Microorganism for degrading sugar source as well as construction method and application thereof

Abstract

The invention discloses a sugar source degrading microorganism and a construction method and application thereof, the recombinant microorganism is characterized in that glucoamylase and / or maltase are / is introduced into the microorganism by using a molecular modification technology, so that related enzymes expressed by the microorganism can continuously hydrolyze disaccharide or polysaccharide in a sugar source into glucose in a fermentation process; microorganisms can simultaneously utilize various sugar sources for fermentation, the utilization rate of the sugar sources is improved, and the fermentation cost is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a microorganism that degrades sugar sources, its construction method, and its application. Background Technology

[0002] In the fermentation industry, carbon sources primarily provide energy for cells, constituting cell bodies and metabolic products. Commonly used carbon sources include sugars, lipids, organic acids, and lower alcohols. For sugars, pure monosaccharides, disaccharides, or polysaccharides can be used, as well as liquid sugars containing these sugars. Liquid sugars, also known as starch sugars, are sugars produced from starchy grains, tubers, etc., through acid, acid-enzyme, or enzymatic processes. These include glucose, maltose, and fructose syrup. Due to their low cost, they are also used in industrial production. However, the difficulty in controlling relevant parameters in the starch sugar preparation process (such as temperature and pH) leads to the presence of non-fermentable sugars, either reduced from monosaccharides or heteroformed from them, significantly affecting the utilization rate and fermentation yield of starch sugars. The traditional solution is to optimize fermentation conditions (such as temperature, pH, aeration rate, and stirring speed) and the fermentation medium. However, this approach suffers from drawbacks such as numerous influencing factors, difficulty in finding and controlling optimal conditions, and a large workload in actual operation. Therefore, developing a method that enables microorganisms to directly and efficiently utilize polysaccharides through fermentation will greatly reduce fermentation costs and improve fermentation efficiency, and has significant industrial application value. Summary of the Invention

[0003] Amylase, also known as glucoamylase, scientifically named α-1,4-glucose hydrolase, hydrolyzes the α-1,4 and α-1,6 glycosidic bonds of starch, dextrin, and oligosaccharides, completely converting them into glucose. Maltase hydrolyzes maltose to produce glucose and fructose. Therefore, the purpose of this invention is to introduce amylase and / or maltase into microorganisms using molecular modification technology, enabling the expressed enzymes to continue hydrolyzing disaccharides or polysaccharides in the sugar source into glucose during fermentation. This allows microorganisms to simultaneously utilize multiple sugar sources during fermentation, improving sugar source utilization and reducing fermentation costs.

[0004] Specifically, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention provides a method for constructing recombinant microorganisms, comprising: conferring or enhancing the activity of saccharifying enzymes and / or maltases in the microorganisms.

[0006] In one embodiment, the activity of saccharifying enzymes and / or maltases in a microorganism is conferred or enhanced by expressing or overexpressing genes encoding saccharifying enzymes and / or maltases in the microorganism.

[0007] In specific embodiments of the present invention, the microorganisms are prokaryotes or eukaryotes, such as *Escherichia coli*, *Corynebacterium glutamicum*, *Bruch's flavobacterium*, yeast, etc. In the embodiments of the present invention, only specific *E. coli* strains are used as examples; however, since the metabolic pathways of other *E. coli* strains are essentially the same, those skilled in the art can expect that any other *E. coli* strain known in the prior art, or any strain capable of fermentation using mixed sugar sources, will have the same or similar effects.

[0008] In one implementation, at least one regulatory element is used to activate or enhance the expression or overexpression of the gene encoding the enzyme.

[0009] In one implementation, the glaA gene is expressed or overexpressed by introducing the saccharifying enzyme gene glaA into a microorganism and integrating a regulatory element before the glaA gene.

[0010] Preferably, the regulatory element is selected from the J23109 promoter to regulate glaA gene expression;

[0011] Preferably, the saccharifying enzyme is derived from Aspergillus niger, more preferably it contains the amino acid sequence shown in SEQ ID NO: 1, and even more preferably it contains the nucleotide sequence shown in EQ ID NO: 3.

[0012] In one implementation, the expression of the glaA gene is not limited to the introduction of exogenous regulatory elements, but can also be regulated by the original promoter at the integration site of the gene.

[0013] In one implementation, the maltase gene malA is expressed or overexpressed by introducing it into a microorganism and integrating a regulatory element before the malA gene.

[0014] Preferably, the regulatory element is selected from the Trc promoter to regulate malA gene expression;

[0015] Preferably, the maltase is derived from Saccharolobus solfataricus, more preferably it contains the amino acid sequence shown in SEQ ID NO: 2, and even more preferably it contains the nucleotide sequence shown in SEQ ID NO: 4.

[0016] In one implementation, the expression of the malA gene is not limited to the introduction of exogenous regulatory elements, but can also be regulated by the original promoter at the integration site of the gene.

