TWI598440B - Gene, composition and method for improving lignocelluloses hydrolyzation - Google Patents

Description

Gene, composition and method for promoting hydrolysis of lignocellulose

The present invention relates to a gene encoding an endo-type lignocellulolytic enzyme I, and more particularly to a gene capable of promoting hydrolysis of lignocellulose.

Lignocellulosic is the world's most abundant carbohydrate, which is mainly composed of glucose covalently bonded by β(1-4) glycosidic bonds. The annual amount of lignocellulosic production produced by the earth's photosynthesis is about 10 ¹¹ metric tons (Delmer and Amor, 1995). However, with the exception of a few microbes, many organisms cannot directly use lignocellulosic energy as an energy source, thus forming environmental waste. A small number of microorganisms have the ability to produce fuels for humans. For example, brewer's yeast can ferment alcohol to replace some of the gasoline; Clostridium and other strains have the ability to ferment to produce butanol or hydrogen (Qureshi and Blaschek, 1999); oil-producing microalgae and yeast can also produce oils for the production of biodiesel (Li, et al., 2008). Although these can be one of the choices for supplying human energy now or in the future, these microorganisms must be converted into energy sources by using simple sugars such as glucose and xylose as carbon sources. Therefore, the provision of rich fermentable sugars will become an important key technology for future energy development and production.

Traditionally, the method of decomposing lignocellulosic material has been carried out by means of strain screening. Lignocellulolytic enzymes are a group of hydrolyzed enzymes that break down undissolved lignocellulose into monosaccharides (Wood, 1985). These hydrolyzed enzymes are classified according to the target, and can be classified into cellulose and hemicellulose. It is known that when lignocellulose is hydrolyzed and saccharified by an enzyme, it is treated with intact lignocellulolytic enzyme, that is, an endo-type lignocellulolytic enzyme (endoglucanase) or an exo-cellulolytic enzyme (exoglucanase or Cellobio-hydrolase, β-glucosidase and xylanase are the four enzymes that can effectively break down cellulose into the final product. However, the current cost of decomposing cellulose hydrolyzing enzymes is much higher than the cost of α-amylase and glucoamylase used in the alcohol industry. This is the most difficult cost to control lignocellulose to alcohol, and it is currently the commercial mass production of this technology. The biggest bottleneck.

Endo-type lignocellulolytic enzyme I of Trichoderma reesei , which is classified as EC 3.2.1.4, and the nucleic acid sequence of the enzyme is published by Penttilae et al. (1986). Arsdell et al. (1987), Aho (1991) and Qin (2008) and other three groups of the selected Trichoderma endo-type lignocellulolytic enzyme I genes were expressed in the Saccharomyces cerevisjae system; Akcapinar et al. reconstituted the Trichoderma endo-type lignocellulolytic enzyme I gene sequence and sent it to the Pichia pastoris system to produce the enzyme. The Republic of China Patent Publication No. I391489 discloses a technique for describing a fibrinolytic enzyme and a recombinant production thereof, and describes a novel fibrillar polypeptide, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide. And the host cell is patented. The Republic of China Announcement Patent Publication No. I386487 discloses an industrial value of industrial application of high heat-resistant cellulase by modifying the gene to improve the activity of the enzyme protein and relatively reducing the process cost. The Republic of China Patent Publication No. I391489 discloses a novel cellulase isolated from the fungus Pilomyces rhizinflata . So far, no one has proposed to optimize the endophytic lignocellulolytic enzyme I gene sequence and transfer it to the Yarrowia lipolytica system, and to transform the transport plastid into double A set of related techniques for endo-type lignocellulolytic enzyme I expression vectors.

The present invention provides a nucleic acid molecule comprising a modified nucleotide sequence encoding an endo-type lignocellulolytic enzyme I. In a specific embodiment of the present invention, the modified nucleotide sequence encoding the endo-type lignocellulosic enzyme I is obtained by modifying SEQ ID NO. 1 with the codon preference of the host cell, or SEQ ID NO.1 is obtained by removing introns. In another embodiment of the invention, the host cell is a yeast. Preferably, the yeast is Yarrowia lipolytica .

In another aspect of the invention, the nucleotide sequence of the modified endo-cut lignocellulolytic enzyme I has at least 70% sequence identity to the SEQ ID NO. In a specific embodiment, the modified nucleotide sequence encoding endo-type lignocellulolytic enzyme I is the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

In another aspect, the present invention further provides a performance carrier, A nucleotide sequence comprising a modified endo-type lignocellulolytic enzyme I. According to a specific embodiment, the nucleic acid molecule has at least two modified nucleotide sequences encoding the endo-type lignocellulolytic enzyme I.

In a specific embodiment of the invention, the expression vector further comprises an inducible promoter sequence that controls the expression of the nucleic acid molecule.

