Patent Grant
11198862
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII Copy, created on May 3, 2021, is named “seq.txt” and is 32,915 bytes in size.
The present invention relates to a method for promoting acetylglucosamine synthesis of Bacillus subtilis, which belongs to the field of genetic engineering.
In human bodies, acetylglucosamine is a synthetic precursor of a glycosaminoglycan disaccharide unit, which plays an important role in repair and maintenance of cartilage and joint tissue function. Therefore, acetylglucosamine is widely added to medicines and nutritional diets to treat and repair joint damage. In addition, acetylglucosamine also has many applications in the fields of cosmetics and pharmacy. At present, acetylglucosamine is mainly produced by acid hydrolysis of chitin in shrimp shells or crab shells. However, waste liquid produced by the method pollutes the environment seriously, and the resulting products are apt to cause allergic reactions, being not suitable for people who are allergic to seafood.
Bacillus subtilis is widely used as a production host for food enzymic preparations and vital nutrient chemicals, and its products are certified as the Generally Regarded as Safe (GRAS) security level by FDA. The reaction formula for producing acetylglucosamine by fermentation of Bacillus subtilis is:
5/2 glucose+ATP+5NAD++2NH3→GlcNAc+glutamate+ADP+5NADH+2CO2+
phosphate
The formula is obtained by calculating three precursors, namely 6-phosphate fructoses, acetyl coenzyme A and glutamine, of the de novo synthesis of acetylglucosamine, it can be seen from the formula that a large amount of NADH would be generated in the synthesis process of acetylglucosamine. The excessively generated NADH has a huge negative effect on the maximum theoretical yield (Yc) of the N-acetylglucosamine pathway. Due to the fact that cells need to maintain the balance of reduction force, the excessively generated NADH will be consumed in two ways: participating in the synthesis of other metabolites (resulting in the generation of by-products), and the other aspect being oxidized to produce ATP (causing a large amount of O2 to be consumed during the fermentation process, and meanwhile, a large amount of bacterial cells is greatly produced). In addition, the use of Bacillus subtilis to synthesize acetylglucosamine is accompanied by metabolic overflow, resulting in the massive synthesis of by-product acetoin. Therefore, effectively treating the NADH concomitantly generated with the acetylglucosamine and the synthesis efficiency of the acetylglucosamine, meanwhile, avoiding the overflow of central carbon metabolism and improving the economical efficiency of carbon atoms, are urgent problems to be solved in the production of acetylglucosamine by the method of microbial fermentation.
In order to solve the foregoing technical problem, the present invention provides a method for eliminating central carbon metabolism overflow of Bacillus subtilis, balancing intracellular reducing force and promoting acetylglucosamine synthesis, and the construction method exogenously introduces pyruvate carboxylase BalpycA derived from Bacillus cereus, eliminates the central carbon metabolism overflow of the Bacillus subtilis and avoids the synthesis of the by-product acetoin. Further, five exogenous reducing force metabolic reactions are introduced to replace the reaction of generating NADH in glycolysis pathway and tricarboxylic acid cycle to reconstruct intracellular reducing force metabolism, which specifically comprise glyceraldehyde-3-phosphate ferredoxin dehydrogenase, isocitrate NAD+ dehydrogenase, a malate quinone dehydrogenase, a ketoacid ferredoxin oxidoreductase and a nitrogenase iron protein. The method is simple to operate and convenient to use, and the constructed recombinant Bacillus subtilis completely avoids central carbon metabolism so as to overflow and balance intracellular reducing power NADH metabolism.
The first objective of the present invention is to provide a recombinant strain of Bacillus subtilis, which integrates and expresses pyruvate carboxylase BalpycA, glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, isocitrate NAD+ dehydrogenase icd, malate quinone dehydrogenase mqo, pyruvate ferredoxin oxidoreductase porAB and nitrogenase ferritin cyh.
In one embodiment of the present invention, the recombinant strain adopts Bacillus subtilis BSGNKAP2 as a starting strain, wherein the Bacillus subtilis BSGNKAP2 is disclosed in the patent application of No. CN201610517961.9.
In one embodiment of the present invention, the pyruvate carboxylase BalpycA is derived from Bacillus cereus, the pyruvate carboxylase BalpycA is shown as NCBI-Protein ID: AAS42897.1.
In one embodiment of the present invention, the pyruvate carboxylase BalpycA derived from Bacillus cereus is exogenously introduced, thus eliminating the central carbon metabolism overflow of the Bacillus subtilis and avoiding the synthesis of the by-product acetoin.
In one embodiment of the present invention, the pyruvate carboxylase BalpycA encoding gene balpycA is expressed by using a strong constitutive promoter P43.
