CN1624104A - Saccharomycetes strain CGMCC No. 1189, its cultivating process and its application - Google Patents
Saccharomycetes strain CGMCC No. 1189, its cultivating process and its application Download PDFInfo
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- CN1624104A CN1624104A CN 200410084047 CN200410084047A CN1624104A CN 1624104 A CN1624104 A CN 1624104A CN 200410084047 CN200410084047 CN 200410084047 CN 200410084047 A CN200410084047 A CN 200410084047A CN 1624104 A CN1624104 A CN 1624104A
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- inner ether
- bacterium
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Abstract
A saccharomycetes Lipomyces starkeyi (CGMCC No.1189( is disclosed, which comes from soil through screening. Its culture medium uses the endotherose as carbon source, which can be used by said lipomyces starkyi to generate somatic protein. It can be used as the bacterial seed and genetic resource to configure the engnieered strain using endoetherose.
Description
The present invention relates to a microorganism strains, cultural method and owing to it can assimilate the purposes that inner ether sugar possesses.
Along with pyrolytic technique makes substantial progress in minority developed countries such as the U.S. and Canada, particularly adopt fast pyrolysis process to make from the pyrolysis product of biomass acquisition, inner ether sugar (levoglucosan, 1,6-dehydration-β-D-Glucopyranose) first mate of output improves (near about 40%), utilizes pyrolysis product to replace grain more and more attractive as the research of the microbial fermentation carbon source and the energy.
Inner ether sugar is very rare at occurring in nature, and also there is certain degree of difficulty in the microorganism that discovery can be assimilated it.Up to the present, only find that abroad three investigators (Nakagawa, Prosen and Nakahara) are engaged in the research of this respect, they find have mould, yeast and joint bacterium can assimilate inner ether sugar.And on this basis, illustrated the metabolic mechanism of inner ether sugar in microorganism cells.All eukaryotic microorganisms that can grow on inner ether sugar all have the inner ether sugar kinases, and become the 6-glucose 1-phosphate1-to enter glycolytic pathway the inner ether sugar catalytic hydrolysis by it.Prokaryotic micro-organisms has significantly differently with eukaryotic microorganisms when utilizing inner ether sugar, and it earlier by three step enzymatic reactions, changes into glucose with inner ether sugar, enters glycolytic pathway then.This three step enzymatic reaction comprises the reduction reaction of dehydrogenation, hydrolysis and the dependence coenzyme NAD of inner ether sugar.Yet up to now, domestic also nobody with the inner ether sugar is that the sole carbon source and the energy were tested the microbial strains that preserves in the strain library, also not from nature screening with separated such microorganism.
The yeast strain that the purpose of this invention is to provide an efficient assimilation inner ether sugar.
The object of the invention also provides a kind of method of cultivating above-mentioned bacterial strains.Another object of the present invention provides a kind of purposes of above-mentioned bacterial strains, promptly as the bacterial classification and the genetic resources that assimilate inner ether sugar.
The yeast strain of assimilation inner ether sugar provided by the present invention is that this reaches saccharomyces oleaginosus Lipomyces starkeyi, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 13rd, 2004, deposit number is CGMCC No.1189, this common micro-organisms centre address is in the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.A pure yeast strain that obtains through a large amount of screenings in up to a hundred the bacterial strains that this bacterial strain system is separated to from the soil of all parts of the country.Examine under a microscope, this mycetocyte sphere, avette, size are (3.0-4.8) * (3.0-4.8) μ m.In the liquid medium within, this bacterium forms collarium.In solid medium, this bacterium bacterium colony is thick, light amber, and smooth surface, glossy, neat in edge, no pseudohypha produces.
The present invention cultivates above-mentioned yeast strain, and this reaches the method for saccharomyces oleaginosus Lipomyces starkeyi, and available usually following described step is carried out.Receive on the inclined-plane with this bacterium thalline of transfering loop picking, under 25-30 ℃, growth is 1-3 days on the slant medium, the picking thalline is under 25-30 ℃, cultivated 1-3 days in the nutrient solution, described substratum consists of inner ether sugar 2%, (NH4) 2,SO4 0.5%, KH2PO4 0.1%, MgSO47H2O 0.05%, pH5.0, agar 2%, liquid nutrient medium do not add agar.
