CN117417874A - Engineering strain HC6-MT and application thereof in low-temperature production of trehalose - Google Patents

Engineering strain HC6-MT and application thereof in low-temperature production of trehalose Download PDF

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CN117417874A
CN117417874A CN202311745292.7A CN202311745292A CN117417874A CN 117417874 A CN117417874 A CN 117417874A CN 202311745292 A CN202311745292 A CN 202311745292A CN 117417874 A CN117417874 A CN 117417874A
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trehalose
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CN117417874B (en
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李春艳
王志源
马添
臧海莲
苗蕾
成毅
徐诚蛟
李宇轩
王德智
张冰
齐家熙
王晨宇
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Northeast Agricultural University
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Abstract

The invention relates to the technical field of microbial engineering. The invention provides an engineering strain HC6-MT and application thereof in low-temperature production of trehalose, wherein the engineering strain HC6-MT is obtained by introducing a promoter enhanced over-expression vector PBBR1-PD-AB into a trehalose degradation gene deletion strain HC 6-M. The invention also provides a Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDThe screening of the low Wen Jiangqi promoter P obtained by the screeningcspDIs used for constructing a promoter enhanced over-expression vector PBBR1-PD-AB. The engineering strain HC6-MT constructed by the technical scheme of the invention can degrade corn stalk hemicellulose to produce trehalose at low temperature, and can also obviously degrade the hemicellulose to produce trehaloseThe yield of the trehalose is improved, an effective scheme is provided for the low-heat, low-cost and low-pollution production of the trehalose, and a new idea is provided for the low-carbon economic development of agricultural wastes.

Description

Engineering strain HC6-MT and application thereof in low-temperature production of trehalose
Technical Field
The invention relates to the technical field of microbial engineering, in particular to an engineering strain HC6-MT and application thereof in low-temperature production of trehalose.
Background
Trehalose is widely available in organisms and often exists as a structural component of organisms, energy metabolism, storage material and stress metabolites. Trehalose is a disaccharide with cytoprotective effect, can effectively maintain the integrity of cell membranes and the tertiary structure of proteins, and improves the tolerance of cells to abiotic stress.
In previous studies, although various methods have been found that can produce trehalose, such as: the trehalose is successfully synthesized by utilizing the gluconic acid conversion, the trehalose is synthesized by utilizing a chemical method, the trehalose is produced by utilizing an enzyme conversion method, and the like. However, the chemical method has the problems of more byproducts, low yield and high energy consumption, and the enzymatic conversion method has the problems of complex process, low catalytic efficiency of enzyme, low substrate utilization rate and the like. In addition, the biological trehalose preparation method adopted at present adopts commercial glucose, maltose and the like as substrates, so that the production cost is higher. Therefore, the search for lower cost and low energy consumption synthetic substrates and synthetic methods becomes the key for refining trehalose.
The straws are the most abundant biomass resources on the earth, waste is caused by improper treatment mode at present, and the enzyme activity of microorganisms is limited by temperature under the low-temperature condition, so that the degradation rate of the straws is low, and the microbial biosynthesis generally requires a temperature of more than 30 ℃, so that the straws in the low-temperature agricultural planting area are accumulated in a large amount and are burnt, and the pollution of the atmosphere and the incomplete utilization of resources are caused. The microorganisms obtained by screening in the low temperature region can be metabolized to produce Cold-active enzyme (Cold-active enzyme) so as to adapt to the low temperature environment. The cold-resistant microorganism has higher biological activity in the temperature range of 0-40 ℃, and can allow the biocatalysis reaction in industrial production to be carried out at low temperature, thereby reducing the energy loss in the industrial production process. Therefore, the novel cold-resistant microorganism is provided for producing the trehalose by taking the corn straw as the raw material, which is very necessary for reducing the cost of trehalose production enterprises for utilizing the agricultural waste in low-temperature areas.
Disclosure of Invention
The invention aims to provide an engineering strain HC6-MT and application thereof in low-temperature production of trehalose, wherein the engineering strain HC6-MT can degrade corn straw hemicellulose to produce trehalose under a low-temperature condition, remarkably improve the yield of the trehalose, provide an effective scheme for low-heat, low-cost and low-pollution production of the trehalose, and provide a new idea for low-carbon economic development of agricultural wastes.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an engineering strain HC6-MT, which is constructed by introducing a promoter enhanced over-expression vector PBBR1-PD-AB into a trehalose degradation gene deletion strain HC 6-M.
Preferably, the promoter enhanced over-expression vector PBBR1-PD-AB comprises a low Wen Jiangqi promoter P separated from Raoulet bacteria HC6cspDKey degradation gene of hemicellulosexynBAnd trehalose synthesis genesotsA。
Preferably, the trehalose degrading gene-deleted strain HC6-M is obtained by: taking Raoul HC6 as an initial strain, knocking out trehalose degradation genes in Raoul HC6 genomeglvA、treCAndTREHone or more of the following.
The invention provides a Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDComprises the following steps: PCR (polymerase chain reaction) is utilized to amplify a promoter sequence of Raoult HC6, a promoter fluorescence intensity expression vector is constructed to carry out recombinant bacterium expression, and recombinant bacterium OD is detected 600nm And fluorescence intensity, screening for fluorescence intensity/OD in the detection range 600nm The promoter with the largest ratio is a low Wen Jiangqi promoter PcspD。
Preferably, the original vector used for constructing the promoter fluorescence intensity expression vector is a pET-RFP vector.
The invention also provides application of the engineering strain HC6-MT in low-temperature production of trehalose.
