CN108866112B - Method for preparing squalene by adopting lignocellulose - Google Patents

Abstract

Aiming at the problems in the aspect of producing squalene by microbial fermentation in the prior art, the invention provides a process for preparing squalene by taking lignocellulose agricultural and forestry waste as a raw material. The process comprises the steps of pretreatment, saccharification, yeast-like fermentation, squalene extraction and the like. The process combines lignocellulose biological saccharification with yeast-like high-density fermentation; by adopting the lignocellulose biomass as the raw material, on one hand, the cost of enzyme and the production cost of squalene microbial fermentation in the saccharification stage are obviously reduced, and on the other hand, the problem of comprehensive utilization of agricultural and forestry wastes is also solved. In addition, the lignocellulose saccharification stage culture medium and the fermentation culture medium can be recycled, so that water can be obviously saved, the use of chemicals can be reduced, and the remarkable effects of reducing wastewater discharge and reducing cost can be achieved. In the process, the yield of squalene can reach 13g/L, the squalene accounts for 5-9% of the dry weight of the thallus, and the purity of the refined squalene is more than 93%.

Description

Method for preparing squalene by adopting lignocellulose

Technical Field

The invention relates to the field of microbial fermentation engineering, in particular to a method for producing squalene by using a lignocellulose raw material.

Background

Squalene (squalene) is a nontoxic marine bioactive substance with disease prevention and treatment effects, has a chemical name of 2,6,10,15,19, 23-hexamethyl-2, 6,10,14,18, 22-tetracosahexaene, has various physiological functions of improving in-vivo superoxide dismutase activity, enhancing organism immunity, improving sexual function, resisting aging, fatigue, resisting tumor and the like, and is widely applied to various fields of medicines, cosmetology, cosmetics, foods and the like.

Currently, the main source of squalene is fish oil, especially shark liver oil. But with the increasing exhaustion of fishery resources and the increasingly perfection of relevant laws and regulations, people are prompted to strive to find other squalene sources; such as olive oil, palm oil and other oil crops, or amaranth, seeds, rice bran, wheat germ, but the squalene content in the plant is very low, approximately 0.1 to 0.7% by weight. In addition, the preparation method of squalene also comprises a chemical synthesis method and a microbial fermentation method. The chemical synthesis method mainly uses succinaldehyde or geranylacetone and the like as raw materials for synthesis, but high-value or dangerous chemicals such as organic lithium compounds or sodium acetylene and the like are needed in the process, so that the problems of high cost, high danger or low product purity are caused. The microbial fermentation method has the advantages of mild conditions, easy large-scale production and the like.

The invention patent application 201410829522 discloses a yeast-like yeast with high squalene yield and application thereof. The application provides the yeast-like yeast with high squalene yield, which can rapidly grow at the temperature of 15-37 ℃ and can greatly accumulate squalene. Patent CN 102666837 a produced squalene using yarrowia lipolytica, in a mass content of 2% of the total lipid in the cell. Patent CN 103266137B uses transgenic Escherichia coli to produce squalene. However, the existing microbial fermentation methods for producing squalene all use glucose derived from corn starch as a main carbon source. Therefore, the supply of glucose and the raw material cost are main limiting factors influencing the productivity and the economy of squalene produced by microbial fermentation, and the development of a low-cost novel carbon source and a matched biological preparation technology has necessity and wide application prospect.

Lignocellulose is a renewable biomass which is rich in supply and environment-friendly, and comprises agricultural and forestry wastes such as straws, waste paper pulp, rice hulls, vinasse and the like. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. Wherein the cellulose is composed of glucose. Therefore, the lignocellulose biomass is a potential cheap glucose raw material, and the production method of squalene by taking the lignocellulose raw material as a substrate is developed, so that the production cost can be obviously reduced, and meanwhile, the problem of comprehensive utilization of agricultural and forestry wastes is solved. At present, cellulase from fungi is generally adopted for bioconversion of lignocellulose, so that the efficiency and the cost are insufficient, the cellulase preparation is mainly prepared in a separate reactor through microbial fermentation, the preparation process is different from the cellulose hydrolysis condition, the production steps are complicated, the cost such as manpower and material resource requirements and equipment investment is obviously increased, and the saccharification process of the lignocellulose has no market competitiveness. Therefore, lignocellulosic bioconversion based on cellulase preparations of fungal origin is difficult to implement for large scale applications, more difficult to integrate with processes for microbial fermentation of squalene.

It is known that cellulosome is a multienzyme complex with complex structure and components produced by anaerobic bacteria such as clostridium thermocellum, and is one of the most efficient cellulose degradation systems known in nature. Despite the great potential of the cellulosome and its production strains for lignocellulose saccharification applications, its industrial application is still limited by a number of factors. Saccharification efficiency is significantly reduced when the lignocellulosic complex substrate has a high content of non-cellulosic components (e.g., hemicellulose, pectin, starch, protein, lignin). The predecessors mainly improve the viability of the cellulosomes and their adaptability to the substrate by adding additional non-cellulosome proteins to the hydrolysis system. However, since the activity and stability of the exogenously added protein are reduced as the saccharification process proceeds, the consumption of the enzyme requires an increase in the amount of enzyme added or the number of times of addition. In addition, these non-cellulosome proteins added to the saccharification system as free enzymes cannot interact with cellulosome, which significantly reduces the synergistic effect with other enzymes in the system, and inevitably results in additional addition of more than required amount of enzyme, leading to high cost of enzyme preparation. Moreover, the saccharification strategy relying on exogenously added free enzyme has complex process and high requirement on equipment, and the conversion efficiency can not meet the requirement of industrial production.

Disclosure of Invention

Aiming at the problems in the aspect of producing squalene by microbial fermentation in the prior art, the invention provides a process using lignocellulose agricultural and forestry waste as a raw material, wherein a cellulase preparation for catalyzing saccharification of lignocellulose is adopted in the process, so that the carbon source cost of microbial fermentation of squalene is reduced.

The technical scheme of the invention is as follows: a method for preparing squalene by adopting lignocellulose comprises the following steps:

(1) pretreatment: pretreating a lignocellulose raw material to obtain a lignocellulose substrate with the lignin content of not higher than 20% and the hemicellulose content of not higher than 25%;

(2) saccharification: transferring the lignocellulose substrate obtained in the step (1) into a saccharification culture medium of an anaerobic fermentation tank according to the solid-liquid weight-volume ratio of 1:2-1:25, adding a cellulase preparation into the lignocellulose substrate, and performing hydrolysis reaction at the temperature of 34-65 ℃ to obtain a sugar solution containing glucose; the saccharification culture medium comprises: 2.9g/L of dipotassium phosphate, 1.5g/L of monopotassium phosphate, 0.8g/L of urea, 0.1g/L of calcium chloride, 1.8g/L of magnesium chloride, 0.0005g/L of ferrous sulfate, 2g/L of sodium sulfide, 4g/L of corn steep liquor and 2g/L, pH 6.5.5-7.5 of trisodium citrate. During saccharification, the pH can be controlled to be 5.8-6.2 by feeding sodium hydroxide.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 25-180g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed liquid, fermenting for 2-5 days at 15-37 deg.C and pH of 6.0-9.0 until the glucose concentration is not higher than 5 g/L; or continuously feeding sugar solution obtained in step (2) in the fermentation process to maintain sugar concentration at 5-10g/L, fermenting at 15-37 deg.C under nitrogen supplementing condition and pH of 6.0-9.0 for 5-10 days to obtain fermentation liquid.

