A method to increase the biomass and protein content of Bacillus methanolicus

Abstract

This invention provides a method for increasing the biomass and protein content of Bacillus methanolis by knocking out the extracellular polysaccharide synthesis gene of Bacillus methanolis. glgC , glgA , glgB , glgD , glgP ) or extracellular polysaccharide synthesis gene clusters ( glgPADCB To reduce the synthesis of extracellular polysaccharides, the biomass and protein content of *Bacillus methanolicus* were increased. Experiments showed that knocking out the extracellular polysaccharide-producing gene increased the cell protein content from 65.2% in the wild type to a maximum of 81.2%, and the protein content of dry matter increased from 44.3% to 58.2%. In addition, it increased the amino acid content of the cell protein; of the 20 amino acids, all except proline were increased. It also increased the fermentation density, with the cell dry weight (DCW) increasing from 14.8 DCW / L to 34.1 DCW / L.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for increasing the biomass and protein content of Bacillus methanolii. Background Technology

[0002] Protein sources include plant and animal proteins, but these are no longer sufficient to meet current needs. Proteins (also called single-cell proteins, SCPs) can also be obtained through microbial fermentation. Currently, microorganisms capable of producing single-cell proteins include microalgae, bacteria, filamentous fungi, and yeast. Microalgae grow by fixing CO2 through photosynthesis. Microalgae protein can be used in health products and livestock feed. Filamentous fungi grow using sugar as a raw material; their hyphae have a fibrous texture similar to meat and are used in meat substitutes. Yeast primarily grows using molasses as a raw material; yeast protein is mainly used in food and feed. Bacteria can grow on a variety of substrates, such as commonly used sugar substrates, and some microorganisms can also utilize CO2 as a substrate.

[0003] Microalgae require photosynthesis to grow, making high-density cultivation difficult and hindering large-scale production. Furthermore, the cell walls of microalgae can impair protein absorption. Filamentous fungi use sugars as a raw material, competing with humans for food. They also face challenges such as slow growth and low protein content, generally not exceeding 50%. Yeasts, being eukaryotic microorganisms, possess complex organelles, resulting in lower protein content compared to bacteria. Bacteria, lacking complex organelles, have high protein content and can be cultivated at high densities, making them ideal substrates for single-cell protein production. Therefore, *Clostridium methylcapsulatum* or *Clostridium alcoholica* have achieved industrial-scale demonstration production of single-cell protein using CO and H2 as substrates. Although these strains utilize substrates that do not compete with humans for food, CO and H2 are difficult to store, transport, and are prone to explosion, limiting their application scenarios. Therefore, it is necessary to explore bacteria that can utilize other non-food substrates to produce single-cell protein.

[0004] Methanol is an important platform compound in the chemical industry, and it can be produced from a variety of abundant raw material resources such as natural gas, coal, and biomass. Considering methanol's low price and good availability, converting methanol into fuels or chemicals through chemical or biomanufacturing has proven to have significant economic, environmental, and social advantages. Therefore, utilizing methyltrophic bacteria to convert methanol into single-cell protein has significant advantages in terms of substrate cost and environmental friendliness. *Bacillus methanolii* (…) Bacillus methanolicus Methanol is a thermophilic bacterium that uses methanol as a carbon source. Due to its rapid growth and efficient protein production capabilities, it has been attempted to be used to produce single-cell protein (SCP) as a feed protein additive.

[0005] Existing techniques for producing single-cell protein using Bacillus methanolicus primarily focus on optimizing culture medium composition and fermentation conditions (such as methanol concentration, pH, and temperature) to increase yield. However, our transmission electron microscopy revealed that this strain secretes large amounts of extracellular polysaccharides (…). Figure 4 Excessive synthesis of extracellular polysaccharides consumes a large amount of carbon and energy, limiting the synthesis efficiency of target proteins. It also reduces the conversion rate of single-cell proteins produced using methanol as a substrate. Excessive extracellular polysaccharide production also increases culture medium viscosity, hindering the transfer of oxygen and nutrients, negatively impacting the production efficiency of single-cell proteins. However, genetic modification strategies targeting the competition between extracellular polysaccharide metabolism and protein synthesis have not yet been developed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for increasing the biomass and protein content of Bacillus methanolii.