[0017] The phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) is widely found in bacteria, fungi, and some archaea. The PTS consists of phosphotransferases such as enzyme I (EI), histidine phosphocarrier protein (HPr or NPr), and enzyme II complex, possessing both catalytic transport functions and a wide range of regulatory functions. The PTS primarily phosphorylates various sugars and their derivatives through a phosphate cascade reaction, transporting them into the cell. It not only participates in carbon and nitrogen metabolism, regulates iron and potassium homeostasis, modulates the virulence of certain pathogens, but also mediates stress responses. However, when microorganisms grow in glucose-limited media, the PTS system consumes approximately 50% of PEP for glucose transport and phosphorylation, directly affecting the synthesis of amino acids that use PEP as a precursor. Therefore, weakening the activity of PTS system proteins, allowing more PEP to flow to amino acid synthesis pathways, increases the synthesis of target amino acids and improves the sugar-acid conversion rate.

[0018] SfsA, a protein that promotes carbohydrate metabolism, is considered a transcriptional activator that can enhance the expression of genes related to starch metabolism. Therefore, overexpression of SfsA can increase amylase activity and the level of maltose-binding protein (encoded by the malE gene, a member of the pathway for maltose uptake and breakdown in E. coli).

[0019] Based on this, in order to improve the ability of Escherichia coli to ferment mixed sugars, the present invention regulates the overexpression of the sugar metabolism promoting protein SfsA and / or modifies the PTS system, including: weakening the activity of PTS system proteins to reduce PEP consumption; and conferring or enhancing the activity of glucokinase to relieve the feedback inhibition effect of glucose metabolites; so that Escherichia coli can utilize mixed sugars for fermentation.

[0020] Specifically, the present invention provides the following preferred solutions:

[0021] In one embodiment, the microorganism confers or enhances the activity of a glucose metabolism-promoting protein.

[0022] In one implementation, the activity of a sugar metabolism-promoting protein in a microorganism is conferred or enhanced by expressing or overexpressing a gene encoding a sugar metabolism-promoting protein in the microorganism.

[0023] In one implementation, the sfsA gene is overexpressed by integrating a regulatory element before the microbial endogenous sugar metabolism-promoting protein gene sfsA.

[0024] Preferably, the regulatory element is selected from the J23109 promoter to regulate the overexpression of the sfsA gene.

[0025] In one embodiment, the microorganism also weakens, if present, the activity of PTS system proteins.

[0026] IIA Glc The enzyme is part of the PTS, and its encoding gene is the crr gene; based on this, in one embodiment, the activity of PTS system proteins is weakened by knocking out the crr gene on the E. coli genome.

[0027] In one embodiment, the microorganism also confers or enhances glucokinase activity.

[0028] In one implementation, the activity of glucokinase in the microorganism is conferred or enhanced by expressing or overexpressing the gene encoding glucokinase in the microorganism.

[0029] In one implementation, the glK gene is expressed or overexpressed by introducing the glucokinase gene glK into a microorganism and integrating regulatory elements before the glK gene.

[0030] Preferably, the regulatory element is selected from the J23109 promoter to regulate glK gene expression;

[0031] Preferably, the glucokinase is derived from E. coli MG1655; more preferably, it contains the amino acid sequence shown in SEQ ID NO: 5, and even more preferably, it contains the nucleotide sequence shown in SEQ ID NO: 6.

[0032] Secondly, the present invention provides a sugar-degrading microorganism, which is constructed by the above method.

[0033] The recombinant microorganism can degrade disaccharides or polysaccharides into monosaccharides, for example, degrading starch, dextrin, oligosaccharides, maltose, isomaltose, etc. into glucose.

[0034] Thirdly, the present invention provides the application of the above-mentioned recombinant microorganisms in any of the following A1)-A3):

[0035] A1) Degrading disaccharides and / or polysaccharides into monosaccharides;

[0036] A2) Improve the utilization rate of sugar sources in the fermentation system, wherein the sugar sources contain disaccharides and / or polysaccharides;

[0037] A3) Fermentation to produce bio-based products, and the sugar source used in the fermentation contains disaccharides and / or polysaccharides;

[0038] The disaccharide or polysaccharide contains α-1,4 glycosidic bonds and / or α-1,6 glycosidic bonds.

[0039] In one embodiment, the product is an amino acid, vitamin, or bio-based material monomer, etc.

[0040] In one embodiment, the disaccharide or polysaccharide contains α-1,4 glycosidic bonds and / or α-1,6 glycosidic bonds, such as maltose, isomaltose, maltulose, etc.

[0041] Fourthly, methods to improve the utilization rate of sugar sources by microorganisms include:

[0042] Genetic modification of microorganisms to give them or enhance their saccharifying enzymes and / or maltases.

[0043] In one embodiment, the sugar source contains disaccharides and / or polysaccharides, which can be hydrolyzed into monosaccharides such as glucose by saccharifying enzymes and / or maltases.

[0044] In one embodiment, the sugar source is starch sugar or liquid sugar.

[0045] In one embodiment, the microorganism also has or has an enhanced sugar metabolism-promoting protein SfsA.

[0046] In one embodiment, the microorganism also has the ability to reduce the activity of PTS system proteins.

