A zymomonas mobilis engineering strain tolerating industrial cane sugar wastewater and high-yield levan, and a construction method and application thereof

Abstract

The application discloses a construction method of an engineered strain of a zymomonas mobilis resistant to industrial cane sugar wastewater and high in levans production, and steps of the method comprise the following steps: (1) knocking out an extracellular sucrose enzyme coding gene and a glucose-fructose oxidoreductase coding gene in the zymomonas mobilis to obtain an engineered strain 1 high in levans production; (2) replacing an ethanol dehydrogenase gene in the engineered strain 1 with a levansucrase gene to obtain an engineered strain 2 high in levans production and low in ethanol byproduct; (3) introducing a heterologous ethanol utilization pathway into the engineered strain 2 obtained in the step (2) to obtain an engineered strain 3; and (4) subjecting the engineered strain 3 obtained in the step (3) to normal-pressure room-temperature plasma mutagenesis and laboratory evolution to obtain the engineered strain of the zymomonas mobilis resistant to industrial cane sugar wastewater and high in levans production. The application realizes resource utilization of high-concentration cane sugar wastewater and synthesis of high-value-added products, and has good industrial application prospect and environmental protection value.

Description

Technical Field

[0001] This application relates to the field of applied microbial technology, specifically to the construction and application of engineered strains of *Mammotrophic motility-fermenting* bacteria in industrial sucrose wastewater with low pH, high osmotic pressure, and strong inhibition. Background Technology

[0002] L-Fructan is a high-value-added β-(2,6)-linked fructan with various biological activities and broad application prospects. It has significant application value in the food, nutritional supplement, pharmaceutical, and cosmetic industries. However, the commercial availability of L-Fructan is still limited by its high production cost. my country has abundant sucrose resources that can be used to produce L-Fructan, which is of great significance for promoting the sustainable development of my country's sugar industry, expanding its functions, and ensuring the supply of national bio-based products. Enzymatic synthesis and microbial fermentation are currently the mainstream methods for producing L-Fructan, but the purification and reuse of enzymes in the enzymatic synthesis process are time-consuming and labor-intensive, making microbial fermentation a more promising method. However, there are currently some challenges in the microbial fermentation production of L-Fructan using sucrose. Many strains in nature cannot utilize sucrose or grow at high sucrose concentrations, and the yield of microorganisms that can naturally utilize sucrose to produce L-Fructan is very low.

[0003] Motile fermentation monoclonal bacteria ( Zymomonas mobilis *Agromonas motilityis* is the only microorganism that ferments glucose via the ED metabolic pathway. Using sucrose as a substrate, it can efficiently convert sucrose into L-glycosyls via various sucrase enzymes, while simultaneously producing glucose for further metabolism. This demonstrates advantages such as high ethanol fermentation efficiency and ethanol tolerance. Furthermore, *Agromonas motilityis* is characterized by ease of gene manipulation, high biosafety (GRAS), strong adaptability, and low byproduct production, making it a more formidable competitor in L-glycosyl production. However, wild-type *Agromonas motilityis* exhibits very low gene editing efficiency, resulting in a low yield of L-glycosyls from sucrose fermentation; with a 100 g / L sucrose substrate, the yield is only 1.4 g / L.

[0004] Currently, most research on *Fermentomonas motilityis* is focused on its use in ethanol production. For example, patent CN116496965A, "Ethanol-producing *Fermentomonas motilityis* and its application," discloses the knockout of *Fermentomonas motilityis*. sacB Genes, to achieve scaCOverexpression of the gene yielded *Factomonas motilityis* F74, a strain obtained by fructose gradient domestication of *Factomonas motilityis* 8b. The *Factomonas motilityis* strain was inoculated into a fermentation medium containing molasses and cultured, and ethanol was harvested from the fermentation broth. The patented invention discloses a gelatinous Bacillus leucosyl sucrase TrLvs and its encoding gene capable of synthesizing a high proportion of high molecular weight leucosyl sucrose. This enzyme exhibits excellent catalytic properties, catalyzing the conversion of sucrose substrates to high molecular weight leucosyl sucrose via transglycosylation at a rate of 84.9%. This invention also discovered a new special functional motif HTL on this enzyme; by retaining or deleting this motif on the leucosyl sucrase, the conversion of the synthesized product leucosyl sucrose within high and low molecular weight ranges can be regulated. From the above utilization of *Factomonas motilityis*, it is found that most current applications use this microorganism for ethanol production by silencing the fructan sucrase gene, preventing the microorganism from converting sucrose to fructan. Currently, a few studies have only focused on knocking out the sucrase gene. sacC However, increasing the content of L-glycosides lacks systematic metabolic engineering modifications, and these strains are intolerant to real sucrose wastewater, mostly utilizing pure sucrose to produce L-glycosides, which lacks practical application value. In summary, there is an urgent need for a method and application for constructing a high-yield L-glycoside-producing motile fermentation monoclonal antibody, providing a method for constructing such an antibody, and significantly improving its ability to produce L-glycosides from sucrose or sucrose wastewater through combined metabolic engineering modifications. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for systematically modifying motile fermentation monotypic bacteria to enable them to produce high yields of levofloxacin (also known as levofloxacin, levofloxacin, etc.) from industrial sucrose wastewater with low pH, high inhibitors, and high sugar concentration.

