Recombinant strain of <i>Bacillus subtilis</i>

Abstract

The invention relates to a recombinant strain of Bacillus subtilis, wherein pyruvate carboxylase BalpycA, glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, isocitrate NAD.sup.+ dehydrogenase icd, malate quinone dehydrogenase mqo, pyruvate ferredoxin oxidoreductase porAB and nitrogenase ferritin cyh are integrated and expressed in the recombinant strain. The invention also discloses use of the recombinant strain in fermentation production of acetylglucosamine. The recombinant Bacillus subtilis of the invention eliminates the central carbon metabolism overflow of the Bacillus subtilis and balances the intracellular reducing force, and the fermentation yield of acetylglucosamine is greatly improved.

Claims

1. A recombinant strain of Bacillus subtilis, wherein pyruvate carboxylase BalpycA, glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, isocitrate NAD.sup.+ dehydrogenase icd, malate quinone dehydrogenase mqo, pyruvate ferredoxin oxidoreductase porAB and nitrogenase ferritin cyh are integrated and expressed in the recombinant strain, wherein the pyruvate carboxylase BalpycA, glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, isocitrate NAD.sup.+ dehydrogenase icd, malate quinone dehydrogenase mqo, pyruvate ferredoxin oxidoreductase porAB and nitrogenase ferritin cyh are respectively expressed by using a strong constitutive promoter.

2. The recombinant strain according to claim 1, wherein the strong constitutive promoter is P.sub.43, P.sub.abrB, P.sub.valS, P.sub.hag, P.sub.spoVG, P.sub.yvyD, P.sub.hemA or P.sub.ffh promoter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a high performance liquid chromatography (HPLC) differential detection chromatograph for producing acetylglucosamine by fermenting Bacillus subtilis, in which, (a) shows HPLC detection results of BSGNKAP2 fermentation broth, and (b) shows HPLC detection results of BSGNKAP3 fermentation broth.

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

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(3) The technical solutions in the embodiments of the present invention will be described clearly and completely in combination with the accompanying drawings. Obviously, the described embodiments are parts of the embodiments of the present invention, instead of all of the embodiments. On the basis of the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

Embodiment 1

(4) Construction of Bacillus subtilis BSGNKAP3

(5) The Bacillus subtilis BSGNKAP2 was B. subtilis 168nagPgamPgamAnagA4nagBldhptaglcK pckA pyk P.sub.43-glmS P43-pycA::lox72, and GNA1 gene was freely expressed by using pP43NMK-GNA1 plasmid. The specific construction method can be referred to the disclosure of Patent Application CN 201610517961.9. Then, based on this, the pyruvate carboxylase BalpycA encoding gene balpycA (NCBI-Protein ID: AAS42897) derived from Bacillus cereus was integrated into malS locus in Bacillus subtilis BSGNKAP2 genome. Upstream sequence (SEQ ID No. 1, 1000 bp) of the integration malS locus, zeocin resistant gene zeo sequence, strong constitutive promoter P.sub.43, target gene sequence balpycA and downstream sequence (SEQ ID No. 2, 1000 bp) of malS locus were used for constructing an integration cassette. The integration cassettes obtained were integrated into the Bacillus subtilis BSGNKAP2 genome by homologous recombination. By screening through zeocin resistance flat plates, carrying out colony PCR verification and sequencing, it was confirmed that integration was successful and the recombinant Bacillus subtilis BSGNKAP3 was obtained.

Embodiment 2

(6) Construction of Bacillus subtilis BSGNKAP4

(7) Bacillus subtilis BSGNKAP3 was used as the host, and GNA1 gene was freely expressed by using pP43NMK-GNA1 plasmid. The glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor (NCBI-ProteinID: CAF30501) was integrated into pyk locus in Bacillus subtilis BSGNKAP3 genome. Upstream sequence (SEQ ID No. 3, 1000 bp) of the integration pyk locus, zeocin resistant gene zeo sequence, strong constitutive promoter P.sub.43, target gene sequence gor and downstream sequence (SEQ ID No. 4, 1000 bp) of pyk locus were used for constructing an integration cassette. The obtained integration cassette was integrated into the Bacillus subtilis BSGNKAP3 genome by homologous recombination. By screening through zeocin resistance flat plates, carrying out colony PCR verification and sequencing, it was confirmed that integration was successful and the recombinant Bacillus subtilis BSGNKAP4 was obtained.