[0047] In one specific implementation, the activity of the PTS system proteins is reduced by knocking out the crr gene.

[0048] In one embodiment, the microorganism also has or has enhanced glucokinase.

[0049] Compared with the prior art, the present invention has the following beneficial technical effects:

[0050] When microorganisms utilize liquid sugar fermentation for production, it was found that a certain amount of reducing sugars remained unutilized in the fermentation broth upon initial fermentation, including maltose, isomaltose, and maltulose. Analysis showed that the residual reducing sugar content after fermentation was approximately 4 g / L. This invention addresses this by genetically modifying microorganisms to efficiently express saccharifying enzymes and / or maltases during fermentation. These enzymes hydrolyze the α-1,4 or α-1,6 glycosidic bonds of disaccharides or polysaccharides in the sugar source, releasing monosaccharides such as glucose. Furthermore, by modifying the microorganisms' sugar-consuming pathways and eliminating the inhibitory effect of glucose catabolism, the strain can simultaneously utilize multiple sugar sources for fermentation. The residual sugar content in the fermentation broth is reduced to approximately 2 g / L, solving the problems of low metabolic efficiency and low conversion rate of multiple sugar sources during fermentation, and lowering production costs. Detailed Implementation

[0051] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0053] The strains and plasmids involved in the following examples are shown in Table 1, and the primers used are shown in Table 2.

[0054] Table 1: Strains and plasmids used in this invention

[0055]

[0056]

[0057] Table 2: Primers used in this invention

[0058]

[0059]

[0060] Example 1: Selection of Escherichia coli genome modification sites

[0061] The selection principle for E. coli genome modification sites is: exogenous integration and multiple copy integration can be stably expressed at the site without affecting the stability of the genome.

[0062] Based on a series of modification sites in the E. coli Sval049 genome that can stably express exogenous genes, as determined in the previous branched-chain amino acid pre-research experiments, this embodiment selects two sites shown in Table 3 below as modification sites for exogenous integration of saccharifying enzyme and maltase.

[0063] Among them, Escherichia coli Sval049 is an L-valine-producing bacterium with accession number CGMCC 19457 and classification name: Escherichia coli. It was submitted for deposit on March 6, 2020, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. For detailed information on strain characteristics, please refer to patent ZL202010460035.9.

[0064] Table 3: Modification sites (upstream and downstream insertion sites) for integrating saccharifying enzymes and maltases on the E. coli genome.

[0065]

[0066] Example 2: Cloning and integration of the glucoamylase gene glaA

[0067] The glaA gene, derived from Aspergillus niger, was integrated into the ykgH_betA site of Escherichia coli Sval049 using CRISPR-Cas9 gene editing technology (with promoter J23109 introduced before the glaA gene) to construct strain Sval049::glaA.

[0068] The specific steps are as follows:

[0069] 2.1 Design the glaA integration site N20 using the CHOPCHOP online website.

[0070] 2.2 Construct the Ptarget-glaA-N20 plasmid.

[0071] Using Ptarget plasmid as a template and glaA-N20-sgF / R as primers, the P plasmid was circulated to obtain plasmid Ptarget-glaA-N20;

[0072] Amplification system: AceTaqa DNA polymerase buffer (2X): 25 μL, primer 1: 1 μL, primer 2: 1 μL, template: 1 μL (50 ng), ddH2O: 22 μL, total volume: 50 μL.

[0073] Among them, AceTaqa DNA polymerase buffer (2X) was purchased from Nanjing Novizan, catalog number P401-d1;

[0074] Amplification program: 95℃ pre-denaturation for 5 min (1×); 95℃ denaturation for 20 s, 57℃ annealing for 20 s, 72℃ extension for 45 s (35×); 72℃ extension for 5 min (1×); store at 4℃.

[0075] The plasmid Ptarget-glaA-N20 was transformed into TOP10 competent cells, identified by PCR using pTarget seq F / R primers, and then sent to Sangon Biotech for sequencing to obtain the circular plasmid Ptarget-glaA-N20.

[0076] 2.3 Construct the Ptarget-glaA-N20-donor plasmid.

[0077] Using the Ptarget-glaA-N20 plasmid as a template and target-CF / R as primers, the plasmid was linearized to obtain the linearized Ptarget-glaA-N20 fragment. The amplification system and procedure were the same as in step 2.2.

[0078] Using the genomic DNA of Sval049 strain as a template and glaA-UF / UR as primers, the UP-1 fragment was amplified;

[0079] Using the genomic DNA of Sval049 strain as a template and glaA-DF / DR as primers, the DOWN-1 fragment was amplified;

[0080] Using the synthetic plasmid ptrc99a-kana-glaA as a template and glaA-F / R as primers, the glaA gene fragment was amplified; the nucleotide sequence of the glaA gene fragment is shown in SEQ ID NO: 3, and the amino acid sequence it encodes is shown in SEQ ID NO: 1;

[0081] The amplification systems and procedures for the UP-1, DOWN-1, and glaA fragments are the same as in step 2.2.

