CN117417874B - Engineering strain HC6-MT and application thereof in low-temperature production of trehalose - Google Patents

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 can degrade corn stalk hemicellulose to produce trehalose at low temperature, and can also remarkably improve the yield of the trehalose, thereby providing an effective scheme for low-heat energy, low-cost and low-pollution production of the trehalose and a new idea for low-carbon economic development of agricultural wastes.

Description

An engineered strain HC6-MT and its application in low temperature production of trehalose

Technical Field

The invention relates to the technical field of microbial engineering, and in particular to an engineering strain HC6-MT and application thereof in producing trehalose at low temperature.

Background technique

Trehalose exists widely in organisms, often as a structural component of organisms, energy metabolism, storage material and stress metabolite. Trehalose is a disaccharide with cell-protective effects, which can effectively maintain the integrity of cell membranes and the tertiary structure of proteins, and improve the tolerance of cells to abiotic stress.

In previous studies, although a variety of methods for producing trehalose have been found, such as: synthesizing trehalose by converting gluconic acid, synthesizing trehalose by chemical method, and producing trehalose by enzyme conversion method, etc. However, the chemical method for preparing trehalose has many by-products, low yield, and high energy consumption, and the enzyme conversion method has complex procedures, low enzyme catalytic efficiency, and low substrate utilization. In addition, the biological preparation of trehalose currently used uses commercial glucose, maltose, etc. as substrates, resulting in high production costs. Therefore, finding a more low-cost, low-energy synthetic substrate and synthesis method has become the key to trehalose refining.

Straw is the most abundant biomass resource on the earth. At present, it is wasted due to improper treatment methods. In addition, the enzyme activity of microorganisms is limited by temperature under low temperature conditions, resulting in a low straw degradation rate. Usually, microbial biosynthesis requires a temperature above 30°C, which leads to a large amount of straw accumulation and burning in low-temperature agricultural planting areas, causing air pollution and incomplete utilization of resources. Microorganisms screened in low-temperature areas can adapt to low-temperature environments by producing cold-active enzymes through metabolism. Cold-resistant microorganisms have high biological activity in the temperature range of 0°C to 40°C, which can allow biocatalytic reactions in industrial production to be carried out at low temperatures, thereby reducing energy loss in the industrial production process. Therefore, it is very necessary to provide a new cold-resistant microorganism to produce trehalose using agricultural waste, corn straw, as raw material, for the utilization of agricultural waste in low-temperature areas and to reduce the cost of trehalose production enterprises.

Summary of the invention

The purpose of the present invention is to provide an engineered strain HC6-MT and its application in low-temperature production of trehalose. The engineered strain HC6-MT can degrade corn straw hemicellulose to produce trehalose under low-temperature conditions and significantly increase the trehalose yield. It also provides an effective solution for the low-energy, low-cost and low-pollution production of trehalose, and provides a new idea for the development of a low-carbon economy of agricultural waste.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an engineering strain HC6-MT, and a promoter-enhanced overexpression vector PBBR1-PD-AB is introduced into a trehalose degradation gene-deficient strain HC6-M to construct the engineering strain HC6-MT.

Preferably, the promoter-enhanced overexpression vector PBBR1-PD-AB includes the low-temperature strong promoter P cspD isolated from Raoultella HC6, the key hemicellulose degradation gene xynB , and the trehalose synthesis gene otsA .

Preferably, the trehalose degradation gene-deficient strain HC6-M is obtained by the following method: using Raoultella HC6 as the initial strain, and knocking out one or more of the trehalose degradation genes glvA , treC and TREH in the Raoultella HC6 genome.

The invention provides a method for screening a low-temperature strong promoter P cspD in Raoultella HC6, comprising the following steps: amplifying a promoter sequence of Raoultella HC6 by PCR, constructing a promoter fluorescence intensity expression vector for recombinant bacterial expression, detecting OD _600nm and fluorescence intensity of the recombinant bacteria, and screening a promoter with the largest fluorescence intensity/OD _600nm ratio within the detection range as the low-temperature strong promoter P cspD .

Preferably, the original vector used to construct the promoter fluorescence intensity expression vector is a pET-RFP vector.

The present invention also provides the use of the engineered strain HC6-MT in producing trehalose at low temperature.

Preferably, the engineered strain HC6-MT is mixed with corn stover hemicellulose and fermented to produce trehalose.

Preferably, the engineered strain HC6-MT is used in the form of a bacterial solution, and the OD600nm of the bacterial solution of the engineered strain HC6-MT is 1.7-2.3.

Preferably, the fermentation temperature is 18-23°C, and the fermentation time is 40-52h.

