CN102433293A - Engineering bacterium of fermenting production of optical pure L-lactate by utilizing xylose and construction thereof - Google Patents
Engineering bacterium of fermenting production of optical pure L-lactate by utilizing xylose and construction thereof Download PDFInfo
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Abstract
The invention discloses an engineering bacterium of fermentation production of optical pure L-lactate by utilizing xylose, as well as a construction method and an application thereof. The engineering bacterium is constructed in the following steps of: synthesizing a kar kanamycin resistant gene, and inserting the kar kanamycin resistant gene into a shuttle vector of escherichia coli and thermophilic anaerobic bacillus; amplifying a certain sequence of an acetokinase coding gene, which is a pta-up sequence; inserting the pta-up sequence into the shuttle vector of the escherichia coli and thermophilic anaerobic bacillus to construct a suicide vector 1; amplifying a certain sequence of a phosphotransacetylase coding gene, which is an ack-down sequence; inserting the ack-down sequence into the suicide vector 1 to obtain a suicide vector pPuKAd; converting the suicide vector pPuKAd into the thermophilic anaerobic bacillus, performing resistance screening to obtain the optical pure L-lactate by utilizing fermentation of xylose. The engineering bacterium of the invention is used for fermentation production of lactate, acetic acid is not generated, and a carbon metabolic flux is distributed once again, so that massive accumulation of the target product L-lactate is promoted, and the optical purity of the L-lactate is over 99.5 percent.
Description
Technical field
The invention belongs to microbial fermentation and genetically engineered field, be specifically related to engineering bacteria and construction process and application a kind of acetate metabolism approach disappearance, that optical pure L-lactic acid is produced in wood-sugar fermentation capable of using.
Background technology
Lactic acid has a wide range of applications in fields such as food, medicine, makeup, leather production, textile industries.Lactic acid still is a kind of important chemical hardware and software platform compound.And because in recent years to the concern of biodegradable POLYACTIC ACID, the cheapness of lactic acid obtains to cause once more investigator's attention.Than chemical synthesis process; Microbial fermentation generates lactic acid and has advantage (Datta such as low temperature, less energy-consumption and low cost; R.and M.Henry., Lactic acid:recent advances in products, processes and technologies-a review.Journal of Chemical Technology and Biotechnology; 2006,81 (7): 1119-1129).And mostly biological fermentation is optical purity L type or D type lactic acid if obtaining, and the optical purity of L type lactic acid is vital to preparing high-quality POLYACTIC ACID.
Along with constantly increasing the weight of of fossil energy crisis, it is that fermenting raw materials is produced lactic acid that the investigator hopes with cheap biomass energy such as molasses, starch or cellulosic waste, thereby further reduces lactic acid fermented production cost.Above-mentioned all kinds of cheap biomass energy; Because fiber biomass has cheapness, wide material sources and can not influence advantages such as grain security, and receives investigator's favor (Tan, T.W. more; F.Shang; Et al.Current development of biorefinery in China.Biotechnology Advances, 2010,28 (5): 543-555).Cellulose series biomass mainly is made up of glucose and wood sugar.But traditional lactobacillus ferment bacterial strain can not utilize wood sugar to be carbon source through fermentation usually.Therefore how to obtain effectively to utilize the bacterial strain of the high lactic acid producing of wood-sugar fermentation; Be that cellulose series biomass is produced a bit (Wang, L.M., B.Zhao most crucial in the lactic acid industry; Etal.; Efficient production of L-lactic acid from corncob molasses, a waste by-productin xylitol production, by a newly isolated xylose utilizing Bacillus sp strain.Bioresource Technology; 2010,101 (20): 7908-7915).Some are like Bacillus sp. (Wang, L.M., B.Zhao; Et al., Efficient production of L-lactic acid from corncob molasses, awaste by-product in xylitol production; By a newly isolated xylose utilizing Bacillussp strain.Bioresource Technology, 2010,101 (20): 7908-7915); Lactobacilluspentosus, Lactobacillus brevis and Pichia stipitis (Ilmen, M.; K.Koivuranta, et al., Efficient production of L-lactic acid from xylose by Pichia stipitis.Applied AndEnvironmental Microbiology; 2007; 73 (1): 117-123) Rhizopus (Skory, C.D.Lacticacid production by Rhizopus oryzae transformants with modified lactatedehydrogenase activity.Applied Microbiology and Biotechnology, 2004; 64 (2): 237-242.Skory; C.D.Isolation and expression of lactate dehydrogenase genes fromRhizopus oryzae.Applied And Environmental Microbiology, 2000,66 (6): 2343-2348); Saccharomyces (Hetenyi; K., A.Nemeth, et al.Role of pH-regulation inlactic acid fermentation:Second steps in a process improvement.ChemicalEngineering And Processing; 2011,50 (3): 293-299) wait that to be in the news in succession can be that fermenting substrate is produced lactic acid with the wood sugar.