In a specific embodiment of the present invention, the expression vector further comprises at least one selected from the group consisting of a marker gene sequence, a reporter gene sequence, an antibiotic resistance gene sequence, a restriction enzyme cleavage position sequence, and polyadenylation Position sequences, enhancer sequences, terminal subsequences, and regulatory subsequences.

In still another aspect, the present invention further provides a host cell for producing a target protein comprising a modified expression vector encoding a nucleic acid molecule of endo-type lignocellulolytic enzyme I.

In a specific embodiment, the host cell line is deposited under the name CPC-2EGI T5 at the Center for Biological Resource Conservation and Research of the Food Industry Development Institute under accession number BCRC 920087. This biomaterial has been tested and passed the viability test.

In another embodiment of the invention, the host cell line is selected from at least one of the group consisting of a virus, a bacterial cell, a fungal cell, an algal cell, a plant cell, and an animal cell. According to a specific embodiment, the fungal cell line is a yeast. In a preferred embodiment, the yeast strain Yarrowia lipolytia .

The present invention further provides an enzyme composition comprising the target protein, wherein the target protein is an endo-type lignocellulolytic enzyme I.

In a specific embodiment of the present invention, the enzyme composition further comprises one or more enzymes selected from the group consisting of exo-type lignocellulolytic enzymes, β-glucosidase, and semi-fibrolytic enzymes.

The present invention provides a method for promoting hydrolysis of lignocellulose by treating the lignocellulose with the above-described expression carrier, host cell or an enzyme composition comprising the endo-type lignocellulolytic enzyme I.

The nucleic acid molecule, the expression carrier and the host cell of the invention can increase the yield of the endo-type lignocellulolytic enzyme I, and are beneficial to promote the hydrolysis of lignocellulose, and are suitable for fermentation to produce biomass fuel such as raw alcohol or butanol or high. Value biochemicals and effectively save production costs.

Figure 1 shows a schematic diagram of the nucleotide sequence (eg1) and pYLSC1 plastid encoding the endo-type lignocellulolytic enzyme I from Trichoderma reesei RUT-C30; wherein, NotI , SfiI and HindIII are Restriction enzyme cleavage site; ori is the replication initiation region; hp4d is the promoter; LEU2 is the marker gene; AP is the antibiotic resistance gene.

Figure 2 shows the endo-type lignocellulolytic enzyme I Congo red activity staining of transformant 1296-eg1/Po1g T1 to T10; wherein pYLSC1 is a transformant of untransformed eg1 gene as a negative control group The standard is a transformant containing an endo-type fibrinolytic enzyme gene as a positive control group.

Figure 3 is a comparison of the nucleotide sequence of the endo-type lignocellulolytic enzyme I encoded by Trichoderma reesei RUT-C30 and its codon-preferred nucleotide sequence; wherein eg1 opt is Codon preference The nucleotide sequence of the SEQ ID NO. 3; eg1 tre is the nucleotide sequence encoding the endo-type lignocellulolytic enzyme I, SEQ ID NO.

Figure 4 is a schematic diagram showing the nucleotide sequence (eg1opt) and pYLSC1 plastid junction of the endo-type lignocellulolytic enzyme I encoding Trichoderma reesei RUT-C30 modified by codon preference; wherein, NotI , SfiI and HindIII is a restriction enzyme cleavage site; ori is a replication initiation region; hp4d is a promoter; LEU2 is a marker gene; and AP is an antibiotic resistance gene.

Figure 5 shows the endo-type lignocellulolytic enzyme I Congo red staining of the transformant 1296-eg1 opt/Po1g T1 to T10; wherein pYLSC1 is a transformant of the untransformed eg1 gene as a negative control group; The product is a transformant containing an endo-type fibrinolytic enzyme gene as a positive control group.

Fig. 6 is a graph showing the enzyme activity and biomass of the endo-type lignocellulolytic enzyme I of the transformant 1296-eg1/Po1g T1 to T10.

Fig. 7 is a graph showing the enzyme activity and biomass of the endo-type lignocellulolytic enzyme I of the transformant 1296-eg1 opt/Po1g T1 to T10.

Figure 8 is a flow chart showing the construction of a two-set gene plastid (ISLT-tc) encoding a nucleotide sequence (EG1 opt) encoding an endo-type lignocellulolytic enzyme I modified by a codon preference; HindIII and XbaI are restriction enzymes; ori is the replication initiation region; hp4d is the promoter; LEU2 is the marker gene; and AP is the antibiotic resistance gene.

Figure 9 shows the restriction enzyme confirmation electrophoresis pattern of ISLT plastids; among them, HindIII1, AvrII and KpnI are restriction enzymes; -1, -3 and -6 are transformations. number.

Figure 10 shows the activity of the endo-type lignocellulolytic enzyme I Congo red in the two sets of transformants 1296-ISLT-tc/Po1g T1 to T10; wherein S is a transformant containing the endo-type fibrolytic enzyme gene Standard; EG1-opt is a single set of transforms 1296-eg1 opt/Po1g T6.