[0006] The objective of this invention is achieved through the following technical solution: On the one hand, a method for constructing an engineered strain of *Bacillus simulans* that is tolerant to industrial sucrose wastewater and produces high levels of levoglucan includes the following steps: (1) Using *M. motile fermentum* as the starting strain, the extracellular sucrase encoding gene and glucose-fructose oxidoreductase encoding gene in *M. motile fermentum* were knocked out to block the fructose metabolism bypass and obtain an engineered strain 1 with increased L-glycan production. (2) Replace the ethanol dehydrogenase gene in the engineered strain 1 with the levo-saccharide sucrase gene to divert carbon to levo-saccharide synthesis and metabolism, thereby obtaining engineered strain 2 with high levo-saccharide production and reduced ethanol by-products. (3) Introduce the heterologous ethanol utilization pathway into the engineered strain 2 obtained in step (2) to convert ethanol into acetyl coenzyme A, improve the balance between cell energy supply and reducing power, and obtain a secondary engineered strain 3 with shortened fermentation time. (4) The engineered strain 3 obtained in step (3) is subjected to atmospheric pressure room temperature plasma mutagenesis and combined with adaptive laboratory evolution to obtain a mutant strain that can tolerate real industrial sucrose wastewater with high sucrose concentration, low pH value and high salt stress, namely the engineered strain of motile fermentation monoclonal bacteria that is tolerant to industrial sucrose wastewater and produces high levels of L-glucan.

[0007] Specifically, the extracellular sucrase encoding gene mentioned in step (1) is *Mammotrophic follicle*. sacC The gene, the sequence of which is shown in SEQ ID NO: 1; the glucose-fructose oxidoreductase encoding gene is *Mammotrophic motility-fermenting*. gfo The gene, whose sequence is shown in SEQ ID NO: 2; The gene-editing plasmid construction method involves: inserting the target gene from the genome... sacC, gfo , adhB Suitable gRNA sequences were selected from the gene and integrated into the gene editing array of the pMini editing backbone plasmid using the Gibson assembly method. Homologous arms of 500 base pairs (bp) upstream and downstream of the DNA sequence to be knocked out were cloned and integrated into the gene editing plasmid using Gibson assembly, resulting in the pMini editing plasmid. ΔsacC pMini- Δgfo pMini- adhB::sacB .

[0008] Specifically, the levo-glycan sucrase gene mentioned in step (2) is from *Mammotrophic follicle*. sacB The gene, whose sequence is shown in SEQ ID NO: 3; the alcohol dehydrogenase gene is from *Fermentomonas motile*. adhB The gene, whose sequence is as shown in SEQ ID NO: 4; The method for constructing the ethanol expression plasmid was as follows: the shuttle plasmid pEZ15Asp plasmid backbone and promoter Ppdc were amplified by PCR. ada Gene, adh2 The gene was separated by 1% agarose gel electrophoresis and purified to obtain the target fragment. After determining the DNA concentration using a Qubit 3 fluorometer, the purified fragment was mixed at a backbone:fragment molar ratio of 1:3. After Gibson assembly, 5 μL of ligation buffer was mixed with 50 μL of E. coli chemicompetent cells DH5α, incubated on ice for 30 min, then heat-shocked at 42℃ for 45 sec, incubated on ice for 2 min, and then 700 μL of LB medium was added. The mixture was incubated at 37℃ and 180 rpm for 40 min with shaking. 50 μL of the culture was spread on LB solid medium containing 100 mg / L spectinomycin and incubated overnight at 37℃. Single colonies were picked, and plasmids were extracted to obtain the correctly expressed plasmid. 500 ng of the plasmid was transformed into *C. motile fermentum* by electroporation.