By adopting the above technical solution, the present invention has the following beneficial effects:

1. The present invention obtains an engineered strain HC6-MT by combining a trehalose degradation gene knockout strain HC6-M and a successfully constructed low-temperature promoter-enhanced overexpression vector PBBR1-PD-AB. The engineered strain HC6-MT can produce trehalose by degrading hemicellulose extracted from corn stalks under low temperature conditions, eliminating the operation of providing heat from the outside. At the same time, the engineered strain HC6-MT constructed after a strong promoter is introduced into wild bacteria HC6 has enhanced utilization of hemicellulose and efficiency of producing trehalose compared with wild strains, thereby reducing the strain inoculation amount and reducing production costs; in addition, the output of toxic and harmful by-products is effectively reduced.

2. The present invention shows that the engineered strain HC6-MT can produce trehalose by degrading corn straw hemicellulose at a low temperature of 20°C, so that the maximum trehalose yield reaches 1.96 g/L, which is 54 times higher than that of the wild strain. After the culture conditions are optimized, the maximum trehalose yield reaches 2.28 g/L in 48 hours.

3. The present invention converts straw into high value-added products through low-temperature microbial fermentation, thereby improving the biological utilization efficiency of straw and reducing environmental pollution pressure, providing a new idea for the development of low-carbon economy of agricultural waste.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an electrophoretic diagram of plasmid PKO3-knoc (M in Figure 1 represents 5000 bp DNA Marker, 1 represents plasmid PKO3- glvA , 2 represents plasmid PKO3- treC , and 3 represents plasmid PKO3- TREH );

Figure 2 is the knockout verification electrophoresis of different trehalose gene-deficient strains (Figure 2 (a) is HC6-g (▲ glvA ), M in (a) represents a 2000bp DNA Marker, 1 represents a gene glvA knockout verification band, and 2 represents a suicide plasmid verification band; Figure 2 (b) is HC6-t (▲ treC ), M in (b) represents a 2000bp DNA Marker, 1 represents a suicide plasmid verification band, and 2 represents a gene treC knockout verification band; Figure 2 (c) is HC6-T (▲ TREH ), M in (c) represents a 2000bp DNA Marker, 1 represents a gene TREH knockout verification band, and 2 represents a suicide plasmid verification band; Figure 2 (d) is HC6-Tg (▲ TREH ▲ glvA ), M in (d) represents a 2000bp DNA Marker, 1 represents the gene glvA knockout verification band, 2 represents the gene TREH knockout verification band, and 3 represents the suicide plasmid verification band; (e) in Figure 2 is HC6-tg (▲ treC ▲ glvA ), and M in (e) represents a 2000bp DNA Marker, 1 represents the suicide plasmid verification band, 2 represents the gene glvA knockout verification band, and 3 represents the gene treC knockout verification band; (f) in Figure 2 is HC6-Tt (▲ TREH ▲ treC ), and M in (f) represents a 2000bp DNA Marker, 1 represents the gene treC knockout verification band, 2 represents the gene TREH knockout verification band, and 3 represents the suicide plasmid verification band; (g) in Figure 2 is HC6-M (▲ glvA ▲ treC ▲ TREH ), and M in (g) represents a 2000bp DNA Marker, 1 represents the suicide plasmid verification band, 2 represents the gene glvA knockout verification band, 3 represents the gene TREH knockout verification band, 4 represents the gene treC knockout verification band);

FIG3 is a diagram showing the construction process of the trehalose gene-deficient strain HC6-M (▲ glvA ▲ treC ▲ TREH );

Figure 4 shows the growth status of the trehalose degradation gene defective strain and the HC6 strain (WT) and the trehalose accumulation (Figure 4 (a) shows the growth curve and trehalose accumulation of the trehalose degradation gene defective strain and the HC6 strain (WT), and Figure 4 (b) shows the growth status of the trehalose degradation gene defective strain and the HC6 strain (WT) at different dilution multiples; significant differences are indicated by different lowercase letters, p <0.05);

Fig. 5 is an electrophoresis diagram of different promoters in strain HC6 (M in Fig. 5 represents 2000 bp DNA Marker, 1 represents cspA gene promoter, 2 represents cspB gene promoter, 3 represents cspC gene promoter, 4 represents cspD gene promoter, 5 represents cspE gene promoter, and 6 represents cspG gene promoter);

Figure 6 is an electrophoretogram of the pET-promoter-RFP vector (M in Figure 6 represents a 5000 bp DNA Marker, 1 represents pET-P cspA -RFP verification, 2 represents pET-P cspB -RFP verification, 3 represents pET-P cspC -RFP verification, 4 represents pET-P cspD -RFP verification, 5 represents pET-P cspE -RFP verification, and 6 represents pET-P cspG -RFP);

Figure 7 is a diagram showing the expression of recombinant bacteria with different promoter fluorescence intensity expression vectors (Figure 7 (a) shows the color change of the culture medium of recombinant bacteria with different promoter fluorescence intensity expression vectors, and Figure 7 (b) shows the fluorescence intensity of recombinant bacteria with different promoter fluorescence intensity expression vectors);

FIG8 is an electrophoresis diagram of different PBBR1-PD-AB recombinant vectors (M in FIG8 represents 2000 bp DNA Marker, 1 represents xynB verification, 2 represents P cspD + xynB , 3 represents PBBR1, 4 represents otsA , and 5 represents P cspD + otsA );