The present invention utilizes a selection and genetic engineering technique to obtain a nucleic acid molecule encoding an endo-type lignocellulolytic enzyme I having high enzyme activity, the nucleic acid molecule comprising one or more modified endo-type lignocellulose-decomposed The nucleotide sequence of the enzyme I. The present invention remodifies the nucleotide sequence of the nucleic acid molecule to construct an expression vector containing the nucleic acid molecule, and delivers the expression vector into the host cell to express the endo-type lignocellulolytic enzyme I. The expression system of the endo-type lignocellulolytic enzyme I provided by the present invention does not require an external inducer, and the target protein can be produced when the host cell grows for a stable growth period. In addition, the performance system provided by the present invention may comprise more than one modified endo-type lignocellulolytic enzyme I gene, which can increase the yield of endo-type lignocellulolytic enzyme I through the performance of multiple sets.

The nucleic acid molecule provided by the present invention, the expression vector containing the nucleic acid molecule, and the host cell containing the expression vector can effectively increase the yield of the endo-cut lignocellulosic enzyme I, and contain the incision type represented by the same The enzyme composition of lignocellulolytic enzyme I can further promote the hydrolysis of lignocellulose, save the process cost of hydrolyzed lignocellulose, and is beneficial to commercial mass production.

The nucleotide sequence of the modified endo-type lignocellulolytic enzyme I used in the present invention is at least 70% identical to the nucleotide sequence of the unmodified endo-type lignocellulolytic enzyme I. And the modification does not substantially affect the activity of the endo-type lignocellulolytic enzyme I. According to a preferred embodiment of the invention, the modified nucleotide sequence encoding the endo-type lignocellulolytic enzyme I can be SEQ ID NO: 2. The modified nucleotide sequence encoding the endo-type lignocellulolytic enzyme I used in the present invention can be obtained by modifying the codon preference of the host cell. According to another preferred embodiment of the present invention, the nucleotide sequence encoding the endo-type lignocellulolytic enzyme I is modified with the codon usage of Yarrowia lipolytica, which is modified The sequence is SEQ ID NO:3.

As used herein, "expression vector" refers to a recombinant expression system that expresses a particular target protein in a host cell. The expression vector includes, in addition to the region of the gene encoding the target protein, a regulatory region, such as a promoter sequence, a terminal subsequence, a regulator sequence, an enhancer sequence, a replication origin, a marker Gene sequences, reporter gene sequences, antibiotic resistance gene sequences, restriction enzyme cleavage position sequences, and polyadenylation position sequences, and the like.

As used herein, "promoter" refers to a DNA sequence that is typically located in front of a gene and provides a location for initiating transcription of the gene to produce mRNA. Promoters can be divided into three classes depending on the mode of action and function: constitutive promoters, inducible promoters, and tissue-specific promoters. (tissue-specific promoter). The promoter suitable for use in the present invention is not particularly limited as long as it can drive the endo-type lignocellulolytic enzyme I to be expressed in a host cell. According to a preferred embodiment of the invention, the promoter is an inducible promoter.

The expression vector provided by the present invention may have a plurality of modified nucleic acid molecules encoding the endo-type lignocellulolytic enzyme I. That is, the nucleic acid molecule has at least two modified nucleotide sequences encoding the endo-type lignocellulolytic enzyme I.

As used herein, "target protein" refers to a protein of interest that is primarily isolated, purified, or analyzed.

The host cell into which the modified nucleic acid molecule encoding the endo-type lignocellulolytic enzyme I (which may be inserted into the expression vector) is introduced is not particularly limited, and for example, a virus, a bacterial cell, a fungal cell, an algal cell, or a plant can be used. Various hosts such as cells and animal cells. When the modified nucleic acid molecule encoding the endo-type lignocellulolytic enzyme I is introduced into a host cell, the host cell can express a large amount of the endo-type lignocellulolytic enzyme I, which can increase the desired production of the enzyme. According to a preferred embodiment of the present invention, the host cell may be a fungal cell; preferably, the host cell is a yeast; more preferably, the host cell is Yarrowia lipolytica.

The host cell line provided by the present invention is deposited under the name of CPC-2EGI T5 at the Center for Biological Resource Conservation and Research of the Food Industry Development Research Institute under accession number BCRC 920087.

The invention also relates to an enzyme composition comprising the endo-type wood represented by the nucleic acid molecule, expression vector and/or host cell provided by the present invention Cellulolytic enzyme I.

In order to effectively hydrolyze the lignocellulose, the enzyme composition comprising the endo-type lignocellulolytic enzyme I provided by the present invention may further comprise exo-type lignocellulolytic enzyme, β-glucosidase and semi-fibrolytic enzyme. .

