CN104278047A - Method for enhancing activity of trans-4-hydroxyproline biosynthesis system containing recombinant DNA - Google Patents
Method for enhancing activity of trans-4-hydroxyproline biosynthesis system containing recombinant DNA Download PDFInfo
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- CN104278047A CN104278047A CN201310283918.7A CN201310283918A CN104278047A CN 104278047 A CN104278047 A CN 104278047A CN 201310283918 A CN201310283918 A CN 201310283918A CN 104278047 A CN104278047 A CN 104278047A
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Abstract
The invention discloses a method for enhancing activity of trans-4-hydroxyproline biosynthesis system containing recombinant DNA. The trans-4-hydroxyproline biosynthesis system containing recombinant DNA is a recombinant microorganism with proline 4-hydroxylase activity, which acts on the free L-proline and produces trans-4-hydroxyproline in the presence of 2-ketoglutarate and ferrous ions. The invention also provides a trans-4-hydroxyproline biosynthesis method.
Description
Technical field
Make containing recombinant DNA trans-method of 4-Hydroxyproline biosynthesis system increased activity, belong to microbiological genetic engineering field.
Background technology
Oxyproline (Hydroxyproline, Hyp) is imino-acid, is the product after proline(Pro) hydroxylation, according to the difference of its hydroxylation position, can be divided into 3-Hydroxyproline (3-Hyp) or 4-Hydroxyproline (4-Hyp).Occurring in nature is trans-and 4-Hydroxyproline is comparatively common, trans-and 4-Hydroxyproline is the important component part of animal tissue protein as collagen etc.Trans-4-Hydroxyproline is widely used in the aspects such as medicine, chemical industry, animal-feed, nutrition and beauty culture.
At present, the method for producing trans-4-Hydroxyproline has chemical synthesis, biological extraction method and microbial enzyme method.Chemical synthesis process synthesis step is more, and cost is high, is not suitable for industrial production.And biological extraction method utilizes animal protein sources such as gelatin, pigskin to be raw material, after acid, alkali or proteolytic enzymes hydrolize, extract trans-4-Hydroxyproline, this method purification step is long, cost is high, waste large content of starting materials, and waste pollution is serious, is easy to by market.Along with the development of DNA recombinant technology and the discovery of Microbial resources, making microbial enzyme method produce trans-4-Hydroxyproline becomes industrial very promising production method.
Proline-4-hydroxylase is in Host Strains, need with a large amount of oxygen for substrate, L-PROLINE catalysis is generated trans-4-Hydroxyproline, be easy to cause the deficiency of Host Strains oxygen during the fermentation, and Vitreoscilla hemoglobin can oxygen under low oxygen conditions in combining environmental, play the effect that collecting oxygen utilizes for host cell, thus improve the oxygen utilization rate of host cell.Therefore, in order to solve the restriction that low dissolved oxygen is produced trans-4-Hydroxyproline, introduce Vitreoscilla hemoglobin gene on a molecular scale, effectively can promote the growth of Host Strains and the catalysis of proline-4-hydroxylase, the final production efficiency improving trans-4-Hydroxyproline.
Summary of the invention
The defect that trans-4-Hydroxyproline cost is high, pollution is large, extraction yield is low is obtained for current biological extraction method, and Production by Microorganism Fermentation trans-4-Hydroxyproline time oxygen requirement large, dissolved oxygen is not enough can limit the shortcoming of trans-4-Hydroxyproline synthesis, the problem to be solved in the present invention be to provide a kind of make containing recombinant DNA trans-method of 4-Hydroxyproline biosynthesis system increased activity.
The present invention is in order to efficiency utilization proline-4-trans-4-Hydroxyproline of hydroxylase production, provide a kind of make in recombinant microorganism after codon optimized trans-copy number of 4-Hydroxyproline synthetic gene increases and in recombinant microorganism, imports the method for Vitreoscilla hemoglobin gene, utilize the method can High-efficient Production trans-4-Hydroxyproline.
The present invention be a kind of make containing recombinant DNA trans-method of 4-Hydroxyproline biosynthesis system increased activity.It is characterized in that: containing recombinant DNA trans-4-Hydroxyproline biosynthesis system to be recombinated on carrier by the gene fragment of the protein by having proline-4-hydroxylase activity and Vitreoscilla hemoglobin gene fragment and proceeds in microorganism cells, having of obtaining be trans-recombinant microorganism of 4-Hydroxyproline biosynthesizing activity, and provide the production method of trans-4-Hydroxyproline.
Above-mentioned proline-4-hydroxylase deposits in case at 2-oxoglutaric acid and ferrous ion, the enzyme of the L-PROLINE dissociated can be made to be converted into trans-4-Hydroxyproline.