Figure 9 is a diagram of corn straw hemicellulose and Fourier transform infrared spectroscopy detection and fermentation results (Figure 9 (a) is an intermediate product in the process of corn straw hemicellulose extraction, 1 in (a) is dry straw powder, 2 in (a) is straw precipitate after alkali solution dissolution, 3 in (a) is hemicellulose solution, and 4 in (a) is hemicellulose precipitate; Figure 9 (b) is the FTIR spectrum of xylan standard (upper curve) and corn straw extracted hemicellulose (lower curve); Figure 9 (c) is the growth curve and xylose yield of strain HC6);

FIG10 is a diagram showing the utilization of substrate hemicellulose by the wild strain HC6 and the engineered strain HC6-MT, i.e., the accumulation of xylose and trehalose (FIG. 10 (a) shows the results of the wild strain HC6, and FIG10 (b) shows the results of the engineered strain HC6-MT);

FIG11 is a graph showing the effect of the interaction between substrate concentration and cold shock time ratio on HC6-MT trehalose production;

FIG12 is a diagram showing the effect of the interaction between pH and cold shock time ratio on HC6-MT trehalose production;

FIG13 is a graph showing the effect of the interaction between phosphate concentration and cold shock time ratio on trehalose production of HC6-M;

FIG14 is a graph showing the effect of the interaction between substrate concentration and pH on HC6-MT trehalose production;

FIG15 is a graph showing the effect of the interaction between substrate concentration and phosphate concentration on trehalose production of HC6-MT;

FIG. 16 is a graph showing the effect of the interaction between phosphate concentration and pH on the trehalose production of HC6-MT.

Biological Deposit Description

The Raoultella sp. HC6 involved in the present invention was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 7, 2020. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 20607.

Detailed ways

The invention provides an engineering strain HC6-MT, and the promoter-enhanced overexpression vector PBBR1-PD-AB is introduced into a trehalose degradation gene-deficient strain HC6-M to construct the engineering strain HC6-MT.

In the present invention, the promoter-enhanced overexpression vector PBBR1-PD-AB includes the low-temperature strong promoter P cspD isolated from Raoultella HC6, the key hemicellulose degradation gene xynB and the trehalose synthesis gene otsA .

In the present invention, the Raoultella sp . HC6 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 7, 2020, with the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 20607. The Raoultella sp. HC6 described in the present invention is a psychrophilic bacterium that can grow and metabolize under low temperature conditions, and the strain itself contains the key hemicellulose degradation gene xynB and the trehalose synthesis gene otsA , so it can use hemicellulose as a substrate to produce trehalose.

In the present invention, the trehalose degradation gene-deficient strain HC6-M is obtained by the following method: using Raoultella HC6 as the initial strain, knocking out one or more of the trehalose degradation genes glvA , treC and TREH in the Raoultella HC6 genome. Further preferably, the trehalose degradation gene-deficient strain HC6-M of the present invention is using Raoultella HC6 as the initial strain, knocking out the trehalose degradation genes glvA , treC and TREH in the Raoultella HC6 genome.

The invention provides a method for screening a low-temperature strong promoter P cspD in Raoultella HC6, comprising the following steps: amplifying a promoter sequence of Raoultella HC6 by PCR, constructing a promoter fluorescence intensity expression vector for recombinant bacterial expression, detecting OD _600nm and fluorescence intensity of the recombinant bacteria, and screening a promoter with the largest fluorescence intensity/OD _600nm ratio within the detection range as the low-temperature strong promoter P cspD .

In the present invention, the original vector used to construct the promoter fluorescence intensity expression vector is the pET-RFP vector.

The present invention also provides the use of the engineered strain HC6-MT in producing trehalose under low temperature conditions.

In the present invention, the engineered strain HC6-MT can produce trehalose by fermenting corn straw hemicellulose. In the present invention, the engineered bacterial strain HC6-MT is mixed with corn straw hemicellulose, and the engineered strain HC6-MT is mixed and applied in the form of bacterial liquid. The OD _600nm of the bacterial liquid of the engineered strain HC6-MT is preferably 1.7-2.3, further preferably 1.8-2.2, and further preferably 1.9-2.1. The fermentation of the present invention is preferably shake flask fermentation, and the rotation speed of the shake flask is preferably 170-250rpm, further preferably 185-220rpm, and further preferably 200rpm; the fermentation temperature is preferably 18-23°C, further preferably 19-21°C, and further preferably 20°C; the fermentation time is preferably 40-52h, further preferably 45-50h, and further preferably 48h.