The invention also relates to a method of promoting hydrolysis of lignocellulose by treating lignocellulose using an expression vector, host cell or enzyme composition provided herein.

As used herein, "lignocellulosic" means any material comprising lignocellulose, such as plant material, such as wood comprising softwood and hardwood, herbaceous crops, agricultural residues, pulp and feed industry waste, and the like.

As used herein, "cultivation" means a method comprising liquid culture or solid culture, as long as the microorganism selected can be cultured, and is not limited to the culture method thereof.

As used herein, "transformation" refers to the manner in which a nucleic acid molecule is introduced into a host cell. Nucleic acid molecules (e.g., recombinant DNA constructs or recombinant expression vectors) can be introduced into a host cell by a variety of techniques known in the art to which the present invention pertains, such as electroporation, microinjection, Liposome-mediated transfection, transfection of calcium phosphate or calcium chloride media, phage transfection or other methods.

The embodiments of the present invention are provided to exemplify the advantages and technical features of the present invention, and the present invention is not intended to limit the present invention. Any one skilled in the art can, without departing from the spirit and scope of the present invention, Make a variety of changes and retouching, therefore, the scope of protection of the present invention, when viewed The scope defined in the patent application is subject to change.

Example Example 1

The gene of endo-type lignocellulolytic enzyme I was selected from the genome of Trichoderma reesei RUT-C30 (purchased from the Center for Bioresource Conservation and Research, number BCRC 32924) and sent to Yarrowia lipolytica. The strain Po1g (Yeastern Co.) performs recombinant enzyme expression.

First, enter the ExPASy Proteomics workstation (http://tw.expasy.org/enzyme/) and search for the endo-type lignocellulolytic enzyme I gene to find Trichoderma reesei P07981 (Swiss-Prot) . Referring to the endo-type lignocellulolytic enzyme I gene of Trichoderma reesei P07981, it was found that the endo-type lignocellulolytic enzyme I gene (SEQ ID NO.1) of Trichoderma reesei RUT-C30 has three explicit Exons are positions 11 to 780, positions 851 to 1440, and positions 1498 to 1517. The intron of the endo-type lignocellulolytic enzyme I gene of Trichoderma reesei RUT-C30 was removed, and the remaining part was 1380 bp, and the translated protein was 459 amino acids in length.

I. Endo-type lignocellulolytic enzyme I gene selection

1. The appropriate amount of Trichoderma reesei RUT-C30 was scraped from the culture medium, suspended in 0.02 N NaOH (100 μl), and the cells were disrupted by heating at 95 ° C for 10 minutes to obtain genomic DNA.

2. Design a pair of primers according to SEQ ID NO. 1: EGI-1F and EGI-2R (the sequences are shown in Table 1), and then use the polymerase chain reaction (PCR) to separate the 11th to the genomic DNA. 1440 position gene fragment (includes two The exon and an intron are amplified, and the sequence is sent to the yT&A selection vector (Yeastern Co.) by TA colony, thereby obtaining endo-type lignocellulose. Decomposes the plastid yT&A-egi (1-2) of the enzyme I gene fragment.

3. Design two primers according to SEQ ID NO. 1: EGI-2F (containing the designed BsaI restriction enzyme cleavage site) and EGI-1R (including the designed BsaI restriction enzyme cleavage site) (the sequences are as shown in Table 1) Show), and use EGI-1F and EGI-2R to amplify the desired gene fragment from yT&A-egi(1-2) by PCR, thereby obtaining two exon fragments of egi, respectively, eg ) and egi (2). The two sequences were then sent to the yT&A selection vector by TA selection, thereby obtaining two plastids with the egi gene fragment, yT&A-egi(1) and yT&A-egi(2), respectively.

4. Exon fragments exi(1) and egi(2) were excised from plastids yT&A-egi(1) and yT&A-egi(2) with restriction enzymes, and egi(1) and egi(2) After the BsaI cleavage is ligated, the EcoRI and BglII nicks on the yT&A selection vector are introduced, thereby obtaining the plastid yT&A-egi (1+2) with the gene fragment egi(1+2).

5. Since the exon egi(3) of the third segment (positions 1498 to 1517) is only 20 bp, the design contains a part of this 20 bp primer EGI-2.5R (the sequence is shown in Table 1), and uses EGI-1F. Two pairs of primers with EGI-2.5R were used to obtain a PCR product by PCR using yT&A-egi (1+2) as a template. Another design of a 21 bp primer EGI-3R (the sequence of which is shown in Table 1) was used, and PCR was carried out using the PCR product as a template. The PCR product thus obtained was the complete egi (1+2+3) gene. (exon sequence only). Finally will The complete egi (1+2+3) gene fragment was sent to the yT&A selection vector by T-A selection to obtain the plastid yT&A-egi (1+2+3), which was confirmed by sequencing.