[0007] The present invention is conceived as follows: Knocking out the gene in *Bacillus methanolii* that enables it to produce extracellular polysaccharides, including... glgC, glgA, glgB, glgD, glgP Or the entire extracellular polysaccharide biosynthesis gene cluster glgPADCB This resulted in mutant strains with reduced extracellular polysaccharides. These mutant strains increased the protein content of dry matter or cell dry weight, improved the amino acid content of bacterial proteins, and increased the fermentation density of the strain.

[0008] To achieve the objectives of this invention, in a first aspect, this invention provides a method for increasing the biomass and protein content of Bacillus methanolis, by knocking out Bacillus methanolis (… Bacillus methanolicus The extracellular polysaccharide synthesis genes or extracellular polysaccharide synthesis gene clusters of Bacillus methylmercury can reduce the synthesis of extracellular polysaccharides, thereby increasing the biomass and protein content of Bacillus methylmercury. The extracellular polysaccharide synthesis gene is selected from glgC , glgA , glgB , glgD , glgP At least one of the genes; said gene glgC , glgA , glgB , glgD , glgP The reference sequence numbers in NCBI are BMMGA3_13730, BMMGA3_13720, BMMGA3_13735, BMMGA3_13725, and BMMGA3_13715, respectively. The extracellular polysaccharide synthesis gene cluster is glgPADCB including the genes glgC , glgA , glgB , glgD andglgP .

[0009] Secondly, the present invention provides a method for constructing engineered Bacillus methanolicus bacteria, which utilizes genetic engineering techniques to construct engineered bacteria by inactivating or weakening the extracellular polysaccharide synthesis genes or extracellular polysaccharide synthesis gene clusters of Bacillus methanolicus.

[0010] Furthermore, the inactivation includes gene knockout.

[0011] Furthermore, the weakening method can be selected from at least one of mutagenesis, site-directed mutagenesis, homologous recombination, etc.

[0012] Preferably, the extracellular polysaccharide synthesis gene or extracellular polysaccharide synthesis gene cluster of Bacillus methylformamide is knocked out by homologous recombination.

[0013] The methods used to construct homologous recombination plasmids include: DNA fragment I containing sfgfp and colEori was amplified from pUB-sfgfp plasmid using primers pBMe01-V1-F and pBMe01-V1-R; simultaneously, DNA fragment II containing the RepB replicon and kanamycin resistance gene was amplified from pZL02 plasmid using primers pBMe01-V2-F and pBMe01-V2-R; after purification, DNA fragments I and II were recombined in vitro using recombinase to obtain homologous recombinant plasmid pBMe01 (sequence shown in SEQ ID NO:1). The sequences of the primers pBMe01-V1-F and pBMe01-V1-R are as follows: pBMe01-V1-F: TGTGCTGCAAGGCGATTAA pBMe01-V1-R:CCATTTTGAACGATGACCTC The sequences of the primers pBMe01-V2-F and pBMe01-V2-R are as follows: pBMe01-V2-F:GAGGTCATCGTTCAAAATGGTATGCGTTTTGACACATCCACTAT pBMe01-V2-R:TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG.

[0014] Furthermore, the homologous recombination plasmid was used to electrotransform Bacillus methanolicus, and the transformed strain was cultured at 60°C to promote homologous recombination.

[0015] Preferably, the starting strain used to construct the engineered Bacillus methanolis is Bacillus methanolis MGA3 (ATCC 53907).

[0016] Thirdly, the present invention provides engineered Bacillus methylformans strain prepared according to the method described above.

[0017] Fourthly, the present invention provides the application of the engineered bacteria in increasing the biomass and protein content of Bacillus methanolii.