The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1 Obtaining Trehalose Degradation Gene Deficient Strain HC6-M

1. Genome sequencing of Raoultella strain HC6

The genome of Raoultella sp. strain HC6 was sequenced, and the results showed that the genome (accession number CP093276.1) was 6243766bp in length, of which the total length of the chromosome was 5955116bp and the GC content was 58.42%. Strain HC6 has three plasmids, plasmid 1 is 167204bp in size and 51.75% in GC content; plasmid 2 is 100650bp in size and 51.54% in GC content; plasmid 3 is 20796bp in size and 57.56% in GC content. The sequencing results of the 16SrDNA gene sequence of strain HC6 were compared with the data in the NCBI ribosomalRNA sequence database, and the results showed that the similarity with the genus Raoultella sp. reached 100%, indicating that strain HC6 did not undergo species variation and could be used for subsequent experiments.

2. Trehalose degradation gene excision

The suicide plasmid PKO3-km (commonly purchased) was digested with endonuclease BamHI at 35°C for 30 min. The digestion reaction system was: PKO3-km 5.0 μL, 10×Ligation Buffer 2.0 μL, Quick cutBamHI 1.0 μL, Sterilized ddH ₂ O Up to 20.0 μL.

Using the genomic DNA of strain HC6 as a template, primers were designed and PCR amplification reaction was performed (PCR amplification system: 5× PrimeSTAR Buffer (Mg ²⁺ Plus) 10 μL, dNTP Mixture (2.5 mM each) 4 μL, Primer F 10-15 pmol, Primer R 10-15 pmol, Template <200 ng, template DNA 2 μL, sterile water up to 50 μL. The PCR amplification program was: 98°C denaturation for 10 s, 60°C annealing for 5 s, 72°C 1 min, and 30 cycles.) The upstream and downstream homology arm sequences of the target genes to be knocked out (trehalose degradation genes glvA , treC and TREH ) were obtained (500 bp sequences upstream and downstream of the target gene sequences to be knocked out were used as homology arms). The primer sequences are shown in Table 1.

Table 1 PCR primer sequences of upstream and downstream homology arms of target genes to be knocked out

After the amplification reaction, the target gene fragment was purified and recovered using the FastPure Gel DNA Extraction Mini Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.). The purified product was directly subjected to a ligation reaction. The ligation reaction products were the upstream and downstream homology arms of the trehalose degradation genes glvA , treC and TREH sequences to be knocked out obtained in the above steps, and then the upstream and downstream homology arm fragments were connected to the linearized PKO3-km plasmid using the In-Fusion seamless cloning connection technology to obtain the plasmid PKO3-knock, specifically PKO3- glvA , PKO3- treC , PKO3- TREH gene knockout recombinant plasmid vector. The In-Fusion seamless cloning connection system is shown in Table 2.

Table 2 In-Fusion seamless cloning connection system

(The "Target gene fragment" indicates the upstream and downstream homology arms of the corresponding gene)

Electrophoresis was performed to verify whether the plasmid PKO3-knock was successfully constructed. The plasmid PKO3-knock obtained above was double-digested with BamHI and SalI to obtain two electrophoresis bands of 1.0 kb and 5.6 kb, which were consistent with the band sizes of PKO3- glvA , PKO3- treC , and PKO3- TREH , indicating that these recombinant plasmids were constructed correctly (as shown in Figure 1).

Using strain HC6 as the host, plasmid PKO3-knock was electroporated into the host bacteria and cultured in SOC medium at 30°C for 24 hours. At this time, plasmid PKO3-knock will undergo homologous recombination with the host genome, thereby knocking out the target gene. Then PCR amplification of specific bands was performed again to verify whether the gene knockout was successful. The primers used are shown in Table 3. The results are shown in Figure 2.

Table 3 PCR primer sequences for testing target gene deletion

3. Verification of the growth of trehalose gene-deficient strains

Based on the above operations, 7 strains with trehalose degradation gene defects were constructed in sequence (as shown in Figure 3), namely HC6-g (▲ glvA ), HC6-t (▲ treC ), HC6-T (▲ TREH ), HC6-Tg (▲ TREH ▲ glvA ), HC6-tg (▲ treC ▲ glvA ), HC6-Tt (▲ TREH ▲ treC ), and HC6-M (▲ glvA ▲ treC ▲ TREH ).

The constructed trehalose degradation gene defective strain and HC6 strain (WT) were inoculated into LB liquid medium for overnight activation, and then the bacterial solution OD _600nm = 2.0 ± 0.1 was adjusted, and the bacterial solution was diluted to 10 ^-1 -10 ^-6 times, and 2.0 μL of bacterial solution was taken for each dilution multiple, and the growth detection and trehalose accumulation were determined in a shake flask fermentation in a hemicellulose liquid medium (20°C, 80h, 200rpm). Similarly, 2.0 μL of bacterial solution was taken for each dilution multiple, and cultured in a 20°C constant temperature incubator on an inorganic salt solid medium with trehalose as the only carbon source, and the growth state of the colony was observed. The results are shown in Figure 4.

Example 2 Obtaining the low temperature promoter enhanced overexpression vector PBBR1-PD-AB

1. Screening of cold shock protein promoters with strong low temperature and prediction of functional regions

There are 6 csp genes in strain HC6. To screen promoters with high expression at low temperature, the promoter sequences of 6 csp genes were obtained from the whole genome of strain HC6. The prokaryotic promoter online analysis and prediction website BPROM (http://linux1.softberry.com/) was used to predict the distances of promoter -10 region, -35 region and spacer region.