[0082] The linearized fragment of Ptarget-glaA-N20, UP-1, DOWN-1, and glaA were cloned and ligated in one step using the ClonExpress Ultra One Step Cloning Kit (purchased from Novizan, catalog number C115-01 / 02). The ligation product was transformed into TOP10 competent cells, and the bacterial culture was identified and sequenced by PCR using pTarget seq F / R as primers to obtain the Ptarget-glaA-N20-donor plasmid.

[0083] 2.4. The glaA gene was integrated into the ykgH_betA site of E. coli Sval049.

[0084] First, Sval049 electroporation competent cells were prepared (refer to Cold Spring Harbor Molecular Cloning Handbook). The pECCas plasmid was electroporated into the Sval049 competent cells to obtain the Sval049-cas strain. After using the Sval049-cas strain as electroporation competent cells, the Ptarget-glaA-N20-donor plasmid was electroporated into the Sval049-cas competent cells. The results were verified by glaA-YZ-F / R primers and sequenced to obtain the Sval049::glaA strain.

[0085] Example 3: Cloning and integration of the maltase gene malA

[0086] Using strain Sval049::glaA as the substrate, the maltase gene malA from Cypripedium unicornuate (with the promoter Trc introduced before the malA gene) was integrated into the ileY_ygaQ site of Escherichia coli Sval049 using CRISPR-Cas9 gene editing technology to construct strain Sval049::glaA::malA. The specific procedures are as follows:

[0087] 3.1 Design the malA integration site N20 using the CHOPCHOP online website.

[0088] 3.2 Construct the Ptarget-malA-N20 plasmid.

[0089] Using the Ptarget plasmid as a template and malA-N20-sgF / R as primers, the circular P plasmid was synthesized to obtain Ptarget-MalA-N20, which was then transformed into TOP10 competent cells. Colony PCR was performed using pTarget seq F / R primers for identification, and the cells were then sent to Sangon Biotech for sequencing to obtain the circular plasmid Ptarget-MalA-N20. The amplification system and procedure for the circular P plasmid were the same as step 2.2 of Example 2.

[0090] 3.3 Construct the Ptarget-MalA-N20-donor plasmid.

[0091] Using the Ptarget-MalA-N20 plasmid as a template and target-CF / R as primers, the plasmid was linearized to obtain the linearized Ptarget-MalA-N20 fragment. The linearization system and procedure are the same as step 2.2 in Example 2.

[0092] Using the genomic DNA of strain Sval049 as a template and MalA-UF / UR as primers, the UP-2 fragment was amplified.

[0093] Using the genomic DNA of strain Sval049 as a template and MalA-DF / DR as primers, the DOWN-2 fragment was amplified.

[0094] Using pACYCDuet-1-malA plasmid as a template and malA-F / R as primers, the malA gene was amplified to obtain the malA fragment; the nucleotide sequence of the malA gene fragment is shown in SEQ ID NO: 4, and the amino acid sequence it encodes is shown in SEQ ID NO: 2.

[0095] The amplification system and amplification procedure for the UP-2 fragment, DOWN-2 fragment, and malA fragment are the same as step 2.2 in Example 2.

[0096] The linearized fragment of Ptarget-MalA-N20, UP-2, DOWN-2, and malA were cloned and ligated in one step using the Novizan ClonExpress Ultra One Step Cloning Kit. The ligation product was transformed into TOP10 competent cells, and the bacterial culture was identified and sequenced by PCR using pTarget seq F / R as primers to obtain the Ptarget-MalA-N20-donor plasmid.

[0097] 3.4. Integrate the malA gene into the ileY_ygaQ site of the basal bacteria.

[0098] First, Sval049::glaA electroporation competent cells were prepared. The pECCas plasmid was electroporated into the Sval049::glaA competent cells to obtain the Sval049::glaA-cas strain. Then, after preparing the Sval049::glaA-cas strain into electroporation competent cells, the Ptarget-MalA-N20-donor plasmid was electroporated into the Sval049::glaA-cas competent cells. After verification with MalA-YZ-F / R primers and sequencing, the Sval049::glaA::malA strain was obtained.

[0099] Example 4: Cloning and integration of the glucokinase gene glk into the crr site (Sval049::glaA)

[0100] Wild-type *Escherichia coli* utilizes the phosphoenolpyruvate-glucose phosphotransferase system (PTS system) to transport and phosphorylate glucose. The PTS system consumes 1 mol of phosphoenolpyruvate (PEP) to transport 1 mol of glucose. When *E. coli* is grown in a glucose-dependent medium, the PTS system consumes approximately 50% of the PEP for glucose transport and phosphorylation, directly affecting the synthesis of amino acids that use PEP as a precursor.

[0101] To enhance the mixed sugar fermentation capability of *E. coli*, this application modifies the PTS system, including: knocking out the crr gene (encoding IIA) on the strain genome using CRISPR-Cas9 gene editing technology. Glc The enzyme (part of PTS) was used to overexpress the glucokinase gene glk at this gene locus (by introducing the promoter J23109 before the glk gene) to relieve the feedback inhibition effect of glucose metabolites, and its growth was almost unaffected and it could utilize mixed sugars for fermentation.

