CN113174355A - Method for improving yield and production intensity of gluconobacter oxydans 1, 3-dihydroxyacetone - Google Patents
Method for improving yield and production intensity of gluconobacter oxydans 1, 3-dihydroxyacetone Download PDFInfo
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Abstract
The invention discloses a method for improving the yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone, and belongs to the technical field of fermentation engineering. According to the invention, a dehydrogenase gene which has potential influence on metabolic flux of 1, 3-dihydroxyacetone is knocked out from gluconobacter oxydans, and the efficiency of converting substrate glycerol into 1, 3-dihydroxyacetone is enhanced, so that the yield and the production intensity of the 1, 3-dihydroxyacetone are improved. Compared with the control strain G.oxydans WSH-003, the recombinant strains G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 have obviously improved 1, 3-dihydroxyacetone yield, conversion rate and production intensity.
Description
Technical Field
The invention relates to a method for improving the yield and production intensity of gluconobacter oxydans 1, 3-dihydroxyacetone, belonging to the technical field of fermentation engineering.
Background
1, 3-Dihydroxyacetone (DHA) is the simplest three-carbon ketose, is white or milk white powdery crystal in appearance, has sweet and cool taste, and is easy to absorb moisture and decompose. 1, 3-dihydroxyacetone forms a film on the skin to prevent water from evaporating, and 1, 3-dihydroxyacetone also reacts with the cells of the stratum corneum of the skin surface to change the color of the skin surface to brown, which can achieve a similar effect to sun exposure, and thus can be used as a sunscreen agent in cosmetics. The effect can also reduce skin diseases such as leukoplakia and vitiligo caused by excessive ultraviolet irradiation.
The production method of 1, 3-dihydroxyacetone mainly comprises a chemical synthesis method and a microbial synthesis method. The chemical synthesis methods are characterized in that it is difficult to find a suitable balance between the production conditions and the original costs, because the raw material costs are high when the production conditions are relatively simple, the production conditions are high when the raw material costs are low, and noble metals are also required in the synthesis process. The method for producing 1, 3-dihydroxyacetone has many disadvantages including low product purity, many byproducts, serious environmental pollution and high product separation and purification cost, so that the chemical method for producing 1, 3-dihydroxyacetone is increasingly limited. In contrast, the microbial synthesis method is mainly used in industry because of its mild reaction conditions, strong specificity and high substrate utilization rate. In addition, the microbial fermentation process has little environmental pollution, the product is relatively easy to separate and purify, and the production cost is lower. From the aspects of technical economy and environmental friendliness, the microbial fermentation method can well avoid the defects of a chemical synthesis method and has relatively more development potential; in addition, 1, 3-dihydroxyacetone can be produced by converting glycerol into 1, 3-dihydroxyacetone by a microbial fermentation method by taking gluconobacter oxydans as a host in the industrial production process, and the method has the advantages of simple operation flow process, easy operation and control, low cost and short production period, so the method becomes a main means for producing 1, 3-dihydroxyacetone at home and abroad at present.
There are many polyol dehydrogenases in gluconobacter oxydans, and these dehydrogenases will also be referred to as glycerol dehydrogenases in general. The enzyme can oxidize sugar alcohol with Bertrand-Hudson conformation, such as glycerol, sorbitol, gluconic acid, etc. Most of these enzymes are ethanol, sorbitol and glycerol dehydrogenases, and most of their prosthetic groups are Pyrroloquinoline (PQQ). Wherein glycerol is the simplest molecule with the conformation, and the production of 1, 3-dihydroxyacetone by taking glycerol as a substrate is one of hot spots of domestic and foreign researches. However, the yield of 1, 3-dihydroxyacetone produced by using glycerol as a substrate is low at present, and the method cannot be suitable for the current industrial production.
Disclosure of Invention
Aiming at the problems that the yield of 1, 3-dihydroxyacetone is lower and the industrial production cannot be met at present, in order to further improve the capability of the gluconobacter oxydans for producing the 1, 3-dihydroxyacetone, a series of recombinant bacteria are constructed by knocking out dehydrogenase genes which have potential relation with the metabolic flux of the gluconobacter oxydans, and the results show that the yield, the conversion rate and the production intensity of the 1, 3-dihydroxyacetone of the recombinant strains are higher than those of the reference. In order to solve the above problems, the present invention provides a method for enhancing the production intensity and conversion rate of 1, 3-dihydroxyacetone fermentation by knocking out a dehydrogenase gene that affects the metabolic flux of 1, 3-dihydroxyacetone.
The first object of the present invention is to provide a genetically engineered bacterium for the production of 1, 3-dihydroxyacetone, which is a knock-out of dehydrogenase genes in gluconobacter oxydans, including genes encoding L-iduronate-5-dehydrogenase I5D, NAD-dependent xylitol dehydrogenase NAD-dependent XD2, alcohol dehydrogenase AD4, aldone dehydrogenase ASD, isocitrate dehydrogenase ID, NAD (P) H dehydrogenase NADH-D2, Zinc-dependent alcohol dehydrogenase Zinc-dependent AD, and/or gluconate dehydrogenase G2D.
