CN108893501A

CN108893501A - The method that DHA is prepared using lignocellulosic

Info

Publication number: CN108893501A
Application number: CN201810939182.7A
Authority: CN
Inventors: 崔球; 刘亚君; 刘世岳; 宋晓金
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2018-08-17
Filing date: 2018-08-17
Publication date: 2018-11-27
Anticipated expiration: 2038-08-17
Also published as: CN108893501B

Abstract

For the problems of in terms of industrialized production DHA, the present invention provides the techniques for producing DHA as raw material using lignocellulose agriculture and forestry organic waste material in the prior art.The technique includes that pretreatment, saccharification, schizochytrium limacinum fermentation and DHA are extracted.Technique of the present invention is combined using lignocellulosic biomass saccharification with schizochytrium limacinum high density fermentation；It is raw material by using lignocellulose biomass, on the one hand significantly reduces the production cost of DHA, on the other hand also solve the problem of complex utilization of agriculture and forestry organic waste material.In technique of the present invention, lignocellulosic saccharification stage culture medium can be recycled with fermentation medium, can significant water-saving and reduction chemicals use.And saccharification of cellulose efficiency is 80%-90%, schizochytrium limacinum biomass can reach 15-80g/L, and DHA yield is 5-25g/L.Compared with prior art, there is higher DHA yield and yield.

Description

The method that DHA is prepared using lignocellulosic

Technical field

The present invention relates to microbial fermentation engineering fields, and in particular to a kind of to utilize lignocellulosic material preparation 22 The method of carbon acid.

Background technique

Docosahexaenoic acid (Docosahexaenoic Acid, DHA) is a kind of polyunsaturated fat needed by human Acid, it is necessary to be supplemented from diet.DHA is the important feature rouge ingredient of central nervous system and retina, has brain tonic, improves note Recall the function of power and eyesight, can especially promote fetus and infant brain cell development to grow, improve teenager's memory, prevent Control senile dementia etc..Traditionally, DHA is mainly derived from deep sea fish oil, but due to by overfishing, environmental pollution, season The influence of variation etc. is very big, is gradually utilized replaced microbial fermentation production.At this stage, industrialization DHA production is main It is fermented and is realized by schizochytrium limacinum (Aurantiochytrium sp.), DHA content is up to 40%-60% in total fatty acids. DHA algal oil using schizochytrium limacinum production in 2010 has been approved as new resource food by the Ministry of Public Health.

The country is related to improving schizochytrium limacinum fermentation preparation DHA grease production capacity and the patent of economy mainly including tripartite at present Face content：1) strain of the high and high DHA content of biomass height, growth rate is acquired by screening schizochytrium limacinum strain breeding thereof, Such as 1,264,967 101,892,160 103,602,591 101575584 B, CN 102199541 of A, CN of B, CN of C, CN of patent CN B, 102,899,254 102888348 A and 102864111 B of CN of A, CN of CN.2) by changing the component in culture medium, such as Full-synthetic culture medium is prepared, electrolytes and minerals content is optimized, reduces chlorinity and addition external source rouge compatible material such as The modes such as trehalose, glycine betaine achieve the purpose that improve DHA yield, such as CN 101993824 A, CN 101831387 B, CN 101886044 101914581 B and 103627640 A of CN of A, CN.3) it is reduced and is produced by optimization for fermentation technology mode This, improves efficiency of pcr product.Such as 101,519,676 101,812,484 101824442 B, CN 102972533 of B, CN of B, CN of CN A, 103614427 A of CN103146584 A and CN.However, the above method be all using the glucose in cornstarch source as Primary carbon source, thus glucose supply and cost of material be influence DHA production capacity and economy key constraints.Therefore, The novel carbon source of developing low-cost and matched biological technology of preparing have necessity and wide application prospect.

Lignocellulosic is a kind of in plentiful supply and environmental-friendly renewable biomass, including stalk, secondary stock, rice The agriculture and forestry organic waste materials such as shell, vinasse.Lignocellulosic is mainly made of cellulose, hemicellulose and lignin three parts.Wherein, Cellulose be by glucose group at.Therefore, lignocellulose biomass is potential cheap glucose feed, is developed with wooden Cellulosic material is the production method of the sodium gluconate of substrate, the production cost of DHA can be significantly reduced, and solve agriculture simultaneously The problem of complex utilization of woods waste.

Currently, typically using the cellulase of originated from fungus in lignocellulosic preparation DHA technology.Patent of invention 2013105714130 provide " using the method for 22 carbon of stalk hydrolyzate fermenting and producing, six diluted acid ".Using originated from fungus Cellulase, galactosidase and pectase pretreated stalk is hydrolyzed, recycle hydrolyzate carry out fragmentation pot Bacterium fermenting and producing DHA.Using the strategy of enzyme preparation of originated from fungus, there are the deficiencies in efficiency and cost, this is primarily due to fiber The needs of plain enzyme preparation are prepared in individual reactor by microbial fermentation, and the technique and cellulose hydrolysising condition prepared Difference, production stage very complicated, the costs such as manpower and material resources demand and equipment investment dramatically increase, so that the sugar of lignocellulosic Change process does not have the market competitiveness, therefore the saccharification based on originated from fungus cellulase preparation and DHA technology of preparing are difficult to Realize large-scale industrial application.Patent of invention 2015108721949 provides one kind and directly bagasse hydrolyzate is utilized to carry out The method of schizochytrium limacinum fermentation, substrate bagasse obtain hydrolyzate rather than enzyme hydrolysis method by dilute acid hydrolysis, and there are environment dirts The potential danger of dye；In addition, it is special to need since the substances such as furfural in hydrolyzate have the function of that strong inhibitions is grown Schizochytrium limacinum strain could directly be grown in the hydrolyzate, and Fungal biodiversity and grease or DHA yield are lower.

Known fiber corpusculum is that a kind of multienzyme with labyrinth and component of the anaerobic bacterias such as Clostridium thermocellum production is multiple Zoarium is one of known most efficient cellulose degradation system in nature.Cellulosome includes that foot prop albumen etc. is non-catalytic Unit and enzyme unit with different catalytically active, and have by multistage foot prop albumen and different cellulosome enzymes more Type group fills the noncovalent interaction of intermodule specificity, and it is more than million dongles that different function ingredients, which are assembled into molecular weight, The supermolecule multienzyme complex to pause.Cellulosome component and structure also have spatiotemporal database characteristic, to adapt to lignocellulosic Complicated ingredient, to ensure that its efficient degradation vigor.Had on foot prop albumen or cellulase in addition, cellulosome also passes through By cellulose binding module and wall built-up module and substrate and cell form ternary complex, cellulosome and intercellular collaboration Effect can be further improved the saccharification efficiency (patent document EP2013355) of lignocellulosic.

Although cellulosome and its production bacterial strain have great potential, industrialization in lignocellulosic saccharification application Using being still limited by various factors.When non-cellulose components in lignocellulosic complex substrate (e.g., hemicellulose, pectin, Starch, protein, lignin) content it is higher when, saccharification efficiency significantly reduce.Forefathers are mainly by additionally adding in hydrolyzation system Add the small body protein of non-fiber to improve the vigor of cellulosome and to the adaptive faculty of substrate.However, due to the albumen of external source addition Activity and stabill can be reduced with the progress of saccharifying, therefore, the consumption of enzyme needs to improve enzyme additive amount or addition Number.In addition, the small body protein of these non-fiber is added in saccharification system as resolvase, it cannot be with cellulosome phase interaction With, the synergistic effect with other enzymes in system can be significantly reduced, unavoidably cause greater than demand enzyme additional addition, lead Cause the high of enzyme preparation cost.Moreover, this saccharification strategy dependent on external source addition resolvase, complex process, The high requirements on the equipment, transformation efficiency are not met by industrialization production requirements.

Summary of the invention

For the problems of in terms of industrialized production DHA, the present invention provides with wood fibre in the prior art Plain class agriculture and forestry organic waste material is the technique that raw material produces DHA, and the technique uses the fiber for being catalyzed lignocellulosic saccharification Plain enzyme preparation, to reduce the carbon source cost of DHA fermentation.

Technical solution of the present invention：The method for preparing DHA using lignocellulosic, includes the following steps：

(1) it pre-processes：Lignocellulosic material is pre-processed, content of lignin is obtained and is not higher than 20%, hemicellulose Cellulose content is not higher than 25% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:2-1:25 are transferred to the lignocellulosic substrate that step (1) obtains In the saccharification culture medium of anaerobic fermentation tank, cellulase preparation is added in the lignocellulosic substrate, in 34-65 DEG C of temperature Reaction is hydrolyzed under the conditions of degree, obtains the liquid glucose containing glucose；The saccharification culture medium is：Dipotassium hydrogen phosphate 2.9g/ L, potassium dihydrogen phosphate 1.5g/L, urea 0.8g/L, calcium chloride 0.1g/L, magnesium chloride 1.8g/L, ferrous sulfate 0.0005g/L, sulphur Change sodium 2g/L, corn pulp 4g/L, trisodium citrate 2g/L, pH 6.5-7.5.Stream adding sodium hydroxide can be passed through in saccharifying Mode makes pH control in 5.8-6.2.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 25-180g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation in 28-38 DEG C of temperature condition, is mended Ferment 36-80h under the conditions of the pH of nitrogen and 6.5-7.0, until fermentation ends when concentration of glucose is not higher than 5g/L；Or it was fermenting The liquid glucose that continuous flow adds step (2) to obtain in journey, makes sugared concentration maintain 5-10g/L, in 28-38 DEG C of temperature condition, mends nitrogen And the 80-120h that ferments under the conditions of the pH of 6.5-7.0, obtain schizochytrium limacinum fermentation liquid.

The fermentation medium is：Corn pulp 5-20g/L, potassium dihydrogen phosphate 2.5g/L, sea crystal 30g/L, vitamin B₁5mg/L, vitamin B₆5mg/L, vitamin B₁₂5mg/L, biotin 5mg/L, pH 6.5-7.5.