[0102] Using Sval049::glaA strain as the substrate bacteria, the E. coli-derived glucokinase gene glk was integrated into the crr site of Sval049::glaA strain using CRISPR-Cas9 gene editing technology, thus constructing the Sval049::glaA-Δcrr::glk strain. The specific procedures are as follows:

[0103] 4.1 Design the CRR integration site N20 using the CHOPCHOP online website.

[0104] 4.2 Construct the Ptarget-Δcrr-N20 plasmid.

[0105] Using the Ptarget plasmid as a template and crr-N20-sgF / R as primers, the circular P plasmid was transformed into Ptarget-Δcrr-N20, which was then converted into TOP10 competent cells. PCR identification was performed using pTarget seq F / R primers, and the cells were then sent to Sangon Biotech for sequencing to obtain the Ptarget-Δcrr-N20 plasmid. The amplification system and procedure for the circular P plasmid were the same as step 2.2 of Example 2.

[0106] 4.3 Construct the Ptarget-Δcrr::glk-N20-donor plasmid.

[0107] Using the Ptarget-Δcrr-N20 plasmid as a template and target-CF / R as primers to linearize the plasmid, the Ptarget-Crr-N20 linearized fragment was obtained.

[0108] Using the genomic DNA of strain Sval049 as a template and Crr-UF / UR as primers, the UP-3 fragment was amplified.

[0109] Using the genomic DNA of strain Sval049 as a template and Crr-DF / DR as primers, the DOWN-3 fragment was amplified.

[0110] Using Escherichia coli MG1655 genomic DNA as a template and glk-F / R as primers, the glk gene was amplified to obtain the glk fragment; the nucleotide sequence of the glk fragment is shown in SEQ ID NO: 6, and the amino acid sequence it encodes is shown in SEQ ID NO: 5.

[0111] The amplification system and amplification procedure for the UP-3 fragment, DOWN-3 fragment, and glk fragment are the same as step 2.2 in Example 2.

[0112] The linearized Ptarget-Crr-N20 fragment, UP-3, DOWN-3, and glk fragments were cloned and ligated in one step using the Novizan ClonExpress Ultra One Step Cloning Kit. The ligation products were transformed into TOP10 competent cells, identified by PCR using pTargetseq F / R primers, and sequenced to obtain the Ptarget-Δcrr::glk-N20-donor plasmid.

[0113] 4.4. Integrate the glk gene into the Δcrr site of the sclerotium.

[0114] First, Sval049::glaA electroporation competent cells were prepared (see Cold Spring Harbor Handbook of Molecular Cloning for details). The pECCas plasmid was electroporated into Sval049::glaA to obtain the Sval049::glaA-cas strain. Then, after electroporating the Sval049::glaA-cas strain into competent cells, the Ptarget-ΔCrr::glk-N20-donor plasmid was electroporated into the strain. After verification with Crr-YZ-F / R primers and sequencing, the Sval049::glaA::Δcrr::glk strain was obtained.

[0115] Example 5: Overexpression of the glucose metabolism-promoting protein gene sfsA (Sval049::glaA:Δcrr::glk)

[0116] SfsA, a protein that promotes glucose metabolism, is considered a transcriptional activator that can enhance the expression of genes related to starch metabolism. Overexpression of SfsA can increase amylase activity and increase the level of maltose-binding protein (encoded by the malE gene, which is a member of the E. coli pathway for maltose absorption and breakdown).

[0117] The J23109 promoter was knocked into the endogenous sfsA gene in *E. coli* Sval049::glaA:Δcrr::glk using CRISPR-Cas9 gene editing technology to overexpress the sfsA gene, thus constructing the *Sval049::glaA-Δcrr::glk-sfsA* strain. The specific procedures are as follows:

[0118] 5.1 Design the sfsA integration site N20 using the CHOPCHOP online website.

[0119] 5.2 Construct the Ptarget-sfsA-N20 plasmid.

[0120] Using the Ptarget plasmid as a template and sfsA-N20-sgF / R as primers, the P target plasmid was looped to obtain Ptarget-SfsA-N20, which was then transformed into TOP10 competent cells. PCR identification was performed using pTarget seq F / R primers, and the result was sent to Sangon Biotech for sequencing to obtain the Ptarget-SfsA-N20 plasmid. The amplification system and procedure for the looped P plasmid were the same as step 2.2 of Example 2.

[0121] 5.3 Construct the Ptarget-SfsA-N20-donor plasmid.

[0122] Using the Ptarget-SfsA-N20 plasmid as a template and target-CF / R as primers to linearize the plasmid, the linearized Ptarget-SfsA-N20 fragment was obtained.

[0123] Using the genomic DNA of Sval049 strain as a template and SfsA-UF / UR as primers, the UP-4 fragment was amplified;

[0124] Using the genomic DNA of strain Sval049 as a template and SfsA-DF / DR as primers, the DOWN-4 fragment was amplified;

[0125] The amplification system and amplification procedure for the UP-4 fragment and the DOWN-4 fragment are the same as step 2.2 in Example 2.

