CN111154705B

CN111154705B - Geobacillus thermoglucosidase engineering bacteria and its construction method and application

Info

Publication number: CN111154705B
Application number: CN202010015867.XA
Authority: CN
Inventors: 李子龙; 杨志恒; 王为善; 张立新
Original assignee: East China University of Science and Technology; Institute of Microbiology of CAS
Current assignee: East China University of Science and Technology; Institute of Microbiology of CAS
Priority date: 2020-01-07
Filing date: 2020-01-07
Publication date: 2021-06-29
Anticipated expiration: 2040-01-07
Also published as: CN111154705A

Abstract

The invention provides a thermoglucosidase Geobacillus engineering bacterium, a construction method and application thereof. The invention constructs a series of high-temperature riboflavin-producing Geobacillus engineering bacteria by carrying out genetic engineering transformation on thermal glucosidase Geobacillus, introducing ribC (G199D) point mutation, sequentially knocking out purR, purA, ccpN and ldh genes, and then introducing an expression vector carrying a riboflavin synthetic gene cluster into the transformed engineering bacteria. Through determination, the yield of riboflavin after the Geobacillus is fermented for 24 hours after being modified by genetic engineering is 520 mg/L; the yield can reach more than 1g/L by carrying out mixed fermentation on the glucose and the xylose on the strain. The engineering bacteria provided by the invention have the advantages of high growth temperature, short generation time, utilization of cheap carbon sources and the like, and can be used for large-scale production of riboflavin at low cost.

Description

Bacillus thermoglucosidasius engineering bacterium and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering and fermentation engineering, in particular to a thermoglucosidase Geobacillus engineering bacterium, a construction method and application thereof.

Background

Riboflavin, vitamin B2, is one of the essential vitamins for bacteria and animals, and is usually present in organisms in the form of FMN and FAD, and participates in intracellular redox reactions. It is mainly applied in the fields of pharmacy, feed additives, food additives, cosmetics and the like, and at present, the riboflavin production process is a microbial fermentation method. In the early days, the production strains were fungi, such as Candida utilis (Candida famata), Ashbya gossypii (Ashbya gossypii), Eremothecium ashbyii, Saccharomyces cerevisiae (Saccharomyces cerevisiae), etc. (Stahmann et al, 2016, Appl Microbiol Biotechnol 100: 2107-. However, the production of riboflavin by fungi has some problems, such as long fermentation period (6-7 days), low yield (less than 5g/L), complex raw material component ratio, high thallus viscosity, difficult later separation, and the need of adding unsaturated fatty acid to promote riboflavin synthesis. Subsequently, genetically engineered bacteria using bacteria as host bacteria were developed. The genetic engineering bacteria have the advantages of simple raw material requirement, high yield, mature prokaryotic cell genetic engineering technology and the like, and quickly replace the production technology utilizing fungi. Currently, Bacillus subtilis (Bacillus subtilis) becomes a main industrial riboflavin production strain (Susanne et al, 2016, applied Microbiol Biotechnol 100: 2107-.

The fermentation production of the bacillus subtilis belongs to normal-temperature fermentation, and compared with the high-temperature fermentation of the geobacillus thermoglucosidases, the fermentation production of riboflavin has the obvious disadvantages that: firstly, the energy consumption is high. Firstly, a large amount of cooling water is needed in the normal-temperature fermentation production process to relieve the biological heat in the fermentation process so as to maintain the optimal growth temperature of the produced strains in the normal-temperature fermentation process, the consumption of the cooling water in the normal-temperature fermentation process accounts for about 30 percent of the production cost, and the high-temperature fermentation can reduce or even not use the cooling water. And secondly, the high-temperature sterilization cooling time of equipment raw materials and the like is short, and energy and water are saved. Secondly, the production period is long, and the growth generation time of the bacillus subtilis reaches 40-120 min. The generation time of the thermal glucosidase Geobacillus can reach 16min at the fastest speed, and is shorter than that of the Bacillus subtilis. Because the reaction rate of enzymes in cells is improved corresponding to the fast growth speed of the Geobacillus, the riboflavin can be produced more quickly due to the fast reaction speed of the enzymes for synthesizing the riboflavin. Thirdly, the patent barrier is obvious, most of the patents of the bacillus subtilis for producing riboflavin are mastered by large foreign companies, and the research and the application of the bacillus subtilis at home are puzzled. And fourthly, the raw materials are expensive. The geobacillus thermoglucosidasius can utilize cheaper carbon source lignocellulose hydrolysate, and is cheaper than raw materials such as glucose and the like used in the fermentation process of bacillus subtilis; fifthly, the fermentation process is simplified. Riboflavin produced by bacillus subtilis and other normal temperature bacteria is fermented at 37 ℃, and a large amount of mixed bacteria can grow at 37 ℃, so that the bacteria are easily infected in the fermentation process. However, few mixed bacteria can grow at the temperature of more than 50 ℃, so the high-temperature fermentation of the geobacillus is a barrier for naturally preventing the mixed bacteria from being infected, a large amount of equipment is not required to be invested to prevent the bacteria from being infected in the fermentation process, and the fermentation process is simplified. Therefore, compared with the bacillus subtilis engineering bacteria, the geobacillus thermophilus has unique advantages and application values. Therefore, the development of high-temperature riboflavin production process is the direction of future riboflavin industry development.

Disclosure of Invention

The invention aims to provide a thermoglucosidase Geobacillus engineering bacterium, a construction method and application thereof.

In order to realize the purpose of the invention, in the first aspect, the invention provides a Geobacillus thermosulfidobacterium engineering bacterium GT-01S, the engineering bacterium GT-01S is constructed by introducing point mutation on the ribC gene of Geobacillus thermosulfidobacterium (Geobacillus thermosulfidosius) through a genetic engineering technology to change the 199 th amino acid residue of the encoded protein from G to D, the modified strain is named as GT-01, and then an expression vector carrying a riboflavin synthetic gene cluster for expressing Geobacillus thermosulfidoensis (Geobacillus modelicinficans) is introduced into the strain GT-01; wherein, the nucleotide sequence of the ribC gene is shown as SEQ ID NO:1 is shown.

Preferably, the geobacillus thermoglucosidase is DSM 2542.

In the second aspect, the invention provides a thermoglucosidase Geobacillus engineering bacterium GT-02S, wherein the engineering bacterium GT-02S is obtained by knocking out purA gene in a strain GT-01 through a genetic engineering technology, naming the modified strain as GT-02 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a Geobacillus thermodenitrificans into the strain GT-02; wherein, the nucleotide sequence of purA gene is shown as SEQ ID NO:2, respectively.