6. Analysis of the sequence of egi (1+2+3) found that it contains a SfiI ginsation ggccatcctggcc, which is not conducive to subsequent colonization. Therefore, site-directed mutagenesis is performed by PCR at the 36th position. SfiI cut bit removed (to ggctatcctggcc). This site-directed mutagenesis does not affect the translated protein sequence. The plastid yT&A-egi(1+2+3)-mutant was obtained after mutation.

7. Design two pairs of primers EGI-SfiI-F and EGI-HindIII-R (the sequences are shown in Table 1), and egi(1+2+3)-mutant automorphism yT&A-egi (1+2) The +3)-mutant amplification was carried out again and sent to the yT&A selection vector to obtain the plastid yT&A-egi(1+2+3)-mutant-SfiI-HindIII.

8. The egi(1+2+3)-mutant was sent to the pYLSC1 plastid (Yeastern Co.) of the Y. YEEX expression kit (Yeastern Co.) by SfiI and HindIII cleavage. Thus, the plastid pYLSC1-egi-mutant with the egi(1+2+3)-mutant gene fragment was obtained, and the sequence was confirmed to be correct after sequencing, and the egi(1+2+3)-mutant type The sequence is SEQ ID NO. 2.

9. The constructed plastid pYLSC1-egi-mutant was sent to Y. lipolytica Po1g, and protein expression and protein activity analysis were performed.

II. Construction, transformation and enzyme activity analysis of new plastids

The constructed plastid pYLSC1-egi-mutant obtained by the above experiment was renamed to 1296-eg1, and the first figure shows a schematic diagram of 1296-eg1. The 1296-eg1 was transformed into Y. lipolytica Po1g, and 10 transformants were screened to express the endo-type lignocellulolytic enzyme I. The 10 transformants were named 1296-EG1/Po1g T1 to T10. The supernatant of each transformant was cultured to analyze the activity of endo-type lignocellulolytic enzyme I. During the analysis, the endo-type lignocellulolytic enzyme I secreted by each transformant was reacted with 1% carboxymethylcellulose (CMC) at different reaction temperatures of 40, 50, 60, 70 ° C for 30 min. The reducing sugar content was analyzed by the DNS method (Miller, 1959) to obtain a difference in the amount of reducing sugar.

As shown in Table 2, the enzyme produced by the T1 to 10 transformant obtained the most reducing sugar amount at 60 ° C, indicating that 60 ° C is the optimum temperature for the endo-type lignocellulolytic enzyme I. Among them, the amount of reducing sugar produced by the reaction between the enzyme secreted by T2 and T6 and the substrate was 506.86 and 510.82 μg/mL, respectively, indicating that the enzyme secreted by T2 and T6 was the best.

Each of the transformants was cultured in Mandels-Reese solid medium at 30 ° C for 4 days, and the enzyme production capacity of the transformants T1 to T10 was confirmed by Congo red staining. Fig. 2 shows that both T1 and T10 have endo-type lignocellulolytic enzyme I activity. According to the results of the reduction of the amount of reducing sugar in the negative control group pYLSC1 and the absence of a transparent ring on Mandels-Reese medium, it can be said that the transformants T1 to T10 have successfully colonized the endo-type lignocellulolytic enzyme I gene. And can produce this enzyme.

Example 2

The nucleotide sequence of the selected endo-type lignocellulolytic enzyme I, SEQ ID NO. 2, is modified according to the Yarrowia lipolytica codon translation preference, and the sequence modified by the codon preference is SEQ ID NO .3. Referring to Figure 3, the optimal sequence of SEQ ID NO. 3 differs from the original sequence by 18%. The optimized sequence was constructed on the pYLSC1 plastid. The constructed plastid was named 1296-eg1-opt, and the fourth figure shows the 1296-eg1-op schematic.

The transformation of 1296-eg1-opt into Po1g was carried out, and the 10 transformants successfully transformed were named 1296-eg1-opt/Po1g T1 to 10, and the supernatant of each transformant was cultured for endo-type lignocellulolytic enzyme I activity. analysis. During the analysis, the endo-type lignocellulolytic enzyme I secreted by each transformant was reacted with 1% carboxymethylcellulose for 30 min at different reaction temperatures of 40, 50, 60, 70 ° C, and the amount of reducing sugar was obtained. as shown in Table 3. Table 3 shows that the maximum amount of reducing sugar can be obtained at 60 ° C. The amount of reducing sugar produced by the reaction between the enzyme secreted by T6 and the substrate is 632.22 μg/mL, indicating that the enzyme activity is the best; and T2 and T5 reach 615.07 and 611.11 μg / mL, indicating that its enzyme activity is second best. Further, at 70 ° C, the amount of reducing sugar catalyzed by the enzyme has decreased, so it is presumed that 60 ° C is a temperature suitable for the reaction of the endo-type lignocellulolytic enzyme I with the substrate.