[0126] The linearized Ptarget-SfsA-N20 fragment, UP-4, and DOWN-4 fragments were cloned and ligated in one step using the Novizan ClonExpress Ultra One Step Cloning Kit. The ligation products were transformed into TOP10 competent cells, identified by PCR using pTargetseq F / R primers, and sequenced to obtain the Ptarget-SfsA-N20-donor plasmid.

[0127] 5.4. Insert the J23109 promoter into the sfsA gene promoter.

[0128] First, Sval049::glaA::Δcrr::glk electroporation competent cells were prepared. The pECCas plasmid was electroporated into Sval049::glaA::Δcrr::glk to obtain the Sval049::glaA::Δcrr::glk-cas strain. Then, the Sval049::glaA::Δcrr::glk-cas strain was further electroporated into competent cells. The Ptarget-SfsA-N20-donor plasmid was electroporated into the strain. After verification with SfsA-YZ-F / R primers and sequencing, the Sval049::glaA-Δcrr::glk-sfsA strain was obtained.

[0129] Example 6: Overexpression of the glucose metabolism-promoting protein gene sfsA (Sval049::glaA::malA)

[0130] The J23109 promoter was knocked into the endogenous sfsA gene of Escherichia coli Sval049::glaA::malA using CRISPR-Cas9 gene editing technology to overexpress the sfsA gene and construct the Sval049::glaA::malA-sfsA strain.

[0131] The specific steps are as follows:

[0132] 6.1 Design the sfsA integration site N20 using the CHOPCHOP online website.

[0133] 6.2 Constructing the Ptarget-sfsA-N20 plasmid: Refer to step 5.2 for construction.

[0134] 6.3 Construct the Ptarget-SfsA-N20-donor plasmid: Refer to step 5.3 for construction.

[0135] 6.4. Insert the J23109 promoter into the sfsA gene promoter.

[0136] First, Sval049::glaA::malA electroporation competent cells were prepared. The pECCas plasmid was electroporated into Sval049::glaA::malA to obtain the Sval049::glaA::malA-cas strain. Then, after preparing Sval049::glaA::malA-cas strain as electroporation competent cells, the Ptarget-SfsA-N20-donor plasmid was electroporated into Sval049::glaA::malA-cas. After verification with SfsA-YZ-F / R primers and sequencing, the Sval049::glaA::malA-sfsA strain was obtained.

[0137] Example 7: Cloning and integration of the glucokinase gene glk into the crr site (Sval049::glaA::malA-sfsA)

[0138] Using Sval049::glaA::malA-sfsA as the substrate bacteria, the glk gene from E. coli was integrated into the crr site of Sval049::glaA::malA-sfsA using CRISPR-Cas9 gene editing technology to construct the Sval049::glaA::malA-Δcrr::glk-sfsA strain.

[0139] The specific steps are as follows:

[0140] 7.1 Design the CRR integration site N20 using the CHOPCHOP online website.

[0141] 7.2 Constructing the Ptarget-crr-N20 plasmid: Refer to step 3.2 for construction.

[0142] 7.3 Constructing the Ptarget-Crr-N20-donor plasmid: Refer to step 4.3 for construction.

[0143] 7.4. The glk gene from E. coli was integrated into the Δcrr position.

[0144] First, Sval049::glaA::malA-sfsA electroporation competent cells were prepared. The pECCas plasmid was electroporated into Sval049::glaA::malA-sfsA to obtain the Sval049::glaA::malA-sfsA-cas strain. Then, using the Sval049::glaA::malA-sfsA-cas strain as electroporation competent cells, the Ptarget-Crr-N20-donor plasmid was electroporated into Sval049::glaA::malA-sfsA-cas. After verification with Crr-YZ-F / R primers and sequencing, the Sval049::glaA::malA-Δcrr::glk-sfsA strain was obtained.

[0145] Example 8: Fermentation production of L-valine by strain

[0146] The strains constructed in Examples 2-7 were fermented to produce L-valine. The fermentation process is as follows:

[0147] ① The strain was cultured in LB liquid medium at 37°C and 220 rpm in a shake flask.

[0148] The composition of LB liquid medium is: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride;

[0149] ② When the OD550 value of the strain in the shake flask reaches about 2.5, it is transferred to a 5L fermenter containing 4L of fermentation medium for anaerobic fermentation. The fermentation conditions are as follows: rotation speed 300rpm, temperature 37℃, pH controlled at 6.9±0.3; the residual sugar and L-valine content of the fermentation broth are tested at regular intervals.

[0150] The fermentation medium consisted of: 20 g / L liquid sugar, 3 g / L ammonium nitrate, 0.5 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.05 g / L FeSO4·7H2O, 0.02 g / L MnSO4·H2O, 0.3 g / L methionine, 0.3 g / L alanine, 0.3 g / L arginine, 2 g / L biotin, 2 g / L nicotinic acid, and 2 g / L pantothenic acid.