(4) DHA is extracted：The schizochytrium limacinum fermentation liquid centrifugal treating that step (3) is obtained, by schizochytrium limacinum somatic cells with Culture medium is separated by solid-liquid separation after fermentation, is extracted from schizochytrium limacinum somatic cells using organic solvent extractionprocess and is obtained DHA.

The method also includes step (5), specially：Culture medium is not dilute after step (4) are separated by solid-liquid separation obtained fermentation It releases or is diluted by 1-3 times, be used for the saccharification culture medium of preparation steps (2).

Wherein, cellulase preparation described in step (2) is by the component in the small body protein of non-fiber and cellulosome Interaction, by non-fiber corpusculum protein binding obtained in the cellulosome complex；The small body protein of non-fiber is wood Dextranase, cellulose restriction endonuclease, cellulose excision enzyme, expansion factor, protease, amylase or pectase；The cellulosome There is the active multienzyme complex of ligocellulose degradation in extracellular to be produced by anaerobic bacteria and being secreted.The fiber is small Group in body is divided into foot prop albumen, the enzyme for having catalysis, assembling module.

The small body protein of non-fiber in cellulosome protein component interaction mode be indirect connect or The connection of person's substantivity；The indirect is connected as：The small body protein of non-fiber passes through covalent phase with the protein component in cellulosome Interaction is attached, and the substantivity is connected as：Component in the small body protein of non-fiber and cellulosome passes through expressing in series It is attached.

Preferably, the small body protein of the non-fiber has sequence table SEQ ID NO:Amino acid shown in 1-3,15-18 Sequence；And with such as SEQ ID NO:The consistency of amino acid sequence shown in 1-3,15-18 95% or more, and with such as SEQ ID NO:The amino acid sequence with the same function of amino acid sequence shown in 1-3,15-18.Wherein：SEQ ID NO:15: Genbank sequence number CRZ35393.1；SEQ ID NO:16:By 1968724 to 1973904 nucleic acid in genome C P001393.1 Sequential coding；SEQ ID NO:17:Genbank Serial No. KC763474.1；SEQ ID NO:18:By genome 2531445 to 2532785 nucleic acid sequence encoding in CP001393.1.

Preferably, the anaerobic bacteria is Clostridium thermocellum (Clostridium thermocellum), the molten fine shuttle of yellow Bacterium (Clostridium clariflavum), thermophilic fiber clostridium (Clostridium cellulovorans) solve fiber clostridium (Clostridium cellulolyticum), Acetivibrio cellulolyticus (Acetivibrio cellulolyticus), molten fiber False bacteroid (Pseudobacteroides cellulosolvens), Ruminococcus albus (Ruminococcus albus), Yellow Ruminococcus (Ruminococcus flavefaciens).Wherein, the Clostridium thermocellum is the β Isosorbide-5-Nitrae-Portugal for expressing excretion The Clostridium thermocellum of polyglycoside enzyme.

Wherein, the small body protein of the non-fiber is attached with the component in cellulosome by covalent interaction, tool Body is：The small body protein of non-fiber and cellulosome component are connect with the polypeptide fragment with covalent interaction respectively, using more The covalent cross-linking of cellulosome component and the small body protein of non-fiber is realized in specific covalent interaction between peptide fragment, utilizes fiber The assembling module of corpusculum component institute band, makes non-fiber corpusculum protein binding in cellulosome complex.It is described small with non-fiber The polypeptide fragment of body protein and cellulosome component covalent interaction is SEQ ID NO:Base sequence or SEQ shown in 4 ID NO:Amino acid sequence shown in 5.Implementing step includes：

1) by the method for gene cloning, the polypeptide fragment centering that valence interacts together of the small body protein of non-fiber is connected One segment (polypeptide fragment I or II, i.e. SEQ ID NO:4 or encoding gene 5)；

2) according to 1), by the method for gene cloning, the polypeptide of cellulosome component protein and covalent interaction is connected Another segment (the polypeptide fragment II or I, i.e. SEQ ID NO of segment centering:5 or encoding gene 4).

3) according to 1), the recombination sequence of the small body protein of non-fiber and polypeptide fragment is connected to and produces cellulosome bacterium Expression plasmid；

4) according to 2), it is thin that the recombination sequence of cellulosome component protein and polypeptide fragment is connected to production cellulosome Bacterium homologous recombination plasmid, and homology arm is designed according to genome sequence.

5) 4) plasmid obtained in is transformed into to produce cellulosome bacterium intracellular, and is realized and is recombinated by homologous recombination screening Replacement of the gene order to pristine fibre corpusculum component protein sequence on genome realizes that fiber is small to construct recombinant bacterial strain Body component protein and polypeptide fragment are producing cellulosome bacterium amalgamation and expression intracellular.

6) 3) the middle plasmid that obtains is transformed into the recombinant bacterial strain obtained in 5), realizes the small body protein of non-fiber and polypeptide Segment is producing cellulosome bacterium amalgamation and expression intracellular.

In the recombinant bacterial strain finally obtained, the cellulosome component protein of genomic expression and the non-fiber of plasmid expression are small Body protein realizes covalent cross-linking by the specific covalent interaction between merged polypeptide fragment I and II, and is assembled in fibre It ties up in small composite.

Wherein, the small body protein of the non-fiber is attached with the component in cellulosome by expressing in series, specially： The assembling module of the small body protein of non-fiber and cellulosome or protein component with assembling module carry out amalgamation and expression, pass through group The noncovalent interaction for filling the specificity of intermodule is realized non-fiber corpusculum protein groups in cellulosome complex. The amalgamation and expression is：The encoding gene of the small body protein of non-fiber be inserted into the sequence of encoding fiber corpusculum component protein N-terminal, On genome among C-terminal or domain sequence.

The module respectively echoed is for adhesion module and to connection module.Described is SEQ ID NO to connection module:6,SEQ ID NO:9-13,SEQ ID NO:Base sequence shown in 14, adhesion module are SEQ ID NO:Amino acid sequence shown in 7 Column.Implementing step includes：

1) by the method for gene cloning, the small body protein of non-fiber and I type are connected to connection module (SEQ ID NO:6) volume Code gene, or

2) by the method for gene cloning, the small body protein of non-fiber and II type adhesion module (SEQ ID NO are connected:7) Encoding gene

1) or 2) 3) the small body protein of non-fiber will be obtained and assemble the recombination sequence of module and be connected to production cellulosome Bacterial expression plasmid

4) 3) plasmid obtained in is transformed into to produce cellulosome bacterium intracellular, realizes the small body protein of non-fiber and I type pair Connection module or II type adhesion module are producing cellulosome bacterium amalgamation and expression intracellular.

In the recombinant bacterial strain finally obtained, the small body protein of the non-fiber of plasmid expression is by merged I type to connection module Or II type adhesion module is realized and carries out specific non-covalent interaction with cellulosome foot prop albumen, so that it is small to be assembled in fiber In composite.

The specific implementation step of the direct amalgamation and expression includes：

1) by the method for gene cloning, it is same that the encoding gene of the small body protein of non-fiber is connected to production cellulosome bacterium In the recombinant plasmid of source, and homology arm is designed according to genome sequence.

2) 1) plasmid obtained in is transformed into to produce cellulosome bacterium intracellular, and passes through the non-fibre of homologous recombination screening realization The encoding gene for tieing up small body protein is inserted into the N-terminal of the sequence of cellulosome component protein or C-terminal or structural domain sequence on genome Column are intermediate, to construct recombinant bacterial strain, realize that the small body protein of non-fiber and cellulosome component protein are producing cellulosome bacterium Amalgamation and expression intracellular.

In the recombinant bacterial strain finally obtained, the small body protein of non-fiber utilizes the cellulosome component protein with its amalgamation and expression Assembling module be integrated in cellulosome complex.

Preferably, lignocellulosic material described in step (1) be corn stover, wheat straw, brush wood branch, wood chip, One of corncob, straw and waste paper or a variety of combinations；It is described pretreatment be alkaline process, diluted acid method, hydro-thermal method, steam explosion method and One of sulfonation method preconditioning technique or a variety of combinations；Pretreated lignocellulosic substrate is that content of lignin is not high In 11%, hemicellulose level is not higher than 12%.

Preferably, the temperature condition in step (2) saccharification step is 55-60 DEG C, the solid and liquid weight body in hydrolyzation system Product is than being 1:3-1:10.

Preferably, it in step (3), after concentration of glucose is 60-150g/L, exports liquid glucose by fermentor and assembles Filter assemblies enter bioreactor.

Preferably, specifically, under the conditions of the pH of 30-35 DEG C of temperature condition, benefit nitrogen and 6.5-7.0 in step (4) Ferment 48-80h, until fermentation ends when concentration of glucose is not higher than 5g/L；Or continuous flow adds step (2) to obtain during the fermentation The liquid glucose obtained, makes sugared concentration maintain 5-10g/L, issues in the pH condition of 30-35 DEG C of temperature condition, benefit nitrogen and 6.5-7.0 Ferment 100-120h, the benefit nitrogen operation are specially：The corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen.

Preferably, organic solvent extractionprocess described in step (5) is specially：Using the mixed liquor of ethyl alcohol and n-hexane Extract DHA.

Beneficial effects of the present invention：

(1) the present invention provides using lignocellulosic as fermenting raw materials produce DHA technique, have raw material sources extensively and Cheap advantage has the extremely strong market competitiveness, will largely solve the increased market demand year by year.

(2) of the present invention compared with the existing glucose using cornstarch source produces DHA as carbon source through fermentation Technique is combined using lignocellulosic biomass saccharification with schizochytrium limacinum high density fermentation；By using lignocellulose biomass For raw material, the production cost of DHA is on the one hand significantly reduced, the comprehensive utilization on the other hand also solving agriculture and forestry organic waste material is asked Topic.Compared with the existing technology for preparing DHA using lignocellulosic, technique of the present invention uses thin based on cellulosome is produced The cellulase preparation of bacterium realizes the saccharification of lignocellulosic, and significantly reduce the saccharification stage uses enzyme cost.

(3) in technique of the present invention, lignocellulosic saccharification stage culture medium can be recycled with fermentation medium, It significantly water-saving can be used with reduction chemicals, there is reduction discharge of wastewater and reduce the remarkable result of cost；This two o'clock is producing Have great importance in industry, not only there is huge economic benefit, and environmental-friendly, what can necessarily be walked is more long-range.