The fermentation medium consists of the following components: 0.6% of corn steep liquor, 0.2% of peptone, 0.8% of potassium dihydrogen phosphate, 0.5% of magnesium sulfate, 3% of sea crystal, 10.02% of vitamin B, 60.02% of vitamin B, 120.005% of vitamin B and 0.005% of biotin. The balance being water, pH 7.0.

(4) Extracting squalene: centrifuging the fermentation liquor obtained in the step (3), and performing solid-liquid separation on the saccharomycete-like thallus cells and the fermented culture medium; and (3) after the obtained saccharomycete-like cell is subjected to enzymolysis and wall breaking, extracting a crude squalene product by using an organic solvent extraction method, and distilling to obtain the refined squalene.

The method further comprises a step (5), specifically: and (3) diluting the culture medium obtained after the solid-liquid separation in the step (4) without dilution or by 1-3 times, and preparing the saccharification culture medium in the step (2).

Wherein, the cellulase preparation in the step (2) is obtained by combining non-cellulosome protein in a cellulosome complex through the interaction of the non-cellulosome protein and components in the cellulosome; the non-cellulosome protein is xylanase, cellulose endonuclease, cellulose exonuclease, swelling factor, protease, amylase or pectinase; the cellulosome is a multienzyme complex with lignocellulose degrading activity produced by anaerobic bacteria and secreted extracellularly. The components in the fibrosome are foot rest protein, enzyme with a catalytic function and an assembly module.

The non-fibrosome protein interacts with the protein components in the fibrosome in a manner that is indirect or direct; the indirect connection is as follows: the non-fibrosome protein is linked to the protein components in the fibrosome by covalent interactions, the direct linkage being: non-fibrosomal proteins are linked to components in the fibrosomes by tandem expression.

Preferably, the non-fibrosomal protein has an amino acid sequence shown in SEQ ID NO 1-3,15-18 of the sequence table; and an amino acid sequence which has more than 95% of consistency with the amino acid sequences shown as SEQ ID NO. 1-3,15-18 and has the same function with the amino acid sequences shown as SEQ ID NO. 1-3, 15-18. Wherein: 15 SEQ ID NO: Genbank SEQ ID NO: CRZ 35393.1; 16 encoded by the 1968724 to 1973904 nucleic acid sequence of genomic CP 001393.1; SEQ ID NO. 17 Genbank sequence No. KC 763474.1; 18 encoded by the 2531445 to 2532785 nucleic acid sequence of genomic CP 001393.1.

Preferably, the anaerobic bacteria are Clostridium thermocellum (Clostridium thermocellum), Clostridium flavum (Clostridium clavatum), Clostridium cellulophilum (Clostridium cellulovorans), Clostridium cellulolyticum (Clostridium cellulolyticum), vibrio cellulolyticus (acetovibrio cellulolyticus), pseudomonas cellulolyticus (pseudomonas cellulolyticus), Ruminococcus albus (Ruminococcus albus), Ruminococcus xanthus (Ruminococcus flavefaciens). Wherein the Clostridium thermocellum is Clostridium thermocellum expressing an secreted beta 1, 4-glucosidase.

Wherein the non-fibrosomal protein is linked to a component in a fibrosome by covalent interactions, in particular: the non-fibrosome protein and the fibrosome component are respectively connected with polypeptide fragments with covalent interaction, the covalent crosslinking of the fibrosome component and the non-fibrosome protein is realized by utilizing the specific covalent interaction between the polypeptide fragments, and the non-fibrosome protein is combined in the fibrosome complex by utilizing an assembly module carried by the fibrosome component. The polypeptide fragment which is covalently interacted with the non-fibrosomal protein and the fibrosomal component is a base sequence shown in SEQ ID NO. 4 or an amino acid sequence shown in SEQ ID NO. 5. The concrete implementation steps comprise:

1) connecting the non-fibrosomal protein with the coding gene of one fragment (polypeptide fragment I or II, namely SEQ ID NO:4 or 5) of the pair of polypeptide fragments with covalent interaction by a gene cloning method;

2) according to 1), the genes encoding the other fragment of the pair of covalently interacting polypeptide fragments (polypeptide fragment II or I, i.e. SEQ ID NO:5 or 4) of the cellulosome component protein are linked by means of gene cloning.

3) According to 1), connecting the recombinant gene sequences of non-fibrosomal proteins and polypeptide fragments to a fibrosomal-producing bacterial expression plasmid;

4) according to 2), the recombinant gene sequences of the cellulosome component proteins and polypeptide fragments are ligated to a cellulosome-producing bacterial homologous recombinant plasmid and homology arms are designed according to the genomic sequence.

5) Transforming the plasmid obtained in the step 4) into a cellulosome-producing bacterial cell, and realizing the replacement of the original cellulosome component protein sequence on the genome by the recombinant gene sequence through homologous recombination screening, thereby constructing a recombinant strain and realizing the fusion expression of the cellulosome component protein and the polypeptide fragment in the cellulosome-producing bacterial cell.

6) Transforming the plasmid obtained in 3) into the recombinant strain obtained in 5), and realizing the fusion expression of the non-fibrosomal protein and the polypeptide fragment in the cell of the fibrosomal-producing bacteria.

In the finally obtained recombinant strain, the cellosome component protein expressed by genome and the non-cellosome protein expressed by plasmid are covalently cross-linked by means of specific covalent interaction between the fused polypeptide fragments I and II, and are assembled in the cellosome complex.

Wherein the non-fibrosomal protein is linked to the components of the fibrosome by tandem expression, specifically: the fusion expression of non-fibrosomal proteins with the assembly modules of the fibrosomes or protein components with the assembly modules, the assembly of non-fibrosomal proteins in the fibrosomal complex is achieved by specific non-covalent interactions between the assembly modules. The fusion is expressed as: genes encoding non-fibrosomal proteins are inserted into the genome at the N-terminus, C-terminus, or in the middle of the domain sequence of the sequence encoding the fibrosomal component proteins.