In one embodiment of the present invention, the pyruvate carboxylase BalpycA encoding gene balpycA is integrated into malS locus in Bacillus subtilis genome.
In one embodiment of the present invention, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase is shown as NCBI-Protein ID: CAF30501.1, the isocitrate NAD+ dehydrogenase is shown as NCBI-Protein ID: AKC61181.1, the malate quinone dehydrogenase is shown as NCBI-Protein ID:ADK05552.1, the pyruvate ferredoxin oxidoreductase porAB are shown as NCBI-Protein ID: ADK06337.1 and NCBI-Protein ID: ADK06336.1, and the nitrogenase iron protein is shown as NCBI-Protein ID: ACV00712.1.
In one embodiment of the present invention, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase, the isocitrate NAD+ dehydrogenase, the malate quinone dehydrogenase, pyruvate ferredoxin oxidoreductase and the nitrogenase iron protein is adopted to replace the reaction of generating NADH in glycolysis pathway and tricarboxylic acid cycle.
In one embodiment of the present invention, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor, the isocitrate NAD+ dehydrogenase encoding gene icd, the malate quinone dehydrogenase encoding gene mqo, the pyruvate ferredoxin oxidoreductase encoding gene porAB and the nitrogenase iron protein gene cyh are expressed by using a strong constitutive promoter P43 respectively.
In one embodiment of the present invention, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor, the isocitrate NAD+ dehydrogenase encoding gene icd, the malate quinone dehydrogenase encoding gene mqo, the pyruvate ferredoxin oxidoreductase encoding gene porAB and the nitrogenase iron protein gene cyh are sequentially integrated into pyk, ywkA, kdgA, melA and pckA loci in Bacillus subtilis genome.
The second objective of the present invention is to provide a method for constructing the recombinant strain, comprising the following steps:
(1) constructing homologous recombination integration cassettes of the pyruvate carboxylase BalpycA encoding gene balpycA, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor, the isocitrate NAD+ dehydrogenase encoding gene icd, the malate quinone dehydrogenase encoding gene mqo, the pyruvate ferredoxin oxidoreductase encoding gene porAB, and the nitrogenase iron protein encoding gene cyh of Bacillus cereus; and
(2) integrating, by carrying out homologous recombination, the integration cassettes obtained in the step (1) into Bacillus subtilis genome.
In one embodiment of the present invention, upstream sequence (1000 bp) of the integration locus, zeocin resistant gene zeo sequence and strong constitutive promoter P43, target gene sequence and upstream sequence (1000 bp) thereof are used for constructing an integration cassette in step (1).
In one embodiment of the present invention, the pyruvate carboxylase BalpycA encoding gene balpycA is derived from Bacillus cereus, and obtained by gene synthesis.
In one embodiment of the present invention, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor is derived from Methanococcus maripaludis KA1, obtained by gene synthesis, and subjected to codon optimization.
In one embodiment of the present invention, the isocitrate NAD+ dehydrogenase encoding gene icd is derived from Clostridium sporogenes, obtained by gene synthesis, and subjected to codon optimization.
In one embodiment of the present invention, the malate quinone dehydrogenase encoding gene mqo is derived from Bacillus cereus, and obtained by gene synthesis.
In one embodiment of the present invention, the pyruvate ferredoxin oxidoreductase encoding gene porAB is derived from Bacillus cereus, obtained by gene synthesis, and subjected to codon optimization.
In one embodiment of the present invention, the nitrogenase iron protein encoding gene cyh is derived from Cyanothece sp. PCC 8802, obtained by gene synthesis, and subjected to codon optimization.
In one embodiment of the present invention, the pyruvate carboxylase BalpycA encoding gene balpycA derived from Bacillus cereus is integrated into malS locus in Bacillus subtilis genome.
In one embodiment of the present invention, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor is integrated into pyk locus in Bacillus subtilis genome.
In one embodiment of the present invention, the isocitrate NAD+ dehydrogenase encoding gene icd is integrated into ywkA locus in Bacillus subtilis genome.
In one embodiment of the present invention, the malate quinone dehydrogenase encoding gene mqo is integrated into kdgA locus in Bacillus subtilis genome.
In one embodiment of the present invention, the pyruvate ferredoxin oxidoreductase encoding gene porAB is integrated into melA locus in Bacillus subtilis genome.
In one embodiment of the present invention, the nitrogenase iron protein encoding gene cyh is integrated into pckA locus in Bacillus subtilis genome.
The present invention also provides a method for preparing acetylglucosamine by the recombinant strain, comprising the following steps: activating the recombinant strain in a seed medium, then transferring activated seeds into a fermentation medium, adding an inducer to carry out fermentation culture and obtaining acetylglucosamine.
