CN112458032B - Construction and application of escherichia coli recombinant bacteria for synthesizing glycine by using glucose - Google Patents

Abstract

The invention discloses construction and application of an escherichia coli recombinant bacterium for synthesizing glycine by using glucose. The invention discloses an escherichia coli recombinant bacterium, which is characterized in that firstly, a plasmid pLB1k-cgAT (Cqu) of a glutamic acid glyoxylate transaminase II gene cgAT, malate thiokinase, malyl-CoA lyase, isocitrate lyase, and a recombinant plasmid pSB1a-mtk (Mca) -mcl (Rsp) -aceAK of isocitrate dehydrogenase kinase/phosphatase are constructed, then, a host bacterium GC05 is constructed by knocking out an inhibitor gene, then, the plasmid pLB1k-cgAT (Cqu) is introduced into GC05 to form a recombinant bacterium GC06, and finally, the plasmid pSB1a-mtk (Mca) -mcl (Rsp) -aceAK is introduced into GC07 to obtain the escherichia coli recombinant bacterium GC07. The invention also provides a method for converting glucose into glycine by using the Escherichia coli recombinant strain GC07. The method has the characteristics of high conversion rate, low cost, simple reaction, small pollution and the like.

Description

Construction and application of escherichia coli recombinant bacteria for synthesizing glycine by using glucose

Technical Field

The invention relates to the field of biotechnology and biochemical engineering of industrial microorganisms, in particular to construction and application of a recombinant escherichia coli engineering strain for synthesizing glycine by taking glucose as a substrate.

Background

In recent years, the development of synthetic biology and metabolic engineering provides favorable conditions for the transformation of escherichia coli engineering bacteria, and cheap substrates can be converted into chemicals, fuels and high-molecular polymers with high added values by directed evolution and reasonable genetic transformation of wild bacteria.

Glycine (also known as Glycine in english) is also known as Glycine, and is the α -amino acid with the simplest structure. It is an important fine chemical synthesis intermediate, and can be widely applied in the fields of medicines, foods, pesticides, feed additives and the like. Glycine is currently commonly used in several areas: (1) Glycine is a synthetic precursor of some important small molecule metabolites in organisms, and a proper amount of glycine is added into diet to effectively treat cardiovascular diseases, inflammation, obesity, tumors and metabolic disorders of diabetes patients (2) glycine is an intermediate synthesized by various medicaments such as delapril hydrochloride, oxalglycine aspirin calcium, paracetamol glycinate, and a limemine injection. (3) Glycine is widely used in the food industry as an amino acid enhancer, a flavoring agent, a sweetening agent and an antioxidant. (4) Glycine is a precursor for the synthetic herbicide glyphosate, which has been evaluated by the U.S. government as the most elegant pesticide.

The existing methods for synthesizing glycine mainly comprise chloroacetic acid ammonolysis, strecker method, catalytic dehydrogenation oxidation method, radiation synthesis method, biosynthesis method and the like. Among them, the biosynthesis method has been conventionally obtained by transformation with microorganisms such as aerobic Agrobacterium, brevibacterium, corynebacterium, etc., using ethanolamine as a substrate, and also has been used for producing glycine by hydrolyzing glycinamide with the use of genera such as Pseudomonas, casein, alcaligenes, etc. Although the above method can produce glycine, it has problems of high raw material cost, low enzyme activity and the need for a large amount of cells. Bioconversion still requires the establishment of a cheaper route to the starting material. Escherichia coli, which is a model microorganism with a clear genetic background and simple genetic manipulation, is often used for metabolic engineering to produce metabolites with high added values. In Escherichia coli, glycine can be obtained from a precursor such as serine or threonine, but there is little research on the synthesis of glycine by metabolic engineering using glucose as a substrate.

Disclosure of Invention

The invention aims to construct an escherichia coli recombinant bacterium, and the recombinant bacterium can efficiently convert glucose into glycine by introducing or knocking out and modifying genes of the recombinant bacterium.

The Escherichia coli recombinant strain provided by the invention has the following modifications:

an escherichia coli recombinant bacterium, wherein the strain comprises the following modifications:

1) Introducing exogenous glutamic glyoxylate transaminase gene cgAT;

2) Introducing exogenous malate thiokinase gene mtk and exogenous malyl-CoA lyase gene mcl;

3) Introducing a glyoxylate pathway aceK gene, namely a gene of isocitrate dehydrogenase kinase/phosphatase;

4) Knocking out aceB gene, namely gene of malate synthase A;

5) Knocking out an iclR gene, namely a gene of a transcription inhibitor of a glyoxylate pathway;

6) Knock-out of the glcB gene, the gene for the protein malate synthase G;

7) Knockout of maeA gene, i.e., gene for malate dehydrogenase (NAD required);

8) The maeB gene, namely the gene of malate dehydrogenase, is knocked out.

The invention also provides a construction method of the escherichia coli recombinant strain, which comprises the following preparation steps:

2) Two plasmids were constructed, one of which was plasmid pLB1k-cgAT (Cqu) of the glutamate glyoxylate transaminase II gene cgAT and the other was recombinant plasmid pSB1a-mtk (Mca) -mcl (R) aceAK of malate thiokinase, malyl-CoA lyase isocitrate lyase and isocitrate dehydrogenase kinase.

2) Constructing a host bacterium, and knocking out a malic acid synthase A gene aceB of a recipient bacterium XY24 to form a recombinant bacterium GC01; knocking out a glyoxylate pathway transcription repression factor gene iclR of the recombinant strain GC01 to form a recombinant strain GC02; knocking out a malic acid synthase G gene glcB of the recombinant strain GC02 to form a recombinant strain GC03; knocking out a malate dehydrogenