CN102199553B - Acid-resistant yeast and method for producing L-lactic acid recombinant strain through metabolic engineering construction - Google Patents
Acid-resistant yeast and method for producing L-lactic acid recombinant strain through metabolic engineering construction Download PDFInfo
- Publication number
- CN102199553B CN102199553B CN2011101167836A CN201110116783A CN102199553B CN 102199553 B CN102199553 B CN 102199553B CN 2011101167836 A CN2011101167836 A CN 2011101167836A CN 201110116783 A CN201110116783 A CN 201110116783A CN 102199553 B CN102199553 B CN 102199553B
- Authority
- CN
- China
- Prior art keywords
- lactic acid
- yeast
- candida magnoliae
- strain
- ldha
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses acid-resistant yeast and a method for producing an L-lactic acid recombinant strain through metabolic engineering construction, which belong to the technical field of genetic engineering. In the invention, the yeast which can grow under the condition that the pH value is 2.5 and is free from L-lactic acid is obtained by screening and separating and is identified as candidamagnoliae C4; the yeast is preserved in the China Center for Type Culture Collection, with the preservation number of CCTCCNO:M2010362; a coding gene (ldhA) of lactate dehydrogenase from rhizopus oryzae As3.819 is inserted into a yeast shuttle vector containing a G418 resistance gene to construct a recombinant plasmid, i.e., pYX212-kanMX-ldhA; and the recombinant plasmid is electrically converted into the candidamagnoliaeC4 to obtain a recombinant strain candidamagnoliae C42 with the capability of producing L-lactic acid, wherein the L-lactic acid yield of the recombinant strain candidamagnoliae C42 reaches 42g/L. The candidamagnoliae C42 constructed by the invention has the remarkable advantage of resisting lactic acid; and the consumption of CaCO3 as a neutralizing agent in the production can be greatly reduced, thus the pollution to the environment is reduced.
Description
Technical field
The present invention relates to the acidproof zymic screening of a strain, and the construction process that produces L-lactic acid reorganization bacterium, gene engineering technology field belonged to.
Background technology
Lactic acid (Lactic acid) is a kind of important organic acid, is present in widely in human body, animal, plant and the mikrobe.Lactic acid is widely used in industry such as food, medicine and makeup because of its security, optical characteristics and specific molecule structure thereof.Lactic acid molecules contains an asymmetric C atom, has enantiomerism, has two kinds on D-type and L-type.Because human body can only utilize L (+)-lactic acid, so the World Health Organization advocates at food and pharmaceutical industries use L (+)-present DL (+)-lactic acid that generally uses of lactic acid replacement; L-lactic acid important use is a synthesizing polylactic acid in addition, and POLYACTIC ACID is because of can be by renewable resources production and can be by one of polymkeric substance that is considered to the most promising that biology is degraded.
The main working method of L-lactic acid is a microbe fermentation method.The existing L-lactic-acid-producing strain of research both at home and abroad mainly concentrate on probiotic lactobacillus (
Lactobacillus), genus bacillus (
Bacillus), lactococcus spp (
Lactococcus) and head mold (
Rhizopus).In lactic fermentation process, the pH value can reduce along with the generation of lactic acid gradually, because bacterium and rhizopus acid resistance are relatively poor, when fermented liquid is in acidity, will influence the growth and the fermentation capacity of bacterial strain.Therefore, lactic fermentation process often adopts CaCO in the industry
3As neutralizing agent, to keep optimum pH.But the lactic acid ca crystallization is tiny, and crystallisation process is wayward, and 30% lactic acid ca remains in the crystalline mother solution, uncrystallizable come out, and a large amount of by product (gypsum) can cause environmental pollution, becomes key point and difficult point that the lactic acid-producing enterprise three wastes are handled.
Therefore both at home and abroad with respect to bacterium and mould, yeast has better acid-resistant property, and the investigator transfers research emphasis gradually to and utilizes gene engineering method to transform genetic background yeast saccharomyces cerevisiae clearly, makes it can produce lactic acid.Though recombinant bacterial strain can be survived and grow under higher acidic conditions, the lactic acid producing ability is generally not strong.Reorganization lactic acid producing yeast ultimate capacity of lactic acid when pH 3.5 of people such as Adachi E research is merely 60% (Adachi E, Torigoe M, Sugiyama M, et al. Modification of metabolic pathways of when pH 4.5
Saccharomyces cerevisiaeBy the expression of lactate dehydrogenase and deletion of pyruvate decarboxylase genes for the lactic acid fermentation at low pH value. J Ferment Bioeng; 1998,86 (3): 284-289.).Afterwards, the optimum pH of the reorganization lactic acid producing yeast of Skory CD institute research and establishment is 5.0, when fermented liquid pH value is lower than 5.0, and lactic acid production (the Skory CD. Lactic acid production by that will decrease
Saccharomyces cerevisiaeExpressing a
Rhizopus oryzaeLactate dehydrogenase gene. J Ind Microbiol Biotechnol (2003) 30:22 – 27.).Therefore the investigator still need add a certain amount of neutralizing agent and regulate fermented liquid pH value when utilizing the reorganization lactic acid producing yeast to ferment in fermented liquid afterwards.
Summary of the invention
The objective of the invention is for overcoming the not strong shortcoming of above-mentioned lactic-acid-producing strain acid resistance; Filter out the dense lactic acid of the anti-height of a strain and do not utilize the acidproof yeast strain of lactic acid; Pass through metabolic engineering on this basis; Increase the lactic acid metabolism approach of this bacterial strain, make it hang down normal fermentation production of L-lactic acid under the pH value.
For realizing the object of the invention, the technical scheme that the present invention adopted is:
One strain can be in the culture environment of regulating pH to 2.5 with lactic acid growth but do not utilize the bacterial strain of lactic acid, called after candida magnoliae C4 (
Candida magnoliaeC4), in China's typical culture collection center preservation, deposit number is CCTCC NO:M2010362.
One strain candida magnoliae C42 (
Candida magnoliaeC42), it obtains candida magnoliae C42 and has the performance of producing L-lactic acid at low pH condition bottom fermentation for described candida magnoliae C4 is carried out recombination.
Described candida magnoliae C42 is the lactic dehydrogenase enzyme coding gene that derives from Rhizopus oryzae As3.819
LdhAInsertion contains the yeast shuttle vector of G418 resistant gene, has made up recombinant plasmid pYX212-
KanMX-
LdhA, and electricity is transformed among the candida magnoliae C4, obtains candida magnoliae C42.
(1) screening of acidproof yeast strain
Take by weighing 5 g nature sample (gathering the soil sample from limit, Wuxi City Taihu Lake), add in the triangular flask that contains 100 mL SPSSs and granulated glass sphere, under 30 ℃, 30 min are broken up in 150 r/min vibration; Sample suspension is carried out serial dilution with SPSS, selects suitable extent of dilution, and separate application is dull and stereotyped in YEPD, cultivates 5 ~ 7 d for 30 ℃, observe the colony growth situation, and discrepant single bacterium colony is chosen on the form that will newly grow.The typical yeast colony of picking is inoculated in respectively in the YEPD liquid nutrient medium with newborn acid for adjusting pH value to 4 from single bacterium colony that separation obtains, and cultivates 48 h for 30 ℃.The selection yeast strain that grows is preferably inserted in the screening culture medium that contains 93.5 g/L lactic acid successively, cultivates 48 h, selects the acidproof yeast of the best strain of growing way, called after C4.Through morphology and molecular biology identification, this acidproof zymic classification position belongs to candida magnoliae
Candida magnoliae(
C. magnoliae), in China's typical culture collection center preservation, deposit number is CCTCC NO:M 2010362.
(2) the lactic dehydrogenase enzyme coding gene of Rhizopus oryzae As3.819 (
LdhA) acquisition
Extracting the chromosomal DNA of Rhizopus oryzae As3.819, is that masterplate carries out pcr amplification with it, and the primer is following:
R1：5′-CGCGGATCCATGGTATT?ACACTCA-3′
R2：5′-CCGAAGCTTTCAACAGCTACTTTTA-3′
PCR method is following: add in the 50 μ L reaction systems: each 1 μ L of the primer R1 of 10mmol/L and R2, and the dNTP 5 μ L of 2 mmol/L, 10 *
ExTaqBuffer 5 μ L, 5 U/ μ L's
ExTaqArchaeal dna polymerase 0.5 μ L, template 1 μ L adds distilled water polishing 50 μ L; Pcr amplification condition: 95 ℃ of preparatory sex change 5 min; 94 ℃ of sex change 30 s, 55 ℃ of annealing 60 s, 72 ℃ of extensions 60 s, totally 30 circulations: last 72 ℃ are extended 10 min.