The corresponding modules are an adhesion module and a butt joint module. The docking module is a base sequence shown in SEQ ID NO 6, SEQ ID NO 9-13 and SEQ ID NO 14, and the adhesion module is an amino acid sequence shown in SEQ ID NO 7. The concrete implementation steps comprise:

1) connecting a non-fibrosomal protein to a gene encoding a type I docking module (SEQ ID NO:6) by gene cloning methods, or

2) Linking non-fibrosomal proteins to the gene encoding the type II adhesion module (SEQ ID NO:7) by means of gene cloning

3) Connecting the recombinant gene sequence of the non-fibrosome protein obtained in 1) or 2) and the assembly module to a fibrosome-producing bacterial expression plasmid

4) Transforming the plasmid obtained in the step 3) into a cellulosome-producing bacterial cell to realize the fusion expression of the non-cellulosome protein and the I-type docking module or the II-type adhesion module in the cellulosome-producing bacterial cell.

In the finally obtained recombinant strain, the non-fibrosome protein expressed by the plasmid is subjected to specific non-covalent interaction with the fibrosome foot-rest protein through the fused I-type docking module or II-type adhesion module, so as to be assembled in a fibrosome complex.

The specific implementation steps of the direct fusion expression comprise:

1) by means of gene cloning, the encoding gene of non-fibrosome protein is connected to the homologous recombinant plasmid of the bacteria producing fibrosome and the homologous arm is designed based on the genome sequence.

2) Transforming the plasmid obtained in the step 1) into a cellulosome-producing bacterial cell, and inserting a coding gene of non-cellulosome protein into the middle of the N end or C end or structural domain sequence of a sequence of the cellulosome component protein on a genome through homologous recombination screening, thereby constructing a recombinant strain and realizing the fusion expression of the non-cellulosome protein and the cellulosome component protein in the cellulosome-producing bacterial cell.

In the finally obtained recombinant strain, the non-fibrosome protein is combined into a fibrosome complex by using an assembly module of a fibrosome component protein fused and expressed with the non-fibrosome protein.

Preferably, the lignocellulose raw material in the step (1) is one or more of corn stalk, wheat straw, shrub twig, wood chip, corncob, rice straw and waste paper; the pretreatment is one or a combination of more of alkaline method, dilute acid method, hydrothermal method, steam explosion method and sulfonation method pretreatment technologies; the pretreated lignocellulose substrate has a lignin content of not higher than 11% and a hemicellulose content of not higher than 12%.

Preferably, the temperature condition in the saccharification step of step (2) is 55-60 ℃, and the solid-liquid weight-volume ratio in the hydrolysis system is 1:3-1: 10.

Preferably, in the step (3), after the glucose concentration is 60-120g/L, the sugar solution is fed into the bioreactor through a filter assembly arranged at the outlet of the fermentation tank. The nitrogen supplementing operation specifically comprises the following steps: 8% (w/v) ammonium sulfate solution is added in a flowing manner for nitrogen supplement.

Preferably, the enzymatic wall breaking in the step (4) is specifically: adopting helicase to break the wall; the organic solvent extraction method specifically comprises the following steps: extracting with organic solvent by using mixed solution of ethanol and n-hexane.

The invention has the beneficial effects that:

(1) the invention provides a process for producing squalene by fermenting yeast with lignocellulose as a raw material, which has the advantages of wide raw material source, low price and strong market competitiveness and can greatly solve the market demand which is increased year by year.

(2) Compared with the existing fermentation process adopting glucose derived from corn starch as a carbon source, the process disclosed by the invention combines lignocellulose biomass saccharification and yeast-like high-density fermentation; by adopting the lignocellulose biomass as the raw material, on one hand, the cost of enzyme and the production cost of squalene microbial fermentation in the saccharification stage are obviously reduced, and on the other hand, the problem of comprehensive utilization of agricultural and forestry wastes is also solved.

(3) In the process, the lignocellulose saccharification stage culture medium and the fermentation culture medium can be recycled, so that water and chemicals can be obviously saved, and the process has the obvious effects of reducing wastewater discharge and cost; the two points have important significance in industry, have great economic benefit and are environment-friendly, and can be carried out for a longer time.

(4) In the process, the yield of squalene can reach 13g/L and accounts for 5-9% of the dry weight of the thalli. The purity of the obtained refined squalene is more than 93 percent by the processes of molecular distillation and the like. Compared with the existing chemical production technology, the process is mild and simple, and compared with the existing fermentation production technology, the production level of squalene is higher.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1: construction of a cellulase preparation based on Clostridium thermocellum cellulosome by means of indirect ligation

The plasmid HR-K was constructed by cloning tdk expression cassette (containing the promoter of gapDH gene) and pyrF expression cassette (containing the pyrF self-promoter) into the downstream of the antibiotic gene cat of plasmid pHK (Mohr, G., Hong, W., Zhang, J., Cui, G., Z., Yang, Y., Cui, Q., et al. (2013) A targeting system for gene targeting in thermophiles and its application in cloning, PLoS One 8: e 699.) by seamless cloning, and by designing primers, adding NheI and XbaI cleavage sites between tdk and pyrF expression cassette, adding eagI and MluI cleavage sites downstream of pyrF for cloning of homologous arm fragments.

The targeting knock-in site was chosen between the enzymatic catalytic domain of cellulase Cel9K (exocellulase, encoded by the 2113813 to 2111293 nucleic acid sequence in genomic CP002416.1) in clostridium thermocellum cellulosome and the docking module. Firstly, a coding gene of beta-1, 4-glucosidase BglA (Genbank serial number is AFO70070.1) is used as a target sequence, and the enzyme cutting sites of MluI and EagI are utilized to clone into a homologous recombinant plasmid pHK-HR, so as to construct the homologous recombinant plasmid pHK-HR-BglA. The upstream homology arm HR-up sequence is the nucleic acid sequence 2111347 to 2112870 in the genome of C.thermocellum DSM1313 (sequence number CP002416.1 in NCBI database), the downstream homology arm HR-down is the nucleic acid sequence 2109848 to 2111354 in the genome of DSM1313, and the intermediate homology arm HR-short is the nucleic acid sequence 2111347 to 2111659 in the genome of DSM 1313. Secondly, the constructed plasmid is transformed into delta pyrF, and the chassis strain is obtained by screening according to three steps. The specific screening method comprises the following steps:

1) the homologous recombinant plasmid pHK-HR was transformed into a pyrF-deleted strain, using G containing thiamphenicolS-2 semi-solid Medium (KH)₂PO₄ 1.5g/L,K₂HPO₄·3H₂O3.8 g/L, urea 2.1g/L, MgCl₂·6H₂O 1.0g/L,CaCl₂·2H₂O 150mg/L,FeSO₄·6H₂O1.25 mg/L, cysteine 1.0g/L, MOPS sodium salt 10g/L, yeast extract 6.0g/L, cellobiose 5.0g/L, trisodium citrate dihydrate 3.0g/L, resazurin 0.1mg/L, pH 7.4) plates were screened to obtain plasmid transformants.