Embodiment 3

(8) Construction of Recombinant Bacillus subtilis BSGNKAP5

(9) BSGNKAP4 was used as the host, and GNA1 gene was freely expressed by using pP43NMK-GNA1 plasmid. The isocitrate NAD.sup.+ dehydrogenase encoding gene icd (NCBI-Protein ID: AKC61181) was integrated into ywkA locus in Bacillus subtilis BSGNKAP4 genome. Upstream sequence (SEQ ID No. 5, 1000 bp) of the integration ywkA locus, zeocin resistant gene zeo sequence, strong constitutive promoter P.sub.43, target gene sequence icd and downstream sequence (SEQ ID No. 6, 1000 bp) of ywkA locus were used for constructing an integration cassette. The obtained integration cassette was integrated into the Bacillus subtilis BSGNKAP4 genome by homologous recombination. By screening through zeocin resistance flat plates, carrying out colony PCR verification and sequencing, it was confirmed that integration was successful and the recombinant Bacillus subtilis BSGNKAP5 was obtained.

Embodiment 4

(10) Construction of Recombinant Bacillus subtilis BSGNKAP6

(11) BSGNKAP5 was used as the host, and GNA1 gene was freely expressed by using pP43NMK-GNA1 plasmid. The malate quinone dehydrogenase encoding gene mqo (NCBI-Protein ID: ADK05552) was integrated into kdgA locus in Bacillus subtilis BSGNKAP5 genome. Upstream sequence (SEQ ID No. 7, 1000 bp) of the integration kdgA locus, zeocin resistant gene zeo sequence, strong constitutive promoter P.sub.43, target gene sequence mqo and downstream sequence (SEQ ID No. 8, 1000 bp) of kdgA locus were used for constructing an integration cassette. The obtained integration cassette was integrated into the Bacillus subtilis BSGNKAP5 genome by homologous recombination. By screening through zeocin resistance flat plates, carrying out colony PCR verification and sequencing, it was confirmed that integration was successful and the recombinant Bacillus subtilis BSGNKAP6 was obtained.

Embodiment 5

(12) Construction of Recombinant Bacillus subtilis BSGNKAP7

(13) BSGNKAP6 was used as the host, and GNA1 gene was freely expressed by using pP43NMK-GNA1 plasmid. The pyruvate ferredoxin oxidoreductase encoding genes porAB (NCBI-Protein ID: ADK06337 and NCBI-Protein ID: ADK06336) were integrated into melA locus in Bacillus subtilis BSGNKAP6 genome. Upstream sequence (SEQ ID No. 9, 1000 bp) of the integration melA locus, zeocin resistant gene zeo sequence, strong constitutive promoter P.sub.43, target gene sequence porAB and downstream sequence (SEQ ID No. 10, 1000 bp) of melA locus were used for constructing an integration cassette. The obtained integration cassette was integrated into the Bacillus subtilis BSGNKAP6 genome by homologous recombination. By screening through zeocin resistance flat plates, carrying out colony PCR verification and sequencing, it was confirmed that integration was successful and the recombinant Bacillus subtilis BSGNKAP7 was obtained.

Embodiment 6

(14) Construction of Bacillus subtilis BSGNKAP8

(15) BSGNKAP7 was used as the host, and GNA1 gene was freely expressed by using pP43NMK-GNA1 plasmid. The nitrogenase ferritin encoding gene cyh (NCBI-Protein ID: ACV00712) was integrated into pckA locus in Bacillus subtilis BSGNKAP7 genome. Upstream sequence (SEQ ID No. 11, 1000 bp) of the integration pckA locus, zeocin resistant gene zeo sequence, strong constitutive promoter P.sub.43, target gene sequence cyh and downstream sequence (SEQ ID No. 12, 1000 bp) of pckA locus were used for constructing an integration cassette. The obtained integration cassette was integrated into the Bacillus subtilis BSGNKAP7 genome by homologous recombination. By screening through zeocin resistance flat plates, carrying out colony PCR verification and sequencing, it was confirmed that integration was successful and the recombinant Bacillus subtilis BSGNKAP7 was obtained.

(16) In the above embodiments of the invention, besides the promoter P.sub.43, other promoters suitable for Bacillus subtilis can also be used, such as, P.sub.abrB, P.sub.valS, P.sub.hag, P.sub.spoVG, P.sub.yvyD, P.sub.hemA or P.sub.ffh.

(17) The pyruvate carboxylase BalpycA encoding gene balpycA also can be integrated into other loci in Bacillus subtilis genome, such as, pyk, ywkA, kdgA, melA, pckA, ctc, yckB, ydgG, speA, bshB, yojF, brxA, yqhS, yqeT, recJ, yvmB, spSC, nupG or rrnO locus.

(18) The glyceraldehyde-3-phosphate ferredoxin dehydrogenase encoding gene gor also can be integrated into other loci in Bacillus subtilis genome, such as, malS, ywkA, kdgA, melA, pckA, ctc, yckB, ydgG, speA, bshB, yojF, brxA, yqhS, yqeT, recJ, yvmB, spSC, nupG or rrnO locus.