Advantages such as thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoence SCUT27) has can reduce raw material pre-treatment process cellulase addition, can effectively utilize wood sugar, meta-bolites is simple; And receive investigator's favor (O-Thong, S., P.Prasertsan; Et al.Thermophilic fermentativehydrogen production by the newly isolated Thermoanaerobacteriumthermosaccharolyticum PSU-2.International Journal of Hydrogen Energy; 200833 (4): 1204-1214., Sommer, P.; T.Georgieva; Et al.Potential for using thermophilicanaerobic bacteria for bioethanol production from hemicellulose.Biochem ical SocietyTransactions, 2004,32:283-289.; Li; S., C.Lai, et al.High efficiency hydrogenproduction from glucose/xylose by the ldh-deleted Thermoanaerobacterium strain.Bioresource Technology; 2010,101 (22): 8718-8724.).Special; Because of the righttest leavening temperature of Thermoanaerobacterium aotearoence SCUT27 is 55 ℃; Can suppress most normal temperature microbial growths, so this bacterial strain can be aseptic less demanding to fermentation technology process, can reduce production costs significantly.The wild-type liquid phase meta-bolites of this bacterial strain comprises lactic acid, acetate and ethanol, though lactic acid is its main metabolites, the lactic acid transformation efficiency still has the raising space.
Summary of the invention
For the shortcoming and deficiency that overcomes prior art, primary and foremost purpose of the present invention is to provide a kind of wood-sugar fermentation capable of using to produce the engineering bacteria of optical pure L-lactic acid.
Another object of the present invention is to provide above-mentioned wood-sugar fermentation capable of using to produce the construction process of the engineering bacteria of optical pure L-lactic acid; The key of this method is the metabolism branch road blocking-up that thermophilic anaerobic bacillus (Thermoanaerobacteriumaotearoence SCUT27) pyruvic acid metabolism is generated acetate; The realization carbon metabolism flow is redistributed, and promotes L-lactic acid to accumulate significantly.
A purpose more of the present invention is to provide above-mentioned wood-sugar fermentation capable of using to produce the purposes of the engineering bacteria of optical pure L-lactic acid.
The object of the invention is realized through following technical proposals:
The engineering bacteria of optical pure L-lactic acid is produced in a kind of wood-sugar fermentation capable of using; Be two key enzymes---the phosphotransacetylase (phosphotransacetylase that thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoence SCUT27) pyruvic acid metabolism is generated the acetate process; Pta) and E.C. 2.7.2.1 (acetatekinase ack) does and obtains behind the gene knockout (knock-out).
The engineering bacteria of optical pure L-lactic acid is produced in a kind of wood-sugar fermentation capable of using, is specifically made up by following steps to obtain:
(1) that resistant gene of Synthesis Card (aph gene), and be inserted in intestinal bacteria and the thermophilic anaerobic bacillus shuttle vectors;
(2) a certain section sequence of pcr amplification E.C. 2.7.2.1 encoding sox is the pta-up sequence; Again the pta-up sequence is inserted in the intestinal bacteria that insert that resistant gene of card and thermophilic anaerobic bacillus shuttle vectors of step (1), makes up the suicide vector 1 that obtains carrying Acetokinase gene portion homologous sequence;
(3) a certain section sequence of pcr amplification phosphotransacetylase enzyme coding gene is the ack-down sequence; Again the ack-down sequence is inserted in the suicide vector 1, obtains to carry the suicide vector pPuKAd of phosphotransacetylase and E.C. 2.7.2.1 portion homologous sequence;
(4) suicide vector pPuKAd is transformed into the thermophilic anaerobic bacillus, and behind resistance screening, obtains the engineering bacteria that optical pure L-lactic acid is produced in wood-sugar fermentation capable of using.