Analyze the purifying reaction product through agarose gel electrophoresis, target stripe occurs, reclaim the target fragment that test kit reclaims out 1.0kb with the PCR fragment at the application of sample swimming lane; The target fragment warp that purifying is good
BamThe H I with
HinD III double digestion reclaims test kit with the PCR fragment and reclaims; PYX212-
KanThe MX process
BamThe H I with
HinD III double digestion reclaims test kit with the PCR fragment and reclaims; Connect two enzymes with the T4 ligase enzyme and cut the good fragment of purifying, connect product transformed into escherichia coli JM109 competent cell, transform the LB agar plate that the back coating contains 30 μ g/mL kantlex, 37 ℃ of overnight cultures; 6 bacterium colonies of random choose are inoculated in 30mL and contain 37 ℃ of overnight cultures in the LB substratum of 30 μ g/mL kantlex, extract plasmid, through
BamThe H I with
HinD III double digestion, agarose gel electrophoresis are identified positive colony, and positive recombinant is kept in the LB substratum that contains 15% glycerine, freezing being stored in-70 ℃; Recombinant plasmid called after pYX212-
KanMX-
LdhA, positive recombinant called after JM109/pYX212-
KanMX-
LdhA, acidproof yeast called after C42 recombinates;
(3) structure of the acidproof yeast C42 of reorganization:
Get the JM109/pYX212-of a freezing preservation
KanMX-
LdhA, be inoculated in 30 mL and contain 37 ℃ of overnight cultures in the LB substratum of 30 μ g/mL kantlex, extract plasmid, transform the acidproof yeast of wild-type with electrotransformation
C. magnoliae, transformant is coated and is contained the antibiotic YEPD of 800 μ g/mL G418 dull and stereotyped (G418 resistant panel), in 30 ℃, cultivated 2 days; The bacterium colony that grows is rule to the G418 resistant panel again, and grow bacterium colony and tentatively confirm as positive transformant, again through bacterium colony PCR, checking recombinant plasmid pYX212-
KanMX-
LdhABe transformed into acidproof yeast
C. magnoliae, again this reorganization bacterium is carried out cytoclasis, carry out the SDS-PAGE electrophoresis behind the cytoclasis liquid dilution proper concn, checking external source lactate dehydrogenase gene is at acidproof yeast
C. magnoliaeIn obtain expressing.
G418 is a kind of aminoglycoside relevant with qingfengmeisu qiong, is a kind of analogue of polygynax, and it can disturb the protein synthesis in ribosomal function of 80 S and the eukaryotic cell, is used as the selective reagents of eukaryotic cell (yeast) usually.For efficiently selecting positive transformant, the present invention's G418 resistance of will encoding
KanMX inserts carrier pYX212, has made up carrier pYX212-
KanMX.
Free type expression vector pYX212-
KanMX-
LdhABe will
LdhABe inserted into carrier pYX212 and go up promotor
TPIDownstream, make it at carrier pYX212-
KanMX is last to express.Gene
LdhAAcquisition, carrier pYX212-
KanMX-
LdhAStructure, gene
LdhAExpression in acidproof yeast sees embodiment for details.
This acidproof yeast of recombinating can detect serum lactic dehydrogenase (LDH) enzyme and live, and successfully will
LdhAIn acidproof yeast, express, and expression of heterologous genes can be that substrate ferments with glucose to not influence of zymic growth, behind 48 h that ferment, can detect L-lactic acid in the fermented liquid.
(4) produce L-lactic acid with the acidproof yeast C42 fermentation of the reorganization that makes up
The fermentation that with glucose is substrate sees embodiment for details.
Beneficial effect of the present invention: the yeast strain that the present invention has anti-lactic acid ability for screening in the nature sample; Make it under low pH condition, have stronger L-lactic acid-producing ability through metabolic engineering; The consumption of neutralizing agent when the L-lactic acid production reaches 42 g/L with the acid of reduction product; Practice thrift cost, this result of study does not still have report at home and abroad.
Utilize the G418 resistant panel to select the carrier pYX212-that has the present invention to make up
KanMX-
LdhAAcidproof yeast, the acidproof yeast of promptly recombinating.Through cultivating the acidproof yeast of reorganization, detect the content of L-lactic acid in the fermented liquid.Cultivation is chosen in 250 mL and shakes on the bottle and carry out, and cultivates in two stages, and the fs is mainly used in the zymic growth, and it is synthetic that subordinate phase is mainly used in product; Can detect L-lactic acid with performance liquid chromatography, can detect the expression of heterologous protein with SDS-PAGE.
Successively to reorganization bacterium C42 fermentation condition: the different vaccination amount is carried out initial optimization when the initial sugared concentration of fermention medium, fermention medium original ph, fermentation culture different time of repose in early stage, fermentation, obtains the L-lactic acid production and reaches 42 g/L.