[0009] Specifically, the heterologous ethanol utilization pathway described in step (3) includes the Digitrophus maize acetaldehyde dehydrogenase gene. ada and the Saccharomyces cerevisiae alcohol dehydrogenase gene adh2 The gene sequences are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. They can convert ethanol into acetyl-CoA without consuming ATP, and each molecule of ethanol is converted into two molecules of NADH. Specifically, the conditions for room temperature plasma mutagenesis combined with adaptive laboratory evolution in step (4) are: high-purity helium ≥ 99.999% plasma irradiation for no more than 60 seconds, flow rate of 10 SLM, radio frequency power of 120 W, and temperature of 22°C. Preferably, the mutant cells are screened in a real industrial sucrose wastewater culture medium to obtain primary mutant strains. The industrial sucrose wastewater culture medium has a pH of 4.5, a sucrose concentration greater than 220 g / L, and oxalate and phosphate concentrations greater than 10 g / L. Specifically, the primary mutant strain underwent two rounds of adaptive evolution, including: the first round of screening using diluted sucrose wastewater as a culture medium, with a concentration gradient of 100-220 g / L, a pH of 4.5, and containing oxalate, phosphate, and other inhibitory factors; the second round of acclimation using simulated sucrose wastewater, through continuous subculturing by pulsed adjustment of stress concentration, to screen for the motile fermentation monoclonal bacteria tolerant to high sucrose concentrations and low pH industrial sugar wastewater, with screening conditions of pH 4.5, sucrose concentration of 30-100 g / L, and containing oxalate and phosphate; The electroporation method for *Mammotrophic Fermentation Monoclonalis* was as follows: Glycerol-treated bacteria stored at -80℃ were streaked onto RM solid plates on ice and incubated at 30℃ for 3 days. Single colonies were picked and placed in 3 mL of RM solid medium and incubated statically at 30℃ for 3-4 h until the OD600 reached approximately 0.3-0.4. The culture was placed on ice for 15 min, centrifuged at 3000 g and 4℃ for 5 min, and the supernatant was discarded. The culture was resuspended in pre-cooled sterile water (20-30 mL) and washed twice, then resuspended in chilled 10% (v / v) glycerol (20-30 mL) and washed three times. Finally, the bacterial cells were resuspended in 700 μL of pre-cooled 10% glycerol. The competent cells were prepared and aliquoted into 50 μL tubes and stored at -80℃. All the above steps were performed gently on ice. Gently mix 500 ng of plasmid with 50 μL of competent cells, incubate on ice for 5 min, and transfer the transformation solution into a pre-cooled 0.1 cm electroporation cuvette. The volume of the plasmid should not exceed 1 / 10 of the competent cell volume. After cleaning the cuvette, place it in an electroporator and set the electroporation parameters as follows: electric field strength 16 kV / cm, capacitance 25 μF, resistance 200 Ω. Immediately after electroporation, add approximately 945 μL of RM liquid medium and incubate at 30°C for 4-6 h. Spread an appropriate amount of the incubated bacterial solution onto an RM plate containing 100 mg / L spectinomycin and incubate at 30°C for 3-5 days. Then, pick a single colony and place it in 500 μL of RM medium containing 100 mg / L spectinomycin and incubate at 30°C.

[0010] On the other hand, the engineered strain of *Mortrophlida* obtained by the construction method described in this application; Specifically, when the engineered strain is fermented in 165 g / L sucrose waste liquor in batch fermentation, the yield of L-polysaccharide is ≥43.9 g / L; when fermented in fed batch fermentation, the yield of L-polysaccharide is ≥76 g / L; and the sucrose conversion rate is >80%.

[0011] On another aspect, the application of the engineered strain of *Mammotrophic Fermentation Monomer* constructed in this application in the production of L-polysaccharides from industrial sucrose wastewater with low pH, high osmotic pressure, and high inhibition. Specifically, the sucrose concentration of the industrial sucrose wastewater is 100~220g / L, the pH value is 4.5-7, and the sucrose components of the industrial sucrose wastewater include oxalate and phosphate. Preferably, the pH of the industrial sugarcane wastewater is 4.5; Specifically, the levo-glycan includes levo-glycans formed by β-2,6 glycosidic bonds.

[0012] The beneficial effects of this invention are: (1) This application is the first to carry out metabolic engineering and irrational (mutation and adaptive evolution) system modification, and constructs a motile fermentation monoclonal strain that is tolerant to high concentration of industrial sucrose wastewater and produces high levels of L-glucan. The sucrose conversion rate exceeds 80%, the L-glucan yield is 76 g / L, reaching 79% of the theoretical conversion rate, and the carbon dioxide emissions are significantly reduced.

