Genetically engineered bacterium for sarcosine production as well as construction method and application

Abstract

The disclosure discloses a genetically engineered strain for sarcosine production as well as a construction method and application. The genetically engineered strain is obtained by using Escherichia coli as a host and by integrating a single copy of imine reductase gene dpkA on its genome; singly copying citrate synthase gene gltA; knocking out glyoxylate cycle inhibitor gene iclR; knocking out malate synthase gene aceB; integrating a single copy of isocitrate lyase gene aceA; integrating a single copy of membrane-bound transhydrogenase gene pntAB; knocking out 2-ketate reductase gene ycdW; integrating a single copy of phosphoenolpyruvate carboxylase gene ppc; and knocking out pyruvate kinase gene pykF. After system metabolism transformation, the engineered strain can synthesize sarcosine with glucose and methylamine as main raw materials. The sarcosine titer can reach 10 g/L after fermentation for 30 h in a 5 L fermenter.

Claims

1. A genetically engineered bacterium for synthesizing Sarcosine, wherein the genetically engineered bacterium is Escherichia coli SAR which is obtained by using Escherichia coli ATCC27325 as a host and through the following transformations: integrating singly copied imine reductase gene dpkA, which is controlled by T7 promoter, at the mbhA site on its genome; singly copying citrate synthase gene gltA, which is controlled by trc promoter, at the ylbE site; knocking out glyoxylate cycle inhibitor gene iclR; knocking out malate synthase gene aceB; singly copying isocitrate lyase gene aceA, which is controlled by trc promoter, at the yeeP site; singly copying membrane-bound transhydrogenase gene pntAB, which is controlled by trc promoter, at the yghE site; knocking out 2-ketate reductase gene ycdW; singly copying phosphoenolpyruvate carboxylase gene ppc, which is controlled by controlled by trc promoter, at the yeeL site; and knocking out pyruvate kinase gene pykF; the imine reductase gene dpkA has a nucleotide sequence of SEQ ID NO: 1; the citrate synthase gene gltA has a nucleotide sequence of SEQ ID NO: 2; the isocitrate lyase gene aceA has a nucleotide sequence of SEQ ID NO: 3; the membrane-bound transhydrogenase gene pntAB has a nucleotide sequence of SEQ ID NO:4; the phosphoenolpyruvate carboxylase gene ppc has a nucleotide sequence of SEQ ID NO:5.

2. A method for constructing a genetically engineered bacterium for synthesizing Sarcosine according to claim 1, wherein the method uses a CRISPR/Cas9-mediated gene editing technology to perform targeted transformation on Escherichia coli, comprising the following steps: (1) introducing a single copy of a imine reductase gene dpkA derived from Brevibacterium linens ATCC 9172 at the mbhA site on Escherichia coli ATCC27325 genome, wherein the dpkA has the sequence of SEQ ID NO:1, and is controlled by T7 promoter; (2) introducing a single copy of the endogenous citrate synthase gene gltA at the ylbE site on Escherichia coli ATCC27325 genome, wherein the qltA has the sequence of SEQ ID NO:2, and is controlled by trc promoter; (3) performing gene knockout at the iclR site on Escherichia coli ATCC27325 genome; (4) performing gene knockout at the aceB site on Escherichia coli ATCC27325 genome; (5) introducing a single copy of the endogenous isocitrate lyase gene aceA at the yeeP site on Escherichia coli ATCC27325 genome, wherein the aceA has the sequence of SEQ ID NO:3 and is controlled by trc promoter; (6) introducing a single copy of the endogenous membrane-bound transhydrogenase gene pntAB at the yghE site on Escherichia coli ATCC27325 genome, wherein the pntAB has the sequence of SEQ ID NO:4 and is controlled by trc promoter; (7) performing gene knockout at the ycdW site on Escherichia coli ATCC27325 genome; (8) introducing a single copy of endogenous phosphoenolpyruvate carboxylase gene ppc at the yeeL site on Escherichia coli ATCC27325 genome, wherein the ppc has the sequence of SEQ ID NO:5, and is controlled by trc promoter; and (9) performing gene knockout at the pykF site on Escherichia coli ATCC27325 genome.

3. A method for producing Sarcosine by fermenting the genetically engineered bacterium according to claim 1, comprising the following steps: Inoculating the fermentation culture, wherein a starter culture of the genetically engineered bacteria of claim 1 is inoculated into a fresh fermentation culture medium in an inoculation amount of 15-20%; performing the fermentation under the following conditions to obtain sarcosine: controlling the pH at 6.8-7.2; maintaining the temperature is maintained at 36.5-37.5° C., and the dissolved oxygen between 25% and 35%; when the glucose in the culture medium is completely consumed, adding glucose via a solution of 700-800 g/L glucose, wherein the concentration of glucose in the fermentation culture medium is maintained to be <3 g/L; when the culture reaches an OD.sub.600=40, feeding a solution of 1.5-1.6 mol/L methylaminehydrochloride at a flow rate of 20-25 mL/h with a feeding amount of 75 mL/L culture medium; and, wherein the fermentation period is 28-32 h; wherein the composition of the fermentation culture medium is: 15-25 g/L of glucose, 1-5 g/L of tryptone, 3-5 g/L of sodium citrate, 1-5 g/L of KH2POs, 0.1-1 g/L of MgSO4*7H2O and the balance of water, and pH 7.0-7.2.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a mbhA::P.sub.T7-dpkA integration electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-target gene; 3-downstream homology arm; 4-overlapped fragments; 5-protobacterium PCR fragment; 6-PCR fragment of target strain.