(19) The isocitrate NAD.sup.+ dehydrogenase encoding gene icd also can be integrated into other loci in Bacillus subtilis genome, such as, pyk, malS, kdgA, melA, pckA, ctc, yckB, ydgG, speA, bshB, yojF, brxA, yqhS, yqeT, recJ, yvmB, spSC, nupG or rrnO locus.

(20) The malate quinone dehydrogenase encoding gene mqo also can be integrated into other loci in Bacillus subtilis genome, such as, pyk, ywkA, malS, melA, pckA, ctc, yckB, ydgG, speA, bshB, yojF, brxA, yqhS, yqeT, recJ, yvmB, spSC, nupG or rrnO locus.

(21) The pyruvate ferredoxin oxidoreductase encoding genes porAB also can be integrated into other loci in Bacillus subtilis genome, such as, pyk, ywkA, kdgA, malS, pckA, ctc, yckB, ydgG, speA, bshB, yojF, brxA, yqhS, yqeT, recJ, yvmB, spSC, nupG or rrnO locus.

(22) The nitrogenase ferritin encoding gene cyh also can be integrated into other loci in Bacillus subtilis genome, such as, pyk, ywkA, kdgA, melA, malS, ctc, yckB, ydgG, speA, bshB, yojF, brxA, yqhS, yqeT, recJ, yvmB, spSC, nupG or rrnO locus.

Embodiment 7

(23) Production of Acetylglucosamine by Fermenting Recombinant Bacillus subtilis

(24) The ingredients of the seed medium included: 10 g/L of peptone, 5 g/L of yeast powder, and 10 g/L of sodium chloride.

(25) The ingredients of the fermentation medium included: 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.

(26) The trace element solution included the following ingredients by 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 aluminium chloride, 0.1 g/L of copper chloride, 0.05 g/L of boric acid, and 5 mol/L of hydrochloric acid.

(27) High performance liquid chromatography (HPLC) was used for detecting content of acetylglucosamine. HPLC test conditions were as follows: instrument model: Agilent 1200, RID detector, column: NH.sub.2 column (2504.6 mm, 5 m); mobile phase: 70% acetonitrile, flow rate: 0.75 mL/min; column temperature: 30 C., and injection volume: 10 L.

(28) Detection of Glucose Concentration in Fermentation Broth: SBA Biosensor Analyzer.

(29) Recombinant Bacillus subtilis BSGNKAP8 was cultured under 37 C. at 220 rpm for 8 h in the seed medium, and then seeds were transferred to the fermentation medium at the inoculum size of 5% and cultured under 37 C. at 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 was measured.

(30) After shake-flask fermentation was completed, the acetyglucosamine yield of BSGNKAP8 was 24.50 g/L, and the acetyglucosamine/glucose yield was 0.469 g/g, respectively 1.97 times and 2.13 times of those of the starting strain BSGNKAP2 (as shown in FIG. 1). Thus, in the invention, increase of the extracellular production of acetylglucosamine in the recombinant Bacillus subtilis is achieved. In addition, acetoin is completely eliminated (as shown in FIG. 1).

(31) TABLE-US-00001 TABLE 1 Comparison of acetylglucosamine and acetylglucosamine/glucose BSGNK BSGNK BSGNK BSGNK BSGNK BSGNK BSGNK Strain AP2 AP3 AP4 AP5 AP6 AP7 AP8 Acetaminoglucose 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 (g/L) Acetylglucosamine/ 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 glucose (g/g)

Embodiment 8

(32) Detection of Intracellular NADH of Recombinant Bacillus subtilis

(33) The detection of intracellular NADH was performed by using the kits from Qingdao Jieshikang Biotechnology Co., Ltd. The thalluses in the logarithmic growth period were collected into centrifuge tubes (10.sup.4), alkaline extract was added at the volume ratio of 500-1000:1. Ultrasonic crushing was performed (ice bath, 20% or 200 W power, ultrasonic for 3 s, interval for 10 s, repeated for 30 times), water bath was carried out at 95 C. for 5 min (tightened to prevent water loss), and after cooling in ice bath, 10000 g of the resulting solution was centrifuged at 4 C. for 10 min, then 500 uL acidic extract was added to 500 uL supernatant for neutralizing, after uniformly mixing, centrifuging was performed on 10000 g of solution at 4 C. for 10 min, the supernatant was taken and placed on the ice to detect NADH according to the standard kit procedures.

(34) Intracellular NADH levels of BSGNKAP3, BSGNKAP4, BSGNKAP5, BSGNKAP6, BSGNKAP7 and BSGNKAP8 are shown in FIG. 2. It shows that the Bacillus subtilis of the invention can completely avoid central carbon metabolism overflow, effectively avoid accumulation of intracellular reducing force NADH, and promote the synthesis of acetylglucosamine.

(35) The abovementioned description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Multiple modifications to these embodiments are obvious to those skilled in the art, and general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to theses embodiments illustrated herein, but needs to be in line with the widest scope consistent with the principles and novel features disclosed herein.