[0013] (2) The motile fermentation monoclonal bacteria obtained by the method of this application have the ability to produce high levels of levograns among microorganisms of the same species.

[0014] (3) The motile fermentation monoclonal bacteria obtained by the method of this application can make efficient use of industrial sugarcane wastewater, save costs, promote the sustainable development of "waste utilization", reduce the discharge of industrial wastewater, and meet the requirements of green production.

[0015] (4) This application uses the endogenous I-F type CRISPR-Cas system of *M. motile fermentation monoclonal* combined with homologous recombination to precisely knock out the target gene. sacC, gfo This increases the success rate of gene modification. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of gene editing in the L-glycan engineered strain of Example 1; Figure 2 Construction of the editing plasmid for the L-glycan engineered strain in Example 1; Figure 3 This is a schematic diagram of the sucrose metabolism pathway of *Mammotrophic motility-fermenting* bacteria in Example 2. Figure 4 This is a graph showing the change in L-glucan content in sucrose produced by continuous fermentation of sucrose using sucrose-fed bacteria in Example 2. Figure 5 The images show the actual fermentation process of L-glycan in Example 2 and the purified L-glycan sample (left image shows the actual fermentation process of 5 liters of continuous L-glycan production; right image shows the purified L-glycan sample). Figure 6 This is an example of the evolutionary process and L-glucan yield of strains that are tolerant to high L-glucan production from industrial sucrose wastewater, as shown in Example 3, which were screened using room temperature plasma mutagenesis combined with laboratory adaptive evolution. Figure 7 For the L-glucan sample produced from fermented sucrose wastewater in Example 4 13 C-NMR spectrum; Figure 8 For the L-glucan sample produced from fermented sucrose wastewater in Example 4 1 H-NMR spectrum; Figure 9 Example 4: The molecular weight of the levofloxacin produced from fermented sucrose wastewater was determined by gel permeation chromatography. Detailed Implementation

[0017] Example 1: Construction of Editing Plasmids and Gene Editing (1) such as Figure 1-2 As shown, the target gene on the genome sacC, gfo , adhB Suitable gRNA sequences were selected from the gene and integrated into the gene editing array of the pMini editing backbone plasmid using the Gibson assembly method. Homologous arms of 500 base pairs (bp) upstream and downstream of the DNA sequence to be knocked out were cloned and integrated into the gene editing plasmid using Gibson assembly, resulting in the pMini editing plasmid. ΔsacC pMini- Δgfo pMini- adhB::sacB .

[0018] Example 2 Construction of engineered strains and evaluation of L-glucan yield Gene editing plasmid pMini- ΔsacC Transformation of *Mammotrophic Fermentatosporum* via electroconversion, knocking out sacC Genes were extracted to obtain the engineered strain MHT-Δ1. Knockout was then performed in MHT-Δ1. gfo Genes were extracted to obtain the engineered strain MHT-Δ2. This double-deletion strain eliminated the major fructose metabolic bypass. In MHT-Δ2, [the following was used]... sacB Gene replacement adhB Genes were used to obtain the engineered strain MHT-Δ3, which enhanced the production of L-glycans while weakening the ethanol metabolic bypass. Expression was performed in MHT-Δ3. ada Genes and adh2 Genes were used to construct an ethanol utilization pathway, resulting in the engineered strain MHT-Δ3-EUP, which converts the main byproduct ethanol into acetyl-CoA, thereby enhancing ATP and reducing power supply performance. Figure 3 ).

[0019] The starting strain had low yield and efficiency of L-glycans, with the carbon stream primarily flowing towards ethanol and CO2. The engineered strain increased L-glycan yield from 1.56 g / L in the starting strain to 36.6 g / L, a 23-fold increase. After fermentation process optimization, in a 5 L fermenter (… Figure 5 Continuous fed-batch fermentation can ultimately reach 76 g / L. sacB replace adhB (MHT-Δ3), although the yield of L-glycan did not increase further, the fermentation time was shortened to 16 hours, and the production efficiency increased significantly to 2.21 g / L / h, an 11-fold increase. After introducing the ethanol utilization pathway into MHT-Δ3-EUP, the L-glycan production efficiency did not increase, but the ethanol yield further decreased to 14.4 g / L. Figure 4 As shown.

