala Val Gln Ala Tyr Val Pro Gly Tyr
225 230 235 240
Arg Leu Lys Gln Gln Val Gln Phe Glu Val Ile Pro Glu Asp Lys Pro
245 250 255
Val Asn Leu Pro Gly Val Gly Cys Phe Ser Gly Leu Lys Thr Ala Val
260 265 270
Tyr Leu Glu Val Glu Gly Ala Ala His Tyr Leu Pro Ala Tyr Ala Gly
275 280 285
Asn Leu Asp Ile Met Thr Ser Ala Ala Leu Ala Thr Ala Glu Gln Met
290 295 300
Ala Gly Ala Met His Ser Ala Ala Gly Ala Thr Ala
305 310 315
<210> 3
<211> 702
<212> DNA
<213> Klebsiella (Klebsiella sp.)
<400> 3
tgtttatcac cggggcgacc tcgggttttg gcgaagcggc agcgcaggtg tttgccgacg 60
cgggctggtc gctggtgctg agcgggcgcc gctatccgcg actgaaggcc ttgcaggaca 120
gactcgccgc cagggtgccg gtgcatatta ttgagcttga cgttcgtgac agcgaggcgg 180
tggctgccgc cgtggcgtcg ctgcccgccg atttcgccga tatcaccacc ctgatcaaca 240
acgccgggct ggcgctgtcg ccgctgccgg cgcaggaggt ggcgctggag gactggaaaa 300
cgatgatcga caccaacgtc accggtctgg tgacagtcac tcacgccctg ctgccgacgc 360
tgatccgcca tggcgccggg gcgagtatta tcaatatcgg ctcgatcgcc gggcagtggc 420
cctatcccgg cagccatgtg tatggcgcca gcaaggcgtt tgtgaagcag ttcagctata 480
acctgcgctg cgatctgctt ggcaccggcg tgcgggtcac cgatctggcg ccggggattg 540
ccgagaccga atttaccctg gtgcgaacca aaggcgacca ggcggcatcg gataagctct 600
atcgcggcac cacgccgctg agcgcgagcg atatcgccga gcagatgttt tatatcgcca 660
cgctgccggc gcatatgaac attaaccggg tggaggtgat gc 702
<210> 4
<211> 951
<212> DNA
<213> Klebsiella (Klebsiella sp.)
<400> 4
atgcgtaaac gtaaagtcgc cattatcggc tccgggaata tcggcaccga tttaatgatt 60
aaaatcctgc gccacggcca gcatctggag atggcggtga tggtcggcat cgacccgcag 120
tcggatggcc tggcccgcgc ccggcgcctg ggcgttgcga ccactcacga aggggtgggc 180
ggcctgatgc agatggcgga atttgccgac atcgattttg tttttgacgc caccagcgcc 240
ggggcgcaca tcaaaaacga tgccgcgctg cgcgaagcga agccggggat ccgcgttatc 300
gatctgacgc cggcggcgat tggcccgtac tgcgtgccgg tggtcaacct cgcggccaac 360
ctccaccagg gcaacgtcaa tatggtgacc tgcggcgggc aggcgaccat cccgatggtc 420
gccgcggtct cgcgagtggc gaaggtgcat tacgcggaga ttgtggcctc gatcgccagc 480
caatccgccg ggccgggcac gcgggccaat attgacgaat tcaccgaaac cacctcgcag 540
gcgattgaaa aagtcggcgg ggcgggtaag ggcaaagcga ttattgtact gaacccggcg 600
gagccgccgc tgatgatgcg cgataccgtc tacgtcctga gcgagctggc gtcgcaggag 660
gcgattgccg cctcgatcgc cgaaatggcg gcagcggtgc aggcttacgt cccgggctac 720
cggctgaagc agcaggtgca gtttgaggtt atccccgagg ataagccggt caacctgccc 780
ggcgtcggct gtttttccgg cctgaaaacc gcggtgtacc tcgaagtgga aggggcggcg 840
cactatctgc cagcgtacgc gggcaacctc gatattatga cctcggcggc gctggcgacg 900
gcggagcaaa tggccggggc aatgcacagc gcggcgggag cgacagcatg a 951
Claims (4)
1. Klebsiella spKlebsiellasp.) engineering bacteria characterized in that the engineering bacteria overexpress large subunits of acetaldehyde dehydrogenasealdhThe amino acid sequence of the large subunit of the acetaldehyde dehydrogenase is shown as SEQ ID NO. 2;
encoding said acetaldehyde dehydrogenase large subunitaldhThe nucleotide sequence of the gene is shown as SEQ ID NO. 4.