Preferably, the engineering strain HC6-MT is mixed with the corn stalk hemicellulose, and the trehalose is produced by fermentation.
Preferably, the engineering strain HC6-MT is applied in the form of bacterial liquid, and the OD600nm of the engineering strain HC6-MT bacterial liquid is 1.7-2.3.
Preferably, the temperature of the fermentation is 18-23 ℃, and the time of the fermentation is 40-52h.
By adopting the technical scheme, the invention has the following beneficial effects:
1. according to the invention, the engineering strain HC6-MT is obtained by jointly knocking out the trehalose degrading gene strain HC6-M and the successfully constructed low-temperature promoter enhanced over-expression vector PBBR1-PD-AB, and the engineering strain HC6-MT can degrade hemicellulose extracted from corn stalks under a low-temperature condition to produce trehalose, so that the operation of providing heat from the outside is omitted, and meanwhile, compared with a wild strain, the engineering strain HC6-MT constructed after the wild strain HC6 is imported by the strong promoter has the advantages that the utilization rate of hemicellulose and the efficiency of producing trehalose are enhanced, so that the strain inoculation amount is reduced and the production cost is reduced; in addition, the yield of toxic and harmful byproducts is effectively reduced.
2. The embodiment of the invention shows that the engineering strain HC6-MT can produce the trehalose by degrading the hemicellulose of the corn straw at the low temperature of 20 ℃ so that the maximum yield of the trehalose reaches 1.96g/L, and is improved by 54 times compared with a wild strain. After that, culture conditions are optimized, and the highest yield of trehalose reaches 2.28g/L in 48 hours.
3. The invention converts the low-temperature microorganism fermentation straw into a product with high added value, improves the straw bioavailability, reduces the environmental pollution pressure and provides a new idea for the low-carbon economic development of agricultural wastes.
Drawings
FIG. 1 is an electrophoretogram of plasmid PKO3-knoc (M in FIG. 1 represents 5000bp DNA Marker,1 represents plasmid PKO3-glvA2 represents plasmid PKO3-treC3 represents plasmid PKO3-TREH)；
FIG. 2 shows the knock-out verification electrophoretogram of different trehalose gene-deficient strains (FIG. 2 (a) is HC6-g (glvA) M in (a) represents 2000bp DNA Marker,1 which represents a geneglvAKnockout verification strip, 2 denotes suicide plasmid verification strip; fig. 2 (b) shows HC6-t (treC) M in (b) represents 2000bp DNA Marker,1, 2 represents a suicide plasmid verification band, and 2 represents a genetreCKnocking out the verification strip; fig. 2 (c) shows HC6-T (TREH) M in (c) represents 2000bp DNA Marker,1 which represents a geneTREHKnockout verification strip, 2 denotes suicide plasmid verification strip; FIG. 2 (d) shows HC6-Tg (TREH▲glvA) M in (d) represents 2000bp DNA Marker,1 representing a geneglvAKnockout verification band, 2 represents a geneTREHKnockout verification strip, 3 denotes suicide plasmid verification strip; the (e) in FIG. 2 is HC6-tg (treC▲glvA) M in (e) represents 2000bp DNA Marker,1, 2 represents a suicide plasmid verification bandGeneglvAKnockout verification band, 3 represents genetreCKnocking out the verification strip; fig. 2 (f) is HC6-Tt (TREH▲treC) M in (f) represents 2000bp DNA Marker,1 which represents a genetreCKnockout verification band, 2 represents a geneTREHKnockout verification strip, 3 denotes suicide plasmid verification strip; the (g) in FIG. 2 is HC6-M (glvA▲treC▲TREH) M in (g) represents 2000bp DNA Marker,1, 2 represents a suicide plasmid verification band, and 2 represents a geneglvAKnockout verification band, 3 represents geneTREHKnockout verification band, 4 represents genetreCA knockout verification strip);
FIG. 3 shows trehalose gene-deficient strain HC6-M (FIGS.)glvA▲treC▲TREH) Is a construction process diagram of (1);
FIG. 4 is a graph showing the growth conditions of the trehalose degrading gene-deficient strain and the HC6 strain (WT) and the accumulation amount of trehalose (FIG. 4 (a) shows the growth curves of the trehalose degrading gene-deficient strain and the HC6 strain (WT) and the accumulation of trehalose, FIG. 4 (b) shows the growth conditions of the trehalose degrading gene-deficient strain and the HC6 strain (WT) at different dilution factors, the difference in significance is expressed in different lower case letters,p<0.05)；
FIG. 5 is an electrophoresis chart of different promoters in the strain HC6 (M in FIG. 5 represents 2000bp DNA Marker,1)cspAGene promoter, 2 representscspBGene promoter, 3 denotescspCGene promoter, 4 meanscspDGene promoter, 5 meanscspEGene promoter, 6 representscspGA gene promoter);
FIG. 6 is an electrophoretogram of pET-master-RFP vector (M in FIG. 6 represents 5000bp DNA Marker,1 represents pET-P)cspARFP validation, 2 represents pET-PcspBRFP validation, 3 represents pET-PcspCRFP validation, 4 represents pET-PcspDRFP validation, 5 represents pET-PcspERFP verification, 6 represents pET-PcspG-RFP)；
FIG. 7 is a graph showing the expression of recombinant bacteria with different promoter fluorescence intensities (FIG. 7 (a) shows the color change of the culture medium of recombinant bacteria with different promoter fluorescence intensities, and FIG. 7 (b) shows the fluorescence intensity of recombinant bacteria with different promoter fluorescence intensities);
FIG. 8 is an electrophoretogram of different PBBR1-PD-AB recombinant vectors (M in FIG. 8 represents 2000bp DNA Marker,1)xynBVerification, 2 denotes PcspD+xynB3 represents PBBR1,4 representsotsA5 represents PcspD+otsA)；