(4) in technique of the present invention, saccharification of cellulose efficiency is 80%-90%, and schizochytrium limacinum biomass can reach 15-80g/L, DHA yield are 5-25g/L.Compared with prior art, there is higher DHA yield and yield.

Specific embodiment

The present invention will be further explained with reference to the examples below.

Embodiment 1：The method connected by indirect constructs the cellulase preparation based on Clostridium thermocellum cellulosome

By seamless clone, (include by tdk expression cassette (promoter comprising gapDH gene) and pyrF expression cassette PyrF own promoter) be cloned into plasmid pHK (Mohr, G., Hong, W., Zhang, J., Cui, G.-Z., Yang, Y., Cui, Q.,et al.(2013)A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum,PLoS One 8:E69032. antibiotic resistance gene cat's) Downstream, and by the design of primer, increase NheI and XbaI enzyme cutting site between tdk and pyrF expression cassette, in the downstream pyrF EagI and MluI restriction enzyme site is added, for the clone of homology arm segment, so that building obtains pHK-HR plasmid.

Select the cellulase Cel9K (exocellulase, by genome C P002416.1 in Clostridium thermocellum cellulosome In 2113813 to 2111293 nucleic acid sequence encodings) enzymatic structural domain and between connection module as targeting knock in site. Firstly, using β-Isosorbide-5-Nitrae-glucuroide BglA (Genbank Serial No. AFO70070.1) encoding gene as target sequence, benefit It is cloned into homologous recombination plasmid pHK-HR with the restriction enzyme site of MluI and EagI, constructs homologous recombination plasmid pHK-HR-BglA. Upstream homology arm HR-up sequence is in Clostridium thermocellum DSM1313 genome (Serial No. CP002416.1 in ncbi database) 2111347 to 2112870 nucleic acid sequences, downstream homology arm HR-down are 2109848 to 2111354 cores in DSM1313 genome Acid sequence, intermediate homology arm HR-short are 2111347 to 2111659 nucleic acid sequences in DSM1313 genome.Secondly, by structure The plasmid built up is transformed into Δ pyrF, and is screened according to three steps and obtained chassis bacterial strain.Specifically screening technique is：

1) homologous recombination plasmid pHK-HR conversion is entered in the bacterial strain of pyrF missing, utilizes the GS-2 containing thiamphenicol Semisolid culturemedium (KH₂PO₄ 1.5g/L,K₂HPO₄·3H₂O 3.8g/L, urea 2.1g/L, MgCl₂·6H₂O 1.0g/L, CaCl₂·2H₂O 150mg/L,FeSO₄·6H₂O 1.25mg/L, cysteine hydrochloric acid 1.0g/L, MOPS sodium salt 10g/L, yeast Extract 6.0g/L, cellobiose 5.0g/L, citrate dihydrate trisodium 3.0g/L, resazurin 0.1mg/L, pH 7.4) plate into Row screening, to obtain plasmid transformant.

2) transformant obtained is in MJ fluid nutrient medium (KH₂PO₄ 1.5g/L,K₂HPO₄·3H₂O 3.8g/L, urea 2.1g/L,MgCl₂·6H₂O 1.0g/L,CaCl₂·2H₂O 150mg/L,FeSO₄·6H₂O 1.25mg/L, cysteine hydrochloric acid 1.0g/L, MOPS sodium salt 10g/L, cellobiose 5.0g/L, citrate dihydrate trisodium 3.0g/L, resazurin 0.1mg/L, hydrochloric acid pyrrole Tremble amine 2mg/L, biotin 0.2mg/L, p-aminobenzoic acid 0.4mg/L, vitamin B12 0.2mg/L, pH 7.4) in switching three Dai Hou, MJ semisolid culturemedium of the coating containing 10 μ g/mL floxuridines (FUDR) carry out the screening of first time homologous recombination. In this step, since Tdk can convert FUDR to the F-dUMP toxic to cell, while chassis cell is in MJ culture medium In must could survive by the pyrF gene chemical synthesis uridylate on plasmid, therefore, this screening strategy ensure that matter While homologous recombination occurs for homologous recombination module and genome on grain, the plasmid after recombination is also lost.According to long same The principle of homologous recombination preferentially occurs for source arm, and with genome homologous recombination occurs for former and later two long homology arms first, obtains at this time Recon prototrophy is reverted back to by the uracil auxotrophy of starting strain.

3) recon that obtains after first time homologous recombination first passes on 3 times in GS-2 fluid nutrient medium, and with identical Culture medium to bacterium solution carry out gradient dilution after coating contain 500 μ g/mL 5- fluororotic acids (FOA) GS-2 Semi-solid cell culture Base is screened, obtain purpose without scar gene knockout/knock in bacterial strain.In this step, the reversed screening effect of PyrF can promote Make brigade commander's homology arm and short homology arm that second of homologous recombination occur to remove pyrF expression cassette from genome.Second same Mutant strain after the recombination of source is mutated into uracil auxotrophy from prototrophy again.To realize target site gene on genome Knockout, knock in or replace.Respectively obtain homologous recombination bacterial strain Δ pyrF-I or Δ pyrF-II, respectively express Cel48S with The fusion protein of polypeptide fragment I or polypeptide fragment II.

Select the cellulose excision enzyme Cel48S of Clostridium thermocellum cellulosome (by genome C P002416.1 3228088 To 3230229 nucleic acid sequence encodings) 5 ' end as targeting knock in site, by polypeptide fragment I (SEQ ID NO:Or polypeptide piece 4) Section II (SEQ ID NO:5) coded sequence of two segments is distinguished as target sequence using the restriction enzyme site of MluI and EagI It is cloned into homologous recombination plasmid pHK-HR, building obtains homologous recombination plasmid pHK-HR-I or pHK-HR-II respectively.Upstream is same Source arm HR-up arrives in Clostridium thermocellum DSM1313 genome (Serial No. CP002416.1 in ncbi database) 3230200 3230700 nucleic acid sequences, downstream homology arm HR-down are 3229699 to 3230199 nucleic acid sequences in DSM1313 genome, in Between homology arm HR-short be DSM1313 genome in 3230200 to 3230500 nucleic acid sequences.The plasmid built is divided again It is not transformed into the chassis bacterial strain of above-mentioned building, and obtains homologous recombination bacterial strain Δ pyrF according to above-mentioned three steps screening technique:: BglA-I or Δ pyrF::BglA-II.

Using the method for overlap-extension polymerase chain reaction, polypeptide fragment II or polypeptide fragment I are connected to xylan Enzyme XynA (SEQ ID NO:1) 3 ' ends.Using BamHI and XbaI enzyme cutting site, using the recombination sequence connected as mesh Mark sequence is cloned on expression plasmid pHK.Expression plasmid containing XynA and the recombination sequence of polypeptide fragment I is transformed into Δ pyrF::BglA-II；Expression plasmid containing XynA and the recombination sequence of polypeptide fragment II is transformed into Δ pyrF::BglA-I, To realize that Cel48S and XynA can be combined by specific covalent interaction between polypeptide fragment I and II.By mentioning The cellulosome discovery for obtaining Clostridium thermocellum recombinant bacterial strain is taken, the XynA with covalent bond module of expression can be with excretion to born of the same parents Outside, and with the Cel48S with covalent bond module it interacts, is assembled into cellulosome complex.By above-mentioned recombinant bacterial strain Culture can be used as full bacterium enzyme to mid-log phase in the GS-2 culture medium using 5 gram per liters of celluloses or cellobiose as carbon source Biology saccharification of the preparation for lignocellulosic.

Embodiment 2：The method connected by indirect constructs the cellulase preparation based on Clostridium thermocellum cellulosome

Unlike the first embodiment, polypeptide fragment II or polypeptide fragment I are connected to cellulosome restriction endonuclease CelZ (SEQ ID NO:15) 3 ' ends.By the recombinant bacterial strain of building using 5 gram per liters of celluloses or cellobiose as the GS-2 culture medium of carbon source Middle culture can be used as biology saccharification of the full bacterial enzyme preparation for lignocellulosic to mid-log phase.

Embodiment 3：By the method for substantivity connection, the cellulase preparation based on Clostridium thermocellum cellulosome is constructed

Using the method for overlap-extension polymerase chain reaction, by cellulose excision enzyme Cel9-48 (SEQ ID NO:16) With sequence (the SEQ ID NO of the II type adhesion module CohIIct of Clostridium thermocellum:7) or I type is to the sequence of connection module DocIct (SEQ ID NO:6) it is directly connected to, wherein the sequence of CohIIct or DocIct is connected to 3 ' ends of Cel9-48 sequence, obtains Obtain Cel9-48-DocIct or Cel9-48-CohIIct sequence.

Using Cel9-48-DocIct or Cel9-48-CohIIct sequence as target sequence, the digestion of MluI and EagI is utilized Site is cloned into homologous recombination plasmid pHK-HR, is targeting replacement sequence, structure with lactic acid dehydrogenase gene clo1313_1160 Build homologous recombination plasmid pHK-HR-cel9-48.Upstream homology arm HR-up is Clostridium thermocellum DSM1313 genome (NCBI data Serial No. CP002416.1 in library) in 1380180 to 1380679 nucleic acid sequences, downstream homology arm HR-down be DSM1313 1380634 to 1381133 nucleic acid sequence in genome, intermediate homology arm HR-short arrive in DSM1313 genome 1380833 1381133 nucleic acid sequences.The plasmid built is transformed into respectively again in the chassis bacterial strain that embodiment 1 constructs, and according to implementation Three step screening techniques of example 1 obtain homologous recombination bacterial strain 1 and 2.By extracting the cellulosome discovery of recombinant bacterial strain, Cel9-48 Can be with excretion to extracellular with the fusion protein of assembling module, and it is small by the interaction mode of non-covalent to be assembled into fiber In composite.Above-mentioned recombinant bacterial strain is cultivated in the GS-2 culture medium using 5 gram per liters of celluloses or cellobiose as carbon source To mid-log phase, biology saccharification of the full bacterial enzyme preparation for lignocellulosic can be used as.