Each of the transformants was cultured in Mandels-Reese solid medium at 30 ° C for 4 days, and the enzyme production capacity of the transformants T1 to T10 was confirmed by Congo red staining. Figure 5 shows that all of the transgenic plants except for T9 have endo-type lignocellulolytic enzyme I activity. In addition, according to the results of the reduction of the amount of reducing sugar in the negative control group pYLSC1 and the absence of a transparent ring on the Mandels-Reese medium, it can be said that T1 to T8 and T10 have successfully colonized the endo-type lignocellulolytic enzyme I. The gene also produces the enzyme.

Example 3

Comparison of the transformation of the endo-type lignocellulolytic enzyme I gene into a transformant 1296-eg1/Po1g T1 to T10 and the transformed endogenous lignocellulolytic enzyme I gene 1296-eg1-opt/ Enzyme production of Po1g T1 to T10.

The body 1296-eg1/Po1g T1 to 10 was re-taken, and after growing at 28 ° C for 4 days in YPD medium, 0.1 ml of the supernatant was taken for activity test. The endo-type lignocellulolytic enzyme I secreted by the transforming body 1296-eg1/Po1g T1 to T10 was reacted with the matrix 1% CMC at 60 ° C for 30 min, and the relationship between the relative absorbance difference and the amount of the obtained cells was as shown in the figure. Figure 6 shows. Figure 6 shows that the enzyme secreted by T1 reacts with the matrix to produce more reducing sugar. When the enzyme activity unit U/mL of the enzyme activity supernatant was expressed, as shown in Table 4, the enzyme activities of the transformants T2 and T6 were 0.93 and 0.94 U/mL, respectively, indicating that the enzyme activities of both were better. However, as shown in Fig. 6, the biomass (biomass) of the T2 cells was 15.03 g/L (dry weight), and the amount of bacteria higher than that of the T6 was 11.52 g/L, indicating that T6 was less than the amount of cells. The enzyme activity close to T2 can be achieved. Therefore, it is speculated that T6 is a more promising transformant, but the growth rate is slower than T2.

The body 1296-eg1-opt/Po1g T1 to T10 was re-taken, and after growing at 28 ° C for 4 days in YPD medium, 0.1 ml of the supernatant was taken for activity test. The relationship between the relative absorbance difference and the amount of bacteria obtained by reacting the endo-type lignocellulolytic enzyme I secreted by the transform 1296-eg1-opt/Po1g T1 to T10 with the matrix 1% CMC at 60 ° C for 30 min. As shown in Figure 7. Figure 7 and Table 5 show that the enzyme activities of T2, T5 and T6 are better, 1.14, 1.13 and 1.17 U/mL, respectively, and the dry weight of the cells is 15.01, 9.62 and 11.11 g/L.

The above comparison results showed that the optimal enzyme activities of the 1296-eg1/Po1g group were 0.93 and 0.94 U/mL, and the other 1296-eg1-opt/Po1g optimal enzyme activities were 1.14, 1.13 and 1.17 U/mL. It can be seen that the enzyme activity analysis value of the transformed body having the optimized endo-type lignocellulolytic enzyme I gene sequence is higher than that of the unmodified gene sequence directly transferred to the same system, and the average activity is improved by about 0.21 U. /mL, the enzyme production efficiency is increased by about 22.7%, so the application of the Yarrowia lipolytica protein expression system and the codon-modified endo-type lignocellulolytic enzyme I gene sequence can effectively improve the enzyme production efficiency. .

Example 4

Using the Yarrowia lipolytica protein expression system, two sets of two-copy codon-adapted endo-type lignocellulolytic enzyme I gene sequences were constructed in pYLSC1 plastids, and then hydrolyzed by NotI restriction enzymes. The plastid is changed from a ring type to a linear type, and then transformed into a preset position on the Po1g gene of Yarrowia lipolytica by homologous recombination, and the enzyme activity is selected after transformation. Shaped body.

I. Construct a plastid with two sets of endo-type lignocellulolytic enzyme I genes modified by codon preference

Refer to Figure 8 for the following experimental procedure:

1. Construction of plastid IS: For the convenience of sequencing, a 50 bp λ DNA gene was added after the EG1 opt gene fragment. First, the λ DNA gene was used as a template, and the primers IS-F and IS-R (the sequences are shown in Table 6) were used for PCR, and the 50 bp fragment IS of the λ DNA gene was selected and added to 1296-EG1 opt. The HindIII and XbaI restriction enzyme cleavage sites. Next, primers IS-F and 6904R (the sequences are shown in Table 6) were used for colony PCR screening, and after confirmation by restriction enzymes (HindIII and XbaI), the constructed plastid was called IS.