Fig. 9 shows corn stalk hemicellulose and fourier infrared spectrum detection and fermentation results (fig. 9 (a) shows intermediate products in the corn stalk hemicellulose extraction process, (a) shows dry stalk powder, (a) shows stalk precipitation after alkali dissolution, (a) shows hemicellulose solution, (a) shows hemicellulose precipitation at 3 and (a) shows hemicellulose precipitation at 4, fig. 9 (b) shows FTIR spectra of xylan standard (upper curve) and corn stalk hemicellulose extraction (lower curve), and fig. 9 (c) shows growth curve of strain HC6 and xylose yield);
FIG. 10 is a graph showing the accumulation of xylose and trehalose, which are the conditions of the wild strain HC6 and the engineering strain HC6-MT, on the substrate hemicellulose (FIG. 10 (a) shows the conditions of the wild strain HC6 and FIG. 10 (b) shows the conditions of the engineering strain HC 6-MT);
FIG. 11 is a graph showing the effect of substrate concentration versus cold shock time ratio interaction on HC6-MT trehalose production;
FIG. 12 is a graph showing the effect of pH-to-cold shock time ratio interaction on HC6-MT trehalose production;
FIG. 13 is a graph showing the effect of phosphate concentration versus cold shock time duty cycle interaction on HC6-M trehalose production;
FIG. 14 is a graph showing the effect of substrate concentration and pH interaction on HC6-MT trehalose production;
FIG. 15 is a graph showing the effect of substrate concentration interaction with phosphate concentration on HC6-MT trehalose production;
FIG. 16 is a graph showing the effect of phosphate concentration and pH interaction on HC6-MT trehalose production.
Description of biological preservation
The invention relates to Raoult fungusRaoultellasp.) HC6 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 09/07 th year 2020, and has a preservation address of No.3 of North Chen Xiyu No. 1 in the Chaoyang area of Beijing city, and a preservation number of CGMCC No.20607.
Detailed Description
The invention provides an engineering strain HC6-MT, which is constructed by introducing a promoter enhanced over-expression vector PBBR1-PD-AB into a trehalose degradation gene deletion strain HC 6-M.
In the invention, the promoter enhanced over-expression vector PBBR1-PD-AB comprises a low Wen Jiangqi promoter P separated from Raoulet bacteria HC6cspDKey degradation gene of hemicellulosexynBAnd trehalose synthesis genesotsA。
In the invention, the Raoult fungus @ isRaoultella sp.) HC6 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 09/07 of 2020, and has a preservation address of CGMCC No.20607, namely, no.3 of North West Lu 1, the Korean region of Beijing. The Raoult HC6 belongs to cold-resistant bacteria, can be used for growth and metabolism under low temperature, and contains hemicellulose key degradation genesxynB、Trehalose synthesis geneotsATherefore, hemicellulose can be used as a substrate for producing trehalose.
In the invention, the trehalose degrading gene deletion strain HC6-M is obtained by the following method: taking Raoul HC6 as an initial strain, knocking out trehalose degradation genes in Raoul HC6 genomeglvA、treCAndTREHone or more of the following. Further preferably, the trehalose degrading gene deletion strain HC6-M of the invention takes Raoult HC6 as an initial strain, and knocks out trehalose degrading genes in Raoult HC6 genomeglvA、treCAndTREH。
the invention provides a Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDComprises the following steps: PCR (polymerase chain reaction) is utilized to amplify a promoter sequence of Raoult HC6, a promoter fluorescence intensity expression vector is constructed to carry out recombinant bacterium expression, and recombinant bacterium OD is detected 600nm And fluorescence intensity, screening for fluorescence intensity/OD in the detection range 600nm The promoter with the largest ratio is a low Wen Jiangqi promoter PcspD。
In the invention, the original vector used for constructing the promoter fluorescence intensity expression vector is a pET-RFP vector.
The invention also provides application of the engineering strain HC6-MT in producing trehalose under a low-temperature condition.
In the invention, the engineering strain HC6-MT can be used for producing trehalose by fermenting corn stalk hemicellulose. The invention mixes the engineering bacterial strain HC6-MT with the corn stalk hemicellulose, the engineering bacterial strain HC6-MT is mixed and applied in the form of bacterial liquid, and the OD of the bacterial liquid of the engineering bacterial strain HC6-MT 600nm Preferably 1.7 to 2.3, more preferably 1.8 to 2.2, and still more preferably 1.9 to 2.1. The fermentation according to the invention is preferably a shake flask fermentation, the rotation speed of the shake flask preferably being 170-250rpm, more preferably 185-220rpm, still more preferably 200rpm; the fermentation temperature is preferably 18-23 ℃, further preferably 19-21 ℃, still further preferably 20 ℃; the fermentation time is preferably 40 to 52 hours, more preferably 45 to 50 hours, still more preferably 48 hours.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 obtaining trehalose degrading Gene-deleted Strain HC6-M
Sequencing of the HC6 genome of Raouli Strain
Sequencing of the HC6 genome of Raouli strain shows that the genome (accession number is CP 093276.1) has the total length of 6243766bp, wherein the total length of chromosome is 595116 bp and the GC content is 58.42%. The strain HC6 has 3 plasmids, the size of the plasmid 1 is 167204bp, and the GC content is 51.75%; plasmid 2 was 100650bp in size and had a GC content of 51.54%; plasmid 3 had a size of 20796bp and a GC content of 57.56%. The sequencing result of the 16SrDNA gene sequence of the strain HC6 is compared with the data in the NCBI ribosomal RNA sequence database, and the result shows that the sequencing result is matched with the genus of the strain HC6Raoultella The sp and similarity reaches 100%, which indicates that the strain HC6 has no species variation and can be subjected to subsequent experiments.