In the third aspect, the invention provides a thermoglucosidase Geobacillus engineering bacterium GT-03S, the engineering bacterium GT-03S is obtained by knocking out purR gene in a strain GT-02 through a genetic engineering technology, naming the modified strain as GT-03 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a Geobacillus thermodenitrificans into the strain GT-03 for construction; wherein, the nucleotide sequence of purR gene is shown as SEQ ID NO:3, respectively.

In a fourth aspect, the invention provides a thermoglucosidase Geobacillus engineering bacterium GT-04S, wherein the engineering bacterium GT-04S is obtained by knocking out the ccpN gene in a strain GT-03 through a genetic engineering technology, naming the modified strain as GT-04, and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a Geobacillus thermodenitrificans into the strain GT-04 for construction; wherein the nucleotide sequence of the ccpN gene is shown as SEQ ID NO:4, respectively.

In the fifth aspect, the invention provides a thermoglucosidase Geobacillus engineering bacterium GT-05S, wherein the engineering bacterium GT-05S is obtained by knocking out ldh gene in a strain GT-04 through a genetic engineering technology, naming the modified strain as GT-05 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a Geobacillus thermodenitrificans into the strain GT-05 for construction; wherein the nucleotide sequence of the ldh gene is shown as SEQ ID NO:5, respectively.

Preferably, in the present invention, the expression vector carrying the riboflavin synthesis gene cluster of Bacillus thermodenitrificans is pUCG3.8-R2. See CN108641992 a. The plasmid pUCG3.8-R2 was constructed in the following manner:

(1) carrying out PCR amplification by using the plasmid pTAC-RiboJ-gfp as a template and primers sgfps and sgfpa to obtain a sequence of sgfp and a terminator T3; carrying out PCR amplification by using a Geobacillus thermodenitrificans genome as a template and using primers Pldhs and Pldha to obtain a promoter pLdh sequence; then obtaining a pLdh-sgfp-T3 sequence through fusion PCR; carrying out EcoRI and BamHI double enzyme digestion on pUCG3.8 and pLdh-sgfp-T3, then carrying out T4 ligase ligation, carrying out transformation, and screening kanamycin resistance to obtain a recombinant plasmid pUCG3.8S;

(2) using pUCG3.8S as a template, and performing PCR amplification by using primers pucG3.81s and pucG3.81a to obtain a vector framework sequence containing a pLdh and a T3 terminator;

(3) using Geobacillus thermosulfinitificans (CGMCC 1.5331) genome as a template, and amplifying by using primers Dnribs and Dnriba to obtain a riboflavin synthesis gene cluster dnrib;

(4) gibson assembly of the vector backbone sequence containing pLdh and T3 terminator in the step (2) and the riboflavin synthetic gene cluster dnrib in the step (3), transformation of JM109, and screening to obtain recombinant plasmid pUCG3.8-R2.

The primer sequences used are shown in table 1:

TABLE 1

Plasmid pTAC-RiboJ-gfp can be found in Lou, C., Stanton, B., Chen, Y.J., Munsky, B., & Voigt, C.A. (2012). Ribozyme-based insulator parts buffer synthetic from genetic engineering, Nature biotechnology, 30(11), 1137.

In a sixth aspect, the invention provides a construction method of a thermoglucosidase Geobacillus engineering bacterium, which comprises the following steps:

A. construction of pUB31-sfGFP plasmid

A1, a backbone sequence of pUB31 plasmid amplified by primers Vm-F and Vm-R with pUB31 plasmid as a template;

a2, taking the genome DNA of Geobacillus thermodenitrificans as a template, and amplifying by using primers Pm-F and Pm-R to obtain a promoter fragment; wherein, the nucleotide sequence of the promoter fragment is shown as SEQ ID NO:6 is shown in the specification;

a3, using pTAC-RiboJ-GFP plasmid as template, amplifying with primers sfGFP-F and sfGFP-R to obtain GFP fragment;

a4, assembling the framework sequence, the promoter fragment and the GFP fragment by Gibson to obtain a plasmid pUB 31-sfGFP;

B. construction of the GT-01 Strain

B1, using the genome DNA of geobacillus thermoglucosidasius DSM2542 as a template, and respectively amplifying an upstream fragment and a downstream fragment of the ribC gene containing the point mutation by using primers ribCm1 and ribCa containing the point mutation and primers ribCs and ribCm2 containing the point mutation;

b2, using plasmid pUB31-sfGFP as a template, and amplifying by using primers pUB31s and pUB31a to obtain a vector fragment;

b3, assembling upstream and downstream fragments of B1 and a vector fragment of B2 through Gibson to obtain a ribC point mutation plasmid pUB31-ribC, introducing the plasmid into geobacillus thermoglucosidase DSM2542, and mutating ribC through a homologous recombination method to obtain a strain GT-01;

C. construction of the GT-02 Strain

C1, respectively amplifying primers purA up-F and purA up-R and purA down-F and purA down-R by using the genome DNA of geobacillus thermoglucosidase DSM2542 as a template to obtain upstream and downstream fragments of the purA gene;

c2, using plasmid pUB31-sfGFP as a template, and amplifying by using primers pUB1-F and pUB31-R to obtain a vector fragment;

c3, assembling upstream and downstream fragments of C1 and a vector fragment of C2 through Gibson to obtain purA gene knockout plasmid pUB31-purA, introducing the plasmid into a strain GT-01, and knocking out purA through a homologous recombination method to obtain a strain GT-02;

D. construction of the GT-03 Strain

D1, respectively amplifying primers purR up-F and purR up-R and purR down-F and purR down-R by using the genome DNA of geobacillus thermoglucosidase DSM2542 as a template to obtain upstream and downstream fragments of purR gene;

d2, using plasmid pUB31-sfGFP as a template, and amplifying by using primers pUB1-F and pUB31-R to obtain a vector fragment;

d3, assembling upstream and downstream fragments of D1 and a vector fragment of D2 through Gibson to obtain purR gene knockout plasmid pUB31-purR, introducing the plasmid into a strain GT-02, and knocking out purR through a homologous recombination method to obtain a strain GT-03;

E. construction of the GT-04 Strain

E1, respectively amplifying primers ccpN up-F and ccpN up-R and primers ccpN down-F and ccpN down-R by using the genome DNA of geobacillus thermoglucosidase DSM2542 as a template to obtain upstream and downstream fragments of a ccpN gene;

e2, using plasmid pUB31-sfGFP as a template, and amplifying by using primers pUB1-F and pUB31-R to obtain a vector fragment;

e3, assembling upstream and downstream fragments of E1 and a vector fragment of E2 through Gibson to obtain purR gene knockout plasmid pUB31-ccpN, introducing the plasmid into a strain GT-03, and knocking out ccpN through a homologous recombination method to obtain a strain GT-04;

F. construction of the GT-05 Strain

F1, respectively amplifying primers ldh up-F and 1dh up-R and ldh down-F and ldh down-R by using the genome DNA of Geobacillus thermosaccharase DSM2542 as a template to obtain the upstream and downstream fragments of the ldh gene;

f2, using plasmid pUB31-sfGFP as a template, and amplifying by using primers pUB1-F and pUB31-R to obtain a vector fragment;

f3, assembling upstream and downstream fragments of F1 and a vector fragment of F2 through Gibson to obtain purR gene knockout plasmid pUB31-ldh, introducing the plasmid into a strain GT-04, and knocking out ldh through a homologous recombination method to obtain a strain GT-05;

G. introducing an expression vector pUCG3.8-R2 carrying a riboflavin synthetic gene cluster of the geobacillus thermosaccharzicola into a strain GT-05 to construct the geobacillus thermosaccharzicola engineering strain.