[0018] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention improves the protein content and fermentation efficiency of single-cell protein production by *Bacillus methanolicus* by inactivating extracellular polysaccharide-related genes to reduce extracellular polysaccharide synthesis and release metabolic resources for single-cell protein synthesis. Experiments show that the protein content of wild-type *Bacillus methanolicus* by cell dry weight is 65.2%, higher than that of eukaryotic yeast strains. To further improve the cell protein content, the extracellular polysaccharide-producing gene was knocked out, further increasing the protein content. Specifically, by knocking out the extracellular polysaccharide-producing gene, the cell protein content increased from 65.2% in the wild type to a maximum of 81.2%, and the protein content of dry matter increased from 44.3% to 58.2%. In addition, it also increases the amino acid content of the cell protein; among 20 amino acids, the content of all amino acids except proline was increased. It also increases the fermentation density, with cell dry weight (DCW) increasing from 14.8 DCW / L to 34.1 DCW / L. Attached Figure Description

[0019] Figure 1 In a preferred embodiment of the present invention glgC A schematic diagram of the structure of the knockout plasmid.

[0020] Figure 2 In a preferred embodiment of the present invention glgC Knockout double crossover verification; M: DNA marker, C: wild-type negative control, 1-8: knockout double crossover verification.

[0021] Figure 3 Knockout in a preferred embodiment of the present invention glgD / glgB / glgA / glgP / glgBCDAP PCR validation gel image.

[0022] Figure 4 The cell dry weights of wild-type and knockout strains in a 5L reactor are shown in a preferred embodiment of the present invention.

[0023] Figure 5The protein content of single cells and dry matter of wild-type and knockout strains in the preferred embodiments of the present invention.

[0024] Figure 6 In a preferred embodiment of the present invention B. methanolicus Amino acid content in the dry matter of wild-type and knockout plants. Detailed Implementation

[0025] This invention provides a method for increasing the biomass and protein content of Bacillus methanolii.

[0026] The present invention adopts the following technical solution: This invention uses Bacillus methanolis (Bacillus) Bacillus methanolicus The starting strain was MGA3. *Bacillus methanolii* can grow using methanol as the sole carbon source, with a growth temperature of 37-60°C. Bioinformatics analysis revealed that *Bacillus methanolii* (MGA3)... Bacillus methanolicus Gene clusters involved in extracellular polysaccharide synthesis were identified in the study. These include... glgC, glgA, glgB, glgD and glgP Five genes. Knocking out these five genes individually or as a whole gene cluster reduces the synthesis of extracellular polysaccharides, alters the allocation of metabolic resources in the strain, and promotes the efficiency of single-cell protein synthesis and bacterial fermentation density.

[0027] Using a thermophilic bacterial editing tool (Zhiheng Yang et al., 2021, Microbialbiotechnology, 14(2), 363-373), the gene producing extracellular polysaccharides was edited. glgC Knockout of the other four genes in the gene cluster or the entire gene cluster was performed. Wild-type and knockout strains were fermented in a 5L fermenter, and the protein content of the fermentation broth was measured after freeze-drying. The cell dry weight of the knockout strain was 22.4% higher than that of the wild-type, and the protein content of the dry matter (directly freeze-dried fermentation broth) was 28.7% higher than that of the wild-type strain. Both cell dry weight and dry matter showed a significant increase in the protein content of the cells. The amino acid content in the dry matter was also measured, revealing that the content of most of the 20 amino acids was increased. The cell density during fermentation was also measured, and the knockout strain also increased the fermentation density. Besides knockout... glgC Alternatively, knocking out the entire gene cluster can achieve the same effect.

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] Example 1: Construction of gene editing plasmids Based on the thermophilic bacteria editing method (Zhiheng Yang et al., 2021, Microbialbiotechnology, 14(2), 363-373), to construct glgC This explanation will take gene knockout editing plasmids as an example. A schematic diagram of the knockout plasmid is shown below. Figure 1 .