Above-mentioned recombinant microorganism, except the microorganism strains that Colibacter, corynebacterium, Rhodopseudomonas, bacillus etc. belong to, can also use yeast etc.
When above-mentioned recombinant microorganism is using intestinal bacteria etc. as Host Strains, proline-4-hydroxylase and Vitreoscilla hemoglobin expression vector independently duplicated in microorganism while, be preferably made up of promotor, ribosome binding sequence, proline-4-'-hydroxylase gene, Vitreoscilla hemoglobin gene, transcription termination sequence.
When above-mentioned recombinant microorganism take intestinal bacteria as Host Strains, expression vector is including, but not limited to plasmids such as pET28a, pKYP10.
In above-mentioned expression vector, promotor can adopt lac promotor, PL promotor, trp promotor, trp Gene expression or T7 promotor etc.
Ribosome binding sequence in above-mentioned expression vector, as long as can express in the hosts such as intestinal bacteria, retains suitable distance between ribosome bind site and initiator codon.
When above-mentioned recombinant microorganism take intestinal bacteria as host, e. coli jm109, bacillus coli DH 5 alpha, e. coli bl21 (DE3), intestinal bacteria SCS110, intestinal bacteria W3310 etc. can be used.
Above-mentioned trans-method of 4-Hydroxyproline biosynthesis system increased activity is by making the copy number of the proline-4-'-hydroxylase gene after codon optimized in recombinant microorganism increase and import Vitreoscilla hemoglobin gene to realize in recombinant microorganism.
The above-mentioned copy number increase of the proline-4-'-hydroxylase gene in Host Strains that makes is by multiple copied plasmid of being recombinated to by the proline-4-'-hydroxylase gene after codon optimized realizes.
Above-mentioned proline-4-'-hydroxylase gene derives from and refers to cyst bacterium RH1 (Dactylosprangium sp.RH1), and above-mentioned Vitreoscilla hemoglobin gene derives from Vitreoscilla (Vitreoscilla sp.).
Above-mentioned trans-production method of 4-Hydroxyproline, when cultivating using microorganisms such as intestinal bacteria, coryneform bacteria, yeast as recombinant microorganism, as long as substratum is containing the utilizable carbon source of microorganism, nitrogenous source, inorganic salt, metal ion, all effectively can cultivate recombinant microorganism, no matter substratum is synthetic medium or natural medium.
Above-mentioned trans-production method of 4-Hydroxyproline in, as long as can utilize as carbon source microorganism, glucose, sucrose, starch, lactose, semi-lactosi and acetic acid, ethanol etc. can be used.
Above-mentioned trans-production method of 4-Hydroxyproline in, can be the various inorganic ammonium salts such as ammoniacal liquor, ammonium sulfate, ammonium chloride, ammonium phosphate as nitrogenous source, can also be other itrogenous organic substances, as gravy extract, yeast extract, corn steep liquor, Tryptones, dregs of beans, soya-bean cake, various fermentation thalli and its digest etc.
Above-mentioned trans-production method of 4-Hydroxyproline in, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, calcium chloride etc. can be used as inorganic salt.
Above-mentioned trans-production method of 4-Hydroxyproline in, can ferrous sulfate, magnesium sulfate etc. be used as metal ion.
Above-mentioned trans-production method of 4-Hydroxyproline in, when recombinant microorganism adopts the expression plasmid of inducible promoter when cultivating, add inductor in the medium as required.If make the expression plasmid of lac promotor or T7 promotor, add lactose or IPTG (isopropyl-beta D-thio galactopyranoside) etc. in the medium, and when using the expression plasmid of trp promotor, indole acrylic acid (IAA) need be added in the medium.
Above-mentioned trans-production method of 4-Hydroxyproline, recombinant microorganism can utilize the carbon source in substratum produce 2-oxoglutaric acid and accumulate 2-oxoglutaric acid in the medium.
Provided by the present invention make containing recombinant DNA trans-method of 4-Hydroxyproline biosynthesis system increased activity, there is important industrial application value.
Accompanying drawing explanation
Fig. 1. the biotransformation pathway of trans-4-Hydroxyproline in Bacillus coli cells.
Fig. 2. trp promoter plasmid map.
Fig. 3. expression plasmid pUC19-ptrp2-Hyp collection of illustrative plates.
Fig. 4. expression plasmid pUC19-ptrp2-Hyp-VHb collection of illustrative plates.
Embodiment
LB substratum: 1% (W/V) Tryptones, 0.5% (W/V) yeast extract, 1% (W/V) NaCl, pH7.0-7.2.