The 6 cold shock protein gene sequences and their promoters are as follows:

Cold shock protein gene HC6_00172 Cold shock protein CspA , the sequence is shown in SEQ ID NO.21, specifically: ATGTCCGGTAAAATGACTGGTATCGTAAAATGGTTCAACGCTGATAAAGGTTTCGGCTTCATTACTCCTGATGACGGTTCTAAAGATGTATTCGTACACTTCTCCGCTATCATGAACGATGGCTACAAATCTCTGGATGAAGGTCAGAAAGTTTCCTTCACCATCGAAAGCGGCGCTAAAGGCCCGGCAGCTGGCAACGTTACCAGCCTGTAA;

The promoter of the cold shock protein gene HC6_00172 Cold shock protein CspA promoter, the sequence of which is shown in SEQ ID NO.22, is specifically: AGTTCTATAATGTTATTGGACACTGATTACGGTCCTGATTAGGGACCGTTTTAATTTCTATGAAAAAAACGTTTGCTATCGCACCTCAAAAATGAGTTAATGCTCGCAGGTTTGATGTACAGACCACAGAGCATTAGTATAGAGTAGTCCTTGAGCGGTTATCCTAGATACCCCGGTAGTGAACTTTCCCTTTATAGCTTCAAATCTGTAGTCCAGACCGTATCGCCGAAAGGTTCATTTATTATTTAAAGGTATTTCGCT;

Cold shock protein gene HC6_02655 Cold shock-like protein CspB , the sequence is shown in SEQ ID NO.23, specifically: ATGTCTGGTAAGATGATTGGTTTAGTAAAATGGTTTAATGAAGGTAAAGGTTTCGGCTTTATTTCTCCAGTAGACGGCAGTAAAGATGTTTTCGTGCATTTTTCTGCGCTGCAGGGCGATGGCTTTAAAACTTTATTTGAAGGGCAGAAGGTCGAATTCACTATCGAGAGCGGCGCCAAAGGCCCTGCTGCCGGTAATGTCGTTCTGCGCGACTAA;

The promoter of the cold shock protein gene HC6_02655 Cold shock-like protein CspB promoter, the sequence of which is shown in SEQ ID NO.24, specifically: TTATTCACGGCTTCTCTGTGGGGGCAGAAAATAATGCTTGCTATCAATGGTTATCTATGTGATCAATAGCAGGTCGGTTTGGTACACAGAACCAATAAAGCGGTTTAGTAAAGCAGTCCTCATCTCAAGCGTTATTCCTAAATAATCCTTTTTTCGAGCCTCCTTATCGTTATTAATTAATTTCTGTGATGCGAACTTTTTCGCCGCAAGGCTTTGAACATTGGGATAATACTT;

Cold shock protein gene HC6_02212 Cold shock-like protein CspC , the sequence is shown in SEQ ID NO.25, specifically: ATGGCAAAGATTAAAGGTCAAGTTAAGTGGTTCAACGAGTCTAAAGGTTTTGGCTTTATTACTCCGGCTGATGGCAGCAAAGACGTGTTCGTACACTTCTCCGCTATCCAGGGTAATGGCTTCAAAACTCTGGCTGAAGGCCAGAACGTTGAGTTCGAAATTCAGGACGGCCAGAAAGGTCCGGCAGCAGTTAACGTAACTGCTATCTGA;

The promoter of the cold shock protein gene HC6_02212 Cold shock-like protein CspC promoter, the sequence of which is shown in SEQ ID NO.26, is specifically:

Cold shock protein gene HC6_03855 Cold shock-like protein CspD , the sequence is shown in SEQ ID NO.27, specifically: ATGGAAATGGGTACTGTTAAGTGGTTCAACAATGCCAAAGGGTTTGGTTTTATTTGCCCTGAGGGCGGAGGCGAAGACATTTTCGCCCATTACTCCACCATCCAGATGGATGGTTACAGAACGCTAAAAGCCGGACAAGCCGTTCGGTTTGATGTTCACCAGGGACCGAAAGGTAATCACGCCAGCGTGATTGTTCCTGTAGAAGCGGAAGCGGCTGCATAA;

The promoter of the cold shock protein gene HC6_03855 Cold shock-like protein CspD promoter, the sequence of which is shown in SEQ ID NO.28, specifically: CGTCAGTATTCATCATCGGTTGCTGTTGCCAACAGGCGGTGGCCTGTCGATGACCAAAAGTTATGCCCATCACAAATCTACAATAGATCATAGATAACTATCATCTATTACTTCCATCCGCGACGTCTGTCACATTCCCCGGCAATAGCGTTAACTGCTTCAAATTTTGACGCATTTTTCGCCTTCCCCTACCGTCAATCGCTTGACGCCTTTTCGTATTTCTCTAAATTGTACTGGCGAGAGTTGGCGAGCATTTGAACAACTCGTCACTCCACTACCGGTTCATTCCATCTTACTTATAAAGAATTACGAAGGATGTCGAAGT;