[0020] Table 1. Assessment of L-glucan production in *M. motile fermentum* Example 3: Room temperature plasma mutagenesis combined with laboratory adaptive evolution screening of strains that produce high levels of L-glucan tolerant to industrial sucrose wastewater. Industrial sucrose wastewater is an excellent raw material for the cost-effective production of L-glucan, but it contains various inhibitory factors that cannot be improved through metabolic engineering. To reduce production costs and achieve high-value utilization of waste sucrose resources, a strategy of random mutagenesis combined with adaptive evolution was employed to enhance the tolerance and production performance of engineered strains.

[0021] MHT-Δ3-EUP was first irradiated with room temperature plasma, and colonies capable of growth were directly screened in solid culture medium containing industrial sucrose wastewater (220 g / L sucrose content). After two rounds of directed laboratory adaptive evolution, the mutant strain ALE-2 was obtained. Although this strain could only tolerate 95 g / L simulated sucrose wastewater under a limited passage time (20 h), in real sucrose wastewater tests, this strain could tolerate low pH (4.5) and highly inhibitory industrial wastewater with sucrose concentrations up to 220 g / L.

[0022] like Figure 6 As shown, MHT-Δ2, ALE1, and ALE2 were inoculated into an industrial sucrose wastewater culture medium to test the growth and fermentation performance of the evolved strains. ALE2 still exhibited good growth and sucrose hydrolysis ability even in actual sucrose wastewater (sugar concentration 165 g / L), fully utilizing sucrose after 3 days of fermentation, with a levan yield of 43.9 g / L. In contrast, the starting strain and MHT-Δ2 could not ferment to produce levans at this level. This case study also revealed that higher sucrose concentrations are not always better. ALE2 could tolerate 220 g / L sucrose wastewater with a sucrose utilization rate as high as 80%, but the product was fructooligosaccharides, not high-molecular-weight levans.

[0023] Example 4 Qualitative Analysis of L-Solids Produced from Fermented Sucrose Wastewater The determination of whether sucrose and sucrose wastewater fermentation products are levans was made using methods such as nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). NMR was used to confirm the chemical structure of the generated levans. 13 C-NMR ( Figure 7 The product shows six main peaks (104.21, 80.30, 76.33, 75.21, 63.40, 59.93 ppm) and a characteristic peak (103-104 ppm) at the C-2 position of the β-(2,6) fructose residue, confirming that the product is a β-(2,6) glycosidic linked levogranose. 1 H-NMR ( Figure 8 The spectral analysis showed seven major proton signals in the cyclic proton region (4.13, 4.04, 3.90, 3.83, 3.71, 3.63, 3.50 ppm), consistent with the mass spectrometric structure of levans produced by most reported microorganisms. NMR spectroscopy revealed that the obtained product was indeed levan. GPC showed the peak pattern of this levan product as follows... Figure 9 In the above figure, the peak molecular weight is 10. 6 Da.

[0024] In summary, this application successfully constructed a highly efficient L-glucan-producing *Syntrophus motility-fermenting* strain through systematic metabolic engineering, random mutagenesis, and adaptive evolution, and then used gene editing technology to knock out... sacC and gfo Genes that block the fructose metabolism bypass, while using sacB Gene replacement adhB Genes were introduced, and an ethanol utilization pathway was added, significantly increasing the yield of L-glycosyltransferase from 1.56 g / L in the starting strain to 36.6 g / L, with a maximum production efficiency of 2.21 g / L / h. The significantly increased L-glycosyltransferase yield was further investigated using room-temperature plasma mutagenesis combined with laboratory adaptive evolution to screen for mutant strains that could grow well in highly inhibitory industrial sucrose wastewater and efficiently ferment to produce L-glycosyltransferase. In 165 g / L sucrose wastewater, the L-glycosyltransferase yield reached 43.9 g / L. Nuclear magnetic resonance and gel permeation chromatography analysis confirmed that the fermentation product was β-(2,6) glycosidic-linked L-glycosyltransferase with a molecular weight of approximately 10. 6 This application provides a feasible technical approach and excellent strain resources for the large-scale production of L-polysaccharides using inexpensive industrial sugarcane wastewater.