(II) trehalose degrading Gene excision
Using suicide plasmid PKO3-km (conventionally purchasedTo), under the condition of 35 ℃, the enzyme digestion reaction system is as follows: PKO3-km 5.0. Mu.L, 10×Lication Buffer 2.0. Mu.L, quick cut BamHI 1.0. Mu.L, sterilized ddH 2 O Up to 20.0μL。
The PCR amplification reaction (PCR amplification system: 5 XPrimeSTAR Buffer (Mg) 2+ Plus) 10μL、dNTP Mixture(2.5 mM each) 4μL、Primer F 10-15pmol、Primer R 10-15pmol、Template <200ng, 2. Mu.L of template DNA, sterilized water up to 50. Mu.L. The PCR amplification procedure was: denaturation at 98℃for 10s, annealing at 60℃for 5s, and circulation at 72℃for 1min for 30 times. ) Obtaining the target gene to be knocked out (trehalose degradation gene)glvA、treCAndTREH) The sequences of the primers are shown in Table 1, wherein the homologous arm sequences of the upstream and downstream 500bp sequences of the target gene sequence to be knocked out are used as homologous arms.
TABLE 1 PCR primer sequences of the upstream and downstream homology arms of the target Gene to be knocked out
After the amplification reaction was completed, the target gene fragment was purified and recovered using FastPure Gel DNA Extraction Mini Kit kit (available from Nanjinozan Biotechnology Co., ltd.). The purified product is directly subjected to a ligation reaction. The connection reactant is the trehalose degradation gene to be knocked out obtained by the stepsglvA、treCAndTREHthe upstream and downstream homology arms of the sequence are then ligated to linearized PKO3-km plasmids using an In-Fusion seamless cloning ligation technique to obtain plasmids PKO3-knock, in particular PKO3-glvA、PKO3-treC、PKO3-TREHThe recombinant plasmid vector is knocked out. The In-Fusion seamless cloning ligation system is shown In Table 2.
Table 2 In-Fusion seamless cloning ligation System
(the term "Target gene fragment" means homologous arms upstream and downstream of the corresponding gene)
Electrophoresis was performed to verify whether plasmid PKO3-knock was constructed successfully. BamHI and SalI double digestion was performed on the plasmid PKO3-knock obtained above to obtain two electrophoresis bands of 1.0kb and 5.6kb, which were in line with PKO3-glvA、PKO3-treC、PKO3-TREHThe size of the bands indicated that these recombinant plasmids were constructed correctly (see FIG. 1).
The bacterial strain HC6 is used as host bacteria, the plasmid PKO3-knock is electrically transformed into the host bacteria, and is cultured for 24 hours in an SOC culture medium at 30 ℃, and at the moment, the plasmid PKO3-knock can undergo homologous recombination with a host genome, so that a target gene is knocked out. The specific bands were then amplified again by PCR to verify that the gene knockdown was successful, and the primers used are shown in Table 3. The results are shown in FIG. 2.
TABLE 3 PCR primer sequences for checking deletion of target genes
(III) verification of growth of trehalose Gene-deficient Strain
Based on the above operations, 7 trehalose degrading gene defect strains (shown in FIG. 3) of HC6-g (, respectively) are constructed in sequenceglvA)、HC6-t(▲treC)、HC6-T(▲TREH)、HC6-Tg(▲TREH▲glvA)、HC6-tg(▲treC▲glvA)、HC6-Tt(▲TREH▲treC)、HC6-M(▲glvA▲treC▲TREH)。
Inoculating the constructed trehalose degrading gene defect strain and HC6 strain (WT) into LB liquid culture medium respectively for overnight activation, and regulating bacterial liquid OD 600nm =2.0±0.1, and the bacterial liquids were diluted to 10 respectively -1 -10 -6 2.0 mu L of bacterial liquid is taken from each dilution multiple, and the growth detection and the trehalose accumulation amount measurement are carried out in a hemicellulose liquid culture medium shaking flask for fermentation (20 ℃,80h,200 rpm). Similarly, 2.0. Mu.L of the bacterial liquid was taken for each dilution factor, and cultured in an incubator at a constant temperature of 20℃on an inorganic salt solid medium containing trehalose as a sole carbon source, and the colony growth state was observed. The results are shown in FIG. 4.
Example 2 obtaining Low temperature promoter enhanced overexpression vector PBBR1-PD-AB
Screening of cold shock protein low-temperature strong promoter and prediction of functional region
A total of 6 strains HC6cspGene, 6 are obtained from the whole genome of the strain HC6 for screening low-temperature high-expression promoterscspThe promoter sequence of the gene was analyzed on line using the prokaryotic promoter to predict the site BPROM (http:// linux1. Software. Com /) predicted promoter-10 region, -35 region and spacer distance.