The primer sequences used are shown in Table 2:

TABLE 2

In a seventh aspect, the invention provides an application of the engineering bacteria or the engineering bacteria constructed according to the method in the production of riboflavin by fermentation.

The application comprises the following steps: carrying out strain fermentation by using an improved ASYE culture medium, wherein the concentration of kanamycin in the culture medium is 12.5 mu g/mL; the fermentation conditions are 50-70 deg.C (preferably 60 deg.C), 250 rpm.

Preferably, the glucose component of the medium is replaced by a mixed sugar obtained by mixing glucose and xylose in a weight ratio of 1:5 to 4: 5. More preferably, the weight ratio of glucose to xylose in the mixed sugar is 1: 2, and the total concentration of the mixed sugar is 0.2M.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention provides a series of Geobacillus geobacillus (Geobacillus geobacillus engineering bacteria of thermal glucosidase) for producing riboflavin at high temperature. The Geobacillus has the advantages of high growth temperature, short generation time, utilization of cheap carbon sources and the like, so that the defects of high energy consumption, long production period, high cost, complex fermentation process and the like in the production of riboflavin by using normal-temperature bacteria are overcome. Thereby achieving the purpose of upgrading the riboflavin industry. The riboflavin concentration in the fermentation broth was estimated by means of a standard established by the riboflavin UV absorption, the genetically engineered strain (GT-05S) G.thermoglucopyranosasius DSM2542(ribC (G199D),^ΔpurR，ΔpurA，^ΔccpN, delta ldh) fermentation yield of riboflavin of 520mg/L after 24 h; the yield can reach more than 1g/L by carrying out mixed fermentation on the glucose and the xylose on the strain.

Drawings

FIG. 1 is a schematic structural diagram of a gene-editing plasmid pUB31-sfGFP in a preferred embodiment of the present invention.

FIG. 2 is a diagram of a validated pyrE gene knock-out gel in a preferred embodiment of the invention. In the experimental groups, 1 to 12 are indicated as experimental groups, "+" indicates a positive control, and "-" indicates a negative control. Wherein 1, 2, 5, 8 and 10 are monoclonal strains with successful knockout.

FIG. 3 is a schematic diagram showing the structure of ribC (G199D) point mutation editing plasmid pUB31-ribC in the preferred embodiment of the present invention.

FIG. 4 shows the sequencing result of ribC (G199D) on the genome of Bacillus thermoglucosidasius engineering bacteria in the preferred embodiment of the present invention. The upper part of the figure is the ribC partial sequence on the genome and the lower part of the figure is the sequencing result. Wherein, the 199 site amino acid is mutated from G to D, and the mutation is successful.

FIG. 5 is a standard curve of riboflavin content and 444nm absorbance for the preferred embodiment of the invention.

FIG. 6 shows the fermentation yields of GT-00S and GT-01S strains containing the riboflavin gene cluster in the preferred embodiment of the present invention. The riboflavin production of GT-00S was 28.7mg/L, and the riboflavin production of GT-01S was 84.5 mg/L.

FIG. 7 is a schematic diagram showing the structure of plasmid pUB31-purA in a preferred embodiment of the present invention.

FIG. 8 is a graph of purA knockout validation gel of strain GT-01 in a preferred embodiment of the invention.

FIG. 9 shows the purA knockout sequencing validation of strain GT-01 in a preferred embodiment of the invention.

FIG. 10 shows the riboflavin production by the strain GT-01S and the strain GT-02S in the preferred embodiment of the present invention.

FIG. 11 is a schematic structural view of plasmid pUB31-purR in accordance with a preferred embodiment of the present invention.

FIG. 12 shows the purR knockout offset validation of the GT-02 strain in a preferred embodiment of the present invention.

FIG. 13 shows the purR knockout sequencing validation of the GT-02 strain in a preferred embodiment of the present invention.

FIG. 14 shows riboflavin production by the GT-02S strain and the GT-03S strain in a preferred embodiment of the present invention.

FIG. 15 is a diagram showing the plasmid structure of pUB31-ccpN in the preferred embodiment of the present invention.

FIG. 16 shows the ccpN knockout offset validation of GT-03 strain in a preferred embodiment of the invention.

FIG. 17 shows the ccpN knockout sequencing validation of GT-03 strain in a preferred embodiment of the invention.

FIG. 18 shows the riboflavin production by the strain GT-03S and the strain GT-04S in the preferred embodiment of the present invention.

FIG. 19 is a schematic diagram showing the plasmid structure of pUB31-ldh in accordance with a preferred embodiment of the present invention.

FIG. 20 is an ldh knockout offset validation of GT-04 strain in a preferred embodiment of the invention.

FIG. 21 is an ldh knockout offset validation of the GT-04 strain in a preferred embodiment of the invention.

FIG. 22 shows the riboflavin production by the strain GT-04S and the strain GT-05S in the preferred embodiment of the present invention.

FIG. 23 shows the riboflavin production of the strain GT-05S in the preferred embodiment of the present invention using glucose or glucose xylose mixed sugar as the carbon source.

Detailed Description

The invention provides a Geobacillus (Geobacillus thermoglucosidasius engineering bacteria) for producing riboflavin at a high temperature, a construction method and application thereof.

The current gene editing tool kit of thermophilic bacteria is not perfect, and only few markers are available (such as high temperature resistant kan)^RResistance), making back-screening of homologous recombination difficult. Kan-containing plasmid obtained by introducing sfGFP into a temperature-sensitive editing plasmid pUB31^RAnd the green fluorescent double Marker editing plasmid pUB 31-sfGFP. The wanted double-exchange strain can be rapidly screened by a fluorescent Marker. The efficiency of gene editing using homologous recombination is greatly improved.