Embodiment 4：By the method for substantivity connection, the cellulase preparation based on Clostridium thermocellum cellulosome is constructed

By expansion factor Epn (SEQ ID NO:2) encoding gene selects Clostridium thermocellum cellulosome as target sequence 5 ' the ends of foot prop Protein S dbA (by 1108113 to 1109912 nucleic acid sequence encodings in genome C P002416.1) are as targeting Knock in site building homologous recombination plasmid pHK-HR-epn.Upstream homology arm HR-up is Clostridium thermocellum DSM1313 genome 1107610 to 1108109 nucleic acid sequences in (Serial No. CP002416.1 in ncbi database), downstream homology arm HR-down For 1109916 to 1110415 nucleic acid sequences in DSM1313 genome, intermediate homology arm HR-short is in DSM1313 genome 1107809 to 1108109 nucleic acid sequences.

Using the method for overlap-extension polymerase chain reaction, by zytase XynA (SEQ ID NO:1) viscous with II type Sequence (the SEQ ID NO of gang mould block CohII:7) it is directly connected to, wherein the sequence of CohII is connected to the 3 ' of XynA sequence End, to obtain the sequence of XynA-CohII.Using XynA-CohII sequence as target sequence, the digestion position of MluI and EagI is utilized Point is cloned into homologous recombination plasmid pHK-HR, is targeting replacement sequence, building with lactic acid dehydrogenase gene clo1313_1878 Homologous recombination plasmid pHK-HR-xynA.Upstream homology arm HR-up is Clostridium thermocellum DSM1313 genome (sequence in ncbi database Row number is CP002416.1) in 2194853 to 2195353 nucleic acid sequences, downstream homology arm HR-down be DSM1313 genome In 2196312 to 2196811 nucleic acid sequences, intermediate homology arm HR-short is 2195053 to arrive in DSM1313 genome 2195353 nucleic acid sequences.

Using the method for overlap-extension polymerase chain reaction, by pectase PelA (SEQ ID NO:18) encoding gene with Sequence (SEQ ID NO of the I type of Clostridium thermocellum to connection module DocIct:6) it is directly connected to, wherein the sequence of DocIct connects 3 ' ends of PelA sequence are connected to, PelA-DocIct target sequence is obtained.BamHI and XbaI enzyme cutting site are recycled, will be connected The recombination sequence come is cloned on expression plasmid pHK as target sequence, obtains expression plasmid pHK-PcelS-PelA- DocIct.Due to the promoter and signal peptide sequence (SEQ ID with the cellulase Cel48S from Clostridium thermocellum on pHK NO:8), the target gene of expression can be extracellular to cell with excretion.

Homologous recombination plasmid pHK-HR-xynA is transformed into the recombinant bacterial strain 1 that embodiment 3 constructs, and according to embodiment 1 The screening technique obtains homologous recombination bacterial strain 3.Homologous recombination plasmid pHK-HR-epn is transformed into recombinant bacterial strain 3 again, together Sample obtains homologous recombination bacterial strain recombinant bacterial strain 4 according to screening technique described in embodiment 1.Finally, by plasmid pHK-PcelS- PelA-DocIct is transformed into recombinant bacterial strain 4, thus obtain simultaneously XynA of the expression with II type adhesion module, with I type pair The Cel9-48 and PelA of connection module and the Clostridium thermocellum recombinant bacterial strain 5 of fusion protein Epn-SdbA.

By extracting the cellulosome discovery of recombinant bacterial strain, zytase XynA, the cellulose excision enzyme Cel9- of expression 48, pectase PelA all substantivity connection by way of be incorporated on Clostridium thermocellum cellulosome, expansion factor Epn by with The amalgamation and expression of foot prop Protein S dbA with excretion and can also be assembled into cellulosome complex.By recombinant bacterial strain 5 every with 5 grams It rises cellulose or cellobiose is that culture can be used as full bacterial enzyme preparation and be used for wood to mid-log phase in the GS-2 culture medium of carbon source The biology saccharification of matter cellulose.

Embodiment 5：By the method for substantivity connection, the cellulase preparation based on Clostridium thermocellum cellulosome is constructed

The recombinant bacterial strain 5 that embodiment 4 is constructed, cultivated in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source to Late log phase or platform early period, sedimentation cell is then removed by low-speed centrifugal, supernatant can be used as cellulosome enzyme preparation Biology saccharification for lignocellulosic.

Embodiment 6：By the method for substantivity connection, the cellulase system based on the molten fine clostridium cellulosome of yellow is constructed Agent

Using the method for overlap-extension polymerase chain reaction, by the molten fine clostridium of cellulose excision enzyme Cel48S and yellow I type docks sequence of modules DocIccl (SEQ ID NO:9) it connects, wherein the sequence of DocIccl is connected to Cel48S sequence 3 ' end, to obtain Cel48S-DocIccl sequence.Recycle BamHI and XbaI enzyme cutting site, the recombination that will be connected Sequence is cloned on expression plasmid pHK as target sequence.The plasmid built is transformed into the molten fine clostridium of yellow The Cel48S recombinant bacterial strain with DocIccl of expression is obtained in (Clostridium clariflavum DSM 19732).It is logical Cross the cellulosome discovery for extracting recombinant bacterial strain, the Cel48S with DocIccl of expression can be with excretion to extracellular, and is assembled into In the molten fine clostridium cellulosome complex of yellow.The bacterial strain is trained in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source It supports to late log phase or platform early period, sedimentation cell is then removed by low-speed centrifugal, supernatant can be used as cellulosome enzyme Biology saccharification of the preparation for lignocellulosic.

Embodiment 7：By the method for substantivity connection, the cellulase system based on Ruminococcus albus cellulosome is constructed Agent

As different from Example 6, cellulose excision enzyme Cel48S is docked into sequence of modules with the I type of Ruminococcus albus DocIra(SEQ ID NO:10) it connects, to obtain Cel48S-DocIra sequence.The plasmid built is transformed into white The recombinant bacterial strain of the Cel48S with DocIra of expression is obtained in Ruminococcus (Ruminococcus albus SY3).Pass through Extract the cellulosome discovery of recombinant bacterial strain, the Cel48S with DocIra of expression can be with excretion to extracellular, and is assembled into white In color Ruminococcus cellulosome complex.The bacterial strain is cultivated in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source To late log phase or platform early period, sedimentation cell is then removed by low-speed centrifugal, supernatant can be used as cellulosome enzyme system Biology saccharification of the agent for lignocellulosic.

Embodiment 8：By the method for substantivity connection, the cellulase system based on yellow Ruminococcus cellulosome is constructed Agent

As different from Example 6, cellulose excision enzyme Cel48S is docked into sequence of modules with the I type of yellow Ruminococcus DocIrf(SEQ ID NO:11) it connects, to obtain Cel48S-DocIrf sequence.The plasmid built is transformed into yellow The recombinant bacterial strain of the Cel48S with DocIrf of expression is obtained in Ruminococcus (Ruminococcus flavefaciens). By extracting the cellulosome discovery of recombinant bacterial strain, the Cel48S with DocIrf of expression can be with excretion to extracellular, and assembles Into yellow Ruminococcus cellulosome complex.By the bacterial strain in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source Culture is to late log phase or platform early period, and then by low-speed centrifugal removal sedimentation cell, supernatant can be used as cellulosome Biology saccharification of the enzyme preparation for lignocellulosic.

Embodiment 9：By the method for substantivity connection, the cellulase based on molten fiber vacation bacteroid cellulosome is constructed Preparation

As different from Example 6, by the I type of cellulose excision enzyme Cel48S and molten fiber vacation bacteroid to connection module sequence Arrange DocIpc (SEQ ID NO:13) it connects, to obtain Cel48S-DocIpc sequence.The plasmid built is transformed into molten Expression is obtained in fiber vacation bacteroid (Pseudobacteroides cellulosolvens DSM 2933) has DocIpc Cel48S recombinant bacterial strain.By extracting the cellulosome discovery of recombinant bacterial strain, the Cel48S with DocIpc of expression can With excretion to extracellular, and it is assembled into molten fiber vacation bacteroid cellulosome complex.By the bacterial strain with 5 gram per liters of celluloses For culture in the GS-2 culture medium of carbon source to late log phase or platform early period, then by low-speed centrifugal removal sedimentation cell, on Clear liquid can be used as biology saccharification of the cellulosome enzyme preparation for lignocellulosic.

Embodiment 10：By the method for substantivity connection, the cellulase system based on thermophilic fiber clostridium cellulosome is constructed Agent

By protease P roL (SEQ ID NO:3) encoding gene selects thermophilic fiber clostridium cellulosome as target sequence Cellulase Clocel_2823 (by 3464080 to 3466140 nucleic acid sequence encodings in genome C P002160.1) 5 ' end Site is knocked in as targeting.It is cloned into homologous recombination plasmid pHK-HR, is constructed homologous using the restriction enzyme site of MluI and EagI Recombinant plasmid pHK-HR-ProL.Homology arm HR-up, HR-down and HR-short are respectively Clostridium In cellulovorans 743B genome 3466141 to 3466640 in (Serial No. CP002160.1 in ncbi database), 3465641 to 3466140 and 3466141 to 3466441 nucleic acid sequences.The plasmid built is transformed into pyrF respectively again to lack In the 743B mutant strain of mistake, and screening acquisition protease P roL and cellulase Clocel_2823 melts according to the method for embodiment 1 Close the homologous recombination bacterium of expression.By extracting the cellulosome discovery of recombinant bacterial strain, the fusion protein can with excretion to extracellular, and It is assembled into cellulosome complex.The bacterial strain is cultivated in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source to right Number later periods or platform early period, sedimentation cell is then removed by low-speed centrifugal, supernatant can be used as cellulosome enzyme preparation and use In the biology saccharification of lignocellulosic.