According to principle of homologous recombination, which partial sequence of amplification phosphotransacetylase and E.C. 2.7.2.1 is unimportant in said step (2) and (3), so long as its homologous sequence gets final product.Preferably, the described a certain section sequence of step (2) the N end parts sequence that is the E.C. 2.7.2.1 encoding sox; The C end parts sequence that the described a certain section sequence of step (3) is the phosphotransacetylase enzyme coding gene;
Described intestinal bacteria of step (1) and thermophilic anaerobic bacillus shuttle vectors are pUC18, pUC19, pBluescript II SK (+) (Li; S.Yang XF.; Et al.Engineering of a thermophilic anaerobicbacterium to produce optically pure L-lactic acid through non-sterilized fermentation; Unpublished results), pSGD8 (Shaw; A.J.; K.K.Podkaminer, et al.Metabolicengineering of a thermophilic bacterium to produce ethanol at high yield.Proceedings of the National Academy of Sciences of the United States of America, 2008; 105 (37): 13769-13774) or pSGD9 (US20100015678A1), preferred pBluescriptII SK (+);
The method of the said conversion of step (4) is existing methods such as heat shock method, electrotransformation, combination conversion, this hybridization of parents;
The described thermophilic anaerobic bacillus of step (4) is the gram-positive microorganism that can fermentating metabolism generates acetate and lactic acid, preferably thermophilic anaerobic Bacillaceae Thermoanaerobacterium sp. or fusobacterium Clostridium sp.; Preferred especially thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoence SCUT27).
The engineering bacteria that optical pure L-lactic acid is produced in above-mentioned wood-sugar fermentation capable of using can be applicable to the fermentative prodn optical pure L-lactic acid.
The present invention has following advantage and effect with respect to prior art:
1, the present invention utilizes the method for homologous recombination, makes pyruvic acid produce the metabolism branch road key enzyme phosphotransacetylase and the E.C. 2.7.2.1 silence of acetate, thereby obtains the engineering bacteria of high lactic acid producing.
2, carry out the fermentative prodn of lactic acid with engineering bacteria of the present invention, do not produce acetate, carbon metabolism flow is redistributed, and has promoted a large amount of accumulation of title product lactic acid.And, also can simplify the back extraction process because the by product kind reduces.
3, be substrate with 50g/L glucose or wood sugar respectively with engineering bacteria of the present invention, by routine fermentation 72 hours, lactic acid concn can reach 47g/L and 38g/L, and substrate conversion efficiency reaches 0.94g/g and 0.76g/g, and the optical purity of L-lactic acid reaches more than 99.5%.When glucose and wood sugar mixing sugar (1: 1) with 50g/L are substrate, substratum is without sterilization direct fermentation 72h, and lactic acid production can reach 45g/L, and substrate conversion efficiency reaches 0.89g/g.
4, the present invention plays a significant role in the suitability for industrialized production of utilizing cellulose series biomass fermentative prodn optical pure L-lactic acid, reducing production costs, and has broad application prospects.
Description of drawings
Fig. 1 is acetate metabolism branch road blocking-up principle schematic; Wherein, A is a pta/ack gene cluster synoptic diagram; Pta/ack gene cluster 1-904bp be phosphotransacetylase (phosphotransacetylase, pta) encoding sox, pta/ack gene cluster 1005-2217bp are E.C. 2.7.2.1 (acetate kinase; Ack) encoding sox; Two homology arm pta-up and the ack-down that is used for homologous recombination lays respectively at gene cluster-275-904bp and 1358-1984bp, and position shown in the probe is the probe location that uses in the Southern blotting process among the figure, is positioned at pta/ack gene cluster 389-874bp; B is a homologous recombination vector pPuKAd synoptic diagram, between homology arm pta-up and ack-down, inserts that resistant gene expression cassette of card.
Fig. 2 is embodiment 1 an engineering bacteria pcr amplification product electrophorogram; Swimming lane M:1 kb DNA marker, swimming lane 1: wild type strain; Swimming lane 2: engineering bacteria.