After the gene editing method was established, gene editing plasmids were constructed, and specifically modified genes included ribC (AOT13_09320), purA (AOT13_03190), purR (AOT13_02815), ccpN (AOT13_ 169900), ldh (AOT13_ 05975). The sequence was obtained from NCBI. According to the annotation, the lengths of the upstream and downstream fragments of purA (AOT13_03190), purR (AOT13_02815), ccpN (AOT13_ 169900) and ldh (AOT13_05975) are about 1kb respectively, primers are designed, and the upstream and downstream fragments of each gene are obtained by PCR. For ribC (AOT 13-09320), the modification site is (G199D), primers are designed to amplify fragments about 1kb each of the upstream and downstream of the mutation site, and the mutation site is introduced by the primers. Then designing primers, carrying out PCR amplification by taking pUB31-sfGFP as a template to obtain a vector fragment, and carrying out Gibson assembly on the vector and 3 fragments of upstream and downstream fragments of each gene respectively to obtain editing plasmids pUB31-ribC, pUB31-purA, pUB31-purR, pUB31-ccpN and pUB 31-ldh. The constructed plasmid is verified to be correct through enzyme digestion.

The gene editing plasmid is transformed into Geobacillus through electrotransformation, and gene editing is carried out through fluorescent protein-assisted homologous recombination. Whether the editing was successful was verified by PCR and sequencing. And constructing a successful strain for fermentation. The fermentation medium is a modified ASYE medium. Fermentation is carried out at 50-70 deg.C (preferably 60 deg.C) and 250 rpm. Because the product riboflavin has an ultraviolet absorption peak at 444nm, a standard curve is established according to the absorption value, and then the riboflavin concentration in the fermentation broth is estimated according to the standard curve (Shi, T., Wang, Y., Wang, Z., Wang, G., Liu, D., Fu, J., & ZHao, X. (2014.) the calibration of the gene pathway in Bacillus subtilis and its use in riboflavin biosynthesis. microbial factors, 13(1), 101). The fermentation result shows that the yield of each step of fermentation is obviously improved compared with the yield of the previous step of modification. Finally, by performing mixed fermentation using glucose and xylose, the yield is further increased.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

In the present invention, the electroporation method and the modified ASYE medium can be referred to Lin, P.P., Rabe, K.S., Takasumi, J.L., Kadisch, M., Arnold, F.H., Liao, J.C (2014), Isobutanol production at extracted temporal in therapeutic Geobacillus thermoglucosylsidus.Metabolic engineering, 24, 1-8.

The primer sequences used in the following examples are shown in Table 2.

Example 1 construction of Geobacillus for high temperature Riboflavin production

1. Establishment of Gene editing method

1.1 construction of pUB31-sfGFP plasmid

Primer Vm-F/Vm-R for amplifying the skeleton of pUB31 plasmid (Acetate amplification in Geobacillus thermoglucosaccharidus and strain engineering for enhanced bioethanol production Hills, C. 1Ju 2015) is designed and amplified by primer6.0 software to obtain pUB31 vector. Geobacillus thermosphakii NG80-2 strain is a laboratory storage strain (Garg, N., Tang, W., Goto, Y., Nair, S.K., & van der Donk, W.A. (2012). Lantibiotics from Geobacillus thermosphakii proceedings of the National Academy of Sciences, 109 (14); 5241) and the genome of G.thermospaticus NG80-2 is extracted by a bacterial genome extraction kit from Beijing Tiangen and the genome is amplified by primer Pm-F/Pm-R designed by primer6.0 software to obtain a promoter fragment (SEQ ID NO: 6). A primer sfGFP-F/sfGFP-R is designed and amplified to obtain a GFP fragment by using pTAC-RiboJ-GFP plasmid (Lou, C., Stanton, B., Chen, Y.J., Munsky, B., & Voigt, C.A. (2012). Ribozyme-based insulator parts buffer synthetic from genetic engineering, Nature biotechnology, 30(11), 1137) as a template, and the three fragments are assembled by Gibson to obtain an edited plasmid pUB31-sfGFP (FIG. 1).

1.2 Gene editing method

Selecting four regions of monoclonals grown from the plates with the resistance after electric transformation, streaking the monoclonals on new plates with the resistance, and putting the plates into an incubator at 68 ℃ for culture. ② selecting the monoclonal without green fluorescence on the fourth area to the non-anti LB liquid culture medium (containing Mg)²⁺，Fe²⁺，Ca²⁺) Shaking at 220rpm at 60 ℃ overnight. ③ taking 100 mu L of bacterial liquid to 900 mu L of water, marking as 10^-1Mixing, adding 100 μ L bacterial liquid into 900 μ L water, and recording as 10^-2Sequentially diluting to 10^-5

Take

10^-4，10^-5Each 100. mu.L of the gradient diluted bacterial solution was uniformly applied to a TSA non-resistant solid plate, and cultured in an incubator at 60 ℃. (iv) selecting the non-fluorescent monoclonal, spreading the monoclonal on the non-anti-TSA solid plate and the anti-TSA solid plate (containing 12.5. mu.g/mL kanamycin) respectively, and culturing in an incubator at 60 ℃. Fifthly, selecting the monoclonal antibody which does not grow on the resistant (containing 12.5 mu g/mL kanamycin) plate, and carrying out colony PCR on the monoclonal antibody which grows on the non-resistant plate. During colony PCR, PCR product gel running with genome as template is used as negative control, and PCR product gel running with editing plasmid as template is used as positive control. PCR products using a single clone as a template were run as an experimental group. The size of the band was verified by running gel and sequencing.

1.3 validation of feasibility of Gene editing methods by knocking out pyrE Gene

1.3.1 construction of the pyrE knock-out plasmid pUB31-pyrE

Genomic information of Geobacillus thermoglucosidasius was obtained from NCBI, pyrE (AOT13_08755) was found by annotation, and compared with the pyrE gene sequence of Bacillus subtilis by BLAST to determine higher similarity, which is similar in gene structure. Geobacillus thermosaccharidus DSM2542 is a laboratory storage strain, a bacterial genome extraction kit of Beijing Tiangen company is used for extracting the genome of Geobacillus thermosaccharidus, and primer6.0 software is used for designing and amplifying a primer pyrE up-F/pyrE up-R at the upstream of pyrE gene and a primer pyrE down-F/pyrE down-R at the downstream of pyrE gene. The upstream and downstream pieces of pyrE gene were obtained by PCR amplification. Primers pUB31-F/pUB31-R were designed and vector fragments were amplified by PCR using plasmid pUB31-sfGFP as template, and then edited plasmid pUB31-pyrE was knocked out by Gibson assembly.

1.3.2 validation of feasibility of Gene editing methods

The pyrE gene on the genome of Geobacillus thermosulfidovum DSM2542 was knocked out by the "gene editing method" described above, and then the knock-out of the pyrE gene on the genome was completed as seen from the gel images by colony PCR of the growing single clone and then gel run verification by agarose gel electrophoresis (FIG. 2).

