Summarize

Based on the problem that food and feed in the planting industry can not meet the needs of breeding, we turned our attention to non-food crops, and finally locked in the straw. Our goal is to construct recombinant saccharomyces cerevisiae strains that can degrade cellulose and hemicellulose in straw and ferment to produce high-protein straw feed.

Idea

1.Why straw is used as feed raw material: straw is the remaining part of many common crops after seed harvest, and the number is large. Direct incineration will increase the inhalable particles in the air, reduce the visibility of the air, and pollute the environment. At the same time, straw is composed of cellulose, hemicellulose and lignin, which is rich in nitrogen, phosphorus, potassium, calcium, magnesium and organic matter. It is a biological resource with rich nutritional value.

2.Why Saccharomyces cerevisiae is used as a chassis cell: Compared to many bacteria and mycelium fungi, the relatively large spherical cells of yeast are often easy to bioprocess. In addition, they can grow at ambient temperatures and their nutritional requirements are relatively simple, and the yeast's ability to multiply at low pH helps reduce the likelihood of contamination in medium and bioreactors in large-scale industrial operations. The two most widely used for recombinant membrane protein production among more than 1,500 yeast species are Saccharomyces cerevisiae and Pichia Pasteuris.

Stage 1 : Construction of high expression engineering strains

Design

It is planned to realize the surface display of three cellulases in yeast respectively, and then carry out molecular transformation of β-glucosidase mainly through genetic engineering technology to optimize enzyme performance, so as to obtain strains that can express β-glucosidase efficiently.

Build

1.Linear PYD1 was used as the carrier, the target gene and vector were cloned with ExnaseII and then mixed with the receptor DH5α and coated on a plate. The colonies with good growth were selected, the green enzyme GreenTaqMix was used for PCR, and the three groups with the most obvious target bands were cultured in liquid by running the adhesive test, and the plasmid was extracted for enzyme digestion verification.

2.The plasmid containing the target fragment was electrically transferred into the receptive yeast cell EBY100, coated with a plate, and then colony PCR verification was performed. The recombinant saccharomyces cerevisiae with the most obvious rubber strip was cultured for two days, and the enzyme activity was measured with an enzyme marker.

Test

Glucose and PNPG standard curves were measured first, and then extracellular enzyme liquid was added to different substrates to measure enzyme activity using enzyme marker. For β-glucosidase (BG), substrate PNPG was added to determine OD405. For exodextranase (CBH), substrate MCC was added and OD540 was determined. For endoglucanase (EG), substrate CMC was added to determine OD540.

Learning

For the plan of molecular modification of β-glucosidase, after conducting an experiment, we found that the original strain in the laboratory could express β-glucosidase efficiently, so we directly used the existing strain in the laboratory. However, the problem in the experiment was endoglucanase: the first time NheI enzyme cut the glue, it was found that the strip was too small, and the enzyme cutting was considered unsuccessful. The strip was normal when repeated, but the glue recovery concentration was low, and the experiment was repeated many times.The surface display experiment of endoglucanase is still not ideal.

Stage 2 : Construction of fibrosomes and assembly

Design

It is planned to combine the highly expressed β-glucosidase, endocellulase and exocellulase through a protein scaffold, anchor them by AGglutinin Aga on the surface of yeast cells, and induce their expression through galactose.

Build

The highly expressed fungal free enzymes β-glucosidase, endocellulase and exocellulase were fused into the corresponding docking module to participate in the construction of fibrosomes. A heterozygous scaffold was formed by connecting the adhesive modules from different sources, and the docking module from the corresponding source was fused with the different cellulase components. According to the specific binding characteristics of the docking module and the adhesion module, cellulase was quantitatively and locatively assembled on the fiber body. Then each unit of the fibrosome is expressed in the same cell, and finally self-assembly is realized in the extracellular. The resulting fibrosomes were anchored on the cell surface by AGglutinin Aga and induced by galactose.

Test

The expression of fibrous bodies was induced by galactose, and two small pieces of WhatmanNo.1 filter paper (about 0.5mg) were added, and OD540 was determined after reaction at 45℃.

Learning

In the advance experiment, it was found that the surface display scheme was more difficult than expected and difficult to carry out. After discussion, we chose to adopt the method of gene fusion expression to achieve the simultaneous expression of three kinds of cellulose.

Stage 3 : The expression strain of saccharomyces cerevisiae was constructed by gene fusion expression

Design

The plan is to use fusion strategies to construct a single gene with three different cellulase functions to reduce complex operations during the expression of multifunctional cellulase, then express the gene in Saccharomyces cerevisiae and screen strains with three cellulase functions.

Build

Using PYD1-BG, PYD1-EG and PYD1-CBH as templates, the CD coding sequences of the catalytic domains of cellulase BG, CBH and EG were obtained by PCR reaction. The resulting PCR products were treated with restriction endonuclides XbaⅠ and EcoRⅠ, and mixed into the linking reaction system according to the mass concentration of the same substance. Using the enzyme-linked mixture as template, BG-SnaB I-F and EG-Eco81 I-R primers were used to amplify the fragment about 3.0kb in length by PCR, which was the fusion gene bce. The fusion gene was verified by enzyme digestion, and then seamlessly cloned and electrocuted into receptive yeast cell EBY100. The plate was coated, colony PCR verification was performed, and the recombinant saccharomyces cerevisiae with the most obvious rubber strip was cultured for two days, and the enzyme activity was determined by enzyme marker.