2) The transformants obtained were cultured in MJ broth (KH)₂PO₄ 1.5g/L,K₂HPO₄·3H₂O3.8 g/L, urea 2.1g/L, MgCl₂·6H₂O 1.0g/L,CaCl₂·2H₂O 150mg/L,FeSO₄·6H₂O1.25 mg/L, cysteine 1.0g/L, MOPS sodium salt 10g/L, cellobiose 5.0g/L, trisodium citrate dihydrate 3.0g/L, resazurin 0.1mg/L, pyridoxamine hydrochloride 2mg/L, biotin 0.2mg/L, p-aminobenzoic acid 0.4mg/L, vitamin B120.2 mg/L, pH 7.4) for three transfers, and then coating MJ semisolid culture medium containing 10 μ g/mL 5-Fluorodeoxyuridine (FUDR) for the first homologous recombination screening. In this step, Tdk can convert FUDR into F-dUMP toxic to cells, and the underpan cells can survive in MJ culture medium only by means of uracil nucleotide synthesized by pyrF gene on plasmid, so that the screening strategy ensures that homologous recombination module on plasmid and genome are homologously recombined and the recombined plasmid is lost. According to the principle that the long homologous arms are preferentially subjected to homologous recombination, the two front and back long homologous arms are firstly subjected to homologous recombination with a genome, and the obtained recombinants are restored to prototrophy from uracil auxotrophs of the starting strain.

3) The recombinants obtained after the first homologous recombination are firstly passaged for 3 times in a GS-2 liquid culture medium, and the bacterial liquid is coated with a GS-2 semisolid culture medium containing 500 mu g/mL 5-fluoroorotic acid (FOA) for screening after being subjected to gradient dilution by using the same culture medium, so that the target scarless gene knockout/knock-in strain is obtained. In this step, the inverse selection of PyrF will drive the upstream long and short homology arms to undergo a second homologous recombination to remove the pyrF expression cassette from the genome. The mutant strain after the second homologous recombination is changed into uracil auxotrophy from prototrophy. Thereby realizing the knockout, knock-in or replacement of the target site gene on the genome. Homologous recombinant strains delta pyrF-I or delta pyrF-II were obtained, respectively, expressing the fusion protein of Cel48S with polypeptide fragment I or polypeptide fragment II, respectively.

The 5' end of cellulose exonuclease Cel48S (coded by 3228088 to 3230229 nucleic acid sequences in genome CP002416.1) of the clostridium thermocellum cellulosome is selected as a target knock-in site, the coding sequences of two fragments of a polypeptide fragment I (SEQ ID NO:4) or a polypeptide fragment II (SEQ ID NO:5) are used as target sequences, the enzyme cutting sites of MluI and EagI are respectively cloned into a homologous recombinant plasmid pHK-HR, and the homologous recombinant plasmid pHK-HR-I or pHK-HR-II is respectively constructed and obtained. The upstream arm HR-up is the nucleic acid sequence 3230200 to 3230700 in the C.thermocellum DSM1313 genome (sequence number CP002416.1 in NCBI database), the downstream arm HR-down is the nucleic acid sequence 3229699 to 3230199 in the DSM1313 genome, and the intermediate arm HR-short is the nucleic acid sequence 3230200 to 3230500 in the DSM1313 genome. The constructed plasmids are respectively transformed into the constructed chassis strains, and homologous recombinant strains delta pyrF, BglA-I or delta pyrF, BglA-II are obtained according to the three-step screening method.

The polypeptide fragment II or the polypeptide fragment I is connected to the 3' end of xylanase XynA (SEQ ID NO:1) by using an overlap extension polymerase chain reaction method. The ligated recombinant sequence was cloned into the expression plasmid pHK using BamHI and XbaI cleavage sites as the target sequence. Transforming an expression plasmid containing a recombinant sequence of XynA and a polypeptide fragment I into delta pyrF, wherein BglA-II; expression plasmids containing recombinant sequences of XynA and polypeptide fragment II were transformed into Δ pyrF:BglA-I, thereby achieving the binding of Cel48S and XynA through specific covalent interactions between polypeptide fragments I and II. By extracting the cellulosome of the recombinant strain of clostridium thermocellum, it was found that the expressed XynA with covalently bound modules can be secreted extracellularly and interact with Cel48S with covalently bound modules, assembling into a cellulosome complex. The recombinant strain is cultured to the middle logarithmic phase in GS-2 culture medium which takes 5g per liter of cellulose or cellobiose as a carbon source, and can be used as a whole bacterial enzyme preparation for biological saccharification of lignocellulose.

Example 2: construction of a cellulase preparation based on Clostridium thermocellum cellulosome by means of indirect ligation

In contrast to example 1, polypeptide fragment II or polypeptide fragment I was ligated to the 3' end of the cellulosome endonuclease CelZ (SEQ ID NO: 15). The constructed recombinant strain is cultured to the middle logarithmic phase in GS-2 culture medium which takes 5g per liter of cellulose or cellobiose as a carbon source, and can be used as a whole bacterial enzyme preparation for biological saccharification of lignocellulose.

Example 3: construction of a cellulase preparation based on Clostridium thermocellum cellulosome by direct ligation

The method of overlap extension polymerase chain reaction is used for directly connecting the cellulose exonuclease Cel9-48(SEQ ID NO:16) with the sequence of the type II adhesion module CohIIct (SEQ ID NO:7) or the type I docking module DocIct (SEQ ID NO:6) of the clostridium thermocellum, wherein the sequence of the CohIIct or the DocIct is connected to the 3' end of the sequence of the Cel9-48, and the sequence of the Cel9-48-DocIct or the sequence of the Cel9-48-CohIIct is obtained.

The sequence Cel9-48-DocIct or Cel9-48-CohIIct is used as a target sequence, the sequence MluI and EagI enzyme cutting sites are cloned into the homologous recombinant plasmid pHK-HR, and the lactate dehydrogenase gene clo1313_1160 is used as a target replacement sequence to construct the homologous recombinant plasmid pHK-HR-Cel 9-48. The upstream arm HR-up is the nucleic acid sequence 1380180 to 1380679 in the C.thermocellum DSM1313 genome (sequence number CP002416.1 in NCBI database), the downstream arm HR-down is the nucleic acid sequence 1380634 to 1381133 in the DSM1313 genome, and the intermediate arm HR-short is the nucleic acid sequence 1380833 to 1381133 in the DSM1313 genome. The constructed plasmids are respectively transformed into the chassis strains constructed in the embodiment 1, and homologous recombinant strains 1 and 2 are obtained according to the three-step screening method of the embodiment 1. By extracting the recombinant strain of the cellulosome found, Cel9-48 and assembly module fusion protein can be secreted extracellular, and through the noncovalent cross-linked interaction mode assembly into the cellulosome complex. The recombinant strain is cultured to the middle logarithmic phase in GS-2 culture medium which takes 5g per liter of cellulose or cellobiose as a carbon source, and can be used as a whole bacterial enzyme preparation for biological saccharification of lignocellulose.