The biological material specimens preservation: a strain can be in the culture environment of regulating pH to 2.5 with lactic acid growth but do not utilize the bacterial strain of lactic acid, called after candida magnoliae C4 (
Candida magnoliaeC4), in China's typical culture collection center preservation, CCTCC is write a Chinese character in simplified form, the address in Chinese typical culture collection center: Chinese Wuhan Wuhan University, and preservation date is on December 22nd, 2010, deposit number is CCTCC NO:M2010362.
Description of drawings
Fig. 1 DNAman software is drawn the homology tree of acidproof yeast strain
Fig. 2 plasmid pYX212-
KanMX
Fig. 3 plasmid pYX212-
KanMX-
LdhA
Fig. 4 plasmid pYX212-
KanMX-
LdhAChecking
Lane1 carrier pYX212-
KanMX
BamH I enzyme is cut;
Lane2 recombinant plasmid pYX212-
KanMX-
LdhAPcr amplification
LdhAChecking;
Lane3 recombinant plasmid pYX212-
KanMX-
LdhABamThe H I with
HinD III double digestion;
Lane4 recombinant plasmid pYX212-
KanMX-
LdhABamH I enzyme is cut;
Lane5：λDNA/
Pst?I?marker。
Recombinate acidproof zymic SDS-PAGE of Fig. 5 analyzes the acidproof yeast of Lane1
C. magnoliaeLane2, the Lane3 acidproof yeast of recombinating; Lane4 molecular weight of albumen standard
The influence that the different initial sugared concentration of Fig. 6 are produced L-lactic acid to the acidproof yeast C42 fermentation of recombinating: 20 g/L (■); 60 g/L (●); 100 g/L (▲); 140 g/L (◆); 180 g/L (★); 200 g/L (
).
The influence that the different original ph of Fig. 7 are produced L-lactic acid to the acidproof yeast C42 fermentation of recombinating: pH2.5 (■); PH3.0 (●); PH3.5 (▲); PH4.0 (◆); PH7.0 (★).
Fig. 8 difference leaves standstill the influence that incubation time produces L-lactic acid to the acidproof yeast C42 fermentation of recombinating: ventilation incubation time 0 h (■) of the thalline that is used to grow; 8 h (●); 16 h (▲); 24 h (◆); 32 h (★); 40 h (
)
The influence that Fig. 9 different vaccination amount is produced L-lactic acid to the acidproof yeast C42 fermentation of recombinating: dense with inoculation secondary fermentation liquid bacterium is mark
OD 600 =5 (■);
OD 600 =10 (●);
OD 600 =15 (▲);
OD 600 =20 (◆);
OD 600 =25 (
)
Embodiment
Embodiment 1: the screening of acidproof yeast strain C4
Take by weighing 5 g nature sample (gathering the soil sample from limit, Wuxi City Taihu Lake), add in the triangular flask that contains 100 mL SPSSs and granulated glass sphere, under 30 ℃, 30 min are broken up in 150 r/min vibration; Sample suspension is carried out serial dilution with SPSS, is 10 with extension rate
-7Diluent coat the YEPD flat board, cultivate 5 ~ 7 d for 30 ℃, observe the colony growth situation, and discrepant single bacterium colony is chosen on the form that will newly grow.The typical yeast colony of picking is inoculated in respectively in the YEPD liquid nutrient medium with newborn acid for adjusting pH value to 4 from single bacterium colony that separation obtains, and cultivates 48 h for 30 ℃.The selection yeast strain that grows is preferably inserted in the screening culture medium that contains 93.5g/L lactic acid successively, cultivates 48 h, selects the acidproof yeast of the best strain of growing way, called after C4.
Embodiment 2: the evaluation of acidproof yeast strain C4
1) morphological specificity
Morphological observation: get acidproof bacterial strain and be cultured to move respectively after the balance period and be connected in the YEPD substratum, cultivate 3 days laggard Xingqi cell morphological characteristics and modes of reproduction observation down for 25 ℃.Yeast colony morphologic observation: get to move respectively after strain culturing to be identified to the balance period and be connected in the YEPD substratum, cultivate after 3 days down for 25 ℃ and carry out the flat-plate bacterial colony morphologic observation.Acidproof zymic colony morphology characteristic is single colony diameter 2 mm, the opaque creaminess that is white in color, and full, the edge is smooth, and is glossy, rat, than thickness, colony colour and quality all compare homogeneous; It is avette or spherical that cell morphological characteristic is that cell is, polygon budding.