Test

Glucose and PNPG standard curves were measured first, and then extracellular enzyme liquid was added to different substrates to measure enzyme activity using enzyme marker. β-glucosidase (BG) activity determination: adding substrate PNPG, determination of OD405; The activity of exodextranase (CBH) was determined by adding substrate MCC to determine OD540. Determination of endoglucanase (EG) activity: adding substrate CMC to determine OD540. Determination of enzyme activity of filter paper: Add two small pieces of WhatmanNo.1 filter paper (about 0.5mg) to determine OD540. The activity of three kinds of cellulase enzyme was compared with that of filter paper enzyme.

Learning

In the specific experiment, the electric transfer failure occurred when saccharomyces cerevisiae EBY100 was used as the receptive cell, and the experiment still failed after repeated for many times. To solve this problem, we changed the type of saccharomyces cerevisiae to INVSc1, continued the experiment, and finally measured the enzyme activity.

Stage 4 : Building degradable xylan module

Design

While building highly efficient cellulose degrading strains, we plan to recombine the gene expressing xylanase into saccharomyces cerevisiae cells, so that saccharomyces cerevisiae can not only degrade cellulose, but also degrade 20% hemicellulost in straw.

Build

1.Linear PYD1 was used as the carrier, the target gene and vector were cloned with ExnaseII and then mixed with the receptor DH5α and coated on a plate. The colonies with good growth were selected, the green enzyme GreenTaqMix was used for PCR, and the three groups with the most obvious target bands were cultured in liquid by running the adhesive test, and the plasmid was extracted for enzyme digestion verification.

2.The plasmid containing the target fragment was electrically transferred into the receptive yeast cell EBY100, coated with a plate, and then colony PCR verification was performed. The recombinant saccharomyces cerevisiae with the most obvious rubber strip was cultured for two days, and the enzyme activity was measured with an enzyme marker.

Learning

In the specific experiment, after the expression of xylanase gene was transferred to saccharomyces cerevisiae INVSc1, the recombinant saccharomyces cerevisiae did not produce hydrolytic rings on a plate containing xylans, and no enzyme activity was detected by enzymoscope. After colony PCR was performed to confirm the size of the target gene fragment and determine the sequence of the gene fragment, it was concluded that xylanase was not expressed in the yeast. We decided to use Escherichia coli to produce inclusion bodies containing xylanase, and at the initial stage we had already constructed Escherichia coli containing the xylanase gene.After extracting the plasmid, the plasmid was transferred to Escherichia coli by errory-prone PCR. The purified protein was obtained after bacteria breaking, Ni column regeneration, Ni column separation and purification, and protein ultrafiltration. The xylanase was purified for protein glue detection, and it was found that the band size was correct and the band corresponding to the purified protein was more obvious. It has high activity.

Stage 5 : The transformation of xylose into xylulose into yeast intracellular carbon cycle

Design

Xylose is obtained after the degradation of xylan, but saccharomyces cerevisiae lacks the ability to fermentation xylose although it has a strong glucose metabolism capacity. We plan to use xylose isomerase to convert xylose to xylulose, which can enter the pentose phosphate pathway and be utilized by yeast. At the same time, by optimizing the promoter of xylose isomerase, we transferred the constructed plasmid into saccharomyces cerevisiae after optimization, and the enzyme was expressed in the cell. After xylose was converted into xylose, it could enter the carbon cycle and be utilized by yeast. In this way, straw resources can be maximized.

Build

Xylose isomerase xylA gene fragment was cut with XbaI and NotI enzymes and cloned into pHM368-PGK-CBH-Ura cut with the same enzyme to obtain pHM368-PPGK-xylA-Ura. The promoters of ADH1, GAPDH, PDC1 and TEF1 were amplified by corresponding primers and inserted into pHM368-PPGK-xylA-Ura cut by NdeI and NotI by seamless and link-independent cloning. The expression plasmids pHM368-PADH1-xylA-Ura, pHM368-PGAPDH-xylA-Ura, pHM368-PPDC1-xylA-Ura and pHM368-PTEF1-xylA-Ura were constructed. The constructed plasmid was linearized with HpaⅠ and transferred into saccharomyces cerevisiae INVSc1 by lithium acetate method. Engineered strains INVSc1/pHM368-PPGK-xylA,INVSc1/pHM368-PADH1-xylA,INVSc1/pHM368-PGAPDH-xylA,INVSc1/pHM368-PPDC1-xylA and INVSc1/pHM36 were obtained, respectively 8-PTEF1-xylA. Strains were selected on an SD board without Ura and then verified by PCR. Finally, the activity of xylose isomerase was determined.

Test

The engineered strains of Saccharomyces cerevisiae containing different promoters were incubated in YPD medium for 48h to obtain crude enzymes. The reaction mixture containing 10mMMnCl2, 100mM xylose and 0.1mL crude enzyme was incubated at 75℃ for 20min, stopped with 50% trichloroacetic acid (TCA) and incubated at room temperature for 20min. Then cysteine-carbazol-sulfuric acid reagent containing 1.5% cysteine, 0.12% carbazole and 13MH2SO4 were added and incubated at 37℃ for 20min. Measure OD540 to determine the amount of xylose released. The most active xylose isomerase was obtained.

Learning

Through xylose isomerase activity detection, strain INVSc1/pHM368-PADH1-xylA was found to be the most effective. It has been confirmed that glucose can protect cells under high stress, and glucose may activate the expression of xylA gene involved in xylose catabolism. Low glucose concentrations promote co-utilization of glucose and xylose by easing catabolism and lead to greatly increased fluxes in different steps of the pentose phosphate pathway.