Fig. 3 is embodiment 1 an engineering bacteria Southern blotting electrophorogram; Swimming lane 1: wild type strain; Swimming lane 2: engineering bacteria.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
The experimental technique that unreceipted actual conditions is arranged in the following example then is to operate according to the described condition of molecular cloning handbook of routine.
The structure of the engineering bacteria of optical pure L-lactic acid is produced in a kind of wood-sugar fermentation capable of using
The structure that the engineering bacteria of optical pure L-lactic acid is produced in a kind of wood-sugar fermentation capable of using is through in the middle of the gene of coding E.C. 2.7.2.1 and phosphotransacetylase; Insert that resistance encoding sox of heat-stable card; Realize knocking out of E.C. 2.7.2.1 and phosphotransacetylase enzyme coding gene; Thereby blocking-up acetate metabolism branch road, and can be through that resistance screening of card, preliminary evaluation positive recombinant.Concrete blocking-up Principle of Process is as shown in Figure 1, and concrete operations may further comprise the steps:
(1) makes up intestinal bacteria and the thermophilic anaerobic bacillus shuttle vectors pBlue-aph that carries that resistance expression's box of card: carry out the synthetic aph gene of full gene according to that resistant gene of card (GenBank:V01547), be inserted among the EcoR I and BamHI site of intestinal bacteria and thermophilic anaerobic bacillus shuttle vectors pBluescript II SK (+).
(2) make up the pBlue-pta-aph carrier that carries the acetokinase homologous sequence
With thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoence SCUT27) genomic dna (SEQ ID No.5) is template, pcr amplification acetokinase encoding sox (GenBank:HM802208, pta) the about 1.2kb of homologous sequence.Primer sequence is following:
Primer 1 (forward primer): 5 '-AACTA
GGTACCAGCGCTGTACGAAATTGCCACTC-3 '.The underscore base is a restriction enzyme Kpn I recognition site.
Primer 2 (reverse primer): 5 '-GTACT
GAATTCCACCCATTCCTTGTGTTATAGG-3 ' underscore base is a restriction enzyme EcoR I recognition site.
Above-mentioned pcr amplification product is connected with the pBlue-aph carrier of same double digestion behind Kpn I and EcoR I double digestion; Be transferred to then in the bacillus coli DH 5 alpha competent cell; The picking transformant is cut the checking positive colony through bacterium colony PCR and enzyme, obtains the pBlue-pta-aph carrier.
(3) make up the recombinant vectors pPuKAd that carries ethanoyl enzyme and phosphotransacetylase homologous sequence simultaneously
With thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoence SCUT27) genomic dna (SEQ ID No.5) is template, pcr amplification acid transacetylase encoding sox (GenBank:HM802208, ack) the about 0.6kb of homologous sequence.Primer sequence is following:
Primer 3 (forward primer): 5 '-TGAGC
GGATCCGCATAGAATTAGCTCCACTGC-3 '.The underscore base is a restriction enzyme BamH I recognition site.
Primer 4 (reverse primer): 5 '-TGACT
GCGGCCGCCGACGCCTCCCATAGCTG-3 '.The underscore base is a restriction enzyme Not I recognition site.
Above-mentioned pcr amplification product is connected with the pBlue-pta-aph carrier of same double digestion behind BamH I and Not I double digestion; Be transferred to then in the bacillus coli DH 5 alpha competent cell; The picking transformant is cut the checking positive colony through bacterium colony PCR and enzyme, obtains the pPuKAd carrier.
(4) with the pPuKAd carrier that builds; Be transferred in the Thermoanaerobacterium aotearoence SCUT27 competent cell through electric method for transformation; Be coated on that microbiotic plate culture medium of card that contains 50 μ g/ml, under 50 ℃ of conditions, cultivated 2-3 days.After growing bacterium colony, identify positive colony through PCR and Southern blotting hybridization, authentication method and result are following:
A, PCR identify
Extraction step (4) blocks the genomic dna of bacterium colony on that microbiotic flat board, with primer 2 (SEQ ID No.2) and primer 3 (SEQ ID No.3) as amplimer.For wild type strain (being Thermoanaerobacterium aotearoence SCUT27), PCR product fragment is about 2.2kb, should be 3.3kb and E.C. 2.7.2.1 and phosphotransacetylase enzyme coding gene knock out the PCR product fragment of the reorganization bacterium of acquisition.PCR result is as shown in Figure 2.