(2) FIG. 2 is a ylbE::P.sub.trc-gltA integration electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-target gene; 3-downstream homology arm; 4-overlapped fragment; 5-protobacterium PCR fragment; 6-PCR fragment of target strain.

(3) FIG. 3 is an iclR knockout electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-downstream homology arm; 3-overlapped fragment; 4-protobacterium PCR fragment; 5-PCR fragment of target strain.

(4) FIG. 4 is an aceB knockout electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-downstream homology arm; 3-overlapped fragment; 4-protobacterium PCR fragments; 5-PCR fragment of target strain.

(5) FIG. 5 is a yeeP::Ptrc-aceA integration electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-target gene; 3-downstream homology arm; 4-overlapped fragment; 5-protobacterium PCR fragment; 6-PCR fragment of target strain.

(6) FIG. 6 is a yghE::P.sub.trc-pntAB integration electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-target gene; 3-downstream homology arm; 4-overlapped fragment; 5-protobacterium PCR fragment; 6-PCR fragment of target strain.

(7) FIG. 7 is an ycdW knockout electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-downstream homology arm; 3-overlapped fragment; 4-protobacterium PCR fragment; 5-PCR fragment of target strain.

(8) FIG. 8 is a yeeL::P.sub.trc-ppc integration electrophortogram in the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-target gene; 3-downstream homology arm; 4-overlapped fragment; 5-protobacterium PCR fragment; 6-PCR fragment of target strain.

(9) FIG. 9 is a pykF knockout electrophortogram of the disclosure; in which, M-1 kb Maker; 1-upstream homology arm; 2-downstream homology arm; 3-overlapped fragment; 4-protobacterium PCR fragment; 5-PCR fragment of target strain.

(10) FIG. 10 is a fermentation process diagram of Example 2 in the disclosure.

(11) FIG. 11 shows an anabolism pathway of sarcosine constructed in Escherichia coli in the disclosure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(12) Next, the disclosure will be further described in combination with embodiments. The following embodiments are narrative but not limiting, and cannot limit the protective scope of the disclosure based on the following embodiments.

(13) The raw materials used in the disclosure, unless otherwise specified, are conventional commercial products. The methods used in the disclosure, unless otherwise specified, are conventional methods in the field, and the quality of each material used in the disclosure is conventionally used quality.

(14) A new high-efficiency imine reductase is derived from Brevibacterium linens ATCC9172, and its coding gene dpkA has a nucleotide sequence of SEQ ID NO:1.

(15) A plasmid-free genetically engineered bacterium for efficiently synthesizing Sarcosine by using a cheap carbon source as a substrate is Escherichia coli SAR, which is obtained by using Escherichia coli ATCC27325 as a host and through the following transformations: integrating singly copied imine reductase gene dpkA, which is controlled by T7 promoter, on its genome; singly copying citrate synthase gene gltA, which is controlled by trc promoter; knocking out glyoxylate cycle inhibitor gene iclR; knocking out malate synthase gene aceB; singly copying isocitrate lyase gene aceA, which is controlled by trc promoter; singly copying membrane-bound transhydrogenase gene pntAB, which is controlled by trc promoter; knocking out 2-ketate reductase gene ycdW; singly copying phosphoenolpyruvate carboxylase gene ppc, which is controlled by controlled by trc promoter; and knocking out pyruvate kinase gene pykF.

(16) Preferably, the imine reductase gene dpkA is derived from Brevibacterium linens ATCC 9172, and has a nucleotide sequence of SEQ ID NO:1.

(17) Preferably, the citrate synthase gene gltA is derived from Escherichia coli ATCC 27325, and has a nucleotide sequence of SEQ ID NO:2.

(18) Preferably, the isocitrate lyase gene aceA is derived from Escherichia coli ATCC27325, and has a nucleotide sequence of SEQ ID NO:3.

(19) Preferably, the membrane-bound transhydrogenase gene pntAB is derived from Escherichia coli ATCC27325, and has a nucleotide sequence of SEQ ID NO:4.

(20) Preferably, the phosphoenolpyruvate carboxylase gene ppc is derived from Escherichia coli ATCC27325, and has a nucleotide sequence of SEQ ID NO:5.