2. Construction of ribC (G199D) Strain

2.1 construction of Gene editing plasmids

Genomic information of geobacillus thermoglucosidase was obtained from NCBI, ribC was found by annotation (AOT13 — 09320), and compared with the ribC gene sequence of bacillus subtilis by BLAST to determine higher similarity, and the gene structure was similar. Geobacillus thermosaccharidus DSM2542 is a laboratory stock strain, the genome of Geobacillus thermosaccharidus is extracted by using a bacterial genome extraction kit of Beijing Tiangen, and primer6.0 software is used for designing and amplifying a primer ribCm1/ribCa upstream of a ribC (G199D) point mutation and a primer ribCs/ribCm2 downstream of the ribC point mutation. Upstream and downstream fragments of ribC (G199D) were obtained by PCR amplification. Primers pUB31s/pUB31a were designed and vector fragments were obtained by PCR amplification using plasmid pUB31-sfGFP as template, followed by Gibson assembly to obtain ribC point mutation editing plasmid pUB31-ribC (see FIG. 3 for plasmid map).

Genome editing of thermoglucopyranosasius DSM2542(ribC (G199D)) 2.2G

The gene editing method is the same as the above-mentioned "gene editing method" in that the point mutation changes only a single base on the genome, and it is impossible to see whether the point mutation is successful or not by looking at the change in the band size through a gel chart. Thus, the strain GT-01 was obtained by sequencing verification, which showed successful point mutation (see FIG. 4).

2.3 introduction of Riboflavin Gene Cluster and fermentation

A plasmid pUCG3.8-R2 (see CN108641992A) containing the G.Thermodernificans NG80-2 riboflavin gene cluster was transferred to a wild-type strain G.Thermogrosidasius DSM2542 by the electroporation method to obtain a strain GT-00S, and was transferred to a strain GT-01 to obtain a strain GT-01S. GT-01S strain was fermented for 24 hours in modified ASYE medium at a kanamycin concentration of 12.5. mu.g/mL. The fermentation conditions were 60 ℃ and 250 rpm.

2.4 Riboflavin yield determination method and fermentation results

Since riboflavin has an ultraviolet absorption at 444nm (Shi, t., Wang, y., Wang, z., Wang, g., Liu, d., Fu, Zhao, X. (2014). specification of a lipid pathway in Bacillus subtilis and an enzyme in riboflavin biochemical factors, 13(1), (101)), the yield of riboflavin can be calculated by establishing a standard curve of riboflavin standard samples and ultraviolet absorption, and then measuring the absorption of the samples at 444nm by an ultraviolet spectrophotometer. The calculation formula obtained from the standard curve is that y is 66.605x-1.3864 (R)²0.9993). The standard curve is shown in FIG. 5. The yield of the strain GT-00S was 28mg/L and the riboflavin yield of the strain GT-01S was 84mg/L as calculated from the absorbance after 24 hours of fermentation as the yield (FIG. 6). As is clear from the fermentation results, the amount of riboflavin produced was increased 3-fold by introducing ribC (G199D) into the gene.

3. Construction of purA knockout strains

3.1 construction of Gene editing plasmids

Genome information of geobacillus thermoglucosidase was obtained from NCBI, purA (AOT13 — 03190) was found by annotation, and compared with purA gene sequence of bacillus subtilis by BLAST to determine higher similarity, and the gene structure was similar. Geobacillus thermosaccharidase G.thermosaccharidasius DSM2542 is a laboratory storage strain, a bacterial genome extraction kit of Beijing Tiangen company is used for extracting the genome of Geobacillus thermosaccharidase, and primer6.0 software is used for designing and amplifying a primer purA up-F/purA up-R at the upstream of purA gene and a primer purA down-F/purA down-R at the downstream of the purA gene. The upstream and downstream fragments of purA gene were obtained by PCR amplification. Primers pUB1-F/pUB31-R were designed and vector fragments were obtained by PCR amplification using plasmid pUB31-sfGFP as a template, and then assembled by Gibson to obtain the purA knockout editing plasmid pUB31-purA (see FIG. 7 for plasmid map).

3.2 purA knockout of GT-01 Strain

The gene editing method was the same as the "gene editing method" described above, and whether the purA gene of the strain GT-01 was knocked out was examined by gel mapping (see FIG. 8). As can be seen from the gel images, monoclonal 2, 3, 5, 6, 7, 8, 9 has successfully completed purA knockout, and further sequence verification (see FIG. 9 for sequence verification) will name GT-02 as the purA knockout GT-01 strain that was correctly sequenced.

3.3 introduction of Riboflavin Gene Cluster and fermentation

The riboflavin gene cluster plasmid pUCG3.8-R2 was transferred into the strain GT-02 by electrotransfer to obtain the strain GT-02S. GT-02S strain was fermented for 24 hours in modified ASYE medium at a kanamycin concentration of 12.5. mu.g/mL. The fermentation conditions were 60 ℃ and 250 rpm.

3.4 Riboflavin production assay

The yield of the strain GT-02S was 150.4mg/L and the riboflavin yield of the strain GT-01S was 84mg/L as calculated from the absorbance after 24 hours of fermentation as the yield (FIG. 10). From the fermentation results, it was found that the knockout of purA (AOT 13-03190) gene increased the yield of riboflavin by 1.79-fold.

4. Construction of purR knockout strains

4.1 construction of Gene editing plasmids

Genomic information of geobacillus thermoglucosidase was obtained from NCBI, purR (AOT13 — 02815) was found by annotation, and compared with purR gene sequence of bacillus subtilis by BLAST, it was confirmed that it has higher similarity and its gene structure is similar. Geobacillus thermosaccharidus DSM2542 is a laboratory storage strain, a bacterial genome extraction kit of Beijing Tiangen company is used for extracting the genome of Geobacillus thermosaccharidus, and primer6.0 software is used for designing and amplifying a primer purR up-F/purR up-R at the upstream of a purR gene and a primer purR down-F/purR down-R at the downstream of the purR gene. The upstream and downstream fragments of purR gene were obtained by PCR amplification. Primers pUB1-F/pUB31-R were designed and amplified by PCR using plasmid pUB31-sfGFP as a template to obtain a vector fragment, which was then assembled by Gibson to obtain purR knock-out editing plasmid pUB31-purR (see FIG. 11 for schematic of plasmid map)

4.2 purR knockout of GT-02 strain

The gene editing method was the same as the "gene editing method" described above, and whether the purR gene of the strain GT-02 was knocked out was examined by gel permeation (see FIG. 12). As can be seen from the gel images, the purR knockout was successfully completed and further verified by sequencing (the sequencing verification result is shown in FIG. 13), and the correctly sequenced purR knocked-out GT-02 strain was named GT-03.

4.3 introduction of Riboflavin Gene Cluster and fermentation

The riboflavin gene cluster plasmid pUCG3.8-R2 was transferred into the strain GT-03 by electrotransfer to obtain the strain GT-03S. GT-03S strain was fermented for 24 hours in modified ASYE medium at a kanamycin concentration of 12.5. mu.g/mL. The fermentation conditions were 60 ℃ and 250 rpm.