Amp resistant panel: 1% (W/V) Tryptones, 0.5% (W/V) yeast extract, 1% (W/V) NaCl, 1.5% (W/V) agarose, penbritin 50 μ g/mL.
SOC substratum (recovery for competent cell): 2% (W/V) Tryptones, 0.5% (W/V) yeast extract, 0.05% (W/V) NaCl, 2.5mM KCl, 10mM MgCl
2, 20mM glucose.
5 × KCM (intestinal bacteria transfer buffer): 0.5M KCl, 0.15M CaCl
2, 0.25M MgCl
2.
The quantitative assay of trans-4-Hydroxyproline: after fermented liquid is centrifugal, get supernatant, 2.5mL is got in 10mL test tube after dilution, add 1mL chloramine-T (chloramine-T solution: be dissolved in by 1.41g chloramine-T in 10mL water, add 10mL n-propyl alcohol and 80mL buffered soln successively, wherein buffered soln formula is: by 50g citric acid, 26.3gNaOH and 146.1g crystallization sodium acetate is water-soluble to 1L, then mix with 200mL water and 300mL n-propyl alcohol), 20min is placed at room temperature after shaking up, then 1mL developer (developer: take 10g paradimethy laminobenzaldehyde is added, use 35mL high chloro acid dissolution, slowly add 65mL Virahol), shake up and rapidly test tube is moved in 60 DEG C of water-baths afterwards, insulation 20min, cool with cold water again, then absorbancy is measured with spectrophotometer at 560nm wavelength place.
Embodiment 1: the design of tryptophane tandem promoter subsequence
Tryptophane Gene expression derives from ptrpL1 plasmid, plasmid map is as Figure of description 2, the trp promoter of this plasmid derives from the tryptophan operon of e. coli k12 strain, it is the strong promoter of an applicable industrial production application, ClaI restriction enzyme site is had with between initiator sequences in SD sequence, meanwhile, there is Hind III digestion site-35 upstreams, district of trp promoter, are convenient to promotor to be connected to like this on other expression vector.Because two trp promoters are together in series and can improve expression intensity, therefore, according to the tryptophan promoter subsequence of ptrpL1 plasmid, full genome combination colour propylhomoserin Gene expression.
By optimizing tryptophane Gene expression, HindIII restriction enzyme site AAGCTT is revised as EcoR I restriction enzyme site GAATTC, simultaneously, in order to make the restriction enzyme site of Gene expression inside single, therefore the Cla I restriction enzyme site ATCGAT of the combining site of two trp promoters is revised as ATGTCGAC, junction is made to become Sal I restriction enzyme site, simultaneously, a Hind III digestion site AAGCTT is added after the Cla I restriction enzyme site of second tryptophan promoter subsequence, enzyme can be utilized like this to cut equimolecular and to operate the distance optimized between SD sequence and initiator codon, goal gene is made to obtain high expression.
The original series of trp promoter gene order in present embodiment, is shown in SEQ ID NO:1.The tryptophane tandem promoter subsequence behaviour work synthesis optimized in present embodiment; The tryptophane tandem promoter subsequence optimized sees SEQ ID NO:2.
Embodiment 2: the design of proline(Pro)-4-'-hydroxylase gene sequence
Optimized gene codon is carried out according to colibacillary codon usage frequency, eliminate the codon of low rate of utilization, utilize synonym method for transformation to eliminate EcoR I restriction enzyme site simultaneously, terminator in original series is revised as the strong terminator of TAAT, for the ease of being connected on other plasmid vectors by proline(Pro)-4-'-hydroxylase gene, therefore after terminator, insert a restriction enzyme site BamH I (GGATCC).
Also to consider the secondary structure of mRNA, first the codon translation pocket of AUG initiator codon and subsequent several based composition to be ensured in opened condition, reduction rrna is attached to the energy gesture on mRNA, and rrna can successfully be translated along initiator codon backward.
The original series of proline-4 in present embodiment-'-hydroxylase gene sequence, is shown in GenbankAccession D78338.1.The proline-4 optimized in present embodiment-'-hydroxylase gene sequence is synthetic; Proline-4-hydroxylase the sequence optimized is shown in SEQ ID NO:3.
Embodiment 3: the structure of proline-4-'-hydroxylase gene expression vector
The tryptophane Gene expression that first compounding design is good respectively and proline-4-'-hydroxylase gene, because tryptophane Gene expression gene order 5 ' terminal sequence is with EcoR I (GAATTC) restriction enzyme site, 3 ' end has Hind III (AAGCTT) restriction enzyme site, and proline-4-'-hydroxylase gene 5 ' terminal sequence is with Hind III (AAGCTT) restriction enzyme site, and 3 ' end has BamH I (GGATCC) restriction enzyme site.