Example 4: construction of a cellulase preparation based on Clostridium thermocellum cellulosome by direct ligation

The homologous recombinant plasmid pHK-HR-epn was constructed by using the gene encoding the swollenin Epn (SEQ ID NO:2) as the target sequence and selecting the 5' end of the horse protein SdbA of Clostridium thermocellum cellulosome (encoded by the nucleic acid sequence 1108113 to 1109912 in genomic CP002416.1) as the targeting knock-in site. The upstream arm HR-up is the nucleic acid sequence 1107610 to 1108109 in the C.thermocellum DSM1313 genome (sequence number CP002416.1 in NCBI database), the downstream arm HR-down is the nucleic acid sequence 1109916 to 1110415 in the DSM1313 genome, and the intermediate arm HR-short is the nucleic acid sequence 1107809 to 1108109 in the DSM1313 genome.

The xylanase XynA (SEQ ID NO:1) is directly linked to the sequence of the type II adhesion module CohII (SEQ ID NO:7) by the overlap extension polymerase chain reaction method, wherein the sequence of CohII is linked to the 3' end of the XynA sequence, thereby obtaining the XynA-CohII sequence. The XynA-CohII sequence is used as a target sequence, the MluI and EagI enzyme cutting sites are cloned into the homologous recombinant plasmid pHK-HR, and the lactate dehydrogenase gene clo1313_1878 is used as a target replacement sequence to construct the homologous recombinant plasmid pHK-HR-xynA. The upstream arm HR-up is the nucleic acid sequence 2194853 to 2195353 in the C.thermocellum DSM1313 genome (sequence number CP002416.1 in NCBI database), the downstream arm HR-down is the nucleic acid sequence 2196312 to 2196811 in the DSM1313 genome, and the intermediate arm HR-short is the nucleic acid sequence 2195053 to 2195353 in the DSM1313 genome.

Directly connecting a pectinase PelA (SEQ ID NO:18) encoding gene with a sequence (SEQ ID NO:6) of a type I docking module DocIct of clostridium thermocellum by using an overlap extension polymerase chain reaction method, wherein the sequence of the DocIct is connected to the 3' end of the PelA sequence to obtain a PelA-DocIct target sequence. And cloning the connected recombinant sequence serving as a target sequence to an expression plasmid pHK by utilizing BamHI and XbaI enzyme cutting sites to obtain the expression plasmid pHK-Pcel S-PelA-DocIct. The pHK carries the promoter and signal peptide sequence (SEQ ID NO:8) of the cellulase Cel48S derived from Clostridium thermocellum, so that the expressed target gene can be secreted to the outside of the cell.

The homologous recombinant plasmid pHK-HR-xynA was transformed into the recombinant strain 1 constructed in example 3, and the homologous recombinant strain 3 was obtained according to the screening method described in example 1. The homologous recombinant plasmid pHK-HR-epn was transformed into recombinant strain 3, and homologous recombinant strain 4 was obtained in the same manner as in the screening method described in example 1. Finally, plasmid pHK-Pcel S-PelA-DocIct was transformed into recombinant strain 4, thereby obtaining recombinant strain 5 of Clostridium thermocellum simultaneously expressing XynA with type II adhesion module, Cel9-48 and PelA with type I docking module and fusion protein Epn-SdbA.

By extracting the cellulosome of the recombinant strain, the expressed xylanase XynA, cellulose exonuclease Cel9-48 and pectinase PelA are combined on the cellulosome of the clostridium thermocellum in a direct connection mode, and the expansion factor Epn can be secreted and assembled into a cellulosome complex through the fusion expression with a foot stool protein SdbA. The recombinant strain 5 is cultured to the middle logarithmic phase in GS-2 culture medium with 5g per liter of cellulose or cellobiose as a carbon source, and can be used as a whole bacterial enzyme preparation for biological saccharification of lignocellulose.

Example 5: construction of a cellulase preparation based on Clostridium thermocellum cellulosome by direct ligation

The recombinant strain 5 constructed in example 4 was cultured in GS-2 medium with 5g per liter of cellulose as carbon source to late log phase or early plateau phase, and then the precipitated cells were removed by low speed centrifugation, and the supernatant could be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 6: construction of cellulase preparations based on C.flavum cellulosomes by means of direct ligation

The sequence of Cel48S-DocIcCl was obtained by ligating the cellulose exonuclease Cel48S to the type I docking module sequence of C.cellulolyticum DocIcCl (SEQ ID NO:9) by the overlap extension polymerase chain reaction method, wherein the sequence of DocIcIcCl was ligated to the 3' end of the sequence of Cel 48S. Then the recombined sequences which are connected are used as target sequences to be cloned on an expression plasmid pHK by utilizing BamHI and XbaI enzyme cutting sites. The constructed plasmid was transformed into Clostridium flavum (Clostridium clariflavum DSM 19732) to obtain an expressed recombinant strain Cel48S with DocIccl. By extracting the recombinant strain's cellulosomes, it was found that the expressed Cel48S with DocIccl can be secreted extracellularly and assembled into the yellow clostridium cellulosome complex. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 7: construction of cellulase preparations based on the cellulosomes of Ruminococcus albus by direct ligation

Different from example 6, the cellulose exonuclease Cel48S and Ruminococcus albus type I docking module sequence DocIra (SEQ ID NO:10) were linked to obtain the Cel48S-DocIra sequence. The constructed plasmid is transformed into rumen coccus albus SY3 to obtain the recombinant strain of Cel48S with DocIra. By extracting the recombinant strain's cellulosomes, it was found that the expressed Cel48S with DocIra was extracellularly secreted and assembled into ruminococcus albus-cellulosome complexes. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 8: construction of cellulase preparations based on cellulosomes of Ruminococcus xanthus by direct ligation

Different from example 6, the cellulose exonuclease Cel48S and Ruminococcus xanthus I docking module sequence DocIrf (SEQ ID NO:11) were linked to obtain the Cel48S-DocIrf sequence. The constructed plasmid is transformed into rumen luteinizing coccus (Ruminococcus flavefaciens) to obtain an expressed recombinant strain of Cel48S with DocIrf. By extracting the recombinant strain's cellulosomes, it was found that the expressed Cel48S with DocIrf can be secreted extracellularly and assembled into the ruminococcus xanthans cellulosome complex. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 9: construction of cellulase preparations based on the cellulosome of Pseudomonas cellulolyticus by direct ligation

In contrast to example 6, the sequence Cel48S-DocIpc was obtained by ligating the cellulose exonuclease Cel48S with the type I docking module sequence DocIpc (SEQ ID NO:13) of Pseudobacteroides cellulolyticus. The constructed plasmid was transformed into Pseudobacteroides cellulolyticus (Pseudomonas cellulosolvalensis DSM 2933) to obtain a recombinant strain of Cel48S with DocPpc expressed. By extracting the recombinant strain's cellulosome, it was found that the expressed Cel48S with DocIpc could be secreted extracellularly and assembled into a bacteroides cellulosome complex. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 10: construction of cellulase preparations based on C.cellulophilus cellulosomes by direct ligation