[0151] The liquid sugar is produced using a dual-enzyme sugar production process. Specifically, starch is first hydrolyzed and liquefied into small-molecule dextrins under the action of amylase, and then further hydrolyzed with saccharifying enzymes to obtain a liquid containing glucose.

[0152] ③ Stop fermentation when the residual glucose in the fermentation broth is detected to be below 2g / L, and record the fermentation cycle and the volume of the lower tank.

[0153] Fermentation effect detection and calculation:

[0154] ① L-valine content detection:

[0155] Take 2 mL of fermentation broth, centrifuge at 12000 rpm for 2 min to collect the supernatant, transfer 100 μL of the supernatant to 900 μL of sterile water, dilute 10 times, filter the supernatant using a 0.22 μm inorganic filter membrane, and inject the supernatant for analysis;

[0156] High-performance liquid chromatography (HPLC) conditions for L-valine detection: chromatographic column: Primesp 100 4.6*250mm, 5µm; mobile phase: 0.04mol / L potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:3 (V / V); wash mobile phase B: 75% acetonitrile; column temperature controlled at 30℃; flow rate: 1mL / min; injection volume: 10µL; detection wavelength: 205nm; retention time of characteristic peak of L-valine: 10.6min.

[0157] ② Glucose residual content detection: The amount of glucose residual in the fermentation broth was detected using a biosensor analyzer.

[0158] ③ Residual reducing sugar content detection: The residual reducing sugar content, including glucose, maltose, lactose, etc. in the fermentation broth was detected in accordance with the "Determination of Reducing Sugars in Food" (GB5009.7-2016).

[0159] Sugar-acid conversion rate = L-valine content (g / L) * lower tank volume (L) / 4 * initial sugar content (g / L);

[0160] The results are shown in Table 4 below.

[0161] Table 4: Fermentation results of each strain

[0162]

[0163]

[0164] As shown in Table 4, by genetically modifying the microorganisms to efficiently express saccharifying enzymes and / or maltases during fermentation, the disaccharides or polysaccharides in the effective hydrolysate sugars are converted into monosaccharides such as glucose, and the residual reducing sugar content is reduced from 4 g / L to 2 g / L, thus improving the sugar-acid conversion rate. Furthermore, by modifying the sugar-consuming pathways of the microorganisms and relieving the inhibitory effect of glucose catabolism, the strains can simultaneously utilize multiple sugar sources for fermentation, and the utilization rate of multiple sugar sources is further improved. This solves the problem of low metabolic efficiency of microorganisms for multiple sugar sources during fermentation, which is beneficial to reducing industrial production costs.

[0165] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

[0166] SEQ ID NO: 1: glaA, derived from Aspergillus niger

[0167] *

[0168] SEQ ID NO:2:malA,from Saccharolobus solfataricus

[0169] MQTIKIYENKGVYKVVIGEPFPPIEFPLEQKISSNKSLSELGLTIVQQGNKVIVEKSLDLKEHIIGLGEKAFELDRKRKRYVMYNVDAGAYKKYQDPLYVSIPLFISVKDGVATGYFFNSASKVIFDVGLEEYDKVIVTIPEDSVEFYVIEGPRIEDVLEKYTELTGKPFLPPMWAFGYMISRYSYYPQDKVVELVDIMQKEGFRVAGVFLDIHYMDSYKLFTWHPYRFPEPKKLIDELHKRNVKLITIVDHGIRVDQNYSPFLSGMGKFCEIESGELFVGKMWPGTTVYPDFFREDTREWWAGLISEWLSQGVDGIWLDMNEPTDFSRAIEIRDVLSSLPVQFRDDRLVTTFPDNVVHYLRGKRVKHEKVRNAYPLYEAMATFKGFRTSHRNEIFILSRAGYAGIQRYAFIWTGDNTPSWDDLKLQLQLVLGLSISGVPFVGCDIGGFQGRNFAEIDNSMDLLVKYYALALFFPFYRSHKATDGIDTEPVFLPDYYKEKVKEIVELRYKFLPYIYSLALEASEKGHPVIRPLFYEFQDDDDMYRIEDEYMVGKYLLYAPIVSKEESRLVTLPRGKWYNYWNGEIINGKSVVKSTHELPIYLREGSIIPLEGDELIVYGETSFKRYDNAEITSSSNEIKFSREIYVSKLTITSEKPVSKIIVDDSKEIQVEKTMQNTYVAKINQKIRGKINLE*

[0170] SEQ ID NO: 3: glaA, derived from Aspergillus niger

[0171]

[0172]

[0173] SEQ ID NO: 5: glk, derived from Escherichia coli MG1655 (P0A6V8)

[0174] MTKYALVGDVGGTNARLALCDIASGEISQAKTYSGLDYPSLEAVIRVYLEEHKVEVKDGCIAIACPITGDWVAMTNHTWAFSIAEMKKNLGFSHLEIINDFTAVSMAIPMLKKEHLIQFGGAEPVEGKPIAVYGAGTLGGVAHLVHVDKRWVSLPGEGGHV DFAPNSEEEAIILEILRAEIGHVSAERVLSGPGLVNLYRAIVKADNRLPENLKPKDITERALADSCTDCRRALSLFCVIMGRFGGNLALNLGTFGGVFIAGGIVPRFLEFFKASGFRAAFEDKGRFKEYVHDIPVYLIVHDNPGLLGSGAHLRQTLGHIL*