By tryptophane Gene expression gene order restriction enzyme EcoRI and Hind III double digestion, by proline-4-'-hydroxylase gene HindIII and BamH I double digestion, meanwhile, plasmid vector pUC19 is also used respectively EcoR I and BamH I digestion process.The two tryptophane Gene expression gene order after enzyme, proline-4-'-hydroxylase gene and pUC19 plasmid vector sepharose test kit cut is reclaimed, then with T4DNA ligase enzyme, three gene fragments are coupled together, connection product is proceeded in e. coli bl21 (DE3) competent cell.
Conversion process is: be added in BL21 (DE3) competent cell of 50 μ L by 6 μ L connecting fluids, and then add 5 × KCM damping fluid of 10 μ L, after mixing, after placing 30min on ice, 42 DEG C of heat shock 90s, then after placing 5min on ice, add the SOC substratum of 500 μ L, 37 DEG C, after 200rpm cultivates 60min, be applied in Amp resistant panel, after cultivating 12h.
Then extract plasmid to verify, whether further sequence verification gene order is correct.Thus obtaining the pUC19-ptrp2-Hyp recombinant plasmid built, plasmid map, as Figure of description 3, obtains recombination bacillus coli BL21 (DE3)/pUC19-ptrp2-Hyp simultaneously.
Embodiment 4: the structure of the recombination bacillus coli containing Vitreoscilla hemoglobin gene
First synthetic Vitreoscilla hemoglobin gene, because Vitreoscilla hemoglobin gene sequence 5 ' terminal sequence is with EcoRI restriction enzyme site, 3 ' end has Nde I restriction enzyme site, by Vitreoscilla hemoglobin gene sequence with after restriction enzyme EcoR I and Nde I double digestion, meanwhile, plasmid vector pUC19-ptrp2-Hyp is also used EcoR I and Nde I digestion process.
The Vitreoscilla hemoglobin gene sequence after enzyme and the recovery of pUC19-ptrp2-Hyp plasmid vector sepharose test kit is cut by two, then with T4DNA ligase enzyme, two gene fragments are coupled together, connection product is proceeded in e. coli bl21 (DE3) competent cell.
Conversion process is: be added in BL21 (DE3) competent cell of 50 μ L by 6 μ L connecting fluids, and then add 5 × KCM damping fluid of 10 μ L, after mixing, after placing 30min on ice, 42 DEG C of heat shock 90s, then after placing 5min on ice, add the SOC substratum of 500 μ L, 37 DEG C, after 200rpm cultivates 60min, be applied in Amp resistant panel, cultivate 12h.
Then extract plasmid to verify, whether further sequence verification gene order is correct.Thus obtaining the pUC19-ptrp2-Hyp-VHb recombinant plasmid built, plasmid map, as Figure of description 4, obtains recombination bacillus coli BL21 (DE3)/pUC19-ptrp2-Hyp-VHb simultaneously.
The original series of Vitreoscilla hemoglobin gene sequence in present embodiment, is shown in GenbankAccession M30794.1.Adding the Vitreoscilla hemoglobin gene sequence after restriction enzyme site in present embodiment is synthetic; Add the Vitreoscilla hemoglobin gene sequence after restriction enzyme site and see SEQ ID NO:4.
Embodiment 5: the fermenting experiment of recombinant bacterial strain
Shake-flask culture: picking recombination bacillus coli list bacterium colony, be inoculated in LB liquid nutrient medium (containing penbritin 50 μ g/mL), 37 DEG C, after 220rpm cultivates 8h, by the access of 6% (V/V) inoculum size containing 30mL fermention medium (1% (W/V) glucose, 0.5% (W/V) glycerine, 0.8% (W/V) Tryptones, 0.5% (W/V) ammonium sulfate, 0.1% (W/V) dipotassium hydrogen phosphate, 0.2% (W/V) sodium-chlor, 3mM ferrous sulfate, 0.2g/L magnesium sulfate, 0.015g/L calcium chloride, 400mM L-PROLINE, pH8.0) in 250mL shaking flask, in rotary shaker 35 DEG C, 220rpm cultivates 48h.Get fermented liquid and detect trans-4-Hydroxyproline concentration.The measuring method of trans-4-Hydroxyproline refers to the general explanation of embodiment.Fermentation results shows, recombination bacillus coli BL21 (DE3)/pUC19-ptrp2-Hyp-VHb trans-4-Hydroxyproline output reaches 4.93g/L.