(21) A method for constructing a genetically engineered bacterium for efficiently synthesizing Sarcosine by using a cheap carbon source as a substrate adopts a CRISPR/Cas9-mediated gene editing technology to perform targeted transformation on Escherichia coli, specifically comprising the following steps:

(22) (1) in order to introduce the anabolism of Sarcosine, imine reductase gene dpkA derived from Brevibacterium linens ATCC 9172 is singly copied at the mbhA site on Escherichia coli ATCC27325 genome, has a sequence of SEQ ID NO:1, is optimized by codons and controlled by T7 promoter;

(23) (2) in order to enhance the metabolism from oxaloacetate to citric acid, the endogenous citrate synthase gene gltA is singly copied at the ylbE site on Escherichia coli ATCC27325 genome, has a sequence of SEQ ID NO:2, and is controlled by trc promoter;

(24) (3) in order to perform glyoxylate cycle on strains under normal culture conditions, gene knockout is performed at the iclR site on Escherichia coli ATCC27325 genome;

(25) (4) in order to block the metabolism from glyoxylic acid to malic acid, gene knockout is performed at the aceB site on Escherichia coli ATCC27325 genome;

(26) (4) in order to enhance the metabolism from isocitrate to glyoxylic acid, endogenous isocitrate lyase gene aceA is singly copied at the yeeP site on Escherichia coli ATCC27325 genome, has a sequence of SEQ ID NO:3 and is controlled by trc promoter;

(27) (5) in order to enhance the metabolism from NADH to NADPH, endogenous membrane-bound transhydrogenase gene pntAB was singly copied at the yghE site on Escherichia coli ATCC27325 genome, has a sequence of SEQ ID NO:4 and is controlled by trc promoter;

(28) (6) in order to block the metabolism from glyoxylic acid to glycolic acid, gene knockout is performed at the ycdW site on Escherichia coli ATCC27325 genome;

(29) (7) in order to enhance the metabolism from phosphoenolpyruvate to oxaloacetate, endogenous phosphoenolpyruvate carboxylase gene ppc is singly copied at the yeeL site on Escherichia coli ATCC27325 genome, has a sequence of SEQ ID NO:5, and is controlled by trc promoter; and

(30) (8) in order to reduce the metabolism from phosphoenolpyruvate to pyruvate, gene knockout is performed at the pykF site on Escherichia coli ATCC27325 genome;

(31) wherein, the construction from steps to is in no order, and needs to be adjusted as required, as shown in FIG. 11.

(32) Provided is application of the above plasmid-free genetically engineered bacterium for efficiently synthesizing Sarcosine by using cheap a carbon source in production of Sarcosine.

(33) A method for producing Sarcosine by fermenting the above genetically engineered bacterium specifically comprises the following steps:

(34) Fermentation culture: the seed liquid of the genetically engineered bacteria is inoculated into a fresh fermentation culture medium in an inoculation volume of 15-20%; during the fermentation, pH is stably controlled at 6.8-7.2, the temperature is maintained at 36.5-37.5° C., and the dissolved oxygen is between 25% and 35%; when the glucose in the culture medium is completely consumed, 700-800 g/L glucose solution is fed for further culture and the concentration of glucose in the fermentation culture medium is maintained to be <3 g/L; when OD.sub.60040, 1.5-1.6 mol/L methylamine hydrochloride solution is fed at a flow rate of 20-25 mL/h with a feeding amount of 75 mL/L culture medium, and a fermentation period is 28-32 h, so that Sarcosine is obtained;

(35) the compositions of the fermentation culture medium: 15-25 g/L of glucose, 1-5 g/L of tryptone, 3-5 g/L of sodium citrate, 1-5 g/L of KH.sub.2PO.sub.4, 0.1-1 g/L of MgSO.sub.4.7H.sub.2O and the balance of water, and pH 7.0-7.2.

(36) Specifically, relevant preparation and detection examples are as follows:

Example 1: Construction of Genetically Engineered Strain Escherichia coli SAR

(37) 1. Gene Editing Method

(38) The disclosure adopted a CRISPR/Cas9-mediated gene editing method, which refers to a literature (Metabolic Engineering, 2015, 31:13-21). Two plasmids used in this method were pGRB and pREDCas9 respectively. The pREDCas9 contained a gRNA plasmid elimination system, a Red recombination system of λ phage and a Cas9 protein expression system. It had spectinomycin resistance (working concentration: 100 mg/L) and was cultured at 32° C. The pGRB plasmid used pUC18 as a backbone, including promoter J23100, a gRNA-Cas9 binding region sequence and a terminator sequence. It had ampicillin resistance (working concentration: 100 mg/L) and was cultured at 37° C.

(39) 2. Specific Process for Strain Construction

(40) 2.1 Integration of P.sub.T7-dpkA (a Fragment Containing dpkA Gene and T7 Promoter) at the mbhA Site

(41) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-mbhA-S(SEQ ID NO:6) and UP-mbhA-A (SEQ ID NO:7) and downstream homology arm primers DN-mbhA-S(SEQ ID NO:8) and DN-mbhA-A (SEQ ID NO:9) were designed according to upstream and downstream sequences of its mbhA gene. The upstream and downstream homology arm fragments were subjected to PCR amplification. Primers dpkA-S(SEQ ID NO:10) and dpkA-A (SEQ ID NO:11) were designed according to the dpkA gene, and the dpkA gene fragment (SEQ ID NO:1) was amplified. Promoter P.sub.T7 was designed in the downstream primer of the upstream homology arm and the upstream primer of the dpkA gene. The integrated fragment (mbhA gene upstream homology arm-P.sub.T7-dpkA-mbhA-gene downstream homology arm) of the dpkA gene was obtained by overlapping PCR of the above fragments. A DNA fragment which was used for constructing the pGRB-mbhA and contained a target sequence was obtained by annealing primers gRNA-mbhA-S(SEQ ID NO:12) and gRNA-mbhA-A (SEQ ID NO:13). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-mbhA. The integrated fragment and pGRB-mbhA were electro transformed into E. coli ATCC27325 competent cells containing pREDCas9. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently, pGRB-mbhA used for gene editing was eliminated, so as to finally obtain strain E. coli SAR7.