[0175] SEQ ID NO: 6: glk, derived from Escherichia coli MG1655 (P0A6V8)

[0176] atgacaaagtatgcattagtcggtgatgtgggcggcaccaacgcacgtcttgctctgtgtgatattgccagtggtgaaatctcgcaggctaagacctattcagggcttgattaccccagcctcgaagcggtcattcgcgtttatcttgaagaacataaggtcgaggtgaaagacggctgtattgccatcgcttgcccaattaccggtgactgggtggcgatgaccaaccatacctgggcgttctcaattgccgaaatgaaaaagaatctcggttttagccatctggaaattattaacgattttaccgctgtatcgatggcgatcccgatgctgaaaaaagagcatctgattcagtttggtggcgcagaaccggtcgaaggtaagcctattgcggtttacggtgccggaacggggcttggggttgcgcatctggtccatgtcgataagcgttgggtaagcttgccaggcgaaggcggtcacgttgattttgcgccgaatagtgaagaagaggccattatcctcgaaatattgcgtgcggaaattggtcatgtttcggcggagcgcgtgctttctggccctgggctggtgaatttgtatcgcgcaattgtgaaagctgacaaccgcctgccagaaaatctcaagccaaaagatattaccgaacgcgcgctggctgacagctgcaccgattgccgccgcgcattgtcgctgttttgcgtcattatgggccgttttggcggcaatctggcgctcaatctcgggacatttggcggcgtgtttattgcgggcggtatcgtgccgcgcttccttgagttcttcaaagcctccggtttccgtgccgcatttgaagataaagggcgctttaaagaatatgtccatgatattccggtgtatctcatcgtccatgacaatccgggccttctcggttccggtgcacatttacgccagaccttaggtcacattctgtaa。

Claims

1. Methods for constructing recombinant microorganisms, including: To confer or enhance the activity of saccharifying enzymes and / or maltases in microorganisms; Preferably, the saccharifying enzyme is derived from Aspergillus niger, and more preferably contains the amino acid sequence shown in SEQ ID NO: 1; the maltase is derived from Saccharolobus solfataricus, and more preferably contains the amino acid sequence shown in SEQ ID NO:

2.

2. The construction method according to claim 1, wherein: Further possessing or having enhanced activity of glucose metabolism-promoting proteins.

3. The construction method according to claim 1, wherein: Reduced activity of PTS system proteins; Preferably, the activity of the PTS system protein is reduced by knocking out the crr gene on the microbial genome.

4. The construction method according to claim 3, wherein: Further possessing or having enhanced glucokinase activity; Preferably, the glucokinase is derived from E. coli MG1655; more preferably, it contains the amino acid sequence shown in SEQ ID NO:

5.

5. The construction method according to any one of claims 1-4, wherein: The expression or overexpression of the gene encoding the enzyme is activated or enhanced using at least one regulatory element to confer or enhance the activity of the enzyme. Preferably, the regulatory element is selected from the J23109 promoter to regulate the expression of the glycated enzyme gene glaA; Preferably, the regulatory element is selected from the Trc promoter to regulate the expression of the maltase gene malA; Preferably, the regulatory element is selected from the J23109 promoter to regulate the expression of the glucokinase gene glK; Preferably, the regulatory element is selected from the J23109 promoter to regulate the expression of the sugar metabolism promoting protein gene sfsA.

6. Sugar-degrading microorganisms, which were constructed using the methods described above; Preferably, the sugar source contains disaccharides and / or polysaccharides, wherein the disaccharides or polysaccharides contain α-1,4 glycosidic bonds and / or α-1,6 glycosidic bonds.

7. The use of the recombinant microorganism according to claim 6 in any of the following A1)-A3): A1) Degrading disaccharides and / or polysaccharides into monosaccharides; A2) Improve the utilization rate of sugar sources in the fermentation system, wherein the sugar sources contain disaccharides and / or polysaccharides; A3) Fermentation to produce bio-based products, wherein the sugar source used in fermentation contains disaccharides and / or polysaccharides; The disaccharide or polysaccharide contains α-1,4 glycosidic bonds and / or α-1,6 glycosidic bonds.

8. Methods to improve the utilization rate of sugar sources by microorganisms, including: Genetic modification of microorganisms to give them or enhance their saccharifying enzymes and / or maltases.

9. The method according to claim 8, wherein: The sugar source contains disaccharides and / or polysaccharides, which are hydrolyzed into monosaccharides by saccharifying enzymes and / or maltases.

10. The method according to claim 8, wherein: The microorganism also has or has an enhanced sugar metabolism promoting protein SfsA; Preferably, the microorganism also has reduced-activity PTS system proteins; More preferably, the activity of the PTS system protein is reduced by knocking out the crr gene; Preferably, the microorganism also has or has enhanced glucokinase.

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