[0030] The specific construction method is as follows: The construction process of the backbone plasmid pBMe01 of the edited plasmid is as follows: First, using primers pBMe01-V1-F / R (TGTGCTGCAAGGCGATTAA / CCATTTTGAACGATGACCTC), the plasmid pUB-sfgfp (provided by Wang Weishan's laboratory at the Institute of Microbiology, Chinese Academy of Sciences) was extracted from the pUB-sfgfp plasmid (this plasmid was provided by Yang Zhiheng's laboratory). Microbial Biotechnology 2021, 14 (2), 363-373) DNA fragments containing the sfgfp and colE ori regions were amplified. Simultaneously, primers pBMe01-V2-F / R (GAGGTCATCGTTCAAAATGGTATGCGTTTTGACACATCCACTAT / TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG) were used to amplify DNA fragments from pZL02 plasmid (provided by the laboratory of Wang Weishan, Institute of Microbiology, Chinese Academy of Sciences) (Yang, Z.; Synth Syst Biotechnol The DNA fragment containing the RepB replicon and kanamycin resistance gene in (2024, 9 (4), 658-666.) was analyzed using KOD One. TM PCR Master Mix was used for amplification, and the amplification reaction program was performed according to the polymerase's instruction manual. The extension time per kb of DNA was set to 1 min. The specific thermal cycling conditions were as follows: initial denaturation phase at 98 °C for 3 min; denaturation temperature at 98 °C for 10 seconds per cycle, annealing temperature at 60 °C for 5 seconds per cycle, extension temperature at 68 °C for 1 min per kb, and the final extension phase at 68 °C for 3 min. After purification, these two DNA fragments were recombined in vitro using NovoRec Plus recombinase to construct the pBMe01 plasmid (sequence shown in SEQ ID NO:1). Construction of the glgC knockout plasmid: B. methanolicusUsing the genome as a template, primer combinations of glgC-LF (ATCTTCACCTAGATCCTTTTAGGAACCTGAACCGCTAAATCG), glgC-LR (GTAAAATCATAATCGGGAGGGACTTC), glgC-RF (CCTCCCGATTATGATTTTACCTAAGCCTGCTCCCTTTCCC), and glgC-RR (ACCAAGTTTACTCATATATACGGCGGTTCGATTCCGTCCC) were used for amplification. glgC Upstream and downstream homologous arms of the gene. The plasmid backbone DNA was amplified using primer pairs donor-VF (CCGTATATATGAGTAAACTTGGTCTGACAG) and donor-VR (TAAAAGGATCTAGGTGAAGATCCTTTTTG), with pBMe01 plasmid as a template. The amplification reaction used KOD One DNA polymerase, and the reaction conditions were the same as before.

[0031] The size of the PCR products was determined by agarose gel electrophoresis. After confirming the correct band size, the target DNA fragment was purified by gel extraction according to the kit instructions. The purified DNA fragment was then added to the NovoRec Plus recombinase system for one-step in vitro recombination. The recombination system and reaction conditions are as follows: 1 μL of NovoRec Plus recombinase, 4 μL of reaction buffer, 2 μL of each DNA fragment, and 11 μL of sterile water were added to the reaction system to bring the total volume to 20 μL. The mixture was then incubated at 50 °C for 30 min to complete the recombination.

[0032] Next, E. coli JM109 competent cells were removed from the -80 °C freezer and thawed on ice. The completed in vitro recombinant reaction mixture was added to these cells, and the cells were treated according to the E. coli transformation method. After single colonies grew, colony PCR was performed using the corresponding validation primers to confirm the correctness of the transformation.