The 6 cold shock protein gene sequences and promoters thereof are as follows:
cold shock protein gene Hc6_00172 Cold shock proteinCspAThe sequence is shown as SEQ ID NO.21, and specifically comprises the following steps: ATGTCCGGTAAAATGACTGGTATCGTAAAATGGTTCAACGCTGATAAAGGTTTCGGCTTCATTACTCCTGATGACGGTTCTAAAGATGTATTCGTACACTTCTCCGCTATCATGAACGATGGCTACAAATCTCTGGATGAAGGTCAGAAAGTTTCCTTCACCATCGAAAGCGGCGCTAAAGGCCCGGCAGCTGGCAACGTTACCAGCCTGTAA;
promoter Hc6_00172 Cold shock protein of cold shock protein geneCspAThe sequence of the promter is shown as SEQ ID NO.22, and specifically comprises the following steps: AGTTCTATAATGTTATTGGACACTGATTACGGTCCTGATTAGGGACCGTTTTAATTTCTATGAAAAAAACGTTTGCTATCGCACCTCAAAAATGAGTTAATGCTCGCAGGTTTGATGTACAGACCACAGAGCATTAGTATAGAGTAGTCCTTGAGCGGTTATCCTAGATACCCCGGTAGTGAACTTTCCCTTTATAGCTTCAAATCTGTAGTCCAGACCGTATCGCCGAAAGGTTCATTTATTATTTAAAGGTATTTCGCT;
cold shock protein gene Hc6_02655 Cold shock-like proteinCspBThe sequence is shown as SEQ ID NO.23, and specifically comprises the following steps: ATGTCTGGTAAGATGATTGGTTTAGTAAAATGGTTTAATGAAGGTAAAGGTTTCGGCTTTATTTCTCCAGTAGACGGCAGTAAAGATGTTTTCGTGCATTTTTCTGCGCTGCAGGGCGATGGCTTTAAAACTTTATTTGAAGGGCAGAAGGTCGAATTCACTATCGAGAGCGGCGCCAAAGGCCCTGCTGCCGGTAATGTCGTTCTGCGCGACTAA;
promoter Hc6_02655 Cold shock-like protein of Cold shock protein geneCspBThe sequence of the promter is shown as SEQ ID NO.24, and specifically comprises the following steps: TTATTCACGGCTTCTCTGTGGGGGCAGAAAATAATGCTTGCTATCAATGGTTATCTATGTGATCAATAGCAGGTCGGTTTGGTACACAGAACCAATAAAGCGGTTTAGTAAAGCAGTCCTCATCTCAAGCGTTATTCCTAAATAATCCTTTTTTCGAGCCTCCTTATCGTTATTAATTAATTTCTGTGATGCGAACTTTTTCGCCGCAAGGCTTTGAACATTGGGATAATACTT；
Cold shock protein gene Hc6_02212 Cold shock-like proteinCspCThe sequence is shown as SEQ ID NO.25, and specifically comprises the following steps: ATGGCAAAGATTAAAGGTCAAGTTAAGTGGTTCAACGAGTCTAAAGGTTTTGGCTTTATTACTCCGGCTGATGGCAGCAAAGACGTGTTCGTACACTTCTCCGCTATCCAGGGTAATGGCTTCAAAACTCTGGCTGAAGGCCAGAACGTTGAGTTCGAAATTCAGGACGGCCAGAAAGGTCCGGCAGCAGTTAACGTAACTGCTATCTGA;
promoter Hc6_02212 Cold shock-like protein of Cold shock protein geneCspCThe sequence of the promter is shown as SEQ ID NO.26, and specifically comprises the following steps: CTGATAAACGGGTGTACACCGCGCTGGTGTACACCTGCCTCATCGCCTTACGCAATAACTTATTCTTATCCTTCCGCGCTTATTGCCACATTTCTTATGTCACTTAATGTGTTAGCGATCGCAATTTTATCCAATAAGCTATGGATTTTTGAGCTATTCAAATAAAATAATAACATCGTGATTTAATTGCATGATGAAAGTGTGTGTCATCGAAATTTCATTATTAAGATGCAATATAAACACTATTTGTCGCGTGGCTAACAGCATTTAATTAACCCTGCATATTCTCGGCCTAAGCCAATTTTCAACTATCAATATGCGCGTTTATGCGCATCGTTATGCAGCTATGCGCACGTAACCCCTCAAAATTGGCAGGGTGCATGCATGGCGGGCTGGACGGAGCGAAGTTGTTGAGCTGTGGTGAAAAAGTTGCAGCTATCATCCATGCATAATCGGATGTTTGCCGCGCGCCCCGGGATTAACAGCGGGCGAGAAAAGGCGCATAAACGTGCAGTTGGTCAAATGATTCCCTTATATTTTGTGCGAAGGATCGAGAGCCGTTTAAAAATGGCTTGCCATTATTAACGTTGTATGTGATAACACCTTTCGGGTTAAACGAGGTACAGTTCTGTTTATGTGTGGCATTTTCAGTAAAGAAGTCCTGAGTAAACACGTTGTCGTTGAATACCGCTTCTCTGCCGAACCTTATATTAGTGCCTCATGCAGTAGTGTGTCAGTTTTATCTATGTAAGCGCCTGCGGGCGAAGAAAACAGTCTAAGGAATTTTGCAA;
cold shock protein gene Hc6_03855 Cold shock-like proteinCspDThe sequence is shown as SEQ ID NO.27, and specifically comprises the following steps: ATGGAAATGGGTACTGTTAAGTGGTTCAACAATGCCAAAGGGTTTGGTTTTATTTGCCCTGAGGGCGGAGGCGAAGACATTTTCGCCCATTACTCCACCATCCAGATGGATGGTTACAGAACGCTAAAAGCCGGACAAGCCGTTCGGTTTGATGTTCACCAGGGACCGAAAGGTAATCACGCCAGCGTGATTGTTCCTGTAGAAGCGGAAGCGGCTGCATAA;