Cold shock protein gene HC6_04170 Cold shock-like protein CspE , the sequence is shown in SEQ ID NO.29, specifically: ATGTCTAAGATTAAAGGTAACGTTAAGTGGTTTAATGAGTCCAAAGGATTCGGTTTCATTACTCCGGAAGATGGCAGCAAAGATGTATTCGTACATTTCTCTGCAATCCAGTCCAACGGTTTCAAAACTCTGGCTGAAGGTCAGCGTGTAGAGTTCGAAATCACTAACGGTGCCAAAGGCCCTTCTGCTGCAAACGTAAACGCTATCTAA;

The promoter of the cold shock protein gene HC6_04170 Cold shock-like protein CspE promoter, the sequence of which is shown in SEQ ID NO.30, is specifically: TCCGCACTAGCTTAGTGATAAAAGAGCTGAGCATTATGTTATGTGGAAAAACAATAACTAAAGCGCAACCACTAAAAAAGATAGCGACTTTTGTCACTTTTTAGCAAAGTTCGACTGGACAAAAGGCACCACAATTGATGTACTGGATCGCGACACAGTATCAGTGTCTTTTTTTCATATAAAGGTAATTTTG;

Cold shock protein gene HC6_02543 Cold shock-like protein CspG , the sequence is shown in SEQ ID NO.31, specifically: TTGTTCCAAAAAATGACAGGCATTGTCAAAAGCTTTGATAATAAAACCGGCAGAGGCCTTATCGTCCCTTCCGACGGTCGTAAAGACGTTCAGGTTCATATTTCCGCATTATCCCCCAACGAGTCCACGCCAATGACGCCCGGTATTCGCGTTGAGTTTCGTCGGGTTAACGGCCTGCGCGGGCCAACCGCGGCAAACGTCTATACCTGCTAG;

The promoter of the cold shock protein gene HC6_02543 Cold shock-like protein CspG promoter, the sequence of which is shown in SEQ ID NO.32, is specifically:

The promoter sequence prediction results are shown in Table 4.

Table 4 Promoter sequence prediction results

(II) Construction of promoter fluorescence intensity expression vector pET-promoter-RFP

Using the genomic DNA of strain HC6 as a template, PCR was used to amplify its promoter DNA sequence, and the size of each promoter sequence was between 200-500 bp. After agarose gel electrophoresis, the amplified bands were recovered and sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were consistent with the genome sequence (as shown in Figure 5).

The amplified promoter DNA sequence was connected to the pUCm-T vector, and the successfully connected vector was named pUCmT-promoter, and was transferred into DH5ɑ bacteria for stable cloning. The pUCmT-promoter vector and the pET-RFP vector were extracted and double-digested with Quick cut BamHI and Quick cut KpnI enzymes, respectively, and the digested pET-RFP vector was connected to the promoter of strain HC6. The successfully connected vector was named pET-promoter-RFP, specifically pET-P cspA -RFP, pET-P cspB -RFP, pET-P cspC -RFP, pET-P cspD -RFP, pET-P cspE -RFP, and pET-P cspG -RFP.

The constructed vector was subjected to electrophoresis to verify whether the construction was successful. The pET-promoter-RFP vector (about 5000 bp) and promoter-RFP gene recombinant fragment (about 1000 bp) bands were consistent with the lengths shown in the electrophoresis results, proving that the vector was successfully constructed (as shown in Figure 6).

3. Identification of low temperature strong promoters

The above-mentioned promoter fluorescence intensity expression vector construction method was used for the traditional T7 strong promoter to construct pET-PT7-RFP, and then pET-P cspA -RFP, pET-P cspB -RFP, pET-P cspC -RFP, pET-P cspD -RFP, pET-P cspE -RFP, pET-P cspG -RFP and pET-PT7-RFP were respectively transferred into E. coli BL31 (DE3) for expression. The relative fluorescence intensity (fluorescence intensity/OD _600nm ) of RFP gene expression was calculated by detecting the biomass and fluorescence intensity of the recombinant bacteria, and this was used to characterize the promoter strength.

After culturing at 37°C for 16 hours in a 48-well plate, the strength of the promoters was ranked as P cspC <P cspB <P cspG <PT7 <P cspA <P cspE <P cspD . After 8 hours of cold induction, except for the P cspC and P cspB groups where no red fluorescence was detected, the order of promoter strength was P cspG <PT7 <P cspA <P cspE <P cspD . At the same time, during the LB liquid culture medium culture of each strain, differences in the color change of the culture medium due to the accumulation of red mCherry protein can be observed (as shown in Figure 7). Based on the above results, the promoter P cspD of the cold shock protein gene cspD was determined to be a low-temperature strong promoter for the construction of a promoter-enhanced overexpression vector.