(42) In the integration process of the P.sub.T7-dpkA fragment, an electrophortogram for the construction of the integrated fragment and the PCR verification of the positive strains is shown in FIG. 1. Wherein, the length of the upstream homology arm is 682 bp, the length of the dpkA gene fragment is 1006 bp, the length of the downstream homology arm is 720 bp, and the length of the overlapped fragment is 2457 bp. When the recons are verified via PCR, the length of the fragment amplified by the positive recons is 2457 bp, and the length of the fragment amplified by protobacteria is 1837 bp.

(43) 2.2 Integration of P.sub.trc-gltA (a Fragment Containing gltA Gene and Trc Promoter) at the Site ylbE

(44) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-ylbE-S(SEQ ID NO:14) and UP-ylbE-A (SEQ ID NO:15) and downstream homology arm primers DN-ylbE-S(SEQ ID NO:16) and DN-ylbE-A (SEQ ID NO:17) were designed according to upstream and downstream sequences of its ylbE gene. The upstream and downstream homology arms of the ylbE gene were amplified. Primers gltA-S(SEQ ID NO:18) and gltA-A (SEQ ID NO:19) were designed according to the gltA gene, and the gltA gene fragment (SEQ ID NO:2) was amplified. Promoter P.sub.trc was designed in the downstream primer of the upstream homology arm of the ylbE gene and the upstream primer of the gltA gene. The integrated fragment (ylbE gene upstream homology arm-P.sub.trc-gltA-ylbE-gene downstream homology arm) of the gltA gene was obtained by overlapping PCR of the above fragments. A DNA fragment which was used for constructing the pGRB-ylbE and contained a target sequence was obtained by annealing primers gRNA-ylbE-S(SEQ ID NO:20) and gRNA-ylbE-A (SEQ ID NO:21). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-ylbE. The integrated fragment and pGRB-ylbE were electro transformed into E. coli SAR7 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-ylbE and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR2.

(45) An electrophortogram for the construction of the P.sub.trc-gltA integrated fragment and the PCR verification of the positive strains is shown in FIG. 2. Wherein, the length of the upstream homology arm is 601 bp, the length of the gltA gene fragment is 1407 bp, the length of the downstream homology arm is 547 bp, and the total length of the integrated fragment is 2474 bp. When PCR verification is carried out, the length of the fragment subjected to PCR amplification by positive bacteria is 2474 bp, and the length of the fragment amplified by protobacteria is 2184 bp.

(46) 2.3 Knockout of iclR Gene

(47) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-iclR-S(SEQ ID NO:22) and UP-iclR-A (SEQ ID NO:23) and downstream homology arm primers DN-iclR-S(SEQ ID NO:24) and DN-iclR-A (SEQ ID NO:25) were designed according to upstream and downstream sequences of its iclR gene. The upstream and downstream homology arms of the iclR gene were amplified. The iclR gene knockout fragment (iclR gene upstream homology arm-iclR gene downstream homology arm) was obtained by overlapping PCR of the above fragments. A DNA fragment which was used for constructing the pGRB-iclR and contained a target sequence was obtained by annealing primers gRNA-iclR-S(SEQ ID NO:26) and gRNA-iclR-A (SEQ ID NO:27). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-iclR. The integrated fragment and pGRB-iclR were electrotransformed into E. coli SAR2 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-iclR and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR3.

(48) An electrophortogram for the construction of the iclR gene knockout fragment and the PCR verification of the positive strains is shown in FIG. 3. Wherein, the length of the upstream homology arm is 595 bp, the length of the downstream homology arm is 532 bp, the total length of the gene knockout fragment is 1086 bp. When PCR verification is carried out, the length of the fragment subjected to PCR amplification by positive bacteria is 1086 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 1745 bp.

(49) 2.4 Knockout of aceB Gene

(50) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-aceB-S(SEQ ID NO:28) and UP-aceB-A (SEQ ID NO:29) and downstream homology arm primers DN-aceB-S(SEQ ID NO:30) and DN-aceB-A (SEQ ID NO:31) were designed according to upstream and downstream sequences of its aceB gene. The upstream and downstream homology arms of the aceB gene were amplified. The aceB gene knockout fragment (aceB gene upstream homology arm-aceB gene downstream homology arm) was obtained by overlapping PCR of the above fragments, A DNA fragment which was used for constructing the pGRB-aceB and contained a target sequence was obtained by annealing primers gRNA-aceB-S(SEQ ID NO:32) and gRNA-aceB-A (SEQ ID NO:33). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-aceB. The integrated fragment and pGRB-iclR were electrotransformed into E. coli SAR3 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-iclR and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR4.

(51) An electrophortogram for the construction of the aceB gene knockout fragment and the PCR verification of the positive strains is shown in FIG. 4. Wherein, the length of the upstream homology arm is 538 bp, the length of the downstream homology arm is 586 bp, the total length of the gene knockout fragment is 1082 bp. When PCR verification is carried out, the length of the fragment amplified by positive bacteria is 1082 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 2397 bp.

