Method for promoting acetylglucosamine synthesis of bacillus subtilis

Abstract

The present invention relates to a method for promoting acetylglucosamine synthesis of Bacillus subtilis, which belongs to the field of genetic engineering. The present invention adopts the recombinant Bacillus subtilis BSGNKAP2 as a starting strain, exogenously introducing pyruvate carboxylase BalpycA derived from Bacillus cereus, 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.sup.+ dehydrogenase, a malate quinone dehydrogenase, a ketoacid ferredoxin oxidoreductase and a nitrogenase ferritin. 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.

Claims

1. A recombinant strain of Bacillus subtilis, wherein the recombinant strain comprises an integrated expression sequence that expresses the following genes: balpycA, which encodes pyruvate carboxylase BalpycA, gor, which encodes a glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, icd, which encodes an isocitrate NAD.sup.+ dehydrogenase, mqo, which encodes a malate quinone dehydrogenase, porAB, which encodes a pyruvate ferredoxin oxidoreductase, and cyh, which encodes a nitrogenase iron protein.

2. The recombinant strain according to claim 1, wherein the pyruvate carboxylase gene is from Bacillus cereus, and comprises the sequence SEQ ID NO:1.

3. The recombinant strain according to claim 1, wherein the balpycA gene is expressed under the control of a strong constitutive promoter P.sub.43.

4. The recombinant strain according to claim 1, wherein the balpycA gene is integrated into a malS locus in a Bacillus subtilis genome of the recombinant strain.

5. The recombinant strain according to claim 1, wherein: the glyceraldehyde-3-phosphate ferredoxin dehydrogenase peptide sequence encoded by the gor gene is SEQ ID NO:2, the isocitrate NAD.sup.+ dehydrogenase peptide sequence encoded by the icd gene is SEQ ID NO:3, the malate quinone dehydrogenase peptide sequence encoded by the mqo gene is SEQ ID NO:4, the pyruvate ferredoxin oxidoreductase peptide sequence encoded by the porAB genes are SEQ ID NO:5 and SEQ ID NO:6, and the nitrogenase iron protein sequence encoded by the cyh gene is SEQ ID NO:7.

6. The recombinant strain according to claim 5, wherein: the gor gene, the icd gene, the mqo gene, the porAB gene, and the cyh gene, are expressed by a strong constitutive promoter P.sub.43.

7. The recombinant strain according to claim 5, wherein the gor gene, the icd gene, the mqo gene, the porAB gene, and the cyh gene, are sequentially integrated into pyk, ywkA, kdgA, melA, and pckA loci in Bacillus subtilis genome, respectively.

8. A method manufacturing the recombinant strain according to claim 1, comprising the following steps: (a) 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 (b) integrating, by carrying out homologous recombination, the integration cassettes obtained in the step (a) into the Bacillus subtilis genome.

9. A method for preparing acetylglucosamine, which comprises: activating the recombinant strain of claim 1 in a seed medium, transferring activated seeds into a fermentation medium, adding an inducer to induce expression from the integrated expression sequence, and fermenting at between 35 and 38° C. to obtain acetylglucosamine, wherein the seed medium comprises: peptone, yeast powder, and sodium chloride, and wherein the fermentation medium comprises: glucose, peptone, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, and a trace elements.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIGS. 1A and 1B are a high performance liquid chromatography (HPLC) differential detection chromatogram for producing acetylglucosamine by fermenting Bacillus subtilis, in which, FIG. 1A shows HPLC detection results of BSGNKAP2 fermentation broth, and FIG. 1B shows HPLC detection results of BSGNKAP3 fermentation broth. The peaks of acetylglucosamine, acetoin and 2,3-butanediol have been marked.

(2) FIG. 2 shows the intracellular NADH levels of Bacillus subtilis engineered strains.

DETAILED DESCRIPTION

(3) 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.

Example I: Construction of Bacillus subtilis BSGNKAP3

(4) The Bacillus subtilis BSGNKAP2 is B. subtilis 168ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔptaΔglcK ΔpckA Δpyk P.sub.43-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.

Example II: Construction of Bacillus subtilis BSGNKAP4

(5) 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.

Example III: Construction of Recombinant Bacillus subtilis BSGNKAP5

(6) BSGNKAP4 is used as the host, and GNA1 gene is freely expressed by using pP43NMK-GNA1 plasmid. Then, based on this, the isocitrate NAD.sup.+ 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.

Example IV: Construction of Recombinant Bacillus subtilis BSGNKAP6

(7) 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.

Example V: Construction of Recombinant Bacillus subtilis BSGNKAP7

(8) 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.

Example VI: Construction of Bacillus subtilis BSGNKAP8

(9) 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.

Example VII: Production of Acetylglucosamine by Fermenting Recombinant Bacillus subtilis

(10) 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.

(11) 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.

(12) 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.

(13) High performance liquid chromatography is used for detecting content of acetylglucosamine. HPLC test conditions are as follows: instrument model Agilent 1200, RID detector, column: NH.sub.2 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.

(14) Detection of glucose concentration in fermentation broth: SBA Biosensor Analyzer.

(15) 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.

Example IIX: Detection of Intracellular NADH of Recombinant Bacillus subtilis

(16) 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 (10.sup.4), 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.

(17) 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 FIG. 1), achieving an increase of the extracellular production of acetylglucosamine in the recombinant Bacillus subtilis. In addition, acetoin is completely eliminated (as shown in FIG. 1), and meanwhile, intracellular NADH levels of BSGNKAP3, BSGNKAP4, BSGNKAP5, BSGNKAP6, BSGNKAP7 and BSGNKAP8 are shown in FIG. 2. This strategy can completely avoid central carbon metabolism overflow, effectively avoid accumulation of intracellular reducing force NADH, and promote the synthesis of acetylglucosamine.

(18) TABLE-US-00001 TABLE I Comparison of acetylglucosamine and acetylglucosamine/glucose Strain BSGNKAP2 BSGNKAP3 BSGNKAP4 BSGNKAP5 BSGNKAP6 BSGNKAP7 BSGNKAP8 Acetaminoglucose (g/L) 12.4 ± 0.56 14.3 ± 0.28 17.5 ± 0.86 19.7 ± 1.11 18.1 ± 0.75 21.5 ± 0.44 24.5 ± 0.68 Acetylglucosamine/glucose (g/g) 0.22 ± 0.01 0.33 ± 0.01 0.35 ± 0.02 0.40 ± 0.02 0.42 ± 0.02 0.39 ± 0.01 0.47 ± 0.01