Claims (10)
1. one kind make containing recombinant DNA trans-method of 4-Hydroxyproline biosynthesis system increased activity, wherein, containing recombinant DNA trans-4-Hydroxyproline biosynthesis system is by carrier of the gene fragment of the protein with proline-4-hydroxylase activity being recombinated to proceeds to microorganism cells, having of obtaining be trans-recombinant microorganism of 4-Hydroxyproline biosynthesizing activity, and the present invention also provides the production method of trans-4-Hydroxyproline.
2. the method making trans-4-Hydroxyproline biosynthesis system increased activity according to claim 1, it is characterized in that the codon by optimizing proline-4-'-hydroxylase gene, and make the copy number of the proline-4-'-hydroxylase gene after codon optimized in recombinant microorganism increase and import Vitreoscilla hemoglobin gene to realize in recombinant microorganism.
3. the copy number increase of the proline-4-'-hydroxylase gene in recombinant microorganism according to claim 2 realizes by being connected in multiple copied plasmid by the proline-4-'-hydroxylase gene after codon optimized.
4. Vitreoscilla hemoglobin gene according to claim 2 derives from Vitreoscilla (Vitreoscilla sp.).
5. multiple copied plasmid according to claim 3 is including, but not limited to pET28a, pKYP10 etc.
6. the production method of trans-4-Hydroxyproline, the recombinant microorganism described in claim 1,2,3 is it is characterized in that to cultivate on substratum, using the culture, thalline or their handled thing that obtain as enzyme source, under 2-oxoglutaric acid and ferrous ion exist, trans-the 4-Hydroxyproline that makes L-PROLINE be converted in aqueous medium, then extract from this aqueous medium generation trans-4-Hydroxyproline.
7. production method according to claim 6, is characterized in that aqueous medium is nutrient solution.
8. production method according to claim 6, is characterized in that adding L-PROLINE in substratum.
9. production method according to claim 6, wherein said recombinant microorganism can utilize the carbon source in substratum to generate 2-oxoglutaric acid, and accumulates 2-oxoglutaric acid in the medium.
10. the recombinant microorganism described in claim 1,2,3,6 is including, but not limited to Colibacter, corynebacterium, Rhodopseudomonas, genus bacillus, yeast etc.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105506018A (en) * | 2015-12-31 | 2016-04-20 | 天津市敬业精细化工有限公司 | Strain culture medium for industrially producing L-hydroxyproline through fermentation method and culture method |
| CN105506019A (en) * | 2015-12-31 | 2016-04-20 | 天津市敬业精细化工有限公司 | Method for industrially producing L-hydroxyproline through fermentation method |
| CN105603017A (en) * | 2016-01-19 | 2016-05-25 | 江南大学 | Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum |
| CN106085931A (en) * | 2016-03-04 | 2016-11-09 | 江苏大成医药科技股份有限公司 | Produce engineering bacteria and the application thereof of trans 4 hydroxyprolines |
| CN107435056A (en) * | 2016-05-27 | 2017-12-05 | 绍兴百茵生物技术有限公司 | The bioconversion method of L- hydroxyprolines |
| CN108102964A (en) * | 2017-12-29 | 2018-06-01 | 河北大学 | A kind of trans-4-hydroxy-l-proline synthesis bacterial strain and its L-PROLINE '-hydroxylase gene and application |
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2013
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| TAKESHI,ET AL: "Enzymatic Production of trans-4-Hydroxy-L-proline by Regio- and Stereospecific Hydroxylation of L-Proline", 《BIOSCI.BIOTECHNOL.BIOCHEM》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506018A (en) * | 2015-12-31 | 2016-04-20 | 天津市敬业精细化工有限公司 | Strain culture medium for industrially producing L-hydroxyproline through fermentation method and culture method |
| CN105506019A (en) * | 2015-12-31 | 2016-04-20 | 天津市敬业精细化工有限公司 | Method for industrially producing L-hydroxyproline through fermentation method |
| CN105603017A (en) * | 2016-01-19 | 2016-05-25 | 江南大学 | Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum |
| CN106085931A (en) * | 2016-03-04 | 2016-11-09 | 江苏大成医药科技股份有限公司 | Produce engineering bacteria and the application thereof of trans 4 hydroxyprolines |
| CN107435056A (en) * | 2016-05-27 | 2017-12-05 | 绍兴百茵生物技术有限公司 | The bioconversion method of L- hydroxyprolines |
| CN108102964A (en) * | 2017-12-29 | 2018-06-01 | 河北大学 | A kind of trans-4-hydroxy-l-proline synthesis bacterial strain and its L-PROLINE '-hydroxylase gene and application |
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