4.4 Riboflavin production assay

The yield of the strain GT-03S was 180.2mg/L and the riboflavin yield of the strain GT-02S was 150.4mg/L, as calculated from the absorbance after 24 hours of fermentation as the yield (FIG. 14). As is clear from the fermentation results, the knockout of purR (AOT 13-02815) gene increased the yield of riboflavin by 1.2-fold.

5. construction of ccpN knockout strains

5.1 construction of Gene editing plasmids

Genome information of geobacillus thermoglucosidase was obtained from NCBI, ccpN (AOT13 — 169900) was found by annotation, and compared with the ccpN gene sequence of bacillus subtilis by BLAST, it was confirmed that it has higher similarity and similar gene structure. Geobacillus thermosaccharidase G.thermosaccharidasius DSM2542 is a laboratory storage strain, a bacterial genome extraction kit of Beijing Tiangen company is used for extracting the genome of Geobacillus thermosaccharidase, and primer6.0 software is used for designing and amplifying a primer ccpN up-F/ccpN up-R at the upstream of a ccpN gene and a primer ccpN down-F/ccpN down-R at the downstream of the ccpN gene. The upstream and downstream fragments of the ccpN gene were obtained by PCR amplification. Primers pUB1-F/pUB31-R were designed and vector fragments were obtained by PCR amplification using plasmid pUB31-sfGFP as a template, followed by Gibson assembly to obtain the ccpN knockout editing plasmid pUB31-ccpN (see FIG. 15 for schematic of plasmid map).

5.2 ccpN knockout of GT-03 Strain

Gene editing methods As in the "Gene editing methods" described above, whether the ccpN gene of the strain GT-03 was knocked out was examined by gel mapping (see FIG. 16). As can be seen from the gel map, the ccpN knockout had been successfully completed and further verified by sequencing (see fig. 17 for sequencing verification results), the correctly sequenced ccpN knockout GT-03 strain was named GT-04.

5.3 introduction of Riboflavin Gene Cluster and fermentation

The riboflavin gene cluster plasmid pUCG3.8-R2 was transferred to the strain GT-04 by electrotransfer to obtain the strain GT-04S. GT-03S strain was fermented for 24 hours in modified ASYE medium at a kanamycin concentration of 12.5. mu.g/mL. The fermentation conditions were 60 ℃ and 250 rpm.

5.4 Riboflavin production assay

The yield of the strain GT-04S was 295.12mg/L as calculated from the absorbance after 24h of fermentation as the yield, while the riboflavin yield of the strain GT-03S was 180.2mg/L (FIG. 18). From the fermentation results, it was found that the deletion of the ccpN (AOT 13-169900) gene increased the production of riboflavin by 1.6-fold.

6. Construction of ldh knockout strains

6.1 construction of Gene editing plasmids

Genomic information of geobacillus thermoglucosidasius was obtained from NCBI, ldh (AOT13 — 05975) was found by annotation, and compared with ldh gene sequence of bacillus subtilis by BLAST to determine higher similarity, and the gene structure was similar. Geobacillus thermosaccharidus DSM2542 is a laboratory storage strain, a bacterial genome extraction kit of Beijing Tiangen company is used for extracting the genome of Geobacillus thermosaccharidus, and primer6.0 software is used for designing and amplifying primer ldh up-F/ldh up-R at the upstream of ldh gene and primer ldh down-F/ldh down-R at the downstream of ldh gene. The upstream and downstream fragments of ldh gene were obtained by PCR amplification. Primers pUB1-F/pUB31-R were designed and amplified by PCR using plasmid pUB31-sfGFP as template to obtain vector fragments, which were then assembled by Gibson to obtain ldh-knocked-out editing plasmid pUB31-ldh (see FIG. 19 for schematic of plasmid map).

6.2 ldh knockout of GT-04 Strain

Gene editing methods As in the "Gene editing methods" described above, whether the ldh gene of the strain GT-04 was knocked out was examined by gel plot (see FIG. 20). As can be seen from the gel images, the ldh knockout had been successfully completed and further verified by sequencing (see FIG. 21 for sequencing verification), the correctly sequenced ldh knocked out GT-04 strain was named GT-05.

6.3 introduction of Riboflavin Gene Cluster and fermentation

The riboflavin gene cluster plasmid pUCG3.8-R2 was transferred into the strain GT-05 by electrotransfer to obtain the strain GT-05S. GT-05S strain was fermented for 24 hours in modified ASYE medium at a kanamycin concentration of 12.5. mu.g/mL. The fermentation conditions were 60 ℃ and 250 rpm.

6.4 Riboflavin production assay

The yield of the strain GT-05S was 553.3mg/L as calculated from the absorbance after 24h of fermentation as the yield, while the yield of riboflavin for the strain GT-04S was 295.12mg/L (FIG. 22). From the fermentation results, it was found that the deletion of ldh (AOT 13-05975) gene increased the riboflavin production by 1.9-fold.

7. Fermentation of xylose and glucose and xylose mixed sugar

Specific formulations of modified ASYE media are described in Lin, P.P., Rabe, K.S., Takasumi, J.L., Kadisch, M., Amold, F.H., & Liao, J.C. (2014). Isobutanol production at extracted temperatures in therapeutic Geobacillus thermoglucosylsidus.Metabolic engineering, 24, 1-8. The medium was used for fermentation, and only glucose in the medium components was changed to xylose and a mixed sugar of glucose and xylose (total concentration of glucose and xylose was 0.2M, weight ratio of glucose to xylose was 1:5 to 4: 5), and the kanamycin concentration was 12.5. mu.g/mL. The fermentation conditions were 60 ℃ and 250 rpm. By measuring the absorbance of the supernatant after 24h of fermentation, the riboflavin production in the medium with different carbon sources was calculated by riboflavin labeling, and the riboflavin production in the medium with xylose (0.2M) as the carbon source was 445.8mg/L for the strain GT-05S, and the riboflavin production in the medium with a mixed sugar of glucose and xylose (glucose: xylose weight ratio of 1: 2) was 1034.4mg/L (FIG. 23).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

East China University of Technology

<120> thermoglucosidase Geobacillus engineering bacterium, construction method and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 984

<212> DNA

<213> Thermosynosidase Geobacillus thermoglucosidasius (Geobacillus thermoglucosidasius)