Embodiment 11：By the method for substantivity connection, the cellulase system based on solution fiber clostridium cellulosome is constructed Agent

As different from Example 10, by amylase AmyA (SEQ ID NO:17) encoding gene is as target sequence, choosing The cellulase Ccel_0729 of solution fiber clostridium cellulosome is selected (by 843122 to 845197 cores in genome C P001348.1 Sequences code) 5 ' end as targeting knock in site.Homology arm HR-up, HR-down and HR-short are respectively In Clostridium cellulolyticum H10 genome 842441 in (Serial No. NC_011898 in ncbi database) To the nucleic acid sequence of 842941,842942 to 843441 and 842641 to 842941.The plasmid built is transformed into respectively again In the H10 mutant strain of pyrF missing, screening obtains the homologous recombination of amylase AmyA and cellulase Ccel_0729 amalgamation and expression Bacterium.By extracting the cellulosome discovery of recombinant bacterial strain, which can be with excretion to extracellular, and is assembled into cellulosome and answers In zoarium.The bacterial strain is cultivated in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source to late log phase or platform early period, Then sedimentation cell is removed by low-speed centrifugal, supernatant can be used as biology of the cellulosome enzyme preparation for lignocellulosic Saccharification.

Embodiment 12：By the method for substantivity connection, the cellulase based on Acetivibrio cellulolyticus cellulosome is constructed Preparation

As different from Example 10, by pectase PelA (SEQ ID NO:18) encoding gene and Acetivibrio cellulolyticus I type docks sequence of modules DocIac (SEQ ID NO:14) it connects, to obtain PelA-DocIac sequence.It builds Plasmid is transformed into Acetivibrio cellulolyticus (Acetivibrio cellulolyticus).It is small by the fiber for extracting recombinant bacterial strain Body discovery, the PelA of expression can be with excretion to extracellular, and passes through the DocIac having and be assembled into Acetivibrio cellulolyticus cellulosome In complex.The bacterial strain is cultivated in the GS-2 culture medium using 5 gram per liters of celluloses as carbon source to late log phase or platform Phase, sedimentation cell is then removed by low-speed centrifugal, supernatant can be used as cellulosome enzyme preparation for lignocellulosic Biology saccharification.

Embodiment 13：DHA is prepared using lignocellulosic

(1) it pre-processes：Corn stover is carried out according to the sulfonation method used in document (Chinese papermaking, 2015,34,1-6) Pretreatment obtains content of lignin not higher than 11%, and hemicellulose level is not higher than 10% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:6, the lignocellulosic substrate that 1kg dry weight step (1) is obtained turns It moves in the 6L culture medium of anaerobic fermentation tank, the lignocellulosic is added in the cellulase preparation for then preparing embodiment 1 In substrate, reaction is hydrolyzed under the conditions of 55 DEG C of temperature, obtains the liquid glucose containing glucose.The saccharification culture medium For：Dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L, urea 0.8g/L, calcium chloride 0.1g/L, magnesium chloride 1.8g/L, sulphur Sour ferrous iron 0.0005g/L, vulcanized sodium 2g/L, corn pulp 4g/L, trisodium citrate 2g/L, pH 6.5-7.5.It can in saccharifying Make pH control in 5.8-6.2 by way of flowing adding sodium hydroxide.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 70-80g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 28 DEG C of temperature conditions, 6.5 Ferment 48h under the conditions of pH, and the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until concentration of glucose is sent out when being not higher than 5g/L Ferment terminates.

The fermentation medium is：Corn pulp 10g/L, corn pulp 5-20g/L, potassium dihydrogen phosphate 2.5g/L, sea crystal 30g/L, vitamin B₁5mg/L, vitamin B₆5mg/L, vitamin B₁₂5mg/L, biotin 5mg/L, pH 6.5-7.5.

(4) DHA is extracted：The schizochytrium limacinum fermentation liquid centrifugal treating that step (3) is obtained, by schizochytrium limacinum somatic cells with Culture medium is separated by solid-liquid separation after fermentation, and the organic solvent extractionprocess extracted using the mixed liquor of ethyl alcohol and n-hexane is from fragmentation pot It is extracted in bacterium somatic cells and obtains DHA.

(5) culture medium recycles：Culture medium does not dilute after step (4) are separated by solid-liquid separation obtained fermentation, is directly used in and matches The saccharification culture medium of step (2) processed.

Embodiment 14：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Wheat stalk is combined in advance according to the hydro-thermal used in patent CN201610133959 with sulfonation method Facture is pre-processed, and obtains content of lignin not higher than 8%, hemicellulose level is not higher than 8% lignocellulosic bottom Object；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:3.5, the lignocellulosic bottom that 100kg dry weight step (1) is obtained Object is transferred in the 350L culture medium of anaerobic fermentation tank, and the cellulase preparation for then preparing embodiment 2 is added described wooden In cellulosic substrate, reaction is hydrolyzed under the conditions of 60 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 140-150g/L in the liquid glucose that step (2) obtains, make sugar Liquid enters bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Obtain fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 30 DEG C of temperature conditions, 6.5 PH under the conditions of ferment 80h, the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until when concentration of glucose is not higher than 5g/L Fermentation ends.The fermentation medium 20g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：For preparing after 1 times of culture medium dilution after the fermentation that step (4) separation of solid and liquid is obtained The saccharification culture medium of step (2).

Embodiment 15：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：To shrub branch according to document (Bin Li, et al.Recent progress on the pretreatment and fractionation of lignocelluloses for Biorefinery at QIBEBT.Journal of Bioresources and Bioproducts, 2017,2 (1), 4-9) in alkaline process pre-process skill Art is pre-processed, and obtains content of lignin not higher than 15%, hemicellulose level is not higher than 7.5% lignocellulosic bottom Object；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:5, the lignocellulosic substrate that 1kg dry weight step (1) is obtained turns It moves in the 5L culture medium of anaerobic fermentation tank, the lignocellulosic is added in the cellulase preparation for then preparing embodiment 3 In substrate, reaction is hydrolyzed under the conditions of 60 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 90-110g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 30 DEG C of temperature conditions, 7.0 Ferment 48h under the conditions of pH, and the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until concentration of glucose is sent out when being not higher than 5g/L Ferment terminates.The fermentation medium 15g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

Embodiment 16：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：To wood chip according to document (Biotechnology for Biofuels, 2014,7:116) alkali in The preconditioning technique that method is combined with hydro-thermal is pre-processed, and obtains content of lignin not higher than 11%, hemicellulose level is not Lignocellulosic substrate higher than 10%；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:8, the lignocellulosic substrate that 100kg dry weight step (1) is obtained It is transferred in the 800L culture medium of anaerobic fermentation tank, the wooden fibre is added in the cellulase preparation for then preparing embodiment 4 It ties up in plain substrate, reaction is hydrolyzed under the conditions of 60 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 60-70g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 30 DEG C of temperature conditions, 7.0 Ferment 100h under the conditions of pH, the liquid glucose that continuous flow adds 14 step of embodiment (2) to obtain, and sugared concentration is made to maintain 5-10g/L, and stream adds The corn pulp solution of 50% (w/v) carries out benefit nitrogen.The fermentation medium 8g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：For preparing after 2 times of culture medium dilution after the fermentation that step (4) separation of solid and liquid is obtained The saccharification culture medium of step (2).

Embodiment 17：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Straw is pre-processed according to the steam explosion in document (Cellulose Science and technology, 2002,3,47-52) Technology is pre-processed, and obtains content of lignin not higher than 20%, hemicellulose level is not higher than 14.5% lignocellulosic Substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:10, the lignocellulosic substrate that 100kg dry weight step (1) is obtained It is transferred in the 1000L culture medium of anaerobic fermentation tank, the wooden fibre is added in the cellulase preparation for then preparing embodiment 5 It ties up in plain substrate, reaction is hydrolyzed under the conditions of 58 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 50-60g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 35 DEG C of temperature conditions, 7.0 Ferment 120h under the conditions of pH, the liquid glucose that continuous flow adds 14 step of embodiment (2) to obtain, and sugared concentration is made to maintain 5-10g/L, and stream adds The corn pulp solution of 50% (w/v) carries out benefit nitrogen.The fermentation medium 8g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

Embodiment 18：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：To waste paper according to the water in document (Bioresource Technology, 2004,91,93-100) Grape berry technology is pre-processed, and obtains content of lignin not higher than 7%, hemicellulose level is not higher than 11% wooden fibre Tie up plain substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:2, the lignocellulosic substrate that 1kg dry weight step (1) is obtained turns It moves in the 2L culture medium of anaerobic fermentation tank, the lignocellulosic is added in the cellulase preparation for then preparing embodiment 6 In substrate, reaction is hydrolyzed under the conditions of 37 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 160-180g/L in the liquid glucose that step (2) obtains, make sugar Liquid enters bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Obtain fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 35 DEG C of temperature conditions, 7.0 PH under the conditions of ferment 115h, the liquid glucose that continuous flow adds step (2) to obtain makes sugared concentration maintain 5-10g/L, stream plus 50% (w/v) corn pulp solution carries out benefit nitrogen.The fermentation medium 20g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：For preparing after 3 times of culture medium dilution after the fermentation that step (4) separation of solid and liquid is obtained The saccharification culture medium of step (2).

Embodiment 19：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Straw is pre-processed according to 13 preconditioning technique of embodiment, content of lignin is obtained and is not higher than 8%, hemicellulose level is not higher than 11% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:15, the lignocellulosic substrate that 0.2kg dry weight step (1) is obtained It is transferred in the 3L culture medium of anaerobic fermentation tank, the wood fibre is added in the cellulase preparation for then preparing embodiment 7 In plain substrate, reaction is hydrolyzed under the conditions of 37 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 30-50g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 30 DEG C of temperature conditions, 6.5 Ferment 36h under the conditions of pH, and the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until concentration of glucose is sent out when being not higher than 5g/L Ferment terminates.The fermentation medium 5g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：It step (4) is separated by solid-liquid separation culture medium after obtained fermentation does not dilute and be directly used in preparation The saccharification culture medium of step (2).