In one embodiment, the nucleotide sequence of the gene encoding L-iduronate-5-dehydrogenase is set forth in SEQ ID No. 1; the nucleotide sequence of the gene for coding the NAD dependent xylitol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for coding the alcohol dehydrogenase is shown as SEQ ID NO. 3; the nucleotide sequence of the gene for coding the aldehyde ketone dehydrogenase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for coding isocitrate dehydrogenase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene for coding NAD (P) H dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the gene for coding the zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO. 7; the nucleotide sequence of the gene for coding the gluconate dehydrogenase is shown as SEQ ID NO. 8.
In one embodiment, g.oxydans WSH-003 is used as the host.
The second purpose of the invention is to provide a method for improving the yield of 1, 3-dihydroxyacetone, wherein the genetically engineered bacterium is a knockout of dehydrogenase genes in gluconobacter oxydans, and the dehydrogenase genes comprise genes for encoding L-iduronate-5-dehydrogenase I5D, NAD-dependent xylitol dehydrogenase NAD-dependent XD2, alcohol dehydrogenase AD4, aldoketone dehydrogenase ASD, isocitrate dehydrogenase ID, NAD (P) H dehydrogenase NADH-D2, Zinc-dependent alcohol dehydrogenase Zinc-dependent AD and/or gluconate dehydrogenase G2D.
In one embodiment, the nucleotide sequence of the gene encoding L-iduronate-5-dehydrogenase is set forth in SEQ ID No. 1; the nucleotide sequence of the gene for coding the NAD dependent xylitol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for coding the alcohol dehydrogenase is shown as SEQ ID NO. 3; the nucleotide sequence of the gene for coding the aldehyde ketone dehydrogenase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for coding isocitrate dehydrogenase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene for coding NAD (P) H dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the gene for coding the zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO. 7; the nucleotide sequence of the gene for coding the gluconate dehydrogenase is shown as SEQ ID NO. 8.
In one embodiment, the gluconobacter oxydans is g.oxydans WSH-003.
The third purpose of the invention is to provide a method for improving the production intensity of gluconobacter oxydans 1, 3-dihydroxyacetone, which is characterized in that dehydrogenase genes in the gluconobacter oxydans are knocked out; the dehydrogenase genes include genes encoding L-iduronate-5-dehydrogenase, NAD-dependent xylitol dehydrogenase, alcohol dehydrogenase, aldone dehydrogenase, isocitrate dehydrogenase, NAD (P) H dehydrogenase, zinc-dependent alcohol dehydrogenase, and/or gluconate dehydrogenase.
In one embodiment, the nucleotide sequence of the gene encoding L-iduronate-5-dehydrogenase is set forth in SEQ ID No. 1; the nucleotide sequence of the gene for coding the NAD dependent xylitol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for coding the alcohol dehydrogenase is shown as SEQ ID NO. 3; the nucleotide sequence of the gene for coding the aldehyde ketone dehydrogenase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for coding isocitrate dehydrogenase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene for coding NAD (P) H dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the gene for coding the zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO. 7; the nucleotide sequence of the gene for coding the gluconate dehydrogenase is shown as SEQ ID NO. 8.
In one embodiment, the gluconobacter oxydans is g.oxydans wsh-003.
The fourth purpose of the invention is to provide a method for producing 1, 3-dihydroxyacetone, which is to use the genetically engineered bacteria to produce 1, 3-dihydroxyacetone by transformation.
In one embodiment, the seed solution of the genetically engineered bacteria is added into a reaction system and reacted at the temperature of 25-35 ℃ and the speed of 200-250rpm for not less than 60 hours.
In one embodiment, the reaction system contains 100 g.L of glycerol-1Yeast powder 15-30 g.L-1、CaCO35.0g·L-1、MgSO4·7H2O 1g·L-1、(NH4)2SO4 2g·L-1、K2HPO4·3H2O 0.131g·L-1、KH2PO40.9g·L-1The pH was 6.2.
The invention also provides application of the genetic engineering bacteria in production of 1, 3-dihydroxyacetone.
The invention has the beneficial effects that:
the method can improve the yield, the conversion rate and the production strength of the 1, 3-dihydroxyacetone. 1, 3-dihydroxyacetone production (g.L.sub.L.) by recombinant strains G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 compared with the control strain G.oxydans WSH-003-1) Respectively increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88 and 16.49; the conversion rate (%) is respectively increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88 and 16.49; production Strength (g.L)-1·h-1) Respectively increased by 0.23, 0.26, 0.37, 0.29, 0.21, 0.24, 0.23 and 0.23.
Drawings
FIG. 1 is a graph showing the effect of G.oxydans WSH-003 knockdown of various dehydrogenases on 1, 3-dihydroxyacetone production.
FIG. 2 is a graph showing the effect of a control group of G.oxydans WSH-003 knock-out dehydrogenases on the production of 1, 3-dihydroxyacetone
Detailed Description
Strain (I): gluconobacter oxydans G.oxydans WSH-003.
Type of culture Medium
Sorbitol base culture Medium (g.L)-1): sorbitol 40 and yeast powder 20, 20g/L agar powder is required to be added for preparing the solid culture medium.
Seed culture Medium (g.L)-1): sorbitol 60 and yeast powder 20.
Fermentation Medium (g.L)-1): 100 parts of glycerol, 15-30 parts of yeast powder and CaCO3 5.0、MgSO4·7H2O 1、(NH4)2SO42、K2HPO4·3H2O 0.131、KH2PO40.9, pH 6.2 was adjusted with sulfuric acid.