(52) 2.5 Integration of P.sub.trc-aceA (a Fragment Containing aceA Gene and Trc Promoter) at the Site yeeP

(53) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-yeeP-S(SEQ ID NO.34) and UP-yeeP-A (SEQ ID NO.35) and downstream homology arm primers DN-yeeP-S(SEQ ID NO.36) and DN-yeeP-A (SEQ ID NO.37) were designed according to upstream and downstream sequences of its yeeP gene. The upstream and downstream homology arms of the yeeP gene were amplified. Primers aceA-S(SEQ ID NO.38) and aceA-A (SEQ ID NO.39) were designed according to the aceA gene, and the aceA gene fragment (SEQ ID. NO: 3) was amplified. Promoter P.sub.trc was designed in the downstream primer of the yeeP gene upstream homology arm and the upstream primer of the aceA gene. The integrated fragment (yeeP gene upstream homology arm-P.sub.trc-aceA-yeeP gene downstream homology arm) of the aceA gene was obtained by overlapping PCR of the above fragments. A DNA fragment which was used for constructing pGRB-yeeP and contained a target sequence was obtained by annealing primers gRNA-yeeP-S(SEQ ID NO: 40) and gRNA-yeeP-A (SEQ ID NO: 41). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-yeeP. The integrated fragment and pGRB-yeeP were electrotransformed into E. coli SAR4 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-yeeP and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR5.

(54) An electrophortogram for the construction of the P.sub.trc-aceA integrated fragment and the PCR verification of the positive strain is shown in FIG. 5. Wherein, the length of the upstream homology arm is 568 bp, the length of the aceA gene fragment is 1428 bp, the length of the downstream homology arm is 576 bp, the total length of the integrated fragment is 2491 bp. When in PCR verification, the length of the fragment subjected to PCR amplification by positive bacteria is 2491 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 1396 bp.

(55) 2.6 Integration of P.sub.trc-pntAB (a Fragment Containing pntAB Gene and Trc Promoter) at the Site yghE

(56) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-yghE-S(SEQ ID NO:42) and UP-yghE-A (SEQ ID NO:43) and downstream homology arm primers DN-yghE-S(SEQ ID NO.44) and DN-yghE-A (SEQ ID NO.45) were designed according to upstream and downstream sequences of its yghE gene. The upstream and downstream homology arms of the yghE gene were amplified. Primers pntAB-S(SEQ ID NO.46) and pntAB-A (SEQ ID NO.47) were designed according to the pntAB gene, and the pntAB gene fragment (SEQ ID. NO: 3) was amplified. Promoter P.sub.trc was designed in the downstream primer of the yghE gene upstream homology arm and the upstream primer of the pntAB gene. The integrated fragment (yghE gene upstream homology arm-P.sub.trc-pntAB-yghE gene downstream homology arm) was obtained by overlapping PCR of the above fragments. A DNA fragment which was used for constructing pGRB-yghE and contained a target sequence was obtained by annealing primers gRNA-yghE-S(SEQ ID NO: 48) and gRNA-yghE-A (SEQ ID NO:49). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-yghE. The integrated fragment and pGRB-yghE were electrotransformed into E. coli SAR5 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-yghE and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR6.

(57) The electrophortogram of the construction of the P.sub.trc-pntAB integrated fragment and the PCR verification of the positive strains was as shown in FIG. 6. Wherein, the length of the upstream homology arm is 559 bp, the length of the pntAB gene fragment is 3050 bp, the length of the downstream homology arm is 549 bp, and the total length of the integrated fragment is 4087 bp. When in PCR verification, the length of the fragment subjected to PCR amplification by positive bacteria is 4087 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 1547 bp.

(58) 2.7 Knockout of ycdW Gene

(59) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-ycdW-S(SEQ ID NO.50) and UP-ycdW-A (SEQ ID NO.51) and downstream homology arm primers DN-ycdW-S(SEQ ID NO:52) and DN-ycdW-A (SEQ ID NO:53) were designed according to upstream and downstream sequences of its ycdW gene. The upstream and downstream homology arms of the ycdW gene were amplified. The ycdW gene knockout fragment (ycdW gene upstream homology arm-ycdW gene downstream homology arm) was obtained by overlapping PCR. A DNA fragment which was used for constructing pGRB-ycdW and contained a target sequence was obtained by annealing primers gRNA-ycdW-S(SEQ ID NO:54) and gRNA-ycdW-A (SEQ ID NO:55). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-ycdW. The integrated fragment and pGRB-ycdW were electrotransformed into E. coli SAR6 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-ycdW and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR7.

(60) The electrophortogram of the construction of the ycdW gene knockout fragment and the PCR verification of the positive strains is as shown in FIG. 7. Wherein, the length of the upstream homology arm is 642 bp, the length of the downstream homology arm is 1428 bp, the total length of the gene knockout fragment is 2024 bp. When in PCR verification, the length of the fragment subjected to PCR amplification by positive bacteria is 2024 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 2604 bp.