Embodiment 20：DHA is prepared using lignocellulosic

As different from Example 13,

(2) it is saccharified：According to solid and liquid weight volume ratio 1:10, the lignocellulosic substrate that 0.2kg dry weight step (1) is obtained It is transferred in the 2L culture medium of anaerobic fermentation tank, the wood fibre is added in the cellulase preparation for then preparing embodiment 8 In plain substrate, reaction is hydrolyzed under the conditions of 65 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 40-60g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 30 DEG C of temperature conditions, 6.5 Ferment 42h under the conditions of pH, and the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until concentration of glucose is sent out when being not higher than 5g/L Ferment terminates.The fermentation medium 8g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13

(5) culture medium recycles：1 times of culture medium dilution is subsequently used for matching after step (4) are separated by solid-liquid separation obtained fermentation The saccharification culture medium of step (2) processed.

Embodiment 21：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Straw is pre-processed according to 14 preconditioning technique of embodiment, content of lignin is obtained and is not higher than 8%, hemicellulose level is not higher than 8% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:4.5, the lignocellulosic substrate that 1kg dry weight step (1) is obtained It is transferred in the 4.5L culture medium of anaerobic fermentation tank, the wooden fibre is added in the cellulase preparation for then preparing embodiment 9 It ties up in plain substrate, reaction is hydrolyzed under the conditions of 34 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 110-130g/L in the liquid glucose that step (2) obtains, make sugar Liquid enters bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Obtain fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 32 DEG C of temperature conditions, 7.0 PH under the conditions of ferment 115h, the liquid glucose that continuous flow adds 14 step of embodiment (2) to obtain makes sugared concentration maintain 5-10g/L, flows The corn pulp solution of 50% (w/v) is added to carry out benefit nitrogen.The fermentation medium 20g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：3 times of culture medium dilution is subsequently used for matching after step (4) are separated by solid-liquid separation obtained fermentation The saccharification culture medium of step (2) processed.

Embodiment 22：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Corncob is located in advance according to the dilute acid hydrolysis in document (biological processing, 2010,3,66-72) Reason technology is pre-processed, and obtains content of lignin not higher than 9%, hemicellulose level is not higher than 18% lignocellulosic bottom Object；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:5, the lignocellulosic substrate that 0.4kg dry weight step (1) is obtained It is transferred in the 2L culture medium of anaerobic fermentation tank, the wood fibre is added in the cellulase preparation for then preparing embodiment 10 In plain substrate, reaction is hydrolyzed under the conditions of 42 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 60-80g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 32 DEG C of temperature conditions, 7.0 Ferment 80h under the conditions of pH, the liquid glucose that continuous flow adds 14 step of embodiment (2) to obtain, and sugared concentration is made to maintain 5-10g/L, and stream adds The corn pulp solution of 50% (w/v) carries out benefit nitrogen.The fermentation medium 10g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：2 times of culture medium dilution is subsequently used for matching after step (4) are separated by solid-liquid separation obtained fermentation The saccharification culture medium of step (2) processed.

Embodiment 23：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Waste paper is pre-processed according to the preconditioning technique of embodiment 17, it is not high to obtain content of lignin In 7%, hemicellulose level is not higher than 9% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:25, the lignocellulosic substrate that 0.2kg dry weight step (1) is obtained It is transferred in the 5L culture medium of anaerobic fermentation tank, the wood fibre is added in the cellulase preparation for then preparing embodiment 11 In plain substrate, reaction is hydrolyzed under the conditions of 40 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 25-35g/L in the liquid glucose that step (2) obtains, make liquid glucose Enter bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 30 DEG C of temperature conditions, 6.5 Ferment 36h under the conditions of pH, and the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until concentration of glucose is sent out when being not higher than 5g/L Ferment terminates.The fermentation medium 5g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

Embodiment 24：DHA is prepared using lignocellulosic

As different from Example 13,

(1) it pre-processes：Corncob is pre-processed according to the preconditioning technique of embodiment 17, obtains content of lignin not Higher than 5%, hemicellulose level is not higher than 25% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:3, the lignocellulosic substrate that 0.2kg dry weight step (1) is obtained It is transferred in the 0.6L culture medium of anaerobic fermentation tank, the wooden fibre is added in the cellulase preparation for then preparing embodiment 12 It ties up in plain substrate, reaction is hydrolyzed under the conditions of 40 DEG C of temperature, obtains the liquid glucose containing glucose.

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 100-120g/L in the liquid glucose that step (2) obtains, make sugar Liquid enters bioreactor by the filter assemblies of fermentor outlet assembly；Nutrient media components are added into bioreactor, are obtained Obtain fermentation medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, in 38 DEG C of temperature conditions, 6.5 PH under the conditions of ferment 50, the corn pulp solution of stream plus 50% (w/v) carry out benefit nitrogen, until concentration of glucose is sent out when being not higher than 5g/L Ferment terminates.The fermentation medium 15g/L containing corn pulp.

(4) DHA is extracted：It is identical as embodiment 13.

(5) culture medium recycles：Culture medium dilutes 3 times after step (4) are separated by solid-liquid separation obtained fermentation, then directly uses In the saccharification culture medium of preparation steps (2).

1. embodiment 13-24 of table prepares the results list of DHA using lignocellulosic

As shown in Table 1, the method for preparing DHA using lignocellulosic in embodiment 13-24, the yield of gained glucose are 27.1-179.2g/L, cellulose sugar rate are 80.10-90.80%, and schizochytrium limacinum biomass is 15.83-81.68g/L, DHA Yield is 5.15-25.03g/L, and the cellulose sugar rate after culture medium recycles is 80.50-89.5%.This explanation, the present invention The method that DHA is prepared using lignocellulosic, the advantage for not only having raw material sources extensive and cheap, and pass through wood The saccharification of matter cellulose biology is combined with schizochytrium limacinum high density fermentation, realizes the yield and yield of high DHA.In addition, with The cellulose sugar rate of the first round is compared, and the cellulose sugar rate after culture medium recycles is very nearly the same, is illustrated of the present invention Technique in, lignocellulosic saccharification stage culture medium can be recycled with fermentation medium, can be significant water-saving with reductionization Product use, and have the remarkable result for reducing discharge of wastewater and reducing cost.

Therefore, the method for the present invention for preparing DHA using lignocellulosic solves prepares DHA institute in the prior art Existing raw material and enzyme preparation is at high cost, preparation process not environmentally, the lower problem of DHA yield, huge economy will be generated Benefit has vast market prospect.

Sequence table

<110>Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

<120>The method that DHA is prepared using lignocellulosic

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 212

<212> PRT

<213>It is pyrolyzed cellulose fruit juice bacillus (Caldicellulosiruptor sp.)

<400> 1

Ala Ile Thr Leu Thr Ser Asn Ala Ser Gly Thr Tyr Asp Gly Tyr Tyr

1 5 10 15

Tyr Glu Leu Trp Lys Asp Ser Gly Asn Thr Thr Met Thr Val Asp Thr

20 25 30

Gly Gly Arg Phe Ser Cys Gln Trp Ser Asn Ile Asn Asn Ala Leu Phe

35 40 45

Arg Thr Gly Lys Lys Phe Asn Thr Ala Trp Asn Gln Leu Gly Thr Val

50 55 60

Lys Ile Thr Tyr Ser Ala Thr Tyr Asn Pro Asn Gly Asn Ser Tyr Leu

65 70 75 80

Cys Ile Tyr Gly Trp Ser Lys Asn Pro Leu Val Glu Phe Tyr Ile Val

85 90 95

Glu Ser Trp Gly Ser Trp Arg Pro Pro Gly Ala Thr Ser Leu Gly Thr

100 105 110

Val Thr Ile Asp Gly Gly Thr Tyr Asp Ile Tyr Lys Thr Thr Arg Val

115 120 125

Asn Gln Pro Ser Ile Glu Gly Thr Thr Thr Phe Asp Gln Tyr Trp Ser

130 135 140

Val Arg Thr Ser Lys Arg Thr Ser Gly Thr Val Thr Val Thr Asp His

145 150 155 160

Phe Lys Ala Trp Ala Ala Lys Gly Leu Asn Leu Gly Thr Ile Asp Gln

165 170 175

Ile Thr Leu Cys Val Glu Gly Tyr Gln Ser Ser Gly Ser Ala Asn Ile

180 185 190

Thr Gln Asn Thr Phe Ser Ile Thr Ser Asp Ser Ser Gly Ser Thr Thr

195 200 205

Pro Thr Thr Thr

210

<210> 2

<211> 327

<212> PRT

<213>The molten fine clostridium (Clostridium clariflavum) of yellow

<400> 2

Met Asn Phe Lys Lys Ile Arg Leu Phe Thr Ala Ile Leu Ile Ile Ala

1 5 10 15

Ala Gln Val Leu Ser Tyr Asn Phe Ile Ser Ser Ala Gln Leu Gln Val

20 25 30

Gly Asp Val Asn Gly Asp Asn Asn Val Asp Ser Ile Asp Phe Ala Leu

35 40 45

Met Lys Ser Phe Ile Leu Lys Ile Ile Asn Thr Leu Pro Ala Glu Asp

50 55 60

Ser Leu Leu Ala Gly Asp Leu Asp Gly Asp Gly Ser Ile Asn Ser Ile

65 70 75 80

Asp Cys Ala Leu Met Lys Gln Tyr Leu Leu Gly Met Ile Lys Val Phe

85 90 95

Pro Lys Thr Gln Ser Pro Ala Pro Thr Pro Thr Asn Thr Pro Leu Pro

100 105 110

Glu Tyr Ser Glu Pro Tyr Pro Gly Trp Asp Lys Ile Arg Ser Gly Tyr

115 120 125

Ala Thr Tyr Thr Gly Ser Gly Tyr Val Gly Gly Ile Ala Leu Leu Asp

130 135 140

Pro Ile Pro Glu Asp Met Glu Ile Val Ala Val Asn Lys Pro Asp Phe

145 150 155 160

Asn Cys Tyr Gly Val Gln Ala Ala Leu Ala Gly Ala Tyr Leu Glu Val

165 170 175

Thr Gly Pro Lys Gly Thr Thr Val Val Tyr Val Thr Asp Cys Tyr Thr

180 185 190

Glu Ala Pro Glu Gly Ala Leu Asp Leu Cys Gly Ile Ser Cys Asp Lys

195 200 205

Ile Gly Asp Thr Asn Val Pro Gly Gly Lys Ile Asp Val Thr Trp Arg

210 215 220

Ile Ile Pro Ala Pro Ile Thr Gly Asn Phe Ile Tyr Arg Ile Leu Pro

225 230 235 240

Ala Ser Ser Lys Trp Trp Phe Ala Ile Gln Val Arg Asn His Lys Tyr

245 250 255

Pro Val Met Lys Met Glu Tyr Phe Lys Asp Gly Glu Trp Val Asp Ile

260 265 270

Pro Lys Asp Arg Cys Asn Tyr Phe Val Ile Asn Asn Leu Asp Thr Ser

275 280 285

Asn Leu Lys Ile Arg Ile Thr Asp Ile Arg Gly Lys Val Val Thr Asp

290 295 300

Ile Ile Asp Pro Ile Pro Asp Asn Leu Met Asn Gly Cys Phe Ile Gln

305 310 315 320

Gly Asn Val Gln Phe Pro Asp

325

<210> 3

<211> 440

<212> PRT

<213>Molten bacillus albuminis (Coprothermobacter proteolyticus)