(III) measurement of 1, 3-dihydroxyacetone: high Performance Liquid Chromatography (HPLC). The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, chromatographic conditions: aminex HPX-87H (Bio-Rad) with dilute H as mobile phase2SO4At a concentration of 5 mmol. L-1The flow rate is 0.5 mL/min-1The column temperature was 40 ℃ and the amount of sample was 10. mu.L. Ultraviolet detector 271 nm: detecting the content of the 1, 3-dihydroxyacetone. Centrifuging the fermentation liquid at 12,000rpm for 2min, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting the yield of 1, 3-dihydroxyacetone by Shimadzu liquid chromatograph system.
Example 1: construction of the dehydrogenase knockout cassette
The genome of G.oxydans WSH-003 is taken as a template to amplify the sequences of 1000bp at the upstream and downstream of a target gene to be knocked out, simultaneously, primers are utilized to amplify kana gene by taking pBBR1MCS-2 as the template, and the genome of G.oxydans WSH-003 is taken as the template to amplify upp gene (the gene sequence is shown as SEQ ID NO. 12). Connecting the four fragments by utilizing a fusion PCR technology to construct a gene knockout frame: left Homologous Arm (HAL) -kana-upp-right Homologous Arm (HAR), and a knock-out box was ligated to the polyclonal restriction site of the pMD19-T vector, transformed into Escherichia coli competent cell JM109, and transformants were plated on LB plates containing kanamycin (kana) (50mg/L) for selection, and the strains with the correct sequencing were preserved. As the dehydrogenase knockout frame is provided with kana (nucleotide sequence of kana is detailed in 1895-2689 th site of Genbank: MH 539767.1) -upp gene, the dehydrogenase knockout frame segment with correct sequencing is transformed into G.oxydans WSH-003 to obtain the strain G.oxydans (knockout gene:: kana-upp) which can be used for normally growing upp gene defect in sorbitol base culture medium of kanamycin kana and cefoxitin, after the first round of kana antibiotic screening is completed, the strain is subjected to the second round of screening in sorbitol base culture medium containing 5-fluorouracil (300mg/L) and cefoxitin (50mg/L), thereby obtaining the target recombinant bacterium.
Example 2: construction of recombinant strain G.oxydans WSH-1
A knock-out frame for knocking out the I5D gene was constructed as I5DL-kana-upp-I5DR according to the method of example 1, the dehydrogenase knock-out frame fragment with the correct sequencing was transformed into G.oxydans WSH-003, and a recombinant strain G.oxydans WSH-1 with the I5D gene knocked out was obtained by screening according to the same method as in example 1.
Example 3: construction of recombinant strain G.oxydans WSH-2
A knock-out frame for knocking out the NAD-dependent XD2 gene is constructed according to the method of example 1 and is NAD-dependent XD2L-kana-upp-NAD-dependent XD2R, a dehydrogenase knock-out frame fragment with correct sequencing is transformed into G.oxydans WSH-003, and a recombinant bacterium G.oxydans WSH-2 with the NAD-dependent XD2 gene knocked out is obtained by screening according to the same method of example 1.
Example 4: construction of recombinant strain G.oxydans WSH-3
A knock-out frame for knocking out the AD4 gene is constructed as AD4L-kana-upp-AD4R according to the method of example 1, a dehydrogenase knock-out frame fragment with correct sequencing is transformed into G.oxydans WSH-003, and a recombinant strain G.oxydans WSH-3 with the AD4 gene knocked out is obtained by screening according to the same method of example 1.
Example 5: construction of recombinant strain G.oxydans WSH-4
A knockout frame for knocking out the ASD gene is constructed as ASDL-kana-upp-ASDR according to the method of example 1, a dehydrogenase knockout frame segment with correct sequencing is transformed into Gluconobacter oxydans WSH-003, and a recombinant strain G.oxydans WSH-4 with the ASD gene knocked out is obtained by screening according to the same method of example 1.
Example 6: construction of recombinant strain G.oxydans WSH-5
A knock-out frame for knocking out the ID gene is constructed as IDL-kana-upp-IDR according to the method of example 1, a dehydrogenase knock-out frame fragment with correct sequencing is transformed into gluconobacter oxydans G.oxydans WSH-003, and a recombinant strain G.oxydans WSH-5 with the ID gene knocked out is obtained by screening according to the same method of example 1.
Example 7: construction of recombinant strain G.oxydans WSH-6
A knock-out frame for knocking out the NADH-D2 gene is constructed into NADH-D2L-kana-upp-NADH-D2R according to the method of example 1, a dehydrogenase knock-out frame segment with correct sequencing is transformed into gluconobacter oxydans G.oxydans WSH-003, and a recombinant bacterium G.oxydans WSH-6 with the NADH-D2 gene knocked out is obtained by screening according to the same method of example 1.
Example 8: construction of recombinant strain G.oxydans WSH-7
A knockout frame for knocking out the Zinc-dependentAD gene is constructed as in example 1, a dehydrogenase knockout frame segment with correct sequencing is transformed into Gluconobacter oxydans WSH-003, and a recombinant bacterium G.oxydans WSH-7 with the Zinc-dependentAD gene knocked out is obtained by screening according to the same method as in example 1.