(61) 2.8 Integration of P.sub.trc-Ppc (a Fragment Containing Ppc Gene and Trc Promoter) at the Site yeeL

(62) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-yeeL-S(SEQ ID NO:56) and UP-yeeL-A (SEQ ID NO:57) and downstream homology arm primers DN-yeeL-S(SEQ ID NO:58) and DN-yeeL-A(SEQ ID NO:59) were designed according to upstream and downstream sequences of its yeeL gene. The upstream and downstream homology arms of the yeeL gene were amplified. Primers ppc-S(SEQ ID NO: 60) and ppc-A(SEQ ID NO: 61) were designed according to the ppc gene, and the ppc gene fragment (SEQ ID NO: 5) was amplified. Promoter P.sub.trc was designed in the downstream primer of the yeeL gene upstream homology arm and the ppc gene upstream primer. The integrated fragment (yeeL gene upstream homology arm-P.sub.trc-ppc-yeeL gene downstream homology arm) of the ppc gene was obtained by overlapping PCR of the above fragments. A DNA fragment which was used for constructing pGRB-yeeL and contained a target sequence was obtained by annealing primers gRNA-yeeL-S(SEQ ID NO:62) and gRNA-yeeL-A (SEQ ID NO:63). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-yghE. The integrated fragment and pGRB-yeeL were electrotransformed into E. coli SAR7 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-yeeL and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR8.

(63) The electrophortogram of the construction of the P.sub.trc-ppc integrated fragment and the PCR verification of the positive strains was as shown in FIG. 8. Wherein, the length of the upstream homology arm is 533 bp, the length of the ppc gene fragment is 2770 bp, the length of the downstream homology arm is 581 bp, and the total length of the integrated fragment is 381 bp. When in PCR verification, the length of the fragment subjected to PCR amplification by positive bacteria is 3813 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 1613 bp.

(64) 2.9 Knockout of pykF Gene

(65) An E. coli ATCC27325 genome was used as a template. Upstream homology arm primers UP-pykF-S(SEQ ID NO: 64) and UP-pykF-A (SEQ ID NO: 65) and downstream homology arm primers DN-pykF-S(SEQ ID NO: 66) and DN-pykF-A (SEQ ID NO: 67) were designed according to upstream and downstream sequences of its pykF gene, and the upstream and downstream homology arms of the pykF gene were amplified. The pykF gene knockout fragment (pykF gene upstream homology arm-pykF gene downstream homology arm) was obtained by overlapping PCR. A DNA fragment which was used for constructing pGRB-pykF and contained a target sequence was obtained by annealing primers gRNA-pykF-S(SEQ ID NO:68) and gRNA-pykF-A (SEQ ID NO:69). The DNA fragment was recombined with a linearized pGRB vector to obtain recombinant pGRB-pykF. The integrated fragment and pGRB-pykF were electrotransformed into E. coli SAR8 competent cells containing a pREDCas9 vector. The resuscitated strains after electrotransformation were coated on an LB plate containing ampicillin and spectinomycin and cultured overnight at 32° C., and the positive recons were verified via PCR. Subsequently pGRB-pykF and pREDCas9 used for gene editing were eliminated, so as to finally obtain strain E. coli SAR9.

(66) The electrophortogram of the construction of the pykF gene knockout fragment and the PCR verification of the positive strains was as shown in FIG. 9. Wherein, the length of the upstream homology arm is 471 bp, the length of the downstream homology arm is 429 bp, and the total length of the gene knockout fragment is 856 bp. When in PCR verification, the length of the fragment subjected to PCR amplification by positive bacteria is 856 bp, and the length of the fragment subjected to PCR amplification by protobacteria is 2180 bp.

(67) 3. Primers Used in the Construction Process of Strains

(68) All primers involved in the construction process of strains are shown in Table as follows:

(69) TABLE-US-00001 SEQ ID NO: Primers Sequence (5′-3′) 6 UP-mbhA-S GCCAGCACGAACATAATCCC 7 UP-mbhA-A TAAAGTTAAACAAAATTATTT CTAGACCCTATAGTGAGTCGT ATTACACGGTGGCAGGTTTT GG 8 DN-mbhA-S TGGGGCCTCTAAACGGGTCT TGAGGGGTTTTTTGGACCAA AAGTGCGTCCGATAC 9 DN-mbhA-A CGGCGTAATCACAAACTGGC 10 dpkA-S TAGGGTCTAGAAATAATTTTG TTTAACTTTAAGAAGGAGATA TACCATGACGAACGAACCGG ACC 11 dpkA-A AGACCCGTTTAGAGGCCCCA AGGGGTTATGCTAGTTATTCG AACAGACTGCGGATG 12 gRNA-mbhA-S AGTCCTAGGTATAATACTAGT TACCGGGCATACCGATGCGA GTTTTAGAGCTAGAA 13 gRNA-mbhA-A TTCTAGCTCTAAAACTCGCAT CGGTATGCCCGGTAACTAGTA TTATACCTAGGACT 14 UP-ylbE-S ACCCAACCTTACGCAACCAG 15 UP-ylbE-A AATTGTTATCCGCTCACAATT CCACACATTATACGAGCCGG ATGATTAATTGTCAATTGTTC GATAACCGCAGCAT 16 DN-ylbE-S AAAGACTGGGCCTTTCGTTT TATCTGTTGTTTGTCGGTGAA CGCTCTCCTGAGTAGGACAA ATCGCTGGCGTGCTTTGAA 17 DN-ylbE-A GGCGTAACTCAGCAGGCAG 18 gltA-S TCCGGCTCGTATAATGTGTGG AATTGTGAGCGGATAACAATT TCACACAGGAAACAGACCAT GGCTGATACAAAAGCAAAAC TC 19 gltA-A CACCGACAAACAACAGATAA AACGAAAGGCCCAGTCTTTC GACTGAGCCTTTCGTTTTATT TGTTAACGCTTGATATCGCTT TTAAAG 20 gRNA-ylbE-S AGTCCTAGGTATAATACTAGT ACACTGGCTGGATGTGCAAC GTTTTAGAGCTAGAA 21 gRNA-ylbE-A TTCTAGCTCTAAAACGTTGCA CATCCAGCCAGTGTACTAGTA TTATACCTAGGACT 22 UP-iclR-S CGTGGAGTTGAAGGTGTTGG T 23 UP-iclR-A TCCTTCGCCGCTTTAATCACC GGCAATCCACTCCAGTAATT 24 DN-iclR-S AATTACTGGAGTGGATTGCC GGTGATTAAAGCGGCGAAGG A 25 DN-iclR-A TAATAGAGGCGTCGCCAGCT 26 gRNA-iclR-S AGTCCTAGGTATAATACTAGT ACGGAACTGGCGCAACAAGC GTTTTAGAGCTAGAA 27 gRNA-iclR-A TTCTAGCTCTAAAACGCTTGT TGCGCCAGTTCCGTACTAGTA TTATACCTAGGACT 28 UP-aceB-S GAGCTGGCGTAGTCACGGTA A 29 UP-aceB-A TTCGCTGGCAATGACTTTCAC AGAAGTTTATTGCGTTGTGGC 30 DN-aceB-S GCCACAACGCAATAAACTTC TGTGAAAGTCATTGCCAGCG AA 31 DN-aceB-A GCACGACGGAAGGTGTTGTT 32 gRNA-aceB-S AGTCCTAGGTATAATACTAGT CCAGCTCAAGCCCAATCCAG GTTTTAGAGCTAGAA 33 gRNA-aceB-A TTCTAGCTCTAAAACCTCGCG GCCAGATACGCATGACTAGTA TTATACCTAGGACT 34 UP-yeeP-S GGTCAGGAGGTAACTTATCA GCG 35 UP-yeeP-A AATTGTTATCCGCTCACAATT CCACACATTATACGAGCCGG ATGATTAATTGTCAAATGGCA GGGCTCCGTTTT 36 DN-yeeP-S AAAGACTGGGCCTTTCGTTT TATCTGTTGTTTGTCGGTGAA CGCTCTCCTGAGTAGGACAA ATGAACTGGATTTTCTTCTGA ACCTGT 37 DN-yeeP-A ACGATGTCAGCAGCCAGCA 38 aceA-S TCCGGCTCGTATAATGTGTGG AATTGTGAGCGGATAACAATT TCACACAGGAAACAGACCAT GAAAACCCGTACACAACAAA TT 39 aceA-A CACCGACAAACAACAGATAA AACGAAAGGCCCAGTCTTTC GACTGAGCCTTTCGTTTTATT TGTTAGAACTGCGATTCTTCA GTGG 40 gRNA-yeeP-S AGTCCTAGGTATAATACTAGT ACAGAATATTCGCGAAAAAA GTTTTAGAGCTAGAA 41 gRNA-yeeP-A TTCTAGCTCTAAAACTTTTTT CGCGAATATTCTGTACTAGTA TTATACCTAGGACT 42 UP-yghE-S GTCAGGCACTGGCGAAAGAT 43 UP-yghE-A AATTGTTATCCGCTCACAATT CCACACATTATACGAGCCGG ATGATTAATTGTCAACGCAAG CCATAAACCCACA 44 DN-yghE-S CTGGGCCTTTCGTTTTATCTG TTGTTTGTCGGTGAACGCTCT CCTGAGTAGGACAAATTTCC GACATCGAAATGCGT 45 DN-yghE-A AGGCGTTGTTGTGGCAGATT 46 pntAB-S TCCGGCTCGTATAATGTGTGG AATTGTGAGCGGATAACAATT TCACACAGGAAACAGACCAT GCGAATTGGCATACCAAGA 47 pntAB-A ACAAACAACAGATAAAACGA AAGGCCCAGTCTTTCGACTG AGCCTTTCGTTTTATTTGTTA CAGAGCTTTCAGGATTGCATC 48 gRNA-yghE-S AGTCCTAGGTATAATACTAGT GCTGAAAAAATATCGCCCAC GTTTTAGAGCTAGAA 49 gRNA-yghE-A TTCTAGCTCTAAAACGTGGG CGATATTTTTTCAGCACTAGT ATTATACCTAGGACT 50 UP-ycdW-S TCCTTCAGCCACTCGGACAC 51 UP-ycdW-A GATAGCAGGAATCCTGATGCT TTATGGATGCGATAATCGTCA AAAC 52 DN-ycdW-S GTTTTGACGATTATCGCATCC ATAAAGCATCAGGATTCCTGC TATC 53 DN-ycdW-A ATTATCCGTTGCAGTTATGAG TGA 54 gRNA-ycdW-S AGTCCTAGGTATAATACTAGT TTGCTCAGAGTCTGCAAACC GTTTTAGAGCTAGAA 55 gRNA-ycdW-A TTCTAGCTCTAAAACGGTTTG CAGACTCTGAGCAAACTAGT ATTATACCTAGGACT 56 UP-yeeL-S TTCATCGGGACGAGTGGAGA 57 UP-yeeL-A AATTGTTATCCGCTCACAATT CCACACATTATACGAGCCGG ATGATTAATTGTCAACCATAG CATCGCCAATCTGA 58 DN-yeeL-S CTGGGCCTTTCGTTTTATCTG TTGTTTGTCGGTGAACGCTCT CCTGAGTAGGACAAATACCC AAAGGTGAAGATAAAGCC 59 DN-yeeL-A CATTCCCTCTACAGAACTAGC CCT 60 ppc-S TCCGGCTCGTATAATGTGTGG AATTGTGAGCGGATAACAATT TCACACAGGAAACAGACCAT GAACGAACAATATTCCGCAT 61 ppc-A ACAAACAACAGATAAAACGA AAGGCCCAGTCTTTCGACTG AGCCTTTCGTTTTATTTGTTA GCCGGTATTACGCATACCT 62 gRNA-yeeL-S AGTCCTAGGTATAATACTAGT AACACAGCAATACGGTACGC GTTTTAGAGCTAGAA 63 gRNA-yeeL-A TTCTAGCTCTAAAACGCGTAC CGTATTGCTGTGTTACTAGTA TTATACCTAGGACT 64 UP-pykF-S ACTGACAACTTCGGCACCAG A 65 UP-pykF-A CAGATGCGGTGTTAGTAGTG CCTCTTCAGATTCGGTTTTCG GTC 66 DN-pykF-S GACCGAAAACCGAATCTGAA GAGGCACTACTAACACCGCA TCTG 67 DN-pykF-A AACCTGCCAGCAGAGTAGAA CC 68 gRNA-pykF-S AGTCCTAGGTATAATACTAGT CGCAACGTGATGAGCAAAAC GTTTTAGAGCTAGAA 69 gRNA-pykF-A TTCTAGCTCTAAAACGTTTTG CTCATCACGTTGCGACTAGTA TTATACCTAGGACT