(IV) Construction of promoter-enhanced overexpression vector PBBR1-PD-AB

The key hemicellulose degradation gene xynB sequence, trehalose synthesis gene otsA sequence and cspD promoter sequence were amplified by PCR, and then connected with the linearized universal expression plasmid PBBR1-MCS2 using the In-Fusion ^® Snap Assembly Master Mix seamless connection kit (purchased from Bao Bioengineering (Dalian) Co., Ltd.), universal restriction endonucleases BmHI and KpnI to complete the construction of PBBR1-PD-AB.

Table 5 PCR primer sequences for xynB, otsA and cspD sequences

To verify whether the recombinant vector was successfully constructed, the PBBR1-PD-AB vector was used as a template to amplify the xynB gene band (1321bp), P cspD + xynB fusion band (1624bp), otsA gene band (896bp), P cspD + otsA fusion band (1199bp) and vector-specific band PBBR1 (346bp). The amplified band electrophoresis gel is shown in Figure 8, and the band sizes are consistent, proving that the PET-PD- xynB and PET-PD- otsA vectors were successfully constructed.

Example 3

(I) Construction of the engineered strain HC6-MT

Take out the prepared trehalose gene-deficient strain HC6-M competent cells from the -80℃ refrigerator and thaw on ice. Take 10μL of the extracted overexpression vector (PBBR1-PD-AB), add it to the competent cells, and continue to place it on ice for 30 minutes. Add the mixture with the overexpression vector (PBBR1-PD-AB) to the pre-cooled electroporation cup, and set the electroporation conditions to: 1500KV, 800ꭥ. After the electroporated bacterial solution is added to 1mL of SOC culture medium for recovery and culture for 1h, the bacterial solution is spread on a Kana antibiotic LB solid culture medium plate with a working concentration of 50ng/mL, cultured overnight at 20℃, pick the colony, and use the overexpression vector (PBBR1-PD-AB) verification primer for PCR verification. The successfully verified strain was named HC6-MT.

2. Extraction of hemicellulose from corn straw

Corn stalks (from Xiangyang Farm, Northeast Agricultural University) were dried, cut into 2.0-3.0 cm small segments, and ground into powder using a grinder. The obtained dry stalk powder was passed through an 80-mesh sieve, and then mixed with 10% NaOH at a ratio of 1:10. After soaking at 70 ° C for 2 hours, the mixture (straw precipitate) was filtered through gauze and the filtrate was taken. The pH of the filtrate was adjusted to 7.0 ± 0.2 with concentrated hydrochloric acid, mixed with activated carbon at a ratio of 10:1 (v/w), and after a 60 ° C water bath for 15 minutes, the activated carbon was removed by centrifugation at 10000 rpm for 10 minutes to obtain a hemicellulose solution. Then, 50% of the volume of the hemicellulose solution was added to anhydrous ethanol to obtain a hemicellulose precipitate. After solid-liquid separation, the filter residue was collected and dried at 50 ° C to obtain hemicellulose.

The results of Fourier transform infrared spectroscopy detection of the obtained hemicellulose and xylan standards are shown in Figure 9. The results show that the absorption peak positions displayed by the corn straw extract and the xylan standard are basically consistent, indicating that the main component of the hemicellulose extracted from corn straw is xylan.

Corn straw hemicellulose was used as the substrate, and 5% (v/v) Raoultella HC6 was inoculated. The culture was fermented at 20°C for 80 hours, and samples were taken every 6 hours to observe the growth curve of Raoultella HC6 and detect the production of xylose. The results are shown in Figure 9, which shows that strain HC6 can grow with hemicellulose extracted from corn straw as the only carbon source, and degrade xylan (the main component of hemicellulose) to produce xylose.

Example 4

1. Preparation of corn straw hemicellulose liquid culture medium

The corn stover hemicellulose liquid culture medium was prepared according to the following formula: 10 g/L of the above-obtained hemicellulose, 2 g/L of NH ₄ NO ₃ , 2 g/L of K ₂ HPO ₄ , and 0.2 g/L of MgSO ₄ .

(two)

The ability of Raoultella HC6 and the engineered strain HC6-MT prepared in Example 3 to produce trehalose was tested, with Raoultella HC6 producing trehalose alone as a control.

Control group: The bacterial liquid of strain HC6 (OD _600nm =2.0±0.1) was inoculated into the above-mentioned corn straw hemicellulose liquid culture medium at a 5% inoculum (v/v), and the culture was shaken at 20°C and 200 rpm for 80 h. Samples were taken every 6 h to detect the utilization of substrate hemicellulose by strain HC6 and the trehalose production.

Experimental group: The engineered bacteria HC6-MT culture liquid (OD _600nm =2.0±0.1) was inoculated into the above-mentioned corn straw hemicellulose liquid culture medium at a 5% inoculum (v/v), and the culture was shaken in a flask at 20°C and 200 rpm for 80 h. Samples were taken every 6 h to detect the utilization of substrate hemicellulose by the strain HC6-MT and the trehalose production.