2. Terminator: In order to reduce the phenomenon of gene recombination, terminal substitutions are performed using different terminals. First, using pINA 1311 as a template, primers LT-F and LT-R (the sequences are shown in Table 6) were used for PCR, and a lipase terminator gene fragment LT was selected and then sent. Among the TA selection vectors, white colonies were selected (blue and white screening), and the plastids of the white colonies were extracted, and the construction of the TA-LT plastids was confirmed by restriction enzymes. The LT gene was further subcloneed to the HindIII restriction enzyme cleavage site of the plastid IS using TA-LT plastids. The primers LT-F and IS-R were used for colony PCR screening and restriction enzymes to confirm the genes contained in each transformant. As shown in Fig. 9, after treatment with HindIII restriction enzyme, the 8635 bp and 127 bp fragments can be excised. The LT gene is transformed with AvrII and KpnI restriction enzymes, and the two fragments of 8858 and 192 bp can be cut into the LT gene and the EG I opt gene is transformed into the same shape. For ISLT.

3. Construct a plastid (ISLT-tc) with 2 sets of EG1 opt:

A. PCR was carried out with the primer EG I mc-F1 (the sequence of which is shown in Table 6) and 6904R, and a nucleotide fragment containing hp4d (promoter) and EG I opt was selected, which was about 2.3 Kb. This fragment was isolated and purified using an electrophoretic film, and this fragment was used as an insert.

B. The plastid ISLT was treated with XbaI restriction enzyme and purified by a reagent group (Probiotics YDF10), and the treated plastid ISLT was used as a carrier.

C. After the above-described insert was ligated with the vector, it was transformed into Escherichia coli (E. coli), and cultured overnight with a plate containing ampicillin (ampicillin), followed by screening.

D. Colony PCR (using primers ISF and 6904R) was used for preliminary screening. The plastid DNA was further extracted according to the results of the preliminary screening, and the orientation and fragment size of the insert were confirmed by restriction enzymes HindIII and XbaI and PCR. will The constructed plastid with two sets of endo-cut lignocellulolytic enzyme I genes was named ISLT-tc, which had a total of 10,863 base pairs.

II. The plastid ISLT-tc was introduced into the Po1g genome, which was named 1296-ISLT-tc/Po1g. III. Performance results of 1296-ISLT-tc/Po1g

1. Congo red active staining: 2 sets of group transformation 1296-ISLT-tc/Po1g T1 to T10 were cultured in Mandels-Reese solid medium at 30 ° C for 4 days, and the enzyme production capacity of T1 to T10 was confirmed by Congo red staining. As shown in Fig. 10, except for T2 and T10, all the other transforming bodies have obvious transparent rings, indicating that they have endo-type lignocellulolytic enzyme I activity.

2. Total protein amount and endo-type lignocellulolytic enzyme I activity assay: The enzyme activity of the 2 sets of transformants 1296-ISLT-tc/Po1g was determined at 60 °C. The results are shown in Table 7, in which T2 and T10 did not have enzyme activity, which was consistent with the results of Congo red activity staining. In addition, enzyme activity analysis was better with transformants T5 and T8, and the optimal enzyme activities were 1.50 and 1.47 U/mL, respectively, while the single set of transformants 1296-eg1 opt/Po1g T6 enzyme activity was only 1.13 U/mL, showing 2 The enzyme activity of the set of transformants increased by an average of 0.36 U/mL, which also increased the enzyme activity by 31.4%.

Compared with the transformant with the endo-type lignocellulolytic enzyme I gene modified without codon preference, the optimal enzyme activity of the transform 1296-EG1/Po1g was 0.93 and 0.94 U/mL, and the transform 1296-EG1- The optimal enzyme activities of opt/Po1g were 1.14, 1.13 and 1.17 U/mL, while the optimal enzyme activities of the 2 set of transformants 1296-ISLT-tc/Po1g were 1.50 and 1.47 U/mL. Comparing the 1296-EG1/Po1g with the gene modified with no codon preference and the 2296-ISLT-tc/Po1g with 2 sets of transformants, the enzyme activity increased by 0.55U/mL, which can increase the enzyme activity by 58.8%. . It can be seen that by modifying the endo-type lignocellulolytic enzyme I gene and using multiple sets of Yarrowia lipolytica protein expression system, the endo-type lignocellulolytic enzyme I production can be increased, and the production cost of the enzyme can be reduced. .

<110> Taiwan Zhongyou Co., Ltd.