Example 2: Production of Sarcosine by Flask Fermentation Using Strain E. coli SAR

(70) Fermentation Experiment of Strain E. coli SAR in a 5 L Fermentor:

(71) Slope activation culture: a ring of bacteria were scrapped off from a preserving tube in a −80° C. refrigerator, evenly coated on an activated slope, cultured for 12 h at 37° C., and then transferred to an eggplant-shaped bottle to conduct further culture for 12 h;

(72) Seed culture: a proper amount of sterile water was placed in the eggplant-shaped bottle, and the bacterial suspension was inoculated into a seed culture medium to culture for 6 h, wherein the pH is stabilized at about 7.0, the temperature is constant at 37° C., and the dissolved oxygen is between 25% and 35%;

(73) Fermentation culture: a fresh fermentation culture medium was inoculated with an inoculation amount of 15%. The loading amount was 60% (v culture medium/v fermentor). During the fermentation, the pH was stably controlled at about 7.0, and the temperature was maintained at 36.5-37.5° C. The dissolved oxygen is between 25% and 35%. When the glucose in the culture medium was completely consumed, 800 g/L glucose solution was fed for further culture, and the glucose concentration in the fermentation medium was maintained to be less than 3 g/L. When OD.sub.600 is 40, 1.6 mol/L methylamine hydrochloride solution was fed at a flow rate of 25 mL/h with a feeding amount of 75 mL/L of culture medium. The fermentation period is 30 h;

(74) The compositions of the slope culture medium: 1 g/L of glucose, 10 g/L of peptone, 10 g/L of beef extract, 5 g/L of yeast powder, 2.5 g/L of NaCl, 25 g/L of agar and the balance of water, and pH 7.0;

(75) The compositions of the seed culture medium: 25/L of glucose, 5 g/L of yeast extract, 5 g/L of tryptone, 5 g/L of KH.sub.2PO.sub.4 5 g/L, 2 g/L of MgSO.sub.4.7H.sub.2O and the balance of water, and pH 7.0.

(76) The compositions of the fermentation culture medium: 20 g/L of glucose, 4 g/L of yeast extract, 5 g/L of tryptone, 5 g/L of sodium citrate, 2 g/L of KH.sub.2PO.sub.4, 1 g/L of MgSO.sub.4.7H.sub.2O and the balance of water, and pH 7.0.

(77) After fermentation for 30 h in the 5 L fermentor, the sarcosine titer can reach 10 g/L. The fermentation process curve is shown in FIG. 10.

(78) Although the embodiments of the disclosure are disclosed for illustrating the purposes, those skilled in the art should understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the disclosure and appended claims, therefore the scope of the disclosure is not limited to the contents disclosed in embodiments.