The 5' end of the cellulase Clocel _2823 (encoded by the 3464080 to 3466140 nucleic acid sequence in genomic CP002160.1) of Clostridium cellulophilus cellulosome was selected as the targeting knock-in site, using the gene encoding the protease ProL (SEQ ID NO:3) as the target sequence. The enzyme cutting sites of MluI and EagI are cloned into the homologous recombinant plasmid pHK-HR to construct the homologous recombinant plasmid pHK-HR-ProL. Homology arms HR-up, HR-down and HR-short are the 3466141 to 3466640, 3465641 to 3466140 and 3466141 to 3466441 nucleic acid sequences, respectively, in the genome of Clostridium cellulovorans 743B (sequence number CP002160.1 in the NCBI database). The constructed plasmids are respectively transformed into pyrF deleted 743B mutant strains, and homologous recombinant bacteria expressed by fusion of protease ProL and cellulase Clocel _2823 are obtained by screening according to the method of the embodiment 1. By extracting the recombinant strain's cellulosome, it was found that the fusion protein can be secreted extracellularly and assembled into a cellulosome complex. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 11: construction of cellulase preparations based on the cellulosomes of Clostridium cellulolyticum by direct ligation

In contrast to example 10, the gene encoding amylase AmyA (SEQ ID NO:17) was used as the target sequence, and the 5' end of cellulase Ccel _0729 (encoded by the nucleic acid sequence 843122 to 845197 in genomic CP 001348.1) of C.cellulolyticum cellulosome was selected as the targeted knock-in site. Homology arms HR-up, HR-down and HR-short are nucleic acid sequences 842441 to 842941, 842942 to 843441 and 842641 to 842941, respectively, in the genome of Clostridium cellulolyticum H10 (SEQ ID NO: NC-011898 in the NCBI database). And respectively transforming the constructed plasmids into pyrF-deleted H10 mutant strains, and screening to obtain homologous recombinant bacteria expressed by fusion of amylase AmyA and cellulase Ccel _ 0729. By extracting the recombinant strain's cellulosome, it was found that the fusion protein can be secreted extracellularly and assembled into a cellulosome complex. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 12: construction of cellulase preparations based on the cellulosome of Vibrio cellulolyticus by direct ligation

In contrast to example 10, the gene encoding pectinase PelA (SEQ ID NO:18) was ligated to the type I docking module sequence DocIac (SEQ ID NO:14) of Vibrio cellulolyticus to obtain a PelA-DocIac sequence. The constructed plasmid was transformed into Vibrio cellulolyticus (Acetivibrio cellulolyticus). By extracting the recombinant strain cellulosome, it was found that the expressed PelA can be secreted extracellularly and assembled into the vibrio cellulosome complex of vibrio cellulolyticus by the possessed DocIac. The strain is cultured in a GS-2 culture medium taking 5g per liter of cellulose as a carbon source to the late logarithmic phase or the early plateau phase, then sediment cells are removed through low-speed centrifugation, and supernatant can be used as a cellulosome enzyme preparation for biological saccharification of lignocellulose.

Example 13: preparation of squalene from lignocellulose

(1) Pretreatment: pretreating corn straws according to a sulfonation method adopted in the literature (Chinese paper making, 2015,34,1-6) to obtain a lignocellulose substrate with the lignin content of not higher than 11% and the hemicellulose content of not higher than 10%;

(2) saccharification: 1kg of the lignocellulosic substrate obtained in the step (1) was transferred to 6L of the medium in an anaerobic fermentation tank in a solid-liquid weight-to-volume ratio of 1:6, and then the cellulase preparation prepared in example 1 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 55 ℃ to obtain a glucose-containing sugar solution. The saccharification culture medium comprises: 2.9g/L of dipotassium phosphate, 1.5g/L of monopotassium phosphate, 0.8g/L of urea, 0.1g/L of calcium chloride, 1.8g/L of magnesium chloride, 0.0005g/L of ferrous sulfate, 2g/L of sodium sulfide, 4g/L of corn steep liquor and 2g/L, pH 6.5.5-7.5 of trisodium citrate. During saccharification, the pH can be controlled to be 5.8-6.2 by feeding sodium hydroxide.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 70-80g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed liquid, fermenting for 5 days under the temperature condition of 20 ℃ and the pH condition of 7.0, feeding 50% (w/v) of corn steep liquor solution for nitrogen supplement, and ending the fermentation when the glucose concentration is not higher than 5 g/L.

The fermentation medium consists of the following components: 0.6% of corn steep liquor, 0.2% of peptone, 0.8% of potassium dihydrogen phosphate, 0.5% of magnesium sulfate, 3% of sea crystal, 10.02% of vitamin B, 60.02% of vitamin B, 120.005% of vitamin B and 0.005% of biotin. The balance being water, pH 7.0.

(4) Extracting squalene: centrifuging the fermentation liquor obtained in the step (3), and performing solid-liquid separation on the saccharomycete-like thallus cells and the fermented culture medium; and (3) after the obtained saccharomycete-like cell is subjected to enzymolysis and wall breaking, extracting a crude squalene product by using an organic solvent extraction method, and distilling to obtain the refined squalene.

(5) Reusing a culture medium: and (3) directly using the culture medium obtained after the solid-liquid separation in the step (4) without dilution to prepare the saccharification culture medium in the step (2).

Example 14: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating wheat straws according to a hydrothermal and sulfonation combined pretreatment method adopted in CN201610133959 to obtain a lignocellulose substrate with the lignin content of not more than 8% and the hemicellulose content of not more than 8%;

(2) saccharification: 100kg of the lignocellulosic substrate obtained in the step (1) was transferred to 350L of the medium in an anaerobic fermentation tank in a solid-liquid weight-to-volume ratio of 1:3.5, and then the cellulase preparation prepared in example 2 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 60 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 140-150g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at the outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 28 deg.C and pH of 7.0 for 7 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor for nitrogen supplement.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 3 times, and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 15: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating shrub branches according to an alkaline pretreatment technology in a document (Bin Li, et al. registration progress on the pretreatment and fractionation for Biorefinery at QIBEBT. journal of biosources and Bioproducts,2017,2(1),4-9) to obtain a lignocellulose substrate with the lignin content of not more than 15% and the hemicellulose content of not more than 7.5%;

(2) saccharification: 100kg of the lignocellulosic substrate obtained in the step (1) was transferred to 800L of a medium in an anaerobic fermentation tank at a solid-liquid weight-to-volume ratio of 1:8, and then the cellulase preparation prepared in example 3 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 60 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 90-110g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 17 deg.C and pH of 7.0 for 8 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor to supplement nitrogen.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 2 times, and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 16: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating the wood chips according to a pretreatment technology combining an alkaline method and hydrothermal method in the literature (Biotechnology for Biofuels, 2014, 7:116) to obtain a lignocellulose substrate with the lignin content of not higher than 11% and the hemicellulose content of not higher than 10%;