<400> 1

atgaaaacac tatttatttc gcatccccat cagatgaaaa aagaagaatt gccgccgacc 60

gttatggcgc ttggctattt tgacggcatc catcttgggc atcagaaagt gattcgcact 120

gcggtccaaa tcgcggcgga gaaaggatat aaaagcgcgg tgatgacatt tcacccccat 180

ccttccgtcg tgcttggaaa gaaggataaa catgttcatt tgataacgcc gcttaagaaa 240

aaagagcagt taattggcga acttggcatc gattatttat atattgtcga gtttacctct 300

tcgtttgcgc aactattccc gcaagaattt gttgatcaat acattatcgg ccttcatgtg 360

aagcatgtcg ttgccgggtt tgactttact tacgggcgcc tcggaaaagg gacgatggaa 420

actttgccgt tccattcgcg cgagcaattt acgcagacgg tgattccaaa attaagcatc 480

gacggcgaga aaataagctc cacttatgtc cggcaattgc tgaaaaatgg cgatgtcgac 540

cagctcccgc gtctgctcgg ccgcttttat gaagtggaag gaacggtggt gggcggtgaa 600

cgccgcggaa gaacgattgg gtttccaacg gcgaatatag cgttaaaaga tgactatttg 660

ttgccggcgc tcggcgtata tgcagtaaaa gtaaaaattg gttcagacat ttttgaagga 720

gtgtgcaacg ttggctataa accgactttt tactcaactc gtgaaggatt gccaagcatc 780

gaagtgcaca ttttcgattt tgccaaagac atttatggag aaacgatgac gatcgagtgg 840

catatgcgct taagaagcga acaaaaattc gcgggtgtgg atgaattaat cgcgcaaatt 900

cagcgggata aggaaaaagc gcaagcatat ttccgcaatt tcgccgaaac tacttgcatt 960

ttatcgcaaa aagaggtatt ctaa 984

<210> 2

<211> 1287

<212> DNA

<400> 2

atgacgtcag tcgtcgttgt cgggacgcaa tggggcgatg aaggaaaagg aaaaattacc 60

gattttctat cagaaaacgc ggaagtgatt gcgagatatc aaggaggaaa caacgcgggg 120

catacgatcg tttttaacgg agaaaaatat aagttacatt taattccatc gggaattttt 180

tataaagata aaatttgcgt catcggaaac ggaatggtaa tcgacccgaa agcattagta 240

agcgagttga attatttgca tgaccacggc atttcaaccg ataatttacg catcagcaac 300

cgcgcgcacg tcattttgcc ttatcattta aaattggatc aattagagga agaacgtaaa 360

ggggcaaaca aaatcggcac gaccaaaaaa ggcatagggc ctgcgtatat ggataaagcg 420

gcgcgcgtcg gcatccgtat cgttgatttg cttgaccgcg aagtgtttga agaaaaatta 480

gcgcgcaact tgcaggaaaa aaatattctt tttgaaaaag tgtacggtgt tgaaggattt 540

aaactggaag atatattaga tgaatactat gaatatggac agcaaattgc caaatatgtt 600

tgcgatacgt ccgttgtgtt aaataatgcg cttgatgaag ggcgccgtgt tctttttgag 660

ggtgcgcaag gcgtcatgct cgatattgac caagggacgt atccgtttgt tacttcatcg 720

aatccagttg ccggcggtgt gacaattggt tccggcgttg ggccaacgaa aattaaacat 780

gtcgtcgggg ttgcgaaagc gtacacgact cgcgttggcg acggcccgtt tccaactgaa 840

ttgcatgatg aaatcggcga ccgcatccgt gaagtcggcc gcgaatatgg aacaacgacg 900

ggacgtccgc gccgcgtcgg ttggttcgac agcgtcgttg ttcgccatgc tcgccgggtt 960

agcggcatca cagatttatc gttaaattcg atcgatgtcc ttactggcat tgaaacatta 1020

aaaatttgtg ttgcttatcg ttataaaggg aaagtgcttg aagagtttcc ggcaagttta 1080

aaagtgctgg cagaatgtga gccgatttac gaagagctgc caggatggtc agaagatatt 1140

accggtgtta agagcttgga tgaactgccg gctaacgccc gccattatgt ggaacgcatt 1200

tcgcaattaa cgggcattcc attatcgatt ttctctgtcg gtccagaccg ttcgcaaaca 1260

aacgttgttc gcagcgtata tgcgtaa 1287

<210> 3

<211> 825

<212> DNA

<400> 3

atgaaattaa ggcgcagcgg ccgtttagtc gatatgactc attatcttct tgaacggcct 60

catcagctta taccgcttac gttttttgca gagcgttatg aatcggcaaa gtcatcgatt 120

agtgaggact tagcgattat aaaacaaacg tttgaacaac aaggaatcgg aacgatcaaa 180

acgctgccag gagcggcggg cggagttcaa tatattccga aaatgtcgag acaggaagca 240

gacgggatcg ttacatattt atgtgagcag ctgtcgcgcc cagaccggct gctgcctggc 300

ggatatttat atatgacgga tattcttggc gaccctcgcg ttgtcaacaa aatcggccgt 360

ctgtatgcat ccatcttcgc tgaccgcccg gtagatgttg tcatgacgat cgcgacaaaa 420

ggaattcccc tcgcttatgc ggtcgcccac tttttgtatg ttcccgtcgt gattgtccgc 480

catgacaaca aagtgacgga aggatcgatg gtcagcatta actatgtttc tggttcttct 540

aaacgaattc aaacgatggt gttggcgaaa cggagtctgg ccgaaggggc aaacgtttta 600

attatcgatg attttatgaa agcgggcggg acggtaaacg gcatgataaa cttattaaat 660

gaatttaacg ccaaactttc cggcatcggc gtcctcgttg aatctgaaga aacgaaagag 720

cggcttgtcg atgaatatat ttcgctcgta aagctgtctt ccgttgatgt gaaagaaaaa 780

caaattaccg taaaagcagg aaattatatt cactttatgg aataa 825

<210> 4

<211> 600

<212> DNA

<400> 4

ttgcaaatcg tcaaagatca tggcccgatt acgggggaga gcattgcgga aaaattgaat 60

ttaacaagag cgacattgcg tccggactta gcgatattaa cgatggccgg atatttggaa 120

gcgcggcccc gggtgggata tttttataca gggaaaaccg gttctcagct gcttgctgat 180

aaaataaaaa agataaaagt agaagattac caatcgattc cagttgttgt caatgaaaat 240

gttagtgtat atgatgcgat tgtcaccatg tttcttgaag atgtcggcac gctgtttgtc 300

gttgatgacg aagcgcttct ggctggcgtg ttatccagaa aagatttatt gcgcgcaagc 360

attggcaaac aagagttaac gacgattcct gtcaatatta ttatgacaag aatgccgaat 420

gtagctgttt gttataaaga tgatccgctt attgaggtag cggagcggtt aattgaaaag 480

cagattgatg cgatgccggt agtgcggaag acagagaagg gatacgaggt cattggccga 540

ataacgaaaa caaatatgac aaaggcgttc gtcgcgctag cgaaagacga tttgttatag 600

<210> 5

<211> 960

<212> DNA

<400> 5

atgaaacaac aaggcatgaa tcgagtagca cttataggaa cggggttcgt tggggccagc 60

tatgcatttg cccttatgaa ccaaggaata gcagatgagt tagtattgat tgatgtaaat 120

aagaataagg cagagggcga tgtgatggat ttaaatcacg gaaaagtatt cgcgccgaag 180

ccgatgaata tttggtttgg agattatcaa gattgccaag acgccgattt ggtggtgatt 240

tgtgcagggg ctaaccaaaa gccgggagaa acaagactgg atcttgttga caaaaatatt 300

aatatcttca aaacgattgt cgattctgtg atgaaatccg gatttgatgg cgtttttctt 360

gtggcaacga acccagtgga tattttaacg tatgctactt ggaaatttag cgggttaccg 420

aaagagcggg taatcggctc aggaacgatt cttgatacag caagattccg cttcttgcta 480

agtgaatatt ttcaagtggc tccgaccaat gtacatgcgt atattattgg cgagcatggg 540

gatacagagc tgcctgtttg gagccatgcg gaaattggaa gcattccagt tgagcaaata 600

ttgatgcaaa acgataacta tagaaaagag gatttagaca atatctttgt taatgttcgt 660