In one embodiment of the present invention, seeds are activated in the seed medium at 35-38° C., and activated seeds are fermented and cultured at 35-38° C.
In one embodiment of the present invention, the seed medium includes the following ingredients: peptone, yeast powder and sodium chloride.
In one embodiment of the present invention, the seed medium includes the following ingredients based on weight: 5-15 g/L of peptone, 5-10 g/L of yeast powder and 5-15 g/L of sodium chloride.
In one embodiment of the present invention, the fermentation medium includes the following ingredients: glucose, peptone, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, and a trace element solution.
In one embodiment of the present invention, the fermentation medium includes the following ingredients based on weight: 15-25 g/L of glucose, 5-8 g/L of peptone, 10-15 g/L of yeast powder, 5˜8 g/L of ammonium sulfate, 10˜15 g/L of dipotassium hydrogen phosphate, 2˜3 g/L of potassium dihydrogen phosphate, 4˜6 g/L of calcium carbonate, and 8˜12 ml/L of trace element solution.
In one embodiment of the present invention, the trace element solution includes: manganese sulfate, cobalt chloride, sodium molybdate, zinc sulfate, aluminum chloride, copper chloride, boric acid and hydrochloric acid.
In one embodiment of the present invention, the trace element solution includes the following ingredients based on weight: 0.8-1.2 g/L of manganese sulfate, 0.2-0.6 g/L of cobalt chloride, 0.1-0.3 g/L of sodium molybdate, 0.1˜0.3 g/L of zinc sulfate, 0.1˜0.3 g/L of aluminum chloride, 0.1˜0.3 g/L of copper chloride, 0.04˜0.06 g/L of boric acid, and 3˜8 mol/L of hydrochloric acid.
In one embodiment of the present invention, the activated seeds are transferred to the fermentation medium at an inoculum size of 5-15% for culture.
In one embodiment of the present invention, the inducer is xylose, and the dosage of xylose for per liter of fermentation medium is 5-10 g.
Compared with the prior art, the present invention has the following advantages:
The present invention provides a method for eliminating central carbon metabolism overflow of Bacillus subtilis, balancing intracellular reducing force, and promoting acetylglucosamine synthesis, and BSGNKAP8 is exogenously introduced pyruvate carboxylase BalpycA of Bacillus cereus, thus eliminating the central carbon metabolism overflow of the Bacillus subtilis and avoiding the synthesis of the by-product acetoin. Further, five exogenous reducing force metabolic reactions are introduced to replace the reaction of generating NADH in glycolysis pathway and tricarboxylic acid cycle to reconstruct intracellular reducing force metabolism, which specifically comprise glyceraldehyde-3-phosphate ferredoxin dehydrogenase, isocitrate NAD+ dehydrogenase, a malate quinone dehydrogenase, a ketoacid ferredoxin oxidoreductase and a nitrogenase iron protein. Compared with the starting strain BSGNKAP2, the central carbon metabolism overflow is avoided and the synthesis of by-product acetoin is eliminated; meanwhile, in the process of producing acetylglucosamine, the intracellular NADH is effectively reduced, and at the same time, the acetylglucosamine synthesis is promoted. In a shake-flask fermentation process using a complex medium, acetylglucosamine yield of the recombinant strain BSGNKAP8 is 24.50 g/L, acetylglucosamine/glucose yield is 0.469 g/g, respectively 1.97 times and 2.13 times of those of the starting strain BSGNKAP2. The construction method of recombinant Bacillus subtilis of the present invention is simple and is convenient to use, and has a good application prospect.
The technical solutions of the present invention are described in further detail below with reference to specific embodiments. The following embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention.
The Bacillus subtilis BSGNKAP2 is B. subtilis 168ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔptaΔglcK ΔpckA Δpyk P43-glmS P43-pycA::lox72, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the pyruvate carboxylase BalpycA encoding gene balpycA (NCBI-Protein ID:AAS42897, SEQ ID NO:1) derived from Bacillus cereus is integrated into malS locus in Bacillus subtilis genome, further screening through zeocin resistance flat plates, carrying out colony PCR verification, sequencing and confirming integration to obtain the recombinant Bacillus subtilis BSGNKAP3.
Bacillus subtilis BSGNKAP3 is used as the host, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor (NCBI-ProteinID: CAF30501, SEQ ID NO:2) is integrated into pyk locus in Bacillus subtilis genome, further screening through zeocin resistance flat plates, carrying out colony PCR verification, sequencing and confirming integration to obtain the recombinant Bacillus subtilis BSGNKAP4.