[0033] Example 2: Implementation of gene knockout The constructed gene knockout plasmid was electroporated to... B. methanolicusTransformed strains were initially screened on TSA medium containing kanamycin at 50°C to obtain single clones containing plasmids. The single clones selected from the antibiotic-resistant plates were then transferred to 60°C for incubation to promote homologous recombination using the temperature-sensitive replicon properties. After incubation until turbid, the culture was diluted and plated on antibiotic-resistant plates. PCR detection and changes in fluorescence signals confirmed that some strains had undergone single crossover events. Next, antibiotic-free relaxation culture was used to further promote the transformation of single crossover events into double crossover events. After serial dilution, strains without fluorescence and without the antibiotic phenotype were selected for PCR verification. Figure 2 Finally, it was confirmed that the target gene had been completely knocked out, and results were obtained. glgC Knockout plants ( Figure 2 The same method was used to complete the same procedure in the wild-type strain. glgA, glgB, glgD, glgP and the entire extracellular polysaccharide biosynthesis gene cluster glgPADCB Knockout ( Figure 3 ).

[0034] Build for knockout glgA, glgB, glgD, glgP The primers for the homologous recombination plasmids of each gene are as follows: glgA-up-F(GAAAACTCACGTTAAGGGATTTTGGTTTGCTGCGATCGGTCTTGTTTG) / glgA-up-R(GTACAAGGGGTGTTGATGAACTCTAAGGAAGAATTTAAAAAAGC), glgA-down-F(GAGTTCATCAACACCCCTTGTACAG) / glgA-down-R(ACCAAGTTTACTCATATATACGGGCTCCCGTTCATTACGGCGG); glgB-up-F(GAAAACTCACGTTAAGGGATTTTGGTCTGTCTTAAGCCAAATAATTCTTGAATAC) / glgB-up-down(GAAATATGAATAGGATGGAGAAACAGTGCACTG ACAAAAAACCTTGCC),glgB-down-F(CTGTTTCTCCATCCTATTCATATT) / glgB-down-R(ACCAAGTTTACTCATATATACGGCGGGGGAGGTTTGGCACCTCGA); glgD-up-F (GAAAACTCACGTTAAGGGATTTTGGTCGATGAAAGCTTTTTCAAAAACGCCG) / glgD-up-R (AATCGGGAGGGACTTCCATAAGTCCTGTTTATTGTTTCCGAATGTG), glgD-down-F (ATGGAAGTCCCTCCCGATTATG) / glgD-down-R (ACCAAGTTTACTCATATATACGGGGGTTGTTTGGGGCACATCTTG); glgP-up-F (GAAAACTCACGTTAAGGGATTTTGGTGAAGATCCGGGTGCTCCGC) / glgP-up-R (GAACTGATCTCAAGGAGTGAAAGCCAACACTGTGAAGGGTTGTAATAAC), glgP-down-F (GGCTTTCACTCCTTGAGATCAGTTC) / glgP-down-R (ACCAAGTTTACTCATATATACGGGGGTTGTTTGGGGCACATCTTG).

[0035] Constructing gene knockout clusters glgPADCB The primers for the homologous recombinant plasmid are: glgP-up-F(GAAAACTCACGTTAAGGGATTTTGGTGAAGATCCGGGTGCTCCGC) / glgP-up-R(GAACTGATCTCAAGGAGTGAAAGCCAACACTGTGAAGGGTTGTAATAAC); and glgB-up-F(GAAAACTCACGTTAAGGGATTTTGGTCTGTCTTAAGCCAAATAATTCTTGAATAC) / glgB-up-down(GAAATATGAATAGGATGGAGAAACAGTGCACTG ACAAAAAACCTTGCC), glgB-down-F (CTGTTTCTCCATCCTATTCATATT) / glgB-down-R (ACCAAGTTTACTCATATATACGGCGGGGAGGTTTGGCACCTCGA).

[0036] Example 3 Fermentation of knockout strains Wild-type strains and ∆ glgC / ∆ glgD / ∆ glgB / ∆ glgA / ∆glgP and knocking out the entire gene cluster ∆ glgBCDAP The knockout strains were subjected to fed-methanol fermentation in 5L fermenters using the same fermentation medium as described in the literature (Frederick J. Schendel et al., 1990, Applied and Environmental Microbiology, 56(4): 963-970). After fermentation, the cell dry weight (DCW) was significantly increased, with ∆ glgC The concentration increased from 14.8 DCW / L in the wild type to 32.5 DCW / L in the knockout strain. Figure 4 The knockout strains exhibited higher fermentation densities under high-density culture conditions. This is because knocking out the extracellular polysaccharides reduces cell adhesion and aggregation that may be caused by the extracellular polysaccharides, thus avoiding their impact on fermentation efficiency.