promoter Hc6_03855 Cold shock-like protein of Cold shock protein geneCspDThe sequence of the promter is shown as SEQ ID NO.28, and specifically comprises the following steps: CGTCAGTATTCATCATCGGTTGCTGTTGCCAACAGGCGGTGGCCTGTCGATGACCAAAAGTTATGCCCATCACAAATCTACAATAGATCATAGATAACTATCATCTATTACTTCCATCCGCGACGTCTGTCACATTCCCCGGCAATAGCGTTAACTGCTTCAAATTTTGACGCATTTTTCGCCTTCCCCTACCGTCAATCGCTTGACGCCTTTTCGTATTTCTCTAAATTGTACTGGCGAGAGTTGGCGAGCATTTGAACAACTCGTCACTCCACTACCGGTTCATTCCATCTTACTTATAAAGAATTACGAAGGATGTCGAAGT；
Cold shock protein gene Hc6_04170 Cold shock-like proteinCspEThe sequence is shown as SEQ ID NO.29, and specifically comprises the following steps: ATGTCTAAGATTAAAGGTAACGTTAAGTGGTTTAATGAGTCCAAAGGATTCGGTTTCATTACTCCGGAAGATGGCAGCAAAGATGTATTCGTACATTTCTCTGCAATCCAGTCCAACGGTTTCAAAACTCTGGCTGAAGGTCAGCGTGTAGAGTTCGAAATCACTAACGGTGCCAAAGGCCCTTCTGCTGCAAACGTAAACGCTATCTAA;
promoter HC 6-04170 Cold shock-like protein of Cold shock protein geneCspEThe sequence of the promter is shown as SEQ ID NO.30, and specifically comprises the following steps: TCCGCACTAGCTTAGTGATAAAAGAGCTGAGCATTATGTTATGTGGAAAAACAATAACTAAAGCGCAACCACTAAAAAAGATAGCGACTTTTGTCACTTTTTAGCAAAGTTCGACTGGACAAAAGGCACCACAATTGATGTACTGGATCGCGACACAGTATCAGTGTCTTTTTTTCATATAAAGGTAATTTTG;
cold shock protein gene Hc6_02543 Cold shock-like proteinCspGThe sequence is shown as SEQ ID NO.31, and specifically comprises the following steps: TTGTTCCAAAAAATGACAGGCATTGTCAAAAGCTTTGATAATAAAACCGGCAGAGGCCTTATCGTCCCTTCCGACGGTCGTAAAGACGTTCAGGTTCATATTTCCGCATTATCCCCCAACGAGTCCACGCCAATGACGCCCGGTATTCGCGTTGAGTTTCGTCGGGTTAACGGCCTGCGCGGGCCAACCGCGGCAAACGTCTATACCTGCTAG;
promoter Hc6_02543 Cold shock-like protein of Cold shock protein geneCspGThe sequence of the promoter is shown as SEQ ID NO.32, and is specifically CCGTCGCGGGGCGGAGGACGCGGCTGCCGCATCGTCCTCCCGCCCCGCCGGGATTGCGTAAATAGCCTTGACCAATCAGACGCTTATTTGCCCGCGGCGAAATCATTTCGAGCTATTGATAGCAGAATGATGTATGGTTGCCGGCCATTACATCAGGAAAAAATCATATGTCAGATCGAAAAGACTCGAAAACTCGCCGTAATTACCTTGTAAAATGCTCCTGCCCTAACTGCTCCCAGGAATCAGAACATAGCTTTAGCCGAGTACAAAAAGGCGCCCAGCTCATCTGCCCTTACTGCCACAAACTCTTCCAGTCATCACCCAGAAACGCCGCCTAATCCCTAAATGCCCGACGCTGATTGTGCCTTGCCCCGTTATTTAGATAATTCTTATTATTGGGTCGAATGAGACAATCGGTTTTATTCCGTTCAGGCAAATAGAAAACCCGCACTCCGGCGGGTTTTAAATAGTTCAATATTGTATTTCTTTGCATAACCCAGGTTATCGCATTCCTGTTTTTACCAGGAGTTTAT.
The predicted promoter sequence results are shown in Table 4.
TABLE 4 promoter sequence prediction results
(II) construction of promoter fluorescence intensity expression vector pET-promoter-RFP
PCR amplification of the promoter DNA sequences of the strain HC6 genome DNA serving as a template, wherein the size of each promoter sequence is 200-500 bp. The amplified band gel was recovered by agarose gel electrophoresis and sent to the Shanghai Co., ltd for sequencing, and the sequencing result was consistent with the genome sequence (FIG. 5).
The amplified promoter DNA sequence was ligated to pUCmT vector, and the successfully ligated vector was named pUCmT-promoter and transferred into DH5 alpha bacteria for stable cloning. Extracting pUCmT-promoter vector and pET-RFP vector, respectively performing double enzyme digestion with Quick cut BamHI and Quick cut KpnI enzyme, and connecting the digested pET-RFP vector with strain HC6 promoter, wherein the successfully connected vector is named pET-promoter-RFP, specifically pET-PcspA-RFP、pET-PcspB-RFP、pET-PcspC-RFP、pET-PcspD-RFP、pET-PcspE-RFP、pET-PcspG-RFP。
And (5) carrying out electrophoresis on the constructed carrier, and verifying whether the construction is successful. The pET-promoter-RFP vector (about 5000 bp) and the promoter-RFP gene recombinant fragment (about 1000 bp) bands are respectively consistent with the lengths shown by the electrophoresis results, and the vector construction is successful (as shown in FIG. 6).