2) molecular biology identification:
The process for extracting of cerevisiae dna is with reference to the molecular cloning laboratory manual, and the primer is a fungi ITS universal primer,
Upper reaches ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ',
Downstream ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ';
Pcr amplification product checks order after electrophoresis detection, and sequencing result is following:
AACCTGCGGA?AGGATCATTT?CTGAACAACC?TTTGCATAGT?GAACAACCTC?TACGGGGGTG?60
AATGCTTCCG?GTTCGCCGGA?CAACAACTAG?TAATTTTGAA?TCTGATACGT?TAAAGAAAAA?120
CTTTCAACAA?CGGATCTCTT?GGTTCTCGCA?TCGATGAAGA?ACGCAGCAAA?GCGCGATAGG?180
TAATGCGAAT?TGCAGACGTG?AGTCATTGAA?TCTTTGAACG?CACATTGCGC?CAGTTTACTG?240
GCATACTGCG?TTGAGCGTCG?GCCTTCCTCC?ACAGGTATTG?CTGTTTGCAA?CAGTCAAAGG?300
CATGGAAGCG?CACCCGTTAG?TAAAACGGTG?CAAAACCACA 340
Institute's calling sequence is carried out the homologous sequence search in nucleic acid sequence data (GenBank); With the ITS regional sequence of relevant barms with bacterial strain sequence to be measured; Draw homology tree (homology trees); Show known saccharomycetic close source relation in strains tested and the sequence library, shown in accompanying drawing 1.
3) Physiology and biochemistry is identified:
Test in assimilation nitrogen (carbon) source: after fresh yeast strain to be measured is washed with sterilized water, coat on the solid medium of assimilation nitrogen (carbon) source, cultivate 2-3 d for 28 ℃, observe the colony growth situation, the result is as shown in table 1.
The physiological and biochemical property of table 1 bacterial strain C4
| Carbon/nitrogenous source | Utilize | Carbon/nitrogenous source | Utilize | Carbon/nitrogenous source | Utilize | Carbon/nitrogenous source | Utilize |
| Glucose | + | The D-wood sugar | + | Raffinose | - | The D-sorbyl alcohol | + |
| The L-sorbose | - | Cellobiose | - | D-ribose | - | Erythritol | - |
| SANMALT-S | - | Synanthrin | - | The D-seminose | + | DL-lactic acid | - |
| Trehalose | - | Methyl alcohol | - | Semi-lactosi | + | Hydrocerol A | - |
| Melibiose | - | Ethanol | - | Lactose | - | Succsinic acid | - |
| Melizitose | - | Inositol | - | Sucrose | - | Maltonic acid | + |
| Zulkovsky starch | - | Melampyrum | - | The L-rhamnosyl | - | L-Methionin | + |
| L-arabinose | - | Ribitol | - | The D-seminose | + | Nitrate salt | + |
| The D-pectinose | - | USP Kosher | + | ? | ? | ? | ? |
Acidproof yeast after morphology, molecular biology and Physiology and biochemistry are identified, the classification position that confirms this acidproof yeast C4 should belong to candida magnoliae (
C. magnoliae), be numbered CCTCC NO:M 2010362 at China typical culture collection center.
Embodiment 3: plasmid pYX212-
KanMX-
LdhAStructure
Extracting the chromosomal DNA of Rhizopus oryzae As3.819, is that masterplate carries out pcr amplification with it, and the primer is following:
R1：5′-CGCGGATCCATGGTATT?ACACTCA-3′
R2：5′-CCGAAGCTTTCAACAGCTACTTTTA-3′
PCR method is following: add in the 50 μ L reaction systems: each 1 μ L of the primer R1 of 10mmol/L and R2, and the dNTP 5 μ L of 2 mmol/L, 10 *
ExTaqBuffer 5 μ L, the ExTaq archaeal dna polymerase 0.5 μ L of 5 U/ μ L, template 1 μ L adds distilled water polishing 50 μ L; Pcr amplification condition: 95 ℃ of preparatory sex change 5 min; 94 ℃ of sex change 30 s, 55 ℃ of annealing 60 s, 72 ℃ are extended 60 s, totally 30 circulations; Last 72 ℃ are extended 10 min.