The trehalose content detection method is as follows: After preparing different concentrations of trehalose standard solutions (0.2, 0.1, 0.075, 0.05, 0.025, 0.0125 mg/mL), take 2.0 mL of the standard solution and add 5.0 mL of 0.2% anthrone-sulfuric acid solution, boil in water bath for 8 minutes, measure the absorbance at 620 nm after cooling, and draw a standard curve for trehalose content at OD _{620 nm} . The blank group is 2.0 mL of deionized water added to 5.0 mL of 0.2% anthrone-sulfuric acid solution, and the same operation is performed after boiling in water bath for 8 minutes.

In the early stage of xylan utilization, a certain amount of xylose will accumulate. Since the depolymerization rate of xylan (i.e., the generation rate of xylose) is greater than the degradation rate of xylose, xylose will accumulate in the early stage of depolymerization. After that, the engineered strain HC6-MT will further utilize xylose and convert it into trehalose.

The test results of trehalose and xylose accumulation of wild strain HC6 and engineered strain HC6-MT are shown in Figure 10. It can be seen from the figure that the maximum trehalose accumulation of the engineered strain HC6-MT is 1.96 g/L, which is 54 times higher than that of the wild strain HC6; the maximum xylose accumulation of the engineered strain HC6-MT is 3.61 g/L, which is 2.3 times higher than that of the wild strain, successfully enhancing the hemicellulose utilization ability and trehalose accumulation ability of the strain HC6.

Example 5 Optimization of fermentation conditions of strain HC6-MT

Minitab2022 software was used to design a four-factor three-level experiment. Based on the results of univariate analysis, with significance p <0.05 as the selection criterion, the 10°C cold shock time after inoculation of the fermentation medium (A), the medium substrate (corn hemicellulose) concentration (B), the medium pH (C) and the phosphate in the medium (D) were used as independent variables, and the trehalose yield was used as the response value to explore the optimal fermentation conditions of the constructed recombinant strain HC6-MT. The four response surface factors and variable levels that affect the fermentation of trehalose by the strain HC6-MT are shown in Table 6.

Table 6 Response surface factors and variable levels for fermentation optimization of strain HC6-MT

As shown in Figures 11 to 16, the engineered strain HC6-MT was inoculated into LB liquid culture medium for activation culture at 20°C for 16 hours, then the OD _600nm was adjusted to 2.0±0.1 with sterile water, and inoculated into corn straw hemicellulose liquid culture medium at an inoculation ratio of 5%. After optimization, under the optimal trehalose production conditions, the maximum trehalose yield of 2.28 g/L was reached in 48 hours.

It can be seen from the above examples that the engineered strain HC6-MT provided in the technical solution of the present invention can produce trehalose by degrading hemicellulose extracted from corn straw under low temperature conditions, saving production raw materials, reducing production costs, and improving the yield, conversion rate and productivity of trehalose.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (3)

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-temperature strong promoter PcspD separated from Raoult bacteria HC6, a hemicellulose key degradation gene xynB and a trehalose synthesis gene otsA;

the sequence of the low-temperature strong promoter PcspD is shown as SEQ ID NO. 28;

the temperature of the low temperature is 20 ℃;

the trehalose degrading gene deletion strain HC6-M is obtained by the following method: taking Raoult bacteria HC6 as an initial strain, and knocking out trehalose degradation genes glvA, treC and TREH in a Raoult bacteria HC6 genome;

the sequences of the PCR primers of the upstream and downstream homology arms of the trehalose degradation gene glvA are a glvA primer 1 group and a glvA primer 2 group, and the specific sequences of the glvA primer 1 group are shown as SEQ ID NO.1 and SEQ ID NO. 2; the specific sequences of the glvA primer 2 group are shown in SEQ ID NO.3 and SEQ ID NO. 4;

the PCR primer sequences of the upstream and downstream homology arms of the trehalose degradation gene treC are treC primer 1 groups and treC primer 2 groups, and the specific sequences of the treC primer 1 groups are shown as SEQ ID NO.5 and SEQ ID NO. 6; the specific sequences of the treC primer 2 group are shown as SEQ ID NO.7 and SEQ ID NO. 8;

the upstream and downstream homology arm PCR primer sequences of the trehalose degradation gene TREH are a TREH primer 1 group and a TREH primer 2 group, and the specific sequences of the TREH primer 1 group are shown as SEQ ID NO.9 and SEQ ID NO. 10; the specific sequences of the TREH primer 2 group are shown as SEQ ID NO.11 and SEQ ID NO. 12.

2. The application of the engineering strain HC6-MT in the production of trehalose, which is characterized in that the engineering strain HC6-MT is mixed with corn stalk hemicellulose, and the mixture is fermented to produce the trehalose;

the fermentation temperature is 18-23 ℃, and the fermentation time is 40-52h.

3. The use according to claim 2, wherein the engineering strain HC6-MT is used in the form of a bacterial liquid, the OD of the bacterial liquid of the engineering strain HC6-MT _600nm 1.7-2.3.