(III) determination of a Low temperature Strong promoter
The method for constructing the fluorescence intensity expression vector of the promoter is adopted for the traditional T7 strong promoter to construct pET-PT7-RFP, and then pET-P is respectively carried outcspA-RFP、pET-PcspB-RFP、pET-PcspC-RFP、pET-PcspD-RFP、pET-PcspE-RFP、pET-PcspGRFP and pET-PT7-RFP transferE.coliBL31 (DE 3) and the relative fluorescence intensity (fluorescence intensity/OD) of RFP gene expression is calculated by detecting the biomass and fluorescence intensity of recombinant bacteria 600nm ) And used to characterize the promoter strength.
After culturing in 48-well plate at 37deg.C for 16h, the promoters were ordered in P-strengthcspC<PcspB<PcspG<PT7<PcspA<PcspE<PcspDAfter 8h cold induction, P is removedcspCAnd PcspBThe intensity sequence of the promoters is P in sequence outside the group in which no red fluorescence is detectedcspG<PT7<PcspA<PcspE<PcspD. Meanwhile, a difference in color change of the medium due to accumulation of red mCherry protein was observed during the culture of each strain LB liquid medium (see fig. 7). By combining the results, the cold shock protein gene is determinedcspDPromoter P of (C)cspDIs a low Wen Jiangqi promoter, and is used for constructing a promoter enhanced over-expression vector.
Construction of promoter-enhanced overexpression vector PBBR1-PD-AB
Amplification of hemicellulose Key degradation Gene by PCRxynBSequence, trehalose synthesis geneotsASequence and sequencecspDPromoter sequence, then use In-Fusion ® Snap Assembly Master Mix seamless connection kit (purchased from Takara Bio-engineering (Dalian) Co., ltd.), universal restriction enzymes BmHI and KpnI are connected with linearized universal expression plasmid PBBR1-MCS2, and construction of PBBR1-PD-AB is completed.
TABLE 5 xynB、otsAA kind of electronic device with high-pressure air-conditioning systemcspDSequence PCR primer sequences
To verify whether the recombinant vector is successfully constructed, the PBBR1-PD-AB vector is used as a template for amplificationxynBGene band (1321 bp), PcspD+xynBFusion band (1624 bp),otsAGene strip (896 bp), PcspD+otsAFusion band (1199 bp) and vector specific band PBBR1 (346 bp). Amplified band electrophoresis gel As shown in FIG. 8, the band size conforms, demonstrating PET-PD-xynBWith PET-PD-otsAThe vector construction was successful.
Example 3
Construction of engineering strain HC6-MT
Taking out the prepared trehalose gene defect strain HC6-M competent cells in a refrigerator at the temperature of-80 ℃, and thawing the competent cells on ice. 10. Mu.L of the extracted overexpression vector (PBBR 1-PD-AB) was added into competent cells, and the mixture was left on ice for 30 min. Adding the mixed solution added with the over-expression vector (PBBR 1-PD-AB) into a precooled electrorotating cup, and setting electrotransformation conditions as follows: 1500KV,800, ꭥ. And (3) adding the bacteria liquid subjected to electrotransformation into 1mL of SOC culture solution for resuscitating and culturing for 1h, coating the bacteria liquid on a Kana antibiotic LB solid culture medium plate with the working concentration of 50ng/mL, culturing overnight at 20 ℃, picking out colonies, and performing PCR (polymerase chain reaction) verification by using an over-expression vector (PBBR 1-PD-AB) verification primer. The strain that was successfully verified was named HC6-MT.
Extraction of corn stalk hemicellulose
Drying corn stalk (from the northeast agricultural university to the sun farm), cutting into 2.0-3.0cm small sections, grinding into powder by using a grinder, sieving the obtained dried stalk powder with a 80-mesh sieve, mixing with 10% NaOH according to a ratio of 1:10, soaking at 70deg.C for 2h, filtering the mixture (stalk precipitation) with gauze, and collecting filtrate. Regulating pH of the filtrate to 7.0+ -0.2 with concentrated hydrochloric acid, mixing with active carbon at a ratio of 10:1 (v/w), water-bathing at 60deg.C for 15min, centrifuging at 10000rpm for 10min, and removing active carbon to obtain hemicellulose solution. And adding 50% of absolute ethyl alcohol of the volume of the hemicellulose solution into the hemicellulose solution to obtain hemicellulose sediment, carrying out solid-liquid separation, collecting filter residues, and drying at 50 ℃ to obtain hemicellulose.
The result of fourier infrared spectrum detection of the obtained hemicellulose and xylan standard is shown in fig. 9, and the result shows that the absorption peak positions of the corn straw extract and the xylan standard are basically consistent, and the hemicellulose extracted from the corn straw is mainly xylan.
Taking corn stalk hemicellulose as a substrate, inoculating 5% (v/v) Raoult HC6, culturing and fermenting at 20 ℃ for 80 hours, sampling every 6 hours, observing the growth curve of the Raoult HC6, and detecting the xylose yield. The results are shown in fig. 9, which shows that the strain HC6 can grow with hemicellulose extracted from corn stover as the only carbon source and degrade xylan (hemicellulose as the main component) to produce xylose.
Example 4
Preparation of corn stalk hemicellulose liquid culture medium
The corn stalk hemicellulose liquid culture medium is prepared according to the following formula: 10g/L, NH of hemicellulose obtained above 4 NO 3 2g/L、K 2 HPO 4 2g/L、MgSO 4 0.2g/L。
(II)
The ability of Raoult HC6 and the engineering strain HC6-MT prepared in example 3 to produce trehalose was examined by using Raoult HC6 alone as a control to produce trehalose.