<120> Gene, composition and method for promoting hydrolysis of lignocellulose

<130> 113210

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 1527

<212> DNA

<213> Trichoderma reesei

<220>

<221> gene

<222> (1)..(1527)

<400> 1

<210> 2

<211> 1380

<212> DNA

<213> Labor

<220>

<223> Modified endo-type lignocellulolytic enzyme I without intron

<220>

<221> Exon

<222> (1)..(1380)

<400> 2

<210> 3

<211> 1380

<212> DNA

<213> Labor

<220>

<223> Modified endo-type lignocellulolytic enzyme I with codon preference modification

<220>

<221> Exon

<222> (1)..(1380)

<400> 3

<210> 4

<211> 24

<212> DNA

<213> Labor

<220>

<223> EGI-1F primer

<220>

<221> misc_difference

<222> (1)..(24)

<400> 4

<210> 5

<211> 24

<212> DNA

<213> Labor

<220>

<223> EGI-2R primer

<220>

<221> misc_difference

<222> (1)..(24)

<400> 5

<210> 6

<211> 27

<212> DNA

<213> Labor

<220>

<223> EGI-1R primer

<220>

<221> misc_difference

<222> (1)..(27)

<400> 6

<210> 7

<211> 27

<212> DNA

<213> Labor

<220>

<223> EGI-2F primer

<220>

<221> misc_difference

<222> (1)..(27)

<400> 7

<210> 8

<211> 27

<212> DNA

<213> Labor

<220>

<223> EGI-2.5R primer

<220>

<221> misc_difference

<222> (1)..(27)

<400> 8

<210> 9

<211> 29

<212> DNA

<213> Labor

<220>

<223> EGI-3R primer

<220>

<221> misc_difference

<222> (1)..(29)

<400> 9

<210> 10

<211> 31

<212> DNA

<213> Labor

<220>

<223> EGI-m1F primer

<220>

<221> misc_difference

<222> (1)..(31)

<400> 10

<210> 11

<211> 31

<212> DNA

<213> Labor

<220>

<223> EGI-m1R primer

<220>

<221> misc_difference

<222> (1)..(31)

<400> 11

<210> 12

<211> 36

<212> DNA

<213> Labor

<220>

<223> EGI-SfiI-F primer

<220>

<221> misc_difference

<222> (1)..(36)

<400> 12

<210> 13

<211> 29

<212> DNA

<213> Labor

<220>

<223> EGI-HindIII-R primer

<220>

<221> misc_difference

<222> (1)..(29)

<400> 13

<210> 14

<211> 24

<212> DNA

<213> Labor

<220>

<223> IS-F primer

<220>

<221> misc_difference

<222> (1)..(24)

<400> 14

<210> 15

<211> 24

<212> DNA

<213> Labor

<220>

<223> IS-R primer

<220>

<221> misc_difference

<222> (1)..(24)

<400> 15

<210> 16

<211> 24

<212> DNA

<213> Labor

<220>

<223> LT-F primer

<220>

<221> misc_difference

<222> (1)..(24)

<400> 16

<210> 17

<211> 23

<212> DNA

<213> Labor

<220>

<223> LT-R primer

<220>

<221> misc_difference

<222> (1)..(23)

<400> 17

<210> 18

<211> 21

<212> DNA

<213> Labor

<220>

<223> EG I mc-F1 primer

<220>

<221> misc_difference

<222> (1)..(21)

<400> 18

<210> 19

<211> 20

<212> DNA

<213> Labor

<220>

<223> 6904R primer

<220>

<221> misc_difference

<222> (1)..(20)

<400> 19

Claims (12)

A nucleic acid molecule comprising a set of modified nucleotide sequences encoding an endo-type lignocellulosic enzyme I, wherein the modified nucleotide sequence encoding the endo-cut lignocellulosic enzyme I SEQ ID NO.1 is obtained by modifying the codon preference of yeast, wherein the modified nucleotide sequence encoding endo-type lignocellulolytic enzyme I is the nucleotide sequence of SEQ ID NO: 3. The nucleic acid molecule according to claim 1, wherein the yeast strain is Yarrowia lipolytica . A performance vector comprising the nucleic acid molecule of claim 1 or 2. The expression vector of claim 3, further comprising an inducible promoter sequence that controls expression of the nucleic acid molecule. The expression vector of claim 3, further comprising at least one selected from the group consisting of a marker gene sequence, a reporter gene sequence, an antibiotic resistance gene sequence, a restriction enzyme cleavage position sequence, polyadenosine Acidic position sequences, enhancer sequences, terminal subsequences, and regulatory subsequences. A host cell for producing a target protein comprising the expression vector of claim 3 of the patent application. The host cell according to claim 6 is at least one selected from the group consisting of a virus, a bacterial cell, a fungal cell, an algal cell, a plant cell, and an animal cell. The host cell according to claim 7, wherein the true Bacteria cell line yeast. The host cell of claim 8, wherein the yeast is Yarrowia lipolytia . The host cell as described in claim 6 is deposited in the Bioresource Conservation and Research Center of the Food Industry Development Institute under the name of CPC-2EGI T5, accession number BCRC 920087. The host cell according to claim 6, wherein the target protein is an endo-type lignocellulosic enzyme I. A method for promoting hydrolysis of lignocellulose, which comprises treating a lignocellulose using a performance vector as described in claim 3 of the patent application or a host cell as described in claim 6 of the patent application.