Analyze the purifying reaction product through agarose gel electrophoresis, target stripe occurs, reclaim the target fragment that test kit reclaims out 1.0kb with the PCR fragment at the application of sample swimming lane; The target fragment warp that purifying is good
BamThe H I with
HinD III double digestion reclaims test kit with the PCR fragment and reclaims; PYX212-
KanThe MX process
BamThe H I with
HinD III double digestion reclaims test kit with the PCR fragment and reclaims; Connect two enzymes with the T4 ligase enzyme and cut the good fragment of purifying, connect product transformed into escherichia coli JM109 competent cell, transform the LB agar plate that the back coating contains 30 μ g/mL kantlex, 37 ℃ of overnight cultures; 6 bacterium colonies of random choose are inoculated in 30mL and contain 37 ℃ of overnight cultures in the LB substratum of 30 μ g/mL kantlex, extract plasmid, through
BamThe H I with
HinD III double digestion, agarose gel electrophoresis are identified positive colony, and positive recombinant is kept among the LB that contains 15% glycerine, freezing being stored in-70 ℃; Recombinant plasmid called after pYX212-
KanMX-
LdhA, positive recombinant called after JM109/pYX212-
KanMX-
LdhA
Embodiment 5: the structure of the acidproof yeast C42 that recombinates
Get the JM109/pYX212-of a freezing preservation
KanMX-
LdhA, be inoculated in 30 mL and contain 37 ℃ of overnight cultures in the LB substratum of 30 μ g/mL kantlex, extract plasmid, transform the acidproof yeast of wild-type with electrotransformation
C. magnoliae, transformant is coated and is contained the antibiotic YEPD of 800 μ g/mL G418 dull and stereotyped (G418 resistant panel), in 30 ℃, cultivated 2 days; The bacterium colony that grows is rule to the G418 resistant panel again, and grow bacterium colony and tentatively confirm as positive transformant, again through bacterium colony PCR, checking recombinant plasmid pYX212-
KanMX-
LdhABe transformed into acidproof yeast
C. magnoliae, again this reorganization bacterium is carried out cytoclasis, carry out the SDS-PAGE electrophoresis behind the cytoclasis liquid dilution proper concn, checking external source lactate dehydrogenase gene is at acidproof yeast
C. magnoliaeIn obtain expressing.The acidproof yeast-inoculated of will recombinating is in 30 mL YEPD substratum (peptones 2%; Glucose 2%, yeast extract paste 1%) in the triangular flask of 250 mL, cultivate 48 h after; Centrifugal collection thalline; Adopt the ultrasonic disruption cell, carry out the SDS-PAGE checking, simultaneously with acidproof yeast as contrast; Find that this strain acidproof yeast C42 that recombinates the protein characteristic band occurs at 36 kD places, (Skory CD. Isolation and expression of lactate dehydrogenase genes from conforms to the target protein size of bibliographical information
Rhizopus oryzae. Applied and Environmental Microbiology, 2000,66 (6): 2343-2348.).
Embodiment 6: the acidproof yeast C42 fermenting characteristic of recombinating
The technology that the present embodiment explanation utilizes the acidproof yeast C42 fermentation of reorganization to produce L-lactic acid.
Seed culture medium: glucose 20 g/L; Peptone 20 g/L; Yeast extract paste 10 g/L; With the tap water preparation, the pH nature.
Fermention medium: peptone 20 g/L; Yeast extract paste 10 g/L; Glucose concn is respectively 20 g/L, 60 g/L, 100 g/L, 140 g/L, 180 g/L and 200 g/L, with tap water preparation, pH value nature.
Seed liquor is switched to 10% inoculum size in the fermention medium that contains different glucose concn, and under 30 ℃, 150 r/min cultivate 48 h.Adopt liquid chromatogram measuring, when the fermention medium initial glucose concentration was 140g/L, the L-lactic acid content was the highest in the fermented liquid, and output reaches 23 g/L, shown in accompanying drawing 6.
The content of L-lactic acid adopts the HPLC method to measure in the fermented liquid.Fermented liquid is through 8000 rpm centrifugal 5 min, get 300 μ L supernatants add 700 μ L absolute ethyl alcohol precipitating proteins, 3 h above after, centrifugal 15 min of 12000 r/min behind the 0.45 μ m millipore filtration, measure through HPLC.Analysis condition: chromatographic column SH1011; Moving phase 0.01 mol/L H
2SO
4Flow velocity 0.8 mL/min; Sample size 5 μ L; 50 ℃ of column temperatures; Detector is a UV-detector, and detecting wavelength is 210 nm.
Embodiment 7: the acidproof yeast fermentation characteristic of recombinating
The technology that the present embodiment explanation utilizes the acidproof yeast C42 fermentation of reorganization to produce L-lactic acid.
Fermention medium: glucose 100 g/L; Peptone 20 g/L; Yeast extract paste 10 g/L; With the tap water preparation, adjust pH is respectively 2.5,3.0,3.5,4.0,7.0;
In the YEPD liquid nutrient medium that contains 100 g/L glucose, drip HCl respectively, make its medium pH value be respectively 2.5,3.0,3.5,4.0,7.0.
Seed liquor is forwarded in the substratum of adjusted pH value with 10% inoculum size respectively, and under 30 ℃, 150 r/min cultivate 48 h; Adopt liquid chromatogram measuring, the fermention medium original ph is 3.5 o'clock, and the L-lactic acid content is the highest in the fermented liquid, and output reaches 15 g/L.Shown in accompanying drawing 7.All the other are with embodiment 6.