Control group: the strain HC6 bacterial liquid (OD 600nm =2.0±0.1) was inoculated into the above-mentioned corn stalk hemicellulose liquid medium in an inoculum size (v/v) of 5%, shake flask fermentation was performed at 20 ℃ and 200rpm for 80 hours, sampling was performed every 6 hours, and the substrate hemicellulose utilization of the strain HC6 and trehalose yield were examined.
Experimental group: engineering bacteria HC6-MT bacterial liquid (OD) 600nm =2.0±0.1) was inoculated into the above-mentioned corn stalk hemicellulose liquid medium in an inoculum size (v/v) of 5%, shake flask fermentation was performed at 20 ℃ and 200rpm for 80 hours, sampling was performed every 6 hours, and the substrate hemicellulose utilization and trehalose yield by the strain HC6-MT were examined.
The trehalose content detection method comprises the following steps: preparing trehalose standard solutions (0.2, 0.1, 0.075, 0.05, 0.025, 0.0125 mg/mL) with different concentrations, respectively taking 2.0mL of standard solution, adding 5.0mL of 0.2% anthrone-sulfuric acid solution, boiling water bath for 8min, cooling, measuring light absorption value at 620nm, and obtaining OD 620nm And drawing a standard curve for the trehalose content. The same procedure was followed for a boiling water bath of 8 minutes with a blank of 2.0mL deionized water added to 5.0mL of a 0.2% anthrone-sulfuric acid solution.
The xylose is accumulated in a certain amount in the earlier stage of the xylan, and the depolymerization rate (namely the xylose generation rate) of the xylan is larger than the xylose degradation rate, so that the xylose is accumulated in the initial stage of depolymerization, and then the xylose is further utilized by the engineering strain HC6-MT to convert the xylose into the trehalose.
The detection results of the accumulation amounts of the wild strain HC6 and the engineering strain HC6-MT trehalose and xylose are shown in figure 10, and the maximum accumulation amount of the engineering strain HC6-MT trehalose is 1.96g/L, which is improved by 54 times compared with the accumulation amount of the wild strain HC6 trehalose; the maximum xylose accumulation of the engineering strain HC6-MT is 3.61g/L, which is enhanced by 2.3 times compared with the wild strain, and the hemicellulose utilization capacity and the trehalose accumulation capacity of the strain HC6 are successfully enhanced.
Example 5 optimization of fermentation conditions for HC6-MT Strain
Four-factor three-level experiments were designed using Minitab2022 software. Based on the result of single factor analysis to significancep<0.05 is a selection standard, and the optimal fermentation conditions of the constructed recombinant strain HC6-MT are explored by taking the cold shock time (A) at 10 ℃ after inoculating a fermentation medium, the concentration (B) of a substrate (corn hemicellulose) of the medium, the pH (C) of the medium and the phosphate (D) in the medium as independent variables and the trehalose yield as a response value. The levels of 4 response surface factors and variables affecting the production of trehalose by fermentation of the strain HC6-MT are shown in Table 6.
TABLE 6 optimization of response surface factor and variable levels for HC6-MT fermentation by strain
As can be seen from FIGS. 11 to 16, the OD was adjusted by sterile water after inoculating the engineering strain HC6-MT into LB liquid medium and performing activation culture at 20℃for 16 hours 600nm =2.0±0.1, and the corn stalk hemicellulose liquid culture medium is inoculated in an inoculation proportion of 5%, and after optimization, the highest yield of trehalose is 2.28g/L in 48 hours under the optimal trehalose production condition.
According to the embodiment, the engineering strain HC6-MT provided by the technical scheme of the invention can be used for producing trehalose by degrading hemicellulose extracted from corn straw under a low-temperature condition, so that the production raw materials are saved, the production cost is reduced, and the yield, the conversion rate and the production rate of the trehalose are improved.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. An engineering strain HC6-MT is characterized in that a promoter enhanced over-expression vector PBBR1-PD-AB is introduced into a trehalose degradation gene deletion strain HC6-M, and the engineering strain HC6-MT is constructed;
the promoter enhanced over-expression vector PBBR1-PD-AB comprises a low Wen Jiangqi promoter P separated from Raouli HC6cspDKey degradation gene of hemicellulosexynBAnd trehalose synthesis genesotsA。
2. The engineered strain HC6-MT according to claim 1, wherein the trehalose degrading gene deletion strain HC6-M is obtained by: taking Raoul HC6 as an initial strain, knocking out trehalose degradation genes in Raoul HC6 genomeglvA、treCAndTREHone or more of the following.
3. Raoult bacterium HC6 medium-low Wen Jiangqi promoter PcspDIs characterized by comprising the following steps: PCR (polymerase chain reaction) is utilized to amplify a promoter sequence of Raoult HC6, a promoter fluorescence intensity expression vector is constructed to carry out recombinant bacterium expression, and recombinant bacterium OD is detected 600nm And fluorescence intensity, screening for fluorescence intensity/OD in the detection range 600nm The promoter with the largest ratio is a low Wen Jiangqi promoter PcspD。
4. The method according to claim 3, wherein the original vector used for constructing the promoter fluorescence intensity expression vector is a pET-RFP vector.
5. Use of the engineering strain HC6-MT according to claim 1 or 2 for low-temperature production of trehalose.
6. The use according to claim 5, wherein the engineering strain HC6-MT is mixed with hemicellulose of corn stalks, and the mixture is fermented to produce trehalose.
7. The use according to claim 5 or 6, wherein the engineered strain HC6-MT is used in the form of a bacterial liquid, the OD of the bacterial liquid of the engineered strain HC6-MT 600nm 1.7-2.3.
8. The use according to claim 6, wherein the fermentation is carried out at a temperature of 18-23 ℃ for a period of 40-52 hours.
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