gatgcggcat atcaaatcat tgagaaaaaa ggggcaacgt attacggcat tgcaatggga 720

ttagtccgta tcactcgtgc tattttgcac aatgaaaatg ccatcttaac cgtttctgct 780

catttggacg gccaatatgg cgaacgaaat gtttatattg gcgtgcctgc cattatcaac 840

cgaaacggta ttcgtgaagt gatggaattg acgctaaatg aaacagaaca acaacaattc 900

catcatagtg taactgtatt aaaagacatt ctttcccgtt attttgatga tgtaaaataa 960

<210> 6

<211> 159

<212> DNA

<213> Geobacillus thermonitrificans (Geobacillus thermonitrificans)

<400> 6

ggagttaact gcctcgtcca tttttttgct taatggaggt tgtcatgaaa atgacaaaca 60

acgtccaaac aattgccata atcgtttacg catagtttcg atttcatcgc gtaaaataat 120

ttgtgaatgt attcacaata ataagaaggg agaatagtg 159

Claims

1. The engineering bacteria GT-01S of Geobacillus thermosaccharidus is characterized in that the engineering bacteria GT-01S is constructed by introducing point mutation on the ribC gene of the Geobacillus thermosaccharidus DSM2542 through a genetic engineering technology to change the 199 th amino acid residue of the encoded protein from G to D, naming the modified strain as GT-01, and then introducing an expression vector carrying a riboflavin synthetic gene cluster expressing Geobacillus thermosulfidothelis into the strain GT-01; wherein, the nucleotide sequence of the ribC gene is shown as SEQ ID NO. 1.

2. The bacillus thermoglucosidasius engineering bacterium GT-02S is characterized in that the engineering bacterium GT-02S is obtained by knocking out purA gene in a strain GT-01 through a genetic engineering technology, naming the modified strain as GT-02 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a bacillus thermodenitrificans into the strain GT-02 to construct; wherein, the nucleotide sequence of the purA gene is shown as SEQ ID NO. 2;

the strain GT-01 is defined in claim 1.

3. The bacillus thermoglucosidasius engineering bacterium GT-03S is characterized in that the engineering bacterium GT-03S is obtained by knocking out purR gene in a strain GT-02 through a genetic engineering technology, naming the modified strain as GT-03 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a bacillus thermodenitrificans into the strain GT-03 for construction; wherein, the nucleotide sequence of the purR gene is shown as SEQ ID NO. 3;

the strain GT-02 is defined in claim 2.

4. The bacillus thermoglucosidasius engineering bacterium GT-04S is characterized in that the engineering bacterium GT-04S is obtained by knocking out a ccpN gene in a strain GT-03 through a genetic engineering technology, naming the modified strain as GT-04 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a bacillus thermodenitrificans into the strain GT-04 for construction; wherein the nucleotide sequence of the ccpN gene is shown as SEQ ID NO. 4;

the strain GT-03 is as defined in claim 3.

5. The bacillus thermoglucosidasius engineering bacterium GT-05S is characterized in that the engineering bacterium GT-05S is obtained by knocking out ldh gene in a strain GT-04 through a genetic engineering technology, naming the modified strain as GT-05 and then introducing an expression vector carrying a riboflavin synthetic gene cluster of a bacillus thermodenitrificans into the strain GT-05 for construction; wherein the nucleotide sequence of the ldh gene is shown as SEQ ID NO. 5;

the strain GT-04 is defined in claim 4.

6. The engineered bacterium of any one of claims 1 to 5, wherein the expression vector carrying a riboflavin synthetic gene cluster of Bacillus thermosphakii is pUCG3.8-R2;

the geobacillus thermoglucosidase is DSM 2542.

7. The construction method of the geobacillus thermoglucosidasius engineering bacteria is characterized by comprising the following steps of:

A. construction of pUB31-sfGFP plasmid

a2, taking the genome DNA of Geobacillus thermodenitrificans as a template, and amplifying by using primers Pm-F and Pm-R to obtain a promoter fragment; wherein, the nucleotide sequence of the promoter fragment is shown as SEQ ID NO. 6;

B. construction of the GT-01 Strain

C. construction of the GT-02 Strain

D. construction of the GT-03 Strain

E. construction of the GT-04 Strain

F. construction of the GT-05 Strain

F1, respectively amplifying primers ldh up-F and ldh up-R and ldh down-F and ldh down-R by using the genome DNA of Geobacillus thermosaccharase DSM2542 as a template to obtain the upstream and downstream fragments of the ldh gene;

G. introducing an expression vector pUCG3.8-R2 carrying a riboflavin synthetic gene cluster of the geobacillus thermosaccharyngii into a strain GT-05 to construct a geobacillus thermosaccharyngii engineering bacterium;

the primer sequences used were as follows:

wherein, the nucleotide sequences of ribC, purA, purR, ccpN and ldh genes are respectively shown as SEQ ID NO. 1-5.

8. Use of the engineered bacterium of any one of claims 1 to 6 or the engineered bacterium constructed according to the method of claim 7 for the fermentative production of riboflavin.

9. Use according to claim 8, wherein the fermentation is carried out using a modified ASYE medium in which kanamycin is present at a concentration of 12.5 μ g/mL; the fermentation conditions were 50-70 deg.C, 250 rpm.

10. The use according to claim 9, wherein the glucose component of the culture medium is replaced by a mixed sugar obtained by mixing glucose and xylose in a weight ratio of 1:5 to 4: 5.