Embodiment 8: the acidproof yeast fermentation characteristic of recombinating
The technology that the present embodiment explanation utilizes the acidproof yeast C42 fermentation of reorganization to produce L-lactic acid.
Fermention medium: glucose 100 g/L; Peptone 20 g/L; Yeast extract paste 10 g/L; With tap water preparation, pH value nature;
Seed liquor is connected to 10% inoculum size and cultivates 96 h in the fermention medium.When beginning fermentation, under 30 ℃, 150 r/min shake-flask culture go to after cultivating 0 h, 8 h, 16 h, 24 h, 32 h and 40 h respectively and leave standstill cultivation (per 8 h shake up 1 time) in 30 ℃ of incubators.Adopt liquid chromatogram measuring, leave standstill when cultivating 8h early stage, and the L-lactic acid content is the highest in the fermented liquid, and output reaches 17 g/L.Shown in accompanying drawing 8.All the other are with embodiment 6.
Embodiment 9: the acidproof yeast fermentation characteristic of recombinating
The technology that the present embodiment explanation utilizes the acidproof yeast C42 fermentation of reorganization to produce L-lactic acid.
Fermention medium: glucose 140 g/L; Peptone 20 g/L; Yeast extract paste 10 g/L; With the tap water preparation, original ph is 3.5;
The bacterium of will recombinating is scraped from the YEPD resistant panel and gets a single bacterium colony to seed culture medium, and under 30 ℃, 150 r/min cultivate 24 h, and centrifugal 5 min of 8000 r/min collect thalline, with the sterilized water washing once, bacterium mud is seeded in the fermention medium, make its inoculation after
OD 600 Be respectively 5,10,15,20,25, cultivate 8 h, transfer 30 ℃ again to and leave standstill cultivation in 30 ℃, 150 r/min.Adopt liquid chromatogram measuring, after the inoculation
OD 600 Be 15 o'clock, the L-lactic acid content is the highest in the fermented liquid, reaches 42 g/L.Shown in accompanying drawing 9.All the other are with embodiment 6.
Sequence table
<210> SEQ?ID?NO:?1
ITS1：5′-TCCGTAGGTGAACCTGCGG-3′
ITS4：5′-TCCTCCGCTTATTGATATGC-3′
<210> SEQ?ID?NO:?2
<211> 340
AACCTGCGGA?AGGATCATTT?CTGAACAACC?TTTGCATAGT?GAACAACCTC?TACGGGGGTG?60
AATGCTTCCG?GTTCGCCGGA?CAACAACTAG?TAATTTTGAA?TCTGATACGT?TAAAGAAAAA?120
CTTTCAACAA?CGGATCTCTT?GGTTCTCGCA?TCGATGAAGA?ACGCAGCAAA?GCGCGATAGG?180
TAATGCGAAT?TGCAGACGTG?AGTCATTGAA?TCTTTGAACG?CACATTGCGC?CAGTTTACTG?240
GCATACTGCG?TTGAGCGTCG?GCCTTCCTCC?ACAGGTATTG?CTGTTTGCAA?CAGTCAAAGG?300
CATGGAAGCG?CACCCGTTAG?TAAAACGGTG?CAAAACCACA 340
<210> SEQ?ID?NO:?3
R1：5′-CGCGGATCCATGGTATT?ACACTCA-3′
R2：5′-CCGAAGCTTTCAACAGCTACTTTTA-3′
Claims (2)
- One strain can be in the culture environment of regulating pH to 2.5 with lactic acid growth but do not utilize the bacterial strain of lactic acid, called after candida magnoliae C4 ( Candida magnoliaeC4), in China's typical culture collection center preservation, deposit number is CCTCC NO:M2010362.