Example 9: construction of recombinant strain G.oxydans WSH-8
A G2DL-kana-upp-G2DR knock-out frame for knocking out the G2D gene is constructed according to the method of example 1, a dehydrogenase knock-out frame fragment with correct sequencing is transformed into gluconobacter oxydans G.oxydans WSH-003, and a recombinant strain G.oxydans WSH-8 with the G2D gene knocked out is obtained by screening according to the same method of example 1.
Example 10: fermentation production of 1, 3-dihydroxyacetone by recombinant bacteria and reference bacteria
The recombinant bacteria G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 prepared in example 2-9 and the control bacteria G.oxydans WSH-003 are picked up, firstly cultured in a sorbitol base culture medium for 2-3d respectively, picked up and singly cloned to a seed culture medium for activation culture for 24h, and then inoculated with 8% (v/v) of the seed liquid obtained by activation culture to a fermentation culture medium respectively, the fermentation culture is carried out at 30 ℃ and 220rpm, and when fermentation is carried out for 72h, substrate glycerol is consumed to finish bundle generation
The fermentation results of the fermentation broth in which 1, 3-dihydroxyacetone was measured are shown in FIG. 1 and Table 1, and the yields of 1, 3-dihydroxyacetone (g.L.L.) of the recombinant strains G.oxydans WSH-1, G.oxydans WSH-2, G.oxydans WSH-3, G.oxydans WSH-4, G.oxydans WSH-5, G.oxydans WSH-6, G.oxydans WSH-7 and G.oxydans WSH-8 were obtained as compared with the control strain G.oxydans WSH-003-1) Respectively increased by 16.36, 18.42, 26.59, 21.00, 15.08, 17.48, 16.88 and 16.49; the conversion (%) was increased by 16.36 and 18, respectively.42. 26.59, 21.00, 15.08, 17.48, 16.88, 16.49; production Strength (g.L)-1·h-1) Respectively increased by 0.23, 0.26, 0.37, 0.29, 0.21, 0.24, 0.23 and 0.23.
TABLE 1 fermentation results of different dehydrogenases with the oxydans WSH-003 knock-out
Comparative example 1
According to the method of example 1, the genes NADH-DTII (NADH dehydration type II, nucleotide sequence is shown in SEQ ID NO. 9), ADLP (Aldehydehydogenase-like protein, nucleotide sequence is shown in SEQ ID NO. 10), NADH-D (Q) (NADH dehydration (quinone), nucleotide sequence is shown in SEQ ID NO. 11) on the G.oxydans WSH-003 genome are knocked out respectively to obtain strains G.oxydans WSH-9, G.oxydans WSH-10 and G.oxydans WSH-11, then, 1, 3-dihydroxyacetone in the production was fermented according to the method of example 10, and the content of 1, 3-dihydroxyacetone was measured, as shown in FIG. 2, the results showed that the production amount, conversion rate and production intensity of 1, 3-dihydroxyacetone of the strains G.oxydans WSH-9, G.oxydans WSH-10 and G.oxydans WSH-11 were not significantly improved as compared with the control.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for improving yield and production intensity of gluconobacter oxydans 1, 3-dihydroxyacetone
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<210> 4
<211> 1539
<212> DNA
<213> Artificial sequence
<400> 4
atgtgtctgc acaatcgcca gaaagacagg gagaagccgt ccatgaacaa gatggcacag 60
aaactggctc catccggagt tgctcgtgac tttggatttt tcatcgatgg tgagtggcgc 120
cacggacgtg agatgttcga gcgcaaatct cccagtcatg atgttgtagt cacgcggatt 180
gcacgttgca cggaagagga tctgaacgac gcagtggctg ctgcccgccg tgcgtttgaa 240
aacggcacat gggctggtct ggcaagttcg gaacgtagtg cgatcctgtt gaagacggca 300
gaactgctga agcagcgccg cgacgatatt gccttctggg aagtgctgga aaacggcaag 360
ccgatctcac aggccaaggc cgagatcgac aactgtattt cctgtttcga gatggctgcg 420