B、Southern?blotting
Extract the genomic dna of wild-type (being Thermoanaerobacterium aotearoence SCUT27) and engineering bacteria (being that step (4) is blocked bacterium colony on that microbiotic flat board), after cutting with Pst I enzyme, with probe (SEQID No.5) hybridization (principle is as shown in Figure 1).There is band wild type strain hybridization colour developing back at the 1.2kb place, and mutant strain hybridization colour developing back stripe size should be 2.2kb (as shown in Figure 3).
PCR and Southern blotting experiment show that all in the engineering bacteria of acquisition, E.C. 2.7.2.1 and phosphotransacetylase gene are successfully knocked out, and the gained positive colony is the engineering bacteria that optical pure L-lactic acid is produced in wood-sugar fermentation capable of using.
That microbiotic plate culture medium of 1L card contains NH
4Cl (ammonium chloride) 0.90g, NaCl (sodium-chlor) 0.90g, MgCl
26H
2O (magnesium chloride) 0.40g, KH
2PO
4(potassium primary phosphate) 0.75g, K
2HPO
4(potassium hydrogenphosphate) 1.50g, pancreatin 2.00g, yeast powder 1.00g, trace element solution 1.00ml, FeCl
36H
2O (iron(ic)chloride) 2.50mg, wood sugar 5.00g, hydrochloric acid-halfcystine 0.75g, Resazurin (resazurin) 0.50mg, agar 10g, surplus is a water, transfers pH to 6.5.
The 1L trace element solution contains 25%HCl (hydrochloric acid) 10ml, FeCl
24H
2O (iron protochloride) 1.5g, ZnCl
2(zinc chloride) 0.07g, MnCl
24H
2O (Manganous chloride tetrahydrate) 0.1g, H
3BO
3(boric acid) 0.006g, CoCl
26H
2O (NSC 51149) 0.19g, CuCl
22H
2O (cupric chloride) 0.002g, NiCl
26H
2O (nickelous chloride) 0.024g, Na
2MoO
42H
2O (Sodium orthomolybdate) 0.036g, surplus is a water.
The engineering bacteria and the contrast of its wild-type lactic acid production of optical pure L-lactic acid produced in wood-sugar fermentation capable of using
(1) seed culture
The engineering bacteria of embodiment 1 is linked in the 20ml serum bottle that the 10ml seed culture medium is housed, and 55 ℃, 150r/min are cultivated 12h, and the OD value is reached more than 0.8.Be inoculated in the 125ml serum bottle that the 50ml seed culture medium is housed with 1: 5 ratio, 55 ℃, 150r/min are cultivated 12h, and the OD value is reached more than 1.0.
(2) shake-flask culture
Seed liquor access by 10% inoculum size is cultivated step (1) is equipped with in the 125ml serum bottle of 50ml fermention medium, and inflated with nitrogen to pressure is 0.04MPa.With the 10g/L wood sugar is substrate cultured continuously 24h.
The operation of wild type strain is the same.
Detect meta-bolites through HPLC, comparative result is as shown in table 1.The result shows, detect acetate in the engineering bacteria nutrient solution, and lactic acid production has improved 1.8 times than wild type strain.
Meta-bolites changed before and after table 1 T.aotearoense SCUT27 acetate metabolism branch road sun was disconnected
The 1L seed culture medium contains glucose 2g, wood sugar 2g, citric acid tri potassium salt 2g, Citric acid monohydrate Food grade 1.25g, sodium sulfate 1g, potassium primary phosphate 1g, sodium hydrogencarbonate 2.5g, ammonium chloride 1.5g, urea 5g, yeast extract 1g, magnesium chloride hexahydrate 1g, four water iron protochloride 0.1g, Calcium dichloride dihydrate 0.2g, a water cysteine hydrochloride 1g, two hydrochloric acid Pyridoxylamine 0.02g, para-amino benzoic acid 0.004g, D-vitamin H 0.002g, cobalamin 0.002g, vitaminB10 .004g, and surplus is a water.