Described in the present invention in the slant medium inner ether sugar recommend to use as carbon source, nitrogenous source is with (NH
4)
2SO
4Recommending pH in described slant medium and the nutrient solution is 5.0, and available mineral acid such as dilute sulphuric acid and mineral alkali such as sodium hydroxide are regulated pH to 5.
Yeast strain of the present invention this to reach saccharomyces oleaginosus Lipomyces starkeyi be that a strain has high vigor, to the bacterial strain that inner ether sugar has high assimilative capacity, its cultural method is simple, fast growth is difficult for variation.Can produce tropina as being main carbon source with inner ether sugar.Also can be from the gene of this bacterial strain clone assimilation inner ether sugar, as the inner ether sugar kinase gene, making up with the inner ether sugar is carbon source, and has the engineering strain of other industrial use.Because the preceding inner ether sugar of having addressed can obtain through fast pyrogenation in a large number from biomass, therefore, this bacterial strain has potential industrial applications prospect.
The present invention will be helped to understand by following embodiment, but content of the present invention can not be limited.
The cultivation of embodiment 1 thalline
This reaches saccharomyces oleaginosus Lipomyces starkeyi and receives on the above-mentioned inclined-plane with transfering loop picking yeast strain, is set in temperature cultivate 1-3 days in 28 ℃ the incubator, the thalline of white will occur.Then, receive the triangular flask that nutrient solution is housed with the well-grown thalline of transfering loop picking from the inclined-plane, on being set to shaking table that 28 ℃, rotating speed are 200rmp, temperature cultivated 1-3 days, gained thalline centrifugal (9000rmp) 10min, thalline washs with stroke-physiological saline solution (0.8%) and sterilized water, centrifugal again, 2-3 time repeatedly, can obtain free of contamination this yeast cell of white.
As previously mentioned, the substratum of cultivating this bacterial strain consists of inner ether sugar 2%, (NH
4)
2SO
40.5%, KH
2PO
40.1%, MgSO
47H
2O 0.05%, pH5.0, and agar 2%, liquid nutrient medium do not add agar.
Above-mentioned substratum and the nutrient solution 20min that all under 121 ℃, 0.1MPa, sterilizes.
The assimilation of embodiment 2 inner ether sugars
This reaches yeast strain saccharomyces oleaginosus Lipomyces starkeyi and is cultured to logarithmic phase by the method for embodiment 1, with pipettor under aseptic condition, pipette nutrient solution, and be added in the ratio of 10% (v/v) in the substratum of test inner ether sugar assimilative capacity, on being set to shaking table that 28 ℃, rotating speed are 200rmp, temperature cultivated two days.With medium centrifugal (9000rmp) 10min, remove cell, measure substratum at the content of cultivating the front and back inner ether sugar with efficient liquid Hunan chromatogram (HPLC), calculate the utilization ratio of inner ether sugar.Utilization ratio (%)=(inner ether sugar content before cultivating-cultivation back inner ether sugar content)/inner ether sugar content * 100 before cultivating.Under this culture condition, yeast strain this to reach saccharomyces oleaginosus Lipomyces starkeyi be 64.1% to the utilization ratio of inner ether sugar.The substratum of test assimilative capacity is: inner ether sugar 4%, (NH
4)
2SO
41%, KH
2PO
40.2%, MgSO
4.7H
2O 0.1%, Yeast extract 0.3%, and Peptone 0.2%, pH5.0.Above-mentioned substratum and the nutrient solution 20min that all under 121 ℃, 0.1MPa, sterilizes.
Embodiment 3 utilizes Protoplast Fusion Technique structure conversion inner ether sugar to be the alcoholic acid engineering strain
This reaches the parent strain that saccharomyces oleaginosus Lipomyces starkeyi can be used as other engineering bacteria of structure yeast strain.Press the cultural method of embodiment 1, cultivate this bacterial strain to logarithmic phase, and centrifugal acquisition somatic cells.With 2% helicase+2% cellulase degradation 4h, obtain protoplastis.Simultaneously with another Wine brewing yeast strain Saccharomyces cerevisiae 2.399 (CGMCC No.2.399, this bacterial strain can not utilize inner ether sugar) on perfect medium, be cultured to logarithmic phase, the somatic cells of results is cultivated 10min with 1% helicase, obtains the protoplastis of this bacterium.The protoplastis of these two yeast cell is merged under the 30%PEG inducing action, and the product that will merge is applied on the regeneration culture medium, cultivated 3-7 days down, obtain the bacterium colony of fusant at 37 ℃.From these bacterium colonies, cultivate through continuous passage, separation screening, the inner ether sugar that can obtain fermenting is the engineering strain of alcohol.