- One strain candida magnoliae C42 ( Candida magnoliaeC42), it is characterized in that the described candida magnoliae C4 of claim 1 is carried out recombination, obtain candida magnoliae C42 and have the performance of producing L-lactic acid at low pH condition bottom fermentation;Described candida magnoliae C42 is the lactic dehydrogenase enzyme coding gene that derives from Rhizopus oryzae As3.819 LdhAInsertion contains the yeast shuttle vector of G418 resistant gene, has made up recombinant plasmid pYX212- KanMX- LdhA, and electricity is transformed among the candida magnoliae C4, obtains candida magnoliae C42.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101167836A CN102199553B (en) | 2011-05-06 | 2011-05-06 | Acid-resistant yeast and method for producing L-lactic acid recombinant strain through metabolic engineering construction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101167836A CN102199553B (en) | 2011-05-06 | 2011-05-06 | Acid-resistant yeast and method for producing L-lactic acid recombinant strain through metabolic engineering construction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102199553A CN102199553A (en) | 2011-09-28 |
| CN102199553B true CN102199553B (en) | 2012-07-11 |
Family
ID=44660542
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101167836A Expired - Fee Related CN102199553B (en) | 2011-05-06 | 2011-05-06 | Acid-resistant yeast and method for producing L-lactic acid recombinant strain through metabolic engineering construction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102199553B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1902319A (en) * | 2003-11-20 | 2007-01-24 | 泰特&莱尔组分美国公司 | Lactic acid producing yeast |
-
2011
- 2011-05-06 CN CN2011101167836A patent/CN102199553B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1902319A (en) * | 2003-11-20 | 2007-01-24 | 泰特&莱尔组分美国公司 | Lactic acid producing yeast |
Non-Patent Citations (4)
| Title |
|---|
| Eri Adachi et al..Modification of metabolic pathways of Saccharomyces cerevisiae by the expression of lactate dehydrogenase and deletion of pyruvate decarboxylase genes for the lactic acid fermentation at low pH value.《Journal of Fermentation and Bioengineering》.1998,第86卷(第3期),284-289. * |
| 李剑唐,等.L-乳糖脱氢酶基因克隆机功能分析.《生物工程学报》.2004,第20卷(第5期),725-729. * |
| 潘丽军，等.分批补料高密度培养米根霉As3.819产L-乳酸的研究.《食品科学》.2009,第30卷(第9期),133-136. * |
| 钱志良,等.工业乳酸发酵的近期进展.《生物加工过程》.2003,第1卷(第1期),23-27. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102199553A (en) | 2011-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108220175B (en) | High-density culture method and pH regulation and control method for saccharomyces cerevisiae | |
| US10472656B2 (en) | Method for fermenting sugars using genetically engineered yeast | |
| CN102311927A (en) | Medium and method for high density fermentation of saccharomyces cerevisiae | |
| CN103249832A (en) | Novel mutant microorganism producing succinic acid simultaneously using sucrose and glycerol, and method for preparing succinic acid using same | |
| CN110791462B (en) | Bacillus subtilis and application thereof in fermentation production of adenosine | |
| CN102586151A (en) | High-yield polysaccharide strain and method for producing polysaccharide by utilizing strain through fermentation | |
| CN103911315B (en) | Bacterial strain and the application thereof of algin catenase are produced in one strain | |
| CN102618478B (en) | Strain producing dynamic controlling recombinant strain and method for preparing D-lactic acid with recombinant strain | |
| RU2539092C1 (en) | RECOMBINANT STRAIN OF YEASTS Schizosaccharomyces pombe - PRODUCENT OF LACTIC ACID | |
| CN105296392A (en) | Fermentation method of bacillus amyloliquefaciens Bam22 for preventing and treating cruciferae plasmodiophora brassicae | |
| CN116751731A (en) | Fermentation process for large-scale plasmid production | |
| CN102199553B (en) | Acid-resistant yeast and method for producing L-lactic acid recombinant strain through metabolic engineering construction | |
| CN109456905A (en) | One plant promotes Cryptococcus and its application of the microalgae using sucrose | |
| CN103275898A (en) | Lipase high-producing strain, screening method thereof and method for producing lipase by using strain through fermentation | |
| CN111849790B (en) | Recombinant cephalosporium acremonium engineering bacteria and construction method and application thereof | |
| Limtong et al. | Three new thermotolerant methylotrophic yeasts, Candida krabiensis sp. nov., Candida sithepensis sp. nov., and Pichia siamensis sp. nov., isolated in Thailand | |
| CN1160467C (en) | Method of producing adenosylmethionine | |
| CN109182154B (en) | Pabuvina rhodotorula strain capable of producing protease at high yield | |
| CN108587923B (en) | Method for improving malic acid fermentation performance | |
| CN108823103B (en) | Penicillium lesinum strain of cold region corn straw rotten fungi as well as fermentation culture method and application thereof | |
| CN105695349A (en) | Method using phosphorus starvation culture to increase intracellular trehalose of yeast cells | |
| RU2650669C1 (en) | Schizosaccharomyces pombe strain - lactic acid producer | |
| CN109554321A (en) | A kind of genetic engineering bacterium of high yield lipopeptid and its application | |
| CN114874950B (en) | Method for improving bacitracin produced by bacillus licheniformis | |
| CN116004750B (en) | Method for producing angustin by using erwinia persicae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120711 Termination date: 20150506 |
|
| EXPY | Termination of patent right or utility model |