(2) saccharification: 100kg of the lignocellulosic substrate obtained in the step (1) was transferred to 1000L of the medium in an anaerobic fermenter at a solid-liquid weight-to-volume ratio of 1:10, and then the cellulase preparation prepared in example 4 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 60 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 60-70g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed liquid, fermenting for 3 days under the conditions of 37 ℃ and 6.0 pH, feeding 50% (w/v) corn steep liquor solution for nitrogen supplement, and ending the fermentation when the glucose concentration is not higher than 5 g/L.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 2 times, and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 17: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating straws according to a steam explosion pretreatment technology in a document (cellulose science and technology, 2002, 3, 47-52) to obtain a lignocellulose substrate with the lignin content of not higher than 20 percent and the hemicellulose content of not higher than 14.5 percent;

(2) saccharification: 1kg of the lignocellulosic substrate obtained in the step (1) was transferred to 5L of the medium in an anaerobic fermenter at a solid-liquid weight-to-volume ratio of 1:5, and then the cellulase preparation prepared in example 5 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 58 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 50-60g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 15 deg.C and pH of 8.0 for 7 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor to supplement nitrogen.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 2 times, and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 18: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating waste paper according to hydrothermal pretreatment Technology in literature (Bioresource Technology, 2004, 91, 93-100) to obtain lignocellulose substrate with lignin content of no more than 7% and hemicellulose content of no more than 11%;

(2) saccharification: 1kg of the lignocellulosic substrate obtained in the step (1) was transferred to 2L of the medium in an anaerobic fermenter at a solid-liquid weight-to-volume ratio of 1:2, and then the cellulase preparation prepared in example 6 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 37 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 160-180g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 20 deg.C and pH of 9.0 for 9 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor for nitrogen supplement.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 3 times, and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 19: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating wheat straws according to the pretreatment technology of the embodiment 13 to obtain a lignocellulose substrate with the lignin content of not higher than 8% and the hemicellulose content of not higher than 11%;

(2) saccharification: 0.2kg of the lignocellulosic substrate obtained in the step (1) was transferred to 3L of the medium in an anaerobic fermenter at a solid-liquid weight-to-volume ratio of 1:15, and then the cellulase preparation prepared in example 7 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 37 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 30-50g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 25 deg.C and pH of 4.0 for 5 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor to supplement nitrogen.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: and (3) directly using the culture medium obtained after the solid-liquid separation in the step (4) without dilution to prepare the saccharification culture medium in the step (2).

Example 20: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating wheat straws according to the pretreatment technology of the embodiment 13 to obtain a lignocellulose substrate with the lignin content of not higher than 8% and the hemicellulose content of not higher than 11%;

(2) saccharification: 0.2kg of the lignocellulosic substrate obtained in the step (1) was transferred to 2L of the medium in an anaerobic fermentation tank in a solid-liquid weight-to-volume ratio of 1:10, and then the cellulase preparation prepared in example 8 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 65 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 40-60g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 30 deg.C and pH of 6.5 for 6 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor for nitrogen supplement.

(4) Extracting squalene: same as in example 13

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 1 time and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 21: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: carrying out pretreatment on wheat straws according to the pretreatment technology in the embodiment 14 to obtain a lignocellulose substrate with the lignin content of not higher than 8% and the hemicellulose content of not higher than 8%;

(2) saccharification: 1kg of the lignocellulosic substrate obtained in the step (1) was transferred to 4.5L of the medium in an anaerobic fermenter at a solid-liquid weight-to-volume ratio of 1:4.5, and then the cellulase preparation prepared in example 9 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 34 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 110-; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 35 deg.C and pH of 6.5 for 10 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor for nitrogen supplement.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 1 time and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 22: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating corncobs according to a dilute acid hydrolysis pretreatment technology in a literature (bioprocessing process, 2010, 3, 66-72) to obtain a lignocellulose substrate with the lignin content of not higher than 9% and the hemicellulose content of not higher than 18%;

(2) saccharification: 0.4kg of the lignocellulosic substrate obtained in the step (1) was transferred to 2L of the medium in an anaerobic fermentation tank in a solid-liquid weight-to-volume ratio of 1:5, and then the cellulase preparation prepared in example 10 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 42 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 60-80g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 22 deg.C and pH of 6.5 for 7 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor for nitrogen supplement.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 1 time and then using the diluted culture medium to prepare the saccharification culture medium in the step (2).

Example 23: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating the waste paper according to the pretreatment technology of the embodiment 17 to obtain a lignocellulose substrate with the lignin content of not more than 7% and the hemicellulose content of not more than 9%;

(2) saccharification: 0.2kg of the lignocellulosic substrate obtained in the step (1) was transferred to 5L of the medium in an anaerobic fermenter at a solid-liquid weight-to-volume ratio of 1:25, and then the cellulase preparation prepared in example 11 was added to the lignocellulosic substrate to conduct hydrolysis reaction at a temperature of 40 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 25-35g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed solution, fermenting at 15 deg.C and pH of 6.5 for 5 days, continuously adding sugar solution obtained in step (2) of example 14 to maintain sugar concentration at 5-10g/L, and adding 50% (w/v) corn steep liquor for nitrogen supplement.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: and (3) directly using the culture medium obtained after the solid-liquid separation in the step (4) without dilution to prepare the saccharification culture medium in the step (2).

Example 24: preparation of squalene from lignocellulose

In contrast to the embodiment 13, in this case,

(1) pretreatment: pretreating the corncobs according to the pretreatment technology of the embodiment 17 to obtain a lignocellulose substrate with the lignin content of not higher than 5% and the hemicellulose content of not higher than 25%;

(2) saccharification: 0.2kg of the lignocellulose substrate obtained in the step (1) was transferred to 0.6L of a medium in an anaerobic fermentation tank in a solid-liquid weight-to-volume ratio of 1:3, and the cellulase preparation prepared in example 12 was added to the lignocellulose substrate to conduct hydrolysis reaction at a temperature of 40 ℃ to obtain a glucose-containing sugar solution.

(3) Fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 100-120g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed liquid, fermenting for 2 days under the temperature condition of 18 ℃ and the pH condition of 6.5, feeding 50% (w/v) corn steep liquor solution for nitrogen supplement, and ending the fermentation when the glucose concentration is not higher than 5 g/L.

(4) Extracting squalene: same as in example 13.

(5) Reusing a culture medium: diluting the culture medium obtained after the solid-liquid separation in the step (4) by 2 times, and then directly using the diluted culture medium to prepare the saccharification culture medium in the step (2).