ggtgccgcac gtcttctgca tggagacagc tttaacaatc ttggcgagag cctgttcggc 480
atggttctgc gggagccggt cggggttgtt ggcctcatta cgccctggaa cttcccgttc 540
atgatcctgt gtgagcgcgt gccattcatt ctggcatccg gctgcacagt tgttgtgaag 600
ccagcggaag tgaccagcgc caccacgctg atgctcgccg atattctgac agaagctggc 660
ctgccaaagg gcgtctataa cgtcgtaacc ggcacgggca aaagcgttgg gcaggccctg 720
acgcagcatc cggatgtaga catgctctcc ttcaccggct ccaccggggt tggccggtcc 780
tgcattcatg cgtccgcaga cagcaacctc aagaagcttg gcctggagct tggtggcaag 840
aacccgatcg tcgtcttcgc ggatagtgat ctggaagatg cggccgatgc ggtggccttc 900
gggatcagct tcaacaccgg gcagtgctgc gtctcgtcca gccgcttgat tgtcgaagag 960
tccgtggccg acaagtttga gaagctggtt gtcgccaaga tggaaaagat ccgcgtgggt 1020
gacccgttcg acccggaaac gcagattggc gccatcacaa cggatgcgca gaacaaaacc 1080
attcttgatt atattgagaa gggtaaggcc gaaggcgcgc gcgttctgtg tggtggcaac 1140
aaggtcgatc tgggtcgtgg gcagtacatc gcgccaacgc tcttcacgaa tgtgaagccg 1200
gacatgtcga ttgcgacaga cgaaatcttt ggtcctgtgc tgtcagtctt ccggttcggc 1260
acccttgaag aagcgatctc cctggccaat gacacggctt atggtctggc ggcgtctgtc 1320
tggacgaagg acatcagcaa ggccctcaag gtgacacgca aggttcaggc cgggcgcttc 1380
tgggtcaaca cgatcatggc tggcggcccc gaaatgccgc tgggtggttt caagcagtcc 1440
ggctggggcc gtgaagcggg gatgtacgga gtggaggaat acacccagat caaatccgtt 1500
cacgtcgatc tcggtaaacg gacgcactgg atctcctga 1539
<210> 5
<211> 1023
<212> DNA
<213> Artificial sequence
<400> 5
atgaccgaca agattcccgc tacactcatc gccggtgacg gtatcggacc tgagatcatg 60
gcctccgtaa cgactgttct ggatgcactc ggcgcaccct tcgtctggga tcatcaaaat 120
gcaggaatgg gcgctctgga aaatcagggc agcgcccttc ccgacgcgac gctcgacagc 180
atcactcgca caggactggt cctgaaaggg cccctgacaa cgcccgtcgg caagggcttt 240
cggtccatca atgtgacgct gcgccagcag ttcgatctgt atgccaatgt ccgcccaacg 300
cagacgatcg ttccgggtgg ccgctacagc aatgtcgatc tggtcgtgat ccgcgagaat 360
gtcgagggtc tttatgccgc catggaacat tacatgcgtg tgggcgacga tccggaagcg 420
gtggcctacg gagcaggatt caacacacgc gcagaatgta atcgtatcgt aaggttcgct 480
ttcgaatacg ccgtgaagaa tggccgcaag aaggtcacgt tggttcacaa ggccaatatt 540
ctcaagatcc tcagcggtat cttcctgcat gaaggtcgtc gcgttgctgc cgaatatgaa 600
ggccgcatcg agcttgaaga acgtattgtc gatgcctgcg cgatggaact cgtcatcaag 660
ccggaaaatt acgatgttat cgtcacgaca aacctgtttg gtgatattct ctcagatctg 720
accgccggcc tcgtgggcgg cctaggcatg gctccgggag ccaatatcgg agagaagatg 780
gccgtattcg aagccgtcca cggatcggct ccggatattg ctggcaaggg tgttgccaat 840
ccgctggcgc tcatgatggc cgccagcatg atgctggccc atgtcggacg tcaggacctg 900
tctgcccgtc ttgatggcgg catgaagcag atcctgacga cggacggcct ccgcacccgc 960
gatctgggcg gcactgccac gacagaagac gttacgcagg ccctcctgcg cgccattcac 1020
taa 1023
<210> 6
<211> 618
<212> DNA
<213> Artificial sequence
<400> 6
atgtttcaga caaagtccct gatcaggaag tgccttgtga aagttcacgt catttttgcc 60
caccctctgg aagacagctt caacgctgcc ctgttcaggc tggtaacaca gacactggaa 120
gcggaagggc acgaagtcga tagcctggac ctgtatcggg acaatttcga ccctgtgctc 180
agcccgcagg atcgcatcga atatcatgat gtcacgatca atcagcaccg cgtggcggat 240
tatgtgaagc gcctgcaaag cacggatgtg ctggttctat gtcatcccgt ctggaacttt 300
ggttggccag cgatcatgaa gggctatttc gatcgtgtat tcctgccgga tgtgtctttc 360
aaactgatag atggcaagct ttcgcccggc ttcacgaaca tcaagaaagt cataactgtc 420
accacatatg gctgcccccg tcacagagct tttgttctgg gagatccgcc acggaagaac 480
ggaacccgct ttctgcgtgc cgtaatgaac cgtcgtgtca gtgtggatta tctgggactt 540
tacaacatga acaacacgac gctggatgcc cgtcgagctt tcatgaaaaa agtgatccgt 600
aagctgaaag ccatctga 618
<210> 7
<211> 1029
<212> DNA
<213> Artificial sequence
<400> 7
atggctgata caatgctggc cgccgtcgtc cgcgagttcg ggaagcctct ctccgttgag 60
aggcttccca tccccgagat ccggcctaac cagatcctcg tgaaagtgga tacctgcggc 120