[0037] Then, the dry matter and single-cell protein content of the wild-type strain and the knockout strains were measured. The results showed that all knockout strains showed improvements, with ∆ glgC The single-cell protein content of the knockout strain increased from 65.2% in the wild type to 79.8%, and the protein content of dry matter increased from 44.3% to 57%. Figure 5 The amino acid content in the dry matter was also determined. Compared with the wild type, the knockout strain showed an increase in the content of all amino acids except for a slight decrease in proline. Figure 6 ).

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for increasing the biomass and protein content of Bacillus methanolicus, characterized in that, By knocking out Bacillus methanol ( Bacillus methanolicus The extracellular polysaccharide synthesis genes or extracellular polysaccharide synthesis gene clusters of Bacillus methylmercury can reduce the synthesis of extracellular polysaccharides, thereby increasing the biomass and protein content of Bacillus methylmercury. The extracellular polysaccharide synthesis gene is selected from glgC , glgA , glgB , glgD , glgP At least one of the genes; said gene glgC , glgA , glgB , glgD , glgP The reference sequence numbers in NCBI are BMMGA3_13730, BMMGA3_13720, BMMGA3_13735, BMMGA3_13725, and BMMGA3_13715, respectively. The extracellular polysaccharide synthesis gene cluster is glgPADCB including the genes glgC , glgA , glgB , glgD and glgP .

2. A method for constructing engineered Bacillus methanolicus strains, characterized in that, Engineered bacteria were constructed by inactivating or weakening extracellular polysaccharide synthesis genes or extracellular polysaccharide synthesis gene clusters in Bacillus methanol using genetic engineering techniques. The inactivation includes gene knockout; The weakening method is selected from at least one of mutagenesis, site-directed mutagenesis, and homologous recombination. The extracellular polysaccharide synthesis gene and extracellular polysaccharide synthesis gene cluster are the same as those described in claim 1.

3. The method according to claim 2, characterized in that, Homologous recombination was used to knock out the extracellular polysaccharide synthesis gene or extracellular polysaccharide synthesis gene cluster of Bacillus methylformamide.

4. The method according to claim 3, characterized in that, The methods used to construct homologous recombination plasmids include: DNA fragment I containing sfgfp and colE ori was amplified from pUB-sfgfp plasmid using primers pBMe01-V1-F and pBMe01-V1-R; simultaneously, DNA fragment II containing RepB replicon and kanamycin resistance gene was amplified from pZL02 plasmid using primers pBMe01-V2-F and pBMe01-V2-R; after purification, DNA fragments I and II were recombined in vitro using recombinase to obtain homologous recombinant plasmids; The sequences of the primers pBMe01-V1-F and pBMe01-V1-R are as follows: pBMe01-V1-F: TGTGCTGCAAGGCGATTAA pBMe01-V1-R:CCATTTTGAACGATGACCTC The sequences of the primers pBMe01-V2-F and pBMe01-V2-R are as follows: pBMe01-V2-F:GAGGTCATCGTTCAAAATGGTATGCGTTTTGACACATCCACTAT pBMe01-V2-R:TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAG.

5. The method according to claim 4, characterized in that, The homologous recombination plasmid was used to electrotransform Bacillus methylmercurate, and the transformed strain was cultured at 60°C to promote homologous recombination.

6. The method according to any one of claims 2-5, characterized in that, The starting strain used to construct the engineered Bacillus methanolis was Bacillus methanolis MGA3.

7. The engineered Bacillus methanolis strain prepared according to any one of claims 2-6.

8. The application of the engineered bacteria according to claim 7 in increasing the biomass and protein content of Bacillus methanolis.

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