Perfect medium (YPD): peptone 2%, glucose 2%, yeast powder 1%, pH5.5;
Regeneration basic medium (RMM): YNB (no amino acid yeast nitrogen, Difco) 0.67%, inner ether sugar 2%, CaCl
20.01mol/l, N.F,USP MANNITOL 0.8mol/l or sorbyl alcohol 0.8mol/l;
Fermention medium (FMM): YNB (no amino acid yeast nitrogen, Difco) 0.67%, inner ether sugar 2%.
Above-mentioned substratum and the nutrient solution 20min that all under 121 ℃, 0.1MPa, sterilizes.
Claims (6)
1. a strain can provide the yeast strain of bacterial classification and genetic resources for the assimilation inner ether sugar, it is characterized in that accession designation number by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation be CGMCC No.1189 yeast strain this reach saccharomyces oleaginosus Lipomyces starkeyi.Examine under a microscope, this mycetocyte sphere, avette, size are (3.0-4.8) * (3.0-4.8) μ m.In the liquid medium within, this bacterium forms collarium.In solid medium, this bacterium bacterium colony is thick, light amber, and smooth surface, glossy, neat in edge, no pseudohypha produces.
Claims 1 described yeast strain this reach the cultural method of saccharomyces oleaginosus Lipomyces starkeyi, it is characterized in that this bacterium thalline is received on the inclined-plane, under 25-30 ℃, growth is 1-3 days on the slant medium, the picking thalline is under 25-30 ℃, cultivated 2-3 days in the nutrient solution, through the centrifugal pure cell that can obtain this bacterial strain.Described substratum consists of inner ether sugar (Levoglucosan) 2%, (NH
4)
2SO
40.5%, KH
2PO
40.1%, MgSO
47H
2O 0.05%, and pH 5.0, and agar 2%, liquid nutrient medium do not add agar.
3. claims 2 described cultural methods is characterized in that described assimilation carbon source is inner ether sugar (Levoglucosan).
Claims 1 described yeast strain this reach the purposes of saccharomyces oleaginosus Lipomyces starkeyi, it is characterized in that as the assimilation inner ether sugar bacterial classification and genetic resources.
5. claims 4 described purposes is characterized in that with this bacterium be bacterial classification, are carbon source with inner ether sugar, produce tropina.
6. claims 4 described purposes is characterized in that from the gene of this bacterium clone assimilation inner ether sugar or are that parent strain makes up the engineering strain that inner ether sugar can be converted into other product such as alcohol fuel with this bacterium directly.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410084047 CN1624104A (en) | 2004-10-19 | 2004-10-19 | Saccharomycetes strain CGMCC No. 1189, its cultivating process and its application |
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|---|---|---|---|
| CN 200410084047 CN1624104A (en) | 2004-10-19 | 2004-10-19 | Saccharomycetes strain CGMCC No. 1189, its cultivating process and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734178A (en) * | 2016-02-18 | 2016-07-06 | 柳州东侯生物能源科技有限公司 | Method for preparing levoglucosan from waste nut shells |
| CN108148850A (en) * | 2016-12-06 | 2018-06-12 | 中国科学院大学 | A kind of construction method of double-mass model colibacillus engineering using inner ether sugar producing and ethanol |
-
2004
- 2004-10-19 CN CN 200410084047 patent/CN1624104A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734178A (en) * | 2016-02-18 | 2016-07-06 | 柳州东侯生物能源科技有限公司 | Method for preparing levoglucosan from waste nut shells |
| CN108148850A (en) * | 2016-12-06 | 2018-06-12 | 中国科学院大学 | A kind of construction method of double-mass model colibacillus engineering using inner ether sugar producing and ethanol |
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