gtctgccata ccgacctgca cgccgcagaa ggggattggc ctgcaaagcc gaacccgccg 180
ttcatcccgg gccatgaagg catcggtcac atcgtggccg ttggcagtca ggtcaatcac 240
gtcaaggttg gcgacgtagt tggcgtgccc tggctctact cggcctgcgg gcactgcgaa 300
cactgcctcg gtggctggga gacgctgtgc aaggatcagg acgacaccgg ctacacggtc 360
aatggctgct tcgccgaata tgtcgtcgca gacccgaact atgtcgcaca tctgcccagc 420
aacattgatc cgcttcaggc tgccccagtc ctttgcgctg gcctgaccgt ctacaagggc 480
ctgaaaatga ccgaggcacg ccctggtcag tggatggcta tttcaggcgt tggcggtctg 540
ggtcagatgg ctgtgcaata cgccgtcgcc atgggcatga atgtcgtggc tgtggatatt 600
gacgacgaca agcttgccac agcaaaaaag cttggcgccg ccctgaccgt caatgccaag 660
gacaaggacc cggcagcctt catccagcag gaagtcggcg gcgctcatgg tgctcttgtt 720
acggccgtgg gccgcaccgc attctcgcag gccatgggct atgcccgccg gggcggcaca 780
atcgttctga acggcctgcc accaggagac ttcccggttt cgatcttcga catggtcatg 840
aacggcacaa ccattcgtgg ctccatcgtc ggcacacggc tggacatgat cgaagccatg 900
gatttcttcg cccgtggcaa agtgaaatcc gtcgtcaccg cagacaagat cgagaacatc 960
aacacgatct tcgacaacct caaaaacggg cgccttcagg gccgtacggt tctggatttc 1020
cggtcctga 1029
<210> 8
<211> 711
<212> DNA
<213> Artificial sequence
<400> 8
gtgcccccga aggaaccgac actgacacgc ccccttcgcc gtagtttcgt caaaggcctt 60
ttgggctcca cgtcattctg gatggtcagc gggcattcgc actgggctga agcgctggct 120
caggaagcgc agaagcccta tcagcccacc ttcttcaccc ctgatgaata ccgtttcgtg 180
gaagccgcag cagagcgtct tttcccgcag gatcagcatg gaccaggagc gcagaccctg 240
ggggttgccg aattcattga tcgacagatg gaatctccat acgggcatgg agacaactgg 300
tacatgtccg caccttttgt gcagggaccg gccaatcttg ggtatcagct cccctttgtc 360
ccgcgcgatc tgtaccgaaa aggaatcgcg ggccttgaga cctatactcg ccagcagcat 420
ggaaaagtct ttgccgatct tccgtgggct gcacaggaac agatactgac ggcgctcgaa 480
ggcggatctg tttcgcttgg agatgttcct ggacgtgttt tctttgagca actgcgcacc 540
aacacgctgg aaggtgcttt tgccgatcct ctctatggcg ggaacaaaag gcttggagga 600
tggctgatgc tcggcttccc aggtgcccgc gccgacttta tggactgggt caatcaggac 660
ggagaggcgt atccctttgg cccgatttcg ctctcgggtg agacggcctg a 711
<210> 9
<211> 1230
<212> DNA
<213> Artificial sequence
<400> 9
atgtctgctg caactcgtgt cgttgttctc ggtggcggag tcggaggtct ggaggccgcg 60
acggcacttg gtcgccgcgg caatatttcc ctgacactcg ttgatcgcag cccggttcac 120
tactggaagc cgtcgctgca tgaatttgca gccggcacga tgcagcatga cggcaactgc 180
attcccttta cggaaactgc cgcgaaattc ggcttcagtt ttacccagag cgttccgaca 240
gctattgatc gcacgaccca gacggtcacg ctcgaaaacg gatcgaccct cccctacgac 300
tatctcgttg tagccctcgg gtcccgcgcg aacgatttcg gcattcccgg aattgtcgag 360
cattgccgct tcattgacag cctcaatgac gccgacactc tgtattccga attccgcaag 420
gcccttcaga cggcacgtgc agccggtcag aaactgagcc tcggcattgt gggtggcggt 480
gcgactggcg ttcagcttgc cgctgaactc tgcaaggcca ttgacgaagc tcccggcttg 540
ggtgtgtctg ttcgcaagac cgggctggat gccgttctga ttgaaaccgg tccacgcatc 600
ctgccagctt tccctgaagc cgtgtccgaa gccgccgcag cacagcttga aaagctgggt 660
attgcggttc gcaccggcgc gatggttgtg ggagctgatg agaatggctt caatctgaag 720
tccggcgaac agatccccgc aacactccgc atctgggcag caggtgttcg cgcttctgac 780
gcaacagccc tgttcgatgg cctggagcgc ggccgtgccg gacagcttgg tgtgacgcag 840
acactccaga ccacagaaga tcccaaaatt ttcgccattg gtgactgtgc ccgcatcgac 900
gcggcccctg ttgcgcctac tgcgcaggca gcccgccagc agggccagta tgtcggacgt 960
attctgccac agatcatcgc cggacagacg cctgctccgt tcgtttataa tgaccgcggt 1020
gctgtcgtgg cccttggtga ttacaacggc tggggcatgc tggatgccaa ccgcagcttc 1080
ggtggcggct tgctctctgg cctttttgcc cgcctgatcc atgaaggact gtaccgccag 1140
catcaggctg gtatcgtagg tctggtaaaa acggcgagca cgacggttcg ggaacatctt 1200
tcgcccgtca agccggacct cggagcctga 1230
<210> 10
<211> 546
<212> DNA
<213> Artificial sequence
<400> 10
atggtggcgg atgtcggtca gagattgcgg cttgttcgtg tcgcaaggaa cctgtcccag 60