<400> 3

Met Lys Lys Ile Leu Leu Thr Leu Val Ile Ala Val Leu Leu Leu Ser

1 5 10 15

Gly Phe Ala Gly Val Lys Ser Ala Glu Leu Leu Phe Val Ser Asn Ser

20 25 30

Thr Thr Thr Asn Gln Glu Asp Pro Glu Asn Glu Ile Ile Val Gly Tyr

35 40 45

Lys Glu Asn Thr Asp Val Ala Val Leu Ser Lys Gln Val Glu Lys Thr

50 55 60

Thr Gly Ala Lys Leu Ser Arg Lys Gly Leu Lys Asn Phe Ala Val Phe

65 70 75 80

Lys Leu Pro Gln Gly Lys Ala Ala Asp Val Val Met Asn Gln Leu Lys

85 90 95

Asn Asp Pro Asn Val Glu Tyr Val Glu Pro Asn Tyr Ile Ala His Ala

100 105 110

Phe Asp Val Pro Asn Asp Thr Phe Phe Asn Pro Tyr Gln Trp Asn Phe

115 120 125

Tyr Asp Tyr Gly Met Thr Ser Asn Gly Tyr Val Ser Asn Tyr Gly Ile

130 135 140

Gln Ala Val Ser Ala Trp Asn Ile Thr Lys Gly Ala Gly Val Lys Val

145 150 155 160

Ala Ile Ile Asp Thr Gly Val Ala Tyr Glu Asn Tyr Gly Ala Tyr Thr

165 170 175

Lys Ala Pro Asp Leu Ala Asn Thr Leu Phe Asp Thr Ala Asn Ala Tyr

180 185 190

Asp Phe Val Asn Asn Asp Thr His Ala Asn Asp Asp Asn Ser His Gly

195 200 205

Thr His Val Ala Gly Thr Ile Ala Gln Ser Thr Asn Asn Gly Met Gly

210 215 220

Ala Ala Gly Ile Ala Tyr Gln Ala Thr Ile Leu Pro Ile Lys Val Leu

225 230 235 240

Asp Ser Glu Gly Ser Gly Thr Tyr Asp Ala Ile Ala Asn Gly Ile Ile

245 250 255

Trp Ala Ala Asp Lys Gly Ala Arg Val Ile Asn Met Ser Leu Gly Gly

260 265 270

Ser Ser Gly Ser Thr Thr Leu Gln Asn Ala Ile Gln Tyr Ala Tyr Asn

275 280 285

Lys Gly Val Val Ile Val Cys Ala Ser Gly Asn Asp Arg Arg Ser Thr

290 295 300

Val Ser Tyr Pro Ala Ala Tyr Thr Gln Cys Ile Ala Val Gly Ser Thr

305 310 315 320

Arg Phe Asp Gly Thr Arg Ala Arg Tyr Ser Asn Tyr Gly Ser Ala Leu

325 330 335

Asp Ile Val Ala Pro Gly Gly Asp Thr Ser Val Asp Gln Asn His Asp

340 345 350

Gly Tyr Gly Asp Gly Ile Leu Gln Gln Thr Phe Ala Glu Gly Ser Pro

355 360 365

Thr Asp Phe Ala Tyr Tyr Phe Phe Gln Gly Thr Ser Met Ala Ser Pro

370 375 380

His Val Ala Gly Val Ala Ala Leu Val Leu Ser Ala His Pro Thr Tyr

385 390 395 400

Thr Asn Glu Gln Val Arg Thr Ala Leu Gln Ser Thr Ala Lys Asp Leu

405 410 415

Gly Thr Ala Gly Trp Asp Lys Tyr Tyr Gly Tyr Gly Leu Val Asn Ala

420 425 430

Tyr Ala Ala Val Asn Trp Thr Pro

435 440

<210> 4

<211> 13

<212> PRT

<213>Streptococcus pyogenes (Streptococcus pyogenes)

<400> 4

Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys

1 5 10

<210> 5

<211> 97

<212> PRT

<213>Streptococcus pyogenes (Streptococcus pyogenes)

<400> 5

Ser Ser Glu Gln Gly Gln Ser Gly Asp Met Thr Ile Glu Glu Asp Ser

1 5 10 15

Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu

20 25 30

Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser

35 40 45

Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly

50 55 60

Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala

65 70 75 80

Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn

85 90 95

Gly

<210> 6

<211> 55

<212> PRT

<213>Clostridium thermocellum (Clostridium thermocellum)

<400> 6

Tyr Gly Asp Val Asn Asp Asp Gly Lys Val Asn Ser Thr Asp Ala Val

1 5 10 15

Ala Leu Lys Arg Tyr Val Leu Arg Ser Gly Ile Ser Ile Asn Thr Asp

20 25 30

Asn Ala Asp Leu Asn Glu Asp Gly Arg Val Asn Ser Thr Asp Leu Gly

35 40 45

Ile Leu Lys Arg Tyr Ile Leu

50 55

<210> 7

<211> 160

<212> PRT

<213>Clostridium thermocellum (Clostridium thermocellum)

<400> 7

Ser Ser Ile Glu Leu Lys Phe Asp Arg Asn Lys Gly Glu Val Gly Asp

1 5 10 15

Ile Leu Ile Gly Thr Val Arg Ile Asn Asn Ile Lys Asn Phe Ala Gly

20 25 30

Phe Gln Val Asn Ile Val Tyr Asp Pro Lys Val Leu Met Ala Val Asp

35 40 45

Pro Glu Thr Gly Lys Glu Phe Thr Ser Ser Thr Phe Pro Pro Gly Arg

50 55 60

Thr Val Leu Lys Asn Asn Ala Tyr Gly Pro Ile Gln Ile Ala Asp Asn

65 70 75 80

Asp Pro Glu Lys Gly Ile Leu Asn Phe Ala Leu Ala Tyr Ser Tyr Ile

85 90 95

Ala Gly Tyr Lys Glu Thr Gly Val Thr Glu Glu Ser Gly Ile Ile Ala

100 105 110

Lys Ile Gly Phe Lys Ile Leu Gln Lys Lys Ser Thr Ala Val Lys Phe

115 120 125

Gln Asp Thr Leu Ser Met Pro Gly Ala Ile Leu Gly Thr Gln Leu Phe

130 135 140

Asp Trp Asp Gly Glu Val Ile Thr Gly Tyr Glu Val Ile Gln Pro Asp

145 150 155 160

<210> 8

<211> 891

<212> DNA

<213>Clostridium thermocellum (Clostridium thermocellum)

<400> 8

tagtactatt aaagtcagac ttggttaaat ataaatttta tgacttgcat acaaacttga 60

tgtgtattat aataaaaata caaaacaaaa tagcaataat ttactcagtt atttttgaaa 120

tgggggtagt attaatatcg tataatgggg ttgcatatct gctgtctttc gaaaaaagca 180

caagaacttc aaatgtttcc atagtgaaat ttaaaaattg gagatttctt tgttgccccc 240

tcaaaaagta tatttttttc gaagatatat atatggaatt tattgattaa tttaagttat 300

taattttggc cttttagggt cgttgaaaac tgaatatgtt aagttgtttt gcgtgattca 360

gctgcatttg acgtaagact tcgccggtct gtttaaattc ccataataag atgtatttat 420

tgtagtaata atctggcatc tacaaatttc agtatttgca atagtctctg ttcaaaaaag 480

caattgtctt ttaaaccttt cagtattgtc ttcgtggcag tttcttttgt tatacgtcgt 540

tccgacaaaa aaatgtaaat ttatgtcaaa tgcgcggctg atttgataaa aaagtttgtt 600

aacacaaatt tattatgtta acacaagtat tttttgggtc cagcttagtt ttatgatgaa 660

aataatgcgt aaaatttatc cgcaaaaagg gggaatgaat ttattgcggg taggttgcat 720

tatttcatca tataacttaa aaagaataaa aaagtatatt tgaaagggga agatggagag 780

atggtaaaaa gcagaaagat ttctattctg ttggcagttg caatgctggt atccataatg 840

atacccacaa ctgcattcgc aggtcctaca aaggcaccta caaaagatgg g 891

<210> 9

<211> 59

<212> PRT

<213>The molten fine clostridium (Clostridium clariflavum) of yellow

<400> 9

Tyr Gly Asp Leu Asn Gly Asp Lys Leu Val Asn Ser Ile Asp Phe Ala

1 5 10 15

Leu Leu Lys Ile Tyr Leu Leu Gly Tyr Ser Lys Glu Phe Pro Tyr Glu

20 25 30

Tyr Gly Ile Lys Ser Ala Asp Leu Asn Arg Asn Gly Glu Val Asp Ser

35 40 45

Ile Asp Phe Ala Ile Leu Arg Ser Phe Leu Leu

50 55

<210> 10

<211> 57

<212> PRT

<213>Ruminococcus albus (Ruminococcus albus)