Fermentative medium formula: except that carbon source (wood sugar) addition is the 10g, all the other compositions are identical with the seed culture based formulas.
The detection of glucose, wood sugar consumption adopts Waters 2695 HPLies (HPLC) to measure
Chromatographic condition is:
Chromatographic column: Aminex HPX-87P (Biorad)
Moving phase: ultrapure water
Flow velocity: 0.6ml/min
Column temperature: 60 ℃
Detector temperature: 40 ℃
Specimen preparation: the 2ml fermented liquid adds 1g CaCO
3, vibration 1min, centrifugal 5min under 16000g gets supernatant with 0.22 μ m membrane filtration.Be used to detect residual sugar.
Sample size: 10 μ l
Detector: differential detector.
The detection of meta-bolites lactic acid, ethanol, acetic acid content adopts Waters 2695 HPLies (HPLC) to measure
Chromatographic condition is:
Chromatographic column: Aminex HPX-87H (Biorad)
Moving phase: 5mM H
2SO
4
Flow velocity: 0.6ml/min
Column temperature: 60 ℃
Detector temperature: 40 ℃
Specimen preparation: the 1.9ml fermented liquid adds 100 μ l 10%H
2SO
4, centrifugal 5min under 16000g gets supernatant with 0.22 μ m membrane filtration.Be used to detect acid and ethanol.Sample size: 10 μ l
Detector: differential detector.
Embodiment 3
Whether substratum sterilizes to the influence of engineering bacterium fermentation product of the present invention
By 10% inoculum size, the seed liquor access of embodiment engineering bacteria is equipped with in the full-automatic reaction kettle of the 5L of NBS company of 3L fermention medium.Before inoculation, reaction kettle feeds nitrogen 30min earlier, feeds nitrogen 30min after the inoculation again, to guarantee the yeasting anaerobic.It is 6.8 that fermenting process uses 5mol/L NaOH control pH value.
Measure lactic acid, acetate, ethanol and sugared concentration (table 2) in the tunning.
With 50g/L glucose is the substrate 72h that continuously ferments, and lactic acid concn can reach 47.2g/L, and transformation efficiency reaches 0.95g/g, and L-lactic acid optical purity surpasses 99.5%.
Glucose and wood sugar mixing sugar with 50g/L are fermenting substrate, and the lactic acid production under medium sterilization and the unsterilised condition reaches 42g/L and 44.8g/L respectively.
The result shows do not have acetate to produce in the fermenting process, shows that the engineering bacteria acetate metabolism branch road that the present invention makes up is successfully blocked.In addition, to have blocked the thermophilic anaerobic bacillus mutant strain fermentation of acetate metabolism branch road, the fermenting process substratum need not sterilization, thereby reduces the requirement to zymotechnique, reduces fermentation costs significantly, has very wide application prospect.
Table 2 is cultivated reorganization bacterium lactic acid production with different fermenting substrate jars
The lactic acid optical purity detects
Lactic acid optical purity (e.e value) calculation formula is following:
Wherein, L-lactic acid uses the glucose-lactic acid analyser (SBA-40C) based on biosensor principle to record.The lactic acid total amount is to adopt HPLC to detect to obtain.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. the engineering bacteria of optical pure L-lactic acid is produced in a wood-sugar fermentation capable of using, it is characterized in that it being to be made up by following steps to obtain:
(1) that resistant gene of Synthesis Card, and be inserted in intestinal bacteria and the thermophilic anaerobic bacillus shuttle vectors;
(2) a certain section sequence of pcr amplification E.C. 2.7.2.1 encoding sox is the pta-up sequence; Again the pta-up sequence is inserted in the intestinal bacteria that insert that resistant gene of card and thermophilic anaerobic bacillus shuttle vectors of step (1), makes up and obtain suicide vector 1;
(3) a certain section sequence of pcr amplification phosphotransacetylase enzyme coding gene is the ack-down sequence; Again the ack-down sequence is inserted in the suicide vector 1, obtains suicide vector pPuKAd;
(4) suicide vector pPuKAd is transformed into the thermophilic anaerobic bacillus, and behind resistance screening, obtains the engineering bacteria that optical pure L-lactic acid is produced in wood-sugar fermentation capable of using.
2. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, it is characterized in that: the N end parts sequence that the described a certain section sequence of step (2) is the E.C. 2.7.2.1 encoding sox.
3. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, it is characterized in that: the C end parts sequence that the described a certain section sequence of step (3) is the phosphotransacetylase enzyme coding gene.
4. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, and it is characterized in that: described intestinal bacteria of step (1) and thermophilic anaerobic bacillus shuttle vectors are pUC18, pUC19, pBluescript II SK (+), pSGD8 or pSGD9.
5. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, and it is characterized in that: described intestinal bacteria of step (1) and thermophilic anaerobic bacillus shuttle vectors are pBluescript IISK (+).
6. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, and it is characterized in that: the method for the said conversion of step (4) is heat shock method, electrotransformation, combination conversion or this hybridization of parents.
7. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, and it is characterized in that: the described thermophilic anaerobic bacillus of step (4) is thermophilic anaerobic Bacillaceae Thermoanaerobacterium sp. or fusobacterium Clostridium sp..
8. the engineering bacteria of optical pure L-lactic acid is produced in wood-sugar fermentation capable of using according to claim 1, and it is characterized in that: the described thermophilic anaerobic bacillus of step (4) is thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoence SCUT27).
9. the application of engineering bacteria in the fermentative prodn optical pure L-lactic acid of each described wood-sugar fermentation production optical pure L-lactic acid capable of using of claim 1-8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154465A (en) * | 2015-07-23 | 2015-12-16 | 昆明理工大学 | Recombinant plasmid for knocking out acetokinase genes and application of recombinant plasmid |
| CN115011536A (en) * | 2022-06-14 | 2022-09-06 | 湖北工业大学 | Engineering bacterium for inducing double anaerobic promoters to produce high-optical-purity D-lactic acid and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100015678A1 (en) * | 2005-10-31 | 2010-01-21 | Shaw Iv Arthur Josephus | Thermophilic Organisms For Conversion of Lignocellulosic Biomass To Ethanol |
-
2011
- 2011-12-29 CN CN2011104536847A patent/CN102433293A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100015678A1 (en) * | 2005-10-31 | 2010-01-21 | Shaw Iv Arthur Josephus | Thermophilic Organisms For Conversion of Lignocellulosic Biomass To Ethanol |
Non-Patent Citations (8)
| Title |
|---|
| 《食品工业科技》 20090831 付晓芬等 微生物利用木糖发酵L-乳酸代谢途径的研究 359-362 1-9 第30卷, 第8期 * |
| NARBERHAUS F,ET AL: "Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE,dnaJ,and a new heat shock gene", 《J BACTERIOL》 * |
| SHAW,A.J.,K.K.PODKAMINER,ET AL: "Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield.", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》 * |
| YANG G,ET AL: "Engineering and metabolic characteristics of a Clostridium tyrobutyricum strain", 《SHENG WU GONG CHENG XUE BAO》 * |
| 付晓芬等: "微生物利用木糖发酵L—乳酸代谢途径的研究", 《食品工业科技》 * |
| 吕福英等: "热纤梭菌分解纤维素生产乙醇的研究进展", 《食品与发酵工业》 * |
| 杨晓锋等: "嗜热厌氧杆菌发酵木糖生产乳酸的代谢调控研究", 《PROCEEDINGS OF 2010 FIRST INTERNATIONAL CONFERENCE ON CELLULAR,MOLECULAR BIOLOGY, BIOPHYSICS AND BIOENGINEERING》 * |
| 王玉华等: "L-乳酸生产菌种选育技术", 《生命的化学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154465A (en) * | 2015-07-23 | 2015-12-16 | 昆明理工大学 | Recombinant plasmid for knocking out acetokinase genes and application of recombinant plasmid |
| CN115011536A (en) * | 2022-06-14 | 2022-09-06 | 湖北工业大学 | Engineering bacterium for inducing double anaerobic promoters to produce high-optical-purity D-lactic acid and preparation method and application thereof |
| CN115011536B (en) * | 2022-06-14 | 2023-06-23 | 湖北工业大学 | Engineering bacterium for producing high optical purity D-lactic acid by double anaerobic promoters and preparation method and application thereof |
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