cgggaactgg ccaagcgcac aggtgtcacc aattctacca tttccctgat tgaatcagga 120
gacatgaatc cctcgatagg cacgttgaag cgtgttcttg acggtattcc tgttacgctt 180
ggcgatttct tcagtttcga gacggaaggg caggaaaaat atttttaccc cgccgaggac 240
ctgacagaga tcggacaaaa aggtgtgtcc ctgcggcagg ttgggggcaa tctgttcggt 300
cgtgcccttc aggtcctgca tgagcgttat gaaccggcaa ctggtaccgg tgttgcatat 360
gctcacgatg gggaggaggc cggtgtcgtt atccgcgggc aggtggaagt caccgtggag 420
aaccagaccc atattcttgg cccgggggat gcgtattact ttgacagccg caagccccat 480
cgcttccgct gtatcagcga agagccctgc gaactgatca gcgcatgtac gccgccgaca 540
ttctga 546
<210> 11
<211> 1467
<212> DNA
<213> Artificial sequence
<400> 11
atgatcatca atgggatttt ccttcctttc cccctctttg tcctgtcagt ggggacaatg 60
cttctgcttg catcagtcac gctgcatcga tcgtccagac tcagtttctc tctggggctg 120
ctaacgctcg ttcttgcaac cgtgatgacc tattatcctg cccccatcat gcctcttgcg 180
gccacgcaga cactcttctt cgcagataca tgggccaact acacagccgc tctgatttta 240
ctctccgcat cctgcatctt cattctttcc tggcaggacg tgacccagcg cagcgcccgg 300
tcggcagacg aatacgccct cctcctactc cttggtgcgt tgggggctac ggccatggta 360
ttcagtgtaa actacatgcc gtttttcctt ggggtggaaa tactctccat tgcattgatt 420
ggactggtga cattcaggag ccgacacacc cagaaaggcc tggaagcggc gatgaaatac 480
ctgatcctgt caggtgtttc atcagccatt cttctctttg gtatcgggtt gtcctattcc 540
gtcaccggat ccttggtttt cgaattttcc accgcgggtc atgaaacggg cacaggtatt 600
gctgccgctg caagcataat ggttctgacc ggaattttct ttaagctctc cgctgtaccg 660
ttccatatgt ggattctcga cgtcatggag ggggcatctg tccctattgc cggtttcata 720
gccgtcgttc ccaagattgg tatattttct gcgctggtgc ggtatttcgg ttctgaaccc 780
gtcacgcctt tcctgcataa tacaatgtca gccatcatta ttctgaccat tctgggtgga 840
aatctgcttg cgctttgcca gaccagtctg atcagactga tggcgtgttc atccatcgcc 900
catgtaggct atctgctcat tgcattctat tcacctggtc atttccagtc tgatacaatg 960
gtgctctatc tggctgccta taccgcagcc acactgggta ctttctcgat tatccaggct 1020
ttcgtaagtc cagaagggct gcagcgcagc acgatttctg actggaaagg actgtttttt 1080
acccatccct ttctcgcggt cgcaatgact gccatgctgc tttccctcgc aggtattcct 1140
cccacaattg gcttttttgc caaatttgaa attgccgctt caggtctgga gcaaagacat 1200
tatatccttc tcacagcgtt aattgtcgga agcatcatag ccttatatta ttatctcaat 1260
attataagat taatgacaac accaaacact tccttaaata ttggacaaaa tgagaaaaca 1320
aatttaataa tttacagtac tgtttttatt ttaacattaa tcgtgtttgt aggaggaatt 1380
tttccttcat tttttatgaa ttccatacat ccggctcttc catcgacaag agctgaatat 1440
cttcaaagcc acattccatt accttga 1467
<210> 12
<211> 639
<212> DNA
<213> Artificial sequence
<400> 12
atgagtcagg cgctgccgcc tatcgttctc acccatccgc tggtgcgcca caagctgacc 60
cgtctgcgtg ataagaacac atccacagcg ggctttcgtc gcctgacccg tgagctcagt 120
ctgcttctgg cttatgaagc cacgcgtaac ctgtctctcg tgccacgtca gatcgaggcg 180
ccaagcgggt tgatggaagg tgaggaactg gacggcaaga agctttgctt cgtctccatt 240
ctgcgtgccg gtaacggcct tctggacgga atgctggatc tcgtaccgtc tgcgcgtgtg 300
gggcatatcg ggctgcggcg cgaccatgag acgctggaag tcagcgaata ctatttcaat 360
atgccgagcg atgttccggg ccggacctgc atcgttctgg acccgatgct ggccaccggg 420
cactcggctg ccgctgctgt aacgcgcgtc aaggaagcgg gagcgattgc gcctgttttt 480
gcctgccttc tggcggctcc ggaaggaatt gctcacatga cggaactgca tccggatgtg 540
caggtcgtga cgtgttgtgt ggatcagaag cttgatgagc acggctatat tgtcccgggt 600
ctgggcgatg ctggcgaccg tctgttcggg acccgctaa 639
Claims (10)
1. Genetically engineered bacterium producing 1, 3-dihydroxyacetone, characterized in that dehydrogenase genes in gluconobacter oxydans are knocked out, the dehydrogenase genes including genes encoding L-iduronate-5-dehydrogenase, NAD-dependent xylitol dehydrogenase, alcohol dehydrogenase, aldone dehydrogenase, isocitrate dehydrogenase, NAD (p) H dehydrogenase, zinc-dependent alcohol dehydrogenase and/or gluconate dehydrogenase.