2. A method for preparing the engineering bacteria of klebsiella as claimed in claim 1, which is characterized by comprising the following specific steps:
(1) Extracted genome DNA of klebsiella;
(2) Amplifying acetaldehyde dehydrogenase large subunit by using genome DNA as template and adopting primer pair ALF1/ALR1aldhA gene;
(3) Connecting the purified gene amplification fragment with a pUCm-T vector, transforming escherichia coli DH5 alpha competent cells, screening positive clones, and sequencing and verifying;
(4) Amplification of cleavage sites by primer pair ALF11/ALR11aldhThe gene is transformed, screened positive clone and sequenced to verify to obtain recombinant plasmidaldh-pUCm-T；
(5) Recombinant plasmidaldh-Connecting a target gene fragment with a pET-28a vector after pUCm-T double enzyme digestion, transforming escherichia coli DH5 alpha competent cells, screening kanamycin resistance, and constructing an overexpression plasmidaldhpET-28a, sequencing verification;
(6) Amplification extractionaldhAnd (3) pET-28a, and further converting the Klebsiella competent cells to obtain the Klebsiella engineering bacteria.
3. The preparation method of the Klebsiella engineering bacterium according to claim 2, which is characterized in that:
aldhthe primer sequences used for gene cloning were as follows:
ALF1：5’-ATGCGTAAACGTAAAGTCGCC-3’
ALR1：5’-TCATGCTGTCGCTCCCGCC-3’
aldhprimer sequences used for gene subcloning were as follows:
ALF11：5’-GCGGCCGC ATGCGTAAACGTAAAGTCGCC-3’
ALR11：5’-CTCGAGTCATGCTGTCGCTCCCGCC-3’。
4. the use of the klebsiella engineering bacterium according to claim 1 in ethanol production, comprising the following specific steps:
inoculating a single colony of the activated Klebsiella engineering bacteria to a 3mL seed culture medium containing kanamycin resistance for overnight culture; inoculating to fresh 30mL seed culture medium the next day, culturing for 2h, adding filtered sterilized IPTG to a final concentration of 1mmol/L, and continuing to induce culture at 37deg.C for 6h; further inoculating fermentation medium with initial sugar concentration of 50g/L, initial pH of 7.5 and IPTG induction concentration of 1mmol/L according to 10% inoculum size, shake culturing at 37deg.C and 180r/min for 12h, transferring into second stage anaerobic fermentation, and culturing at 37deg.C to 72h; the fermentation supernatant was collected by centrifugation and used to detect ethanol, glucose and xylose concentrations.
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| CN101775363A (en) * | 2000-10-06 | 2010-07-14 | 埃尔斯沃思生物技术有限公司 | Ethanol production |
| CN104126011A (en) * | 2011-11-30 | 2014-10-29 | 帝斯曼知识产权资产有限公司 | Yeast strains engineered to produce ethanol from acetic acid and glycerol |
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| CN1065915C (en) * | 1991-03-18 | 2001-05-16 | 佛罗里达大学 | Ethanol production by recombinant hosts |
| AU4744199A (en) * | 1991-03-18 | 1999-11-04 | Bioenergy International, L.C. | Ethanol production by recombinant hosts |
| US6280986B1 (en) * | 1997-12-01 | 2001-08-28 | The United States Of America As Represented By The Secretary Of Agriculture | Stabilization of pet operon plasmids and ethanol production in bacterial strains lacking lactate dehydrogenase and pyruvate formate lyase activities |
| WO2008021141A2 (en) * | 2006-08-09 | 2008-02-21 | The University Of Florida Research Foundation, Inc. | Re-engineering bacteria for ethanol production |
| EP2334799B1 (en) * | 2008-07-24 | 2012-06-06 | Biogasol IPR APS | Increased ethanol production in recombinant bacteria |
| WO2010062707A1 (en) * | 2008-10-30 | 2010-06-03 | Joule Unlimited, Inc. | Methods and compositions for producing carbon-based products of interest in micro-organisms |
| DK2446019T3 (en) * | 2009-06-23 | 2015-06-15 | Bp Corp North America Inc | Recombinant ethanologist bacterium |
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| CN101775363A (en) * | 2000-10-06 | 2010-07-14 | 埃尔斯沃思生物技术有限公司 | Ethanol production |
| CN104126011A (en) * | 2011-11-30 | 2014-10-29 | 帝斯曼知识产权资产有限公司 | Yeast strains engineered to produce ethanol from acetic acid and glycerol |
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