BSGNKAP4 is used as the host, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the isocitrate NAD+ dehydrogenase encoding gene icd (NCBI-Protein ID: AKC61181, SEQ ID NO:3) is integrated into ywkA locus in Bacillus subtilis genome, further screening through zeocin resistance flat plates, carrying out colony PCR verification, sequencing and confirming integration to obtain the recombinant Bacillus subtilis BSGNKAP5.
BSGNKAP5 is used as the host, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the malate quinone dehydrogenase encoding gene mqo (NCBI-Protein ID: ADK05552, SEQ ID NO:4) is integrated into kdgA locus in Bacillus subtilis genome, further screening through zeocin resistance flat plates, carrying out colony PCR verification, sequencing and confirming integration to obtain the recombinant Bacillus subtilis BSGNKAP6.
BSGNKAP6 is used as the host, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the pyruvate ferredoxin oxidoreductase encoding genes porAB (NCBI-Protein ID: ADK06337, SEQ ID NO:5; and, NCBI-Protein ID: ADK06336, SEQ ID NO:6) are integrated into melA locus in Bacillus subtilis genome, further screening through zeocin resistance flat plates, carrying out colony PCR verification, sequencing and confirming integration to obtain the recombinant Bacillus subtilis BSGNKAP7.
BSGNKAP7 is used as the host, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the nitrogenase iron protein encoding gene cyh (NCBI-Protein ID: ACV00712, SEQ ID NO:7) is integrated into pckA locus in Bacillus subtilis genome, further screening through zeocin resistance flat plates, carrying out colony PCR verification, sequencing and confirming integration to obtain the recombinant Bacillus subtilis BSGNKAP8.
The ingredients of the seed medium include: 10 g/L of peptone, 5 g/L of yeast powder, and 10 g/L of sodium chloride.
The ingredients of the fermentation medium include: 20 g/L of glucose, 6 g/L of peptone, 12 g/L of yeast powder, 6 g/L of ammonium sulfate, 12.5 g/L of dipotassium hydrogen phosphate, 2.5 g/L of potassium dihydrogen phosphate, 5 g/L of calcium carbonate, and 10 ml/L of trace element solution.
The trace element solution includes the following ingredients based on weight: 1.0 g/L of manganese sulfate, 0.4 g/L of cobalt chloride, 0.2 g/L of sodium molybdate, 0.2 g/L of zinc sulfate, 0.1 g/L of aluminum chloride, 0.1 g/L of copper chloride, 0.05 g/L of boric acid, and 5 mol/L of hydrochloric acid.
High performance liquid chromatography is used for detecting content of acetylglucosamine. HPLC test conditions are as follows: instrument model Agilent 1200, RID detector, column: NH2 column (250×4.6 mm, 5 μm), mobile phase: 70% acetonitrile, flow rate: 0.75 mL/min, column temperature: 30° C., and injection volume: 10 μL.
Detection of glucose concentration in fermentation broth: SBA Biosensor Analyzer.
Recombinant Bacillus subtilis BSGNKAP1 is cultured at the conditions of 37° C. and 220 rpm for 8 h in the seed medium, and then seed is transferred to the fermentation medium at the inoculum size of 5% and cultured at the conditions of 37° C. and 220 rpm for 48 h in a 500 ml shake flask. At the end of the fermentation, the content of acetylglucosamine in the fermentation supernatant will be measured.
The detection of intracellular NADH is performed by using the kits from Qingdao Jieshikang Biotechnology Co., Ltd. Collecting the thalluses in the logarithmic growth period into centrifuge tubes (104), adding alkaline extract volume (mL) to the ratio of 500-1000:1, performing ultrasonic crushing (ice bath, 20% or 200 W power, ultrasonic for 3 s, interval for 10 s, repeated for 30 times), performing water bath at 95° C. for 5 min (tightened to prevent water loss), and after cooling in ice bath, centrifuging at 10000 g and 4° C. for 10 min, adding 500 μl supernatant to 500 μl acidic extract to neutralize, uniformly mixing, centrifuging at 10000 g and 4° C. for 10 min, taking the supernatant, and placing the supernatant on the ice to detect NADH according to the standard kit procedures.
After shake-flask fermentation is completed, the acetylglucosamine yield of BSGNKAP8 is 24.50 g/L, and the acetylglucosamine/glucose yield is 0.469 g/g, respectively 1.97 times and 2.13 times of those of the starting strain BSGNKAP2 (as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810299583.0 | Apr 2018 | CN | national |
| Number | Date | Country |
|---|---|---|
| 201610517961 | Nov 2016 | CN |
| Number | Date | Country | |
|---|---|---|---|
| 20190309281 A1 | Oct 2019 | US |