<400> 10

Arg Gly Asp Val Asn Gly Asp Gly Val Val Asn Val Thr Asp Val Ala

1 5 10 15

Lys Ile Ala Ala His Val Lys Gly Lys Lys Ile Leu Thr Gly Asp Ser

20 25 30

Leu Lys Asn Ala Asp Val Asn Phe Asp Gly Ser Val Asn Ile Thr Asp

35 40 45

Ile Thr Arg Ile Ala Ala Phe Val Lys

50 55

<210> 11

<211> 56

<212> PRT

<213>Yellow Ruminococcus (Ruminococcus flavefaciens)

<400> 11

Tyr Gly Asp Ala Asn Cys Asp Gly Asn Val Ser Ile Ala Asp Ala Thr

1 5 10 15

Ala Ile Leu Gln His Leu Gly Asn Arg Asp Lys Tyr Gly Leu Arg Ala

20 25 30

Gln Gly Met Leu Asn Ala Asp Val Asp Gly Gln Ser Gly Val Thr Ala

35 40 45

Asn Asp Ala Leu Val Leu Gln Lys

50 55

<210> 12

<211> 54

<212> PRT

<213>It solves fiber clostridium (Clostridium cellulolyticum)

<400> 12

Tyr Gly Asp Tyr Asn Asn Asp Gly Ser Ile Asp Ala Leu Asp Phe Ser

1 5 10 15

Ser Phe Lys Met Tyr Leu Met Asn Pro Val Arg Thr Tyr Thr Glu Val

20 25 30

Leu Asp Leu Asn Ser Asp Asn Thr Val Asp Ala Ile Asp Phe Ala Ile

35 40 45

Met Lys Gln Tyr Leu Leu

50

<210> 13

<211> 57

<212> PRT

<213>Molten fiber vacation bacteroid (Pseudobacteroides cellulosolvens)

<400> 13

Tyr Gly Asp Val Thr Gly Asp Gln Leu Val Thr Asp Ala Asp Lys Thr

1 5 10 15

Lys Val Ser Asn Tyr Ile Leu Gly Ser Val Tyr Leu Thr Ser Arg Glu

20 25 30

Phe Ala Ala Ala Asp Val Asn Gly Asp Gln Val Val Asn Ser Gly Asp

35 40 45

Leu Thr Leu Ile Asn Arg His Ile Leu

50 55

<210> 14

<211> 58

<212> PRT

<213>Acetivibrio cellulolyticus (Acetivibrio cellulolyticus)

<400> 14

Lys Gly Asp Val Asp Leu Asp Gly Ala Ala Asn Ser Ile Asp Phe Gly

1 5 10 15

Lys Met Arg Leu Cys Leu Leu Gly Lys Ser Pro Ala Phe Thr Gly Gln

20 25 30

Ala Leu Asp Asn Ala Asp Leu Asn Asp Asp Gly Ala Phe Asn Ser Ile

35 40 45

Asp Phe Gly Tyr Met Arg Lys Lys Leu Leu

50 55

Claims

1. the method for preparing DHA using lignocellulosic, it is characterised in that：Include the following steps：

(1) it pre-processes：Lignocellulosic material is pre-processed, obtains content of lignin not higher than 15%, hemicellulose contains Amount is not higher than 20% lignocellulosic substrate；

(2) it is saccharified：According to solid and liquid weight volume ratio 1:2-1:The lignocellulosic substrate that step (1) obtains is transferred to anaerobism by 25 In the saccharification culture medium of fermentor, cellulase preparation is added in the lignocellulosic substrate, in 34-65 DEG C of temperature strip Reaction is hydrolyzed under part, obtains the liquid glucose containing glucose；

(3) schizochytrium limacinum fermentation：After concentration of glucose reaches 25-180g/L in the liquid glucose that step (2) obtains, pass through liquid glucose The filter assemblies of fermentor outlet assembly enter bioreactor；Nutrient media components are added into bioreactor, are fermented Culture medium, and autoclave sterilization；Then the schizochytrium limacinum seed liquor for accessing activation, 28-38 DEG C of temperature condition, mend nitrogen and Ferment 36-80h under the conditions of the pH of 6.5-7.0, until fermentation ends when concentration of glucose is not higher than 5g/L；Or during the fermentation Continuous flow add step (2) obtain liquid glucose, so that sugared concentration is maintained 5-10g/L, 28-38 DEG C of temperature condition, mend nitrogen and Ferment 80-120h under the conditions of the pH of 6.5-7.0, obtains schizochytrium limacinum fermentation liquid；

(4) DHA is extracted：The schizochytrium limacinum fermentation liquid centrifugal treating that step (3) is obtained, by schizochytrium limacinum somatic cells and fermentation Culture medium is separated by solid-liquid separation afterwards, is extracted from schizochytrium limacinum somatic cells using organic solvent extractionprocess and is obtained DHA.

2. the method according to claim 1 for preparing DHA using lignocellulosic, it is characterised in that：It further include step (5), specially：Step (4) are separated by solid-liquid separation culture medium after obtained fermentation not dilute or dilute by 1-3 times, for preparing The saccharification culture medium of step (2).

3. the method according to claim 2 for preparing DHA using lignocellulosic, it is characterised in that：The cellulase Preparation is interacted by the component in the small body protein of non-fiber and cellulosome, and non-fiber corpusculum protein binding is small in fiber It is obtained in composite；The small body protein of non-fiber be zytase, cellulose restriction endonuclease, cellulose excision enzyme, expansion because Son, protease, amylase or pectase；The cellulosome is to be produced by anaerobic bacteria and secreted to have wooden fibre in extracellular Tie up the multienzyme complex of plain degrading activity.

4. the method according to claim 3 for preparing DHA using lignocellulosic, it is characterised in that：The anaerobic bacteria For the molten fine clostridium of Clostridium thermocellum, yellow, thermophilic fiber clostridium, solution fiber clostridium, Acetivibrio cellulolyticus, molten fiber vacation bacteroid, white Color Ruminococcus or yellow Ruminococcus；The Clostridium thermocellum is the Clostridium thermocellum for expressing the β 1,4- glucuroide of excretion.

5. the method according to claim 3 or 4 for preparing DHA using lignocellulosic, it is characterised in that：The non-fiber Small body protein has sequence table SEQ ID NO:Amino acid sequence shown in 1-3,15-18；And with such as SEQ ID NO:1-3, The consistency of amino acid sequence shown in 15-18 95% or more, and with such as SEQ ID NO:Amino acid shown in 1-3,15-18 Sequence amino acid sequence with the same function.

6. the method according to claim 5 for preparing DHA using lignocellulosic, it is characterised in that：The non-fiber is small Body protein is that indirect is connect or substantivity connects with the mode of the protein component interaction in cellulosome；It is described indirect Property is connected as：The small body protein of non-fiber is attached with the protein component in cellulosome by covalent interaction, described straight Connecing property is connected as：The small body protein of non-fiber is attached with the component in cellulosome by expressing in series.

7. the method according to claim 6 for preparing DHA using lignocellulosic, it is characterised in that：The non-fiber is small Component in body protein and cellulosome by covalent interaction be attached for：The small body protein of non-fiber and cellulosome group Divide and connect respectively with the polypeptide fragment with covalent interaction, specific covalent interaction between polypeptide fragment is utilized to realize fibre The covalent cross-linking of dimension corpusculum component and the small body protein of non-fiber makes non-fiber using the assembling module of cellulosome component institute band Corpusculum protein binding is in cellulosome complex；

Component in the small body protein of non-fiber and cellulosome by expressing in series be attached for：The small body protein of non-fiber Amalgamation and expression is carried out with the assembling module of cellulosome or the protein component with assembling module, passes through the special of assembling intermodule Property noncovalent interaction, realize non-fiber corpusculum protein groups in the cellulosome complex；The amalgamation and expression is： The encoding gene of the small body protein of non-fiber is inserted into N-terminal, C-terminal or the domain sequence of the sequence of encoding fiber corpusculum component protein On intermediate genome.

8. the method according to claim 7 for preparing DHA using lignocellulosic, it is characterised in that：Described in step (2) Saccharification culture medium be：Dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L, urea 0.8g/L, calcium chloride 0.1g/L, magnesium chloride 1.8g/L, ferrous sulfate 0.0005g/L, vulcanized sodium 2g/L, corn pulp 4g/L, trisodium citrate 2g/L, pH 6.5-7.5；

Fermentation medium described in step (3) is：Corn pulp 5-20g/L, potassium dihydrogen phosphate 2.5g/L, sea crystal 30g/L, dimension life Plain B₁5mg/L, vitamin B₆5mg/L, vitamin B₁₂5mg/L, biotin 5mg/L, pH 6.5-7.5.

9. the method according to claim 7 for preparing DHA using lignocellulosic, it is characterised in that：Described in step (1) Lignocellulosic material be one of corn stover, wheat straw, brush wood branch, wood chip, corncob, straw and waste paper or a variety of Combination；The pretreatment is one of alkaline process, diluted acid method, hydro-thermal method, steam explosion method and sulfonation method preconditioning technique or a variety of Combination；Pretreated lignocellulosic substrate is that content of lignin is not higher than 11%, and hemicellulose level is not higher than 12%； Temperature condition in step (2) saccharification step is 55-60 DEG C, and the solid and liquid weight volume ratio in hydrolyzation system is 1:3-1:10.

10. the method according to claim 7 for preparing DHA using lignocellulosic, it is characterised in that：In step (3), when After concentration of glucose is 60-150g/L, liquid glucose is made to enter bioreactor by the filter assemblies of fermentor outlet assembly；? 30-35 DEG C of temperature condition mends the 48-80h that ferments under the conditions of nitrogen and the pH of 6.5-7.0, until when concentration of glucose is not higher than 5g/L Fermentation ends, or the liquid glucose that continuous flow adds step (2) to obtain during the fermentation, make sugared concentration maintain 5-10g/L, 30-35 DEG C of temperature condition mends the 100-120h that ferments under the conditions of nitrogen and the pH of 6.5-7.0；The benefit nitrogen operates：Stream adds The corn pulp solution of 50% (w/v) carries out benefit nitrogen；Organic solvent extractionprocess described in step (4) is specially：Using ethyl alcohol and The mixed liquor of n-hexane extracts DHA.