2. The genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the gene encoding L-iduronate-5-dehydrogenase is represented by SEQ ID No. 1; the nucleotide sequence of the gene for coding the NAD dependent xylitol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for coding the alcohol dehydrogenase is shown as SEQ ID NO. 3; the nucleotide sequence of the gene for coding the aldehyde ketone dehydrogenase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for coding isocitrate dehydrogenase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene for coding NAD (P) H dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the gene for coding the zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO. 7; the nucleotide sequence of the gene for coding the gluconate dehydrogenase is shown as SEQ ID NO. 8.
3. The genetically engineered bacterium of claim 1, wherein g.oxydans wsh-003 is used as a host.
4. A method for improving the yield of 1, 3-dihydroxyacetone of gluconobacter oxydans is characterized in that a dehydrogenase gene in the gluconobacter oxydans is knocked out; the dehydrogenase genes include genes encoding L-iduronate-5-dehydrogenase, NAD-dependent xylitol dehydrogenase, alcohol dehydrogenase, aldone dehydrogenase, isocitrate dehydrogenase, NAD (P) H dehydrogenase, zinc-dependent alcohol dehydrogenase, and/or gluconate dehydrogenase.
5. The method according to claim 4, wherein the nucleotide sequence of the gene encoding L-iduronate-5-dehydrogenase is represented by SEQ ID No. 1; the nucleotide sequence of the gene for coding the NAD dependent xylitol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for coding the alcohol dehydrogenase is shown as SEQ ID NO. 3; the nucleotide sequence of the gene for coding the aldehyde ketone dehydrogenase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for coding isocitrate dehydrogenase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene for coding NAD (P) H dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the gene for coding the zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO. 7; the nucleotide sequence of the gene for coding the gluconate dehydrogenase is shown as SEQ ID NO. 8.
6. A method for improving the production intensity of gluconobacter oxydans 1, 3-dihydroxyacetone is characterized in that a dehydrogenase gene in the gluconobacter oxydans is knocked out; the dehydrogenase genes include genes encoding L-iduronate-5-dehydrogenase, NAD-dependent xylitol dehydrogenase, alcohol dehydrogenase, aldone dehydrogenase, isocitrate dehydrogenase, NAD (P) H dehydrogenase, zinc-dependent alcohol dehydrogenase, and/or gluconate dehydrogenase.
7. The method according to claim 4, wherein the nucleotide sequence of the gene encoding L-iduronate-5-dehydrogenase is represented by SEQ ID No. 1; the nucleotide sequence of the gene for coding the NAD dependent xylitol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for coding the alcohol dehydrogenase is shown as SEQ ID NO. 3; the nucleotide sequence of the gene for coding the aldehyde ketone dehydrogenase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for coding isocitrate dehydrogenase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene for coding NAD (P) H dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the gene for coding the zinc-dependent alcohol dehydrogenase is shown as SEQ ID NO. 7; the nucleotide sequence of the gene for coding the gluconate dehydrogenase is shown as SEQ ID NO. 8.
8. A method for producing 1, 3-dihydroxyacetone, which comprises transforming the genetically engineered bacterium of any one of claims 1 to 3 to produce 1, 3-dihydroxyacetone.
9. The method as claimed in claim 8, wherein the seed solution of the genetically engineered bacteria is added into the reaction system and reacted at 25-35 ℃ and 200-250rpm for not less than 60 h.
10. Use of the genetically engineered bacterium of any one of claims 1 to 3 for the production of 1, 3-dihydroxyacetone.
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| CN202211217751.XA CN115927145A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1,3-dihydroxyacetone |
| CN202211208253.9A CN115975896A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1,3-dihydroxyacetone |
| CN202211208240.1A CN116121159A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
| CN202110445471.3A CN113174355B (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
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| CN202110445471.3A CN113174355B (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
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| CN202211208240.1A Division CN116121159A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
| CN202211208253.9A Division CN115975896A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1,3-dihydroxyacetone |
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| CN202110445471.3A Active CN113174355B (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
| CN202211208253.9A Pending CN115975896A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1,3-dihydroxyacetone |
| CN202211208240.1A Pending CN116121159A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
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| CN202211208240.1A Pending CN116121159A (en) | 2021-04-25 | 2021-04-25 | Method for improving yield and production strength of gluconobacter oxydans 1, 3-dihydroxyacetone |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392056A (en) * | 2011-12-09 | 2012-03-28 | 华东理工大学 | Genetically engineered strain and method for producing dihydroxyacetone by using the same |
| CN109628367A (en) * | 2019-01-30 | 2019-04-16 | 江南大学 | A method of improving Gluconobacter oxvdans sorb candy output and production intensity |
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- 2021-04-25 CN CN202110445471.3A patent/CN113174355B/en active Active
- 2021-04-25 CN CN202211208253.9A patent/CN115975896A/en active Pending
- 2021-04-25 CN CN202211208240.1A patent/CN116121159A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115975896A (en) | 2023-04-18 |
| CN113174355B (en) | 2023-06-13 |
| CN116121159A (en) | 2023-05-16 |
| CN115927145A (en) | 2023-04-07 |
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