[0025] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for constructing an engineered strain of *Bacillus simulans* that is tolerant to industrial sucrose wastewater and produces high levels of levoglucan, characterized in that... Includes the following steps: (1) Using *M. motile fermentum* as the starting strain, the extracellular sucrase encoding gene and glucose-fructose oxidoreductase encoding gene in *M. motile fermentum* were knocked out to block the fructose metabolism bypass and obtain an engineered strain 1 with increased L-glycan production. (2) Replace the ethanol dehydrogenase gene in the engineered strain 1 with the levo-saccharide sucrase gene to divert carbon to levo-saccharide synthesis and metabolism, thereby obtaining engineered strain 2 with high levo-saccharide production and reduced ethanol by-products. (3) Introduce the heterologous ethanol utilization pathway into the engineered strain 2 obtained in step (2) to convert ethanol into acetyl coenzyme A, improve the balance of cell energy supply and reducing power, and obtain engineered strain 3 with shortened fermentation time. (4) The engineered strain 3 obtained in step (3) is subjected to atmospheric pressure room temperature plasma mutagenesis and combined with adaptive laboratory evolution to obtain a mutant strain that can tolerate real industrial sucrose wastewater with high sucrose concentration, low pH value and high salt stress, namely the engineered strain of motile fermentation monoclonal bacteria that is tolerant to industrial sucrose wastewater and produces high levels of L-glucan.

2. The construction method according to claim 1, characterized in that: The extracellular sucrase encoding gene mentioned in step (1) is *Mammotrophic ferruginosa*. sacC The gene, the sequence of which is shown in SEQ ID NO: 1; the glucose-fructose oxidoreductase encoding gene is *Mammotrophic motility-fermenting*. gfo The gene, whose sequence is shown in SEQ ID NO:

2.

3. The construction method according to claim 1, characterized in that: The L-glycan sucrase gene mentioned in step (2) is from *Mammotrophic motility*. sacB The gene, whose sequence is shown in SEQ ID NO: 3; the alcohol dehydrogenase gene is from *Fermentomonas motile*. adhB The gene, whose sequence is shown in SEQ ID NO:

4.

4. The construction method according to claim 1, characterized in that: The heterologous ethanol utilization pathway described in step (3) includes the acetaldehyde dehydrogenase gene of Digitrophus maize. ada and the Saccharomyces cerevisiae alcohol dehydrogenase gene adh2 The gene sequences are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. They can convert ethanol into acetyl-CoA without consuming ATP, and each molecule of ethanol is converted into two molecules of NADH.

5. The construction method according to claim 1, characterized in that: The conditions for room temperature plasma mutagenesis combined with adaptive laboratory evolution described in step (4) are: high-purity helium ≥ 99.999% plasma irradiation for no more than 60 seconds, flow rate of 10 SLM, radio frequency power of 120 W, and temperature of 22°C. Mutant cells were screened by coating them in a real industrial sucrose wastewater culture medium to obtain mutant strain 1. The industrial sucrose wastewater culture medium had a pH of 4.5, a sucrose concentration greater than 220 g / L, and oxalate and phosphate concentrations greater than 10 g / L. The mutant strain 1 underwent two rounds of adaptive evolution, specifically including: the first round used diluted sucrose wastewater as a culture medium for screening, with a concentration gradient set at 100-220 g / L, pH value at 4.5, containing oxalate, phosphate and other inhibitory factors; the second round used simulated sucrose wastewater for acclimatization, and through continuous subculturing by pulsed adjustment of stress concentration, the engineered strain of *Mammotrophic motility-fermenting monotypic bacteria* resistant to high sucrose concentration and low pH industrial sugar wastewater was screened, with screening conditions of pH 4.5, sucrose concentration of 30-100 g / L, containing oxalate and phosphate.

6. The engineered strain of *Mammotrophic Fermentation Monoclonalella* obtained by the construction method according to any one of claims 1-5.

7. The engineered strain of *Morphozoa motilityis* according to claim 6, characterized in that: When the engineered strain was fermented in 165 g / L sucrose waste liquor in batch fermentation, the yield of L-polysaccharide was ≥43.9 g / L; when fermented in fed batch fermentation, the yield of L-polysaccharide was ≥76 g / L; and the sucrose conversion rate was >80%.

8. The application of the engineered strain of *Mammotrophic Fermentatosporium* as described in claim 6 or 7 in the production of L-polysaccharides from industrial sucrose wastewater with low pH, high osmotic pressure, and high inhibition.

9. The application according to claim 8, characterized in that: The industrial sucrose wastewater has a sucrose concentration of 100-220 g / L and a pH value of 4.5-7. The sucrose components of the industrial sucrose wastewater include oxalate and phosphate.

10. The application according to claim 8, characterized in that: The levo-polysaccharide comprises levo-polysaccharides linked by β-2,6 glycosidic bonds.

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