TABLE 1 tabulated results for examples 13-24 preparation of squalene using lignocellulose

As can be seen from Table 1, in the methods for producing squalene using lignocellulose in examples 13 to 24, the cellulose saccharification rate is 80.10 to 90.80%, the biomass of the yeast is 33.72 to 178.68g/L, and the squalene accounts for 5.2 to 9.0% of the dry weight of the cells; the yield of squalene is 2.91-12.98g/L, the purity of squalene is 93.1-95.0%, and the saccharification rate of cellulose after the culture medium is reused is 80.90-91.5%. The method for preparing squalene by adopting lignocellulose has the advantages of wide raw material source and low cost, and realizes high yield and yield of squalene by combining lignocellulose biomass saccharification and yeast-like high-density fermentation.

In addition, compared with the cellulose saccharification rate of the first round, the cellulose saccharification rate after the culture medium is reused is almost the same, which shows that in the process of the invention, the culture medium and the fermentation culture medium in the lignocellulose saccharification stage can be recycled, so that water and chemicals can be obviously saved, and the process has the obvious effects of reducing wastewater discharge and reducing cost. Compared with the existing chemical production technology, the process is mild and simple, and compared with the existing fermentation production technology, the production level of squalene is higher.

Sequence listing

<110> Kingsheng Biotech Co., Ltd in Qingdao

<120> method for preparing squalene by using lignocellulose

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 212

<212> PRT

<213> cellulose-pyrolyzing strain of fruit juice bacillus (Caldicellulosisruptor 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> Clostridium yellow (Clostridium clarifiavum)

<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> Proteobacteria (Coprotobacter proteoliticus)

<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> Clostridium yellow (Clostridium clarifiavum)

<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> Ruminococcus flavefaciens (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> 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> Pseudobacteroides cellulolyticus (Pseudomonas cellulosolvans)

<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> Vibrio acetobacter 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 (5)

1. The method for preparing squalene by adopting lignocellulose is characterized by comprising the following steps: the method comprises the following steps:

(1) pretreatment: pretreating a lignocellulose raw material to obtain a lignocellulose substrate with the lignin content of not higher than 20% and the hemicellulose content of not higher than 25%;

(2) saccharification: transferring the lignocellulose substrate obtained in the step (1) into a saccharification culture medium of an anaerobic fermentation tank according to the solid-liquid weight-volume ratio of 1:2-1:25, adding a cellulase preparation into the lignocellulose substrate, and performing hydrolysis reaction at the temperature of 34-65 ℃ to obtain a sugar solution containing glucose; the cellulase preparation is obtained by combining non-cellulosome protein in a cellulosome complex through interaction of the non-cellulosome protein and components in the cellulosome; the cellulosome is a multienzyme complex with lignocellulose degrading activity produced by clostridium thermocellum and secreted extracellularly;

wherein the non-fibrosome protein is specifically: cellulose exonuclease Cel9-48 encoded by nucleic acid sequence 1968724 to 1973904 in genome CP001393.1, bulking factor Epn encoded by SEQ ID NO 2, xylanase XynA encoded by SEQ ID NO 1 and pectinase PelA encoded by nucleic acid sequence 2531445 to 2532785 in genome CP 001393.1;

wherein the 3' end of the cellulose exonuclease Cel9-48 is directly connected with the sequence SEQ ID NO. 6 of the I-type docking module DocIct of the clostridium thermocellum; the expansion factor Epn is expressed by fusion with the 5' end of a foot stool protein SdbA, and the sequence of the foot stool protein SdbA is encoded by 1108113 to 1109912 nucleic acid sequences in a genome CP 002416.1; the 3' end of the xylanase XynA is directly connected with a sequence SEQ ID NO. 7 of a II type adhesion module CohII; the 3' end of the pectinase PelA is directly connected with the sequence SEQ ID NO of the I-type docking module DocIct of the clostridium thermocellum;

(3) fermenting yeast-like yeast: when the concentration of glucose in the sugar solution obtained in the step (2) reaches 25-180g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; adding culture medium components into a bioreactor to obtain a fermentation culture medium, and sterilizing at high temperature and high pressure; then inoculating activated yeast-like yeast seed liquid, fermenting for 2-5 days at 15-37 deg.C and pH of 4.0-9.0 until the glucose concentration is not higher than 5 g/L; or continuously feeding the sugar solution obtained in step (2) in the fermentation process to maintain the sugar concentration at 5-10g/L, and fermenting at 15-37 deg.C under nitrogen supplementing condition and pH of 4.0-9.0 for 5-10 days to obtain fermentation liquid;

(4) extracting squalene: centrifuging the fermentation liquor obtained in the step (3), and performing solid-liquid separation on the saccharomycete-like thallus cells and the fermented culture medium; and (3) after the obtained saccharomycete-like cell is subjected to enzymolysis and wall breaking, extracting a crude squalene product by using an organic solvent extraction method, and distilling to obtain the refined squalene.

2. The method for preparing squalene according to claim 1, wherein: further comprising the step (5), specifically: and (3) diluting the culture medium obtained after the solid-liquid separation in the step (4) without dilution or by 1-3 times, and preparing the saccharification culture medium in the step (2).

3. The method for preparing squalene according to claim 2, wherein: the saccharification culture medium in the step (2) is as follows: 2.9g/L dipotassium phosphate, 1.5g/L monopotassium phosphate, 0.8g/L urea, 0.1g/L calcium chloride, 1.8g/L magnesium chloride, 0.0005g/L ferrous sulfate, 2g/L sodium sulfide, 4g/L corn steep liquor and 2g/L, pH 6.5.5-7.5 trisodium citrate; the fermentation medium in the step (3) consists of the following components: corn steep liquor 0.6%, peptone 0.2%, potassium dihydrogen phosphate 0.8%, magnesium sulfate 0.5%, sea crystal 3%, vitamin B10.02%, vitamin B60.02%, vitamin B120.005%, biotin 0.005%, and water in balance, and pH 7.0.

4. The method for preparing squalene according to claim 2, wherein: the lignocellulose raw material in the step (1) is one or a combination of a plurality of corn stalks, wheat straws, shrub branches, wood chips, corncobs, straws and waste paper; the pretreatment is one or a combination of more of alkaline method, dilute acid method, hydrothermal method, steam explosion method and sulfonation method pretreatment technologies; the pretreated lignocellulose substrate has the lignin content of not higher than 11 percent and the hemicellulose content of not higher than 12 percent; the temperature condition in the saccharification step in the step (2) is 55-60 ℃, and the solid-liquid weight-volume ratio in a hydrolysis system is 1:3-1: 10.

5. The method for preparing squalene according to claim 2, wherein: in the step (3), after the concentration of the glucose is 60-120g/L, enabling the sugar solution to enter a bioreactor through a filtering component assembled at an outlet of a fermentation tank; the nitrogen supplementing operation specifically comprises the following steps: 8% (w/v) ammonium sulfate solution is added in a flowing way to supplement nitrogen; the organic solvent extraction method in the step (4) specifically comprises the following steps: extracting with organic solvent by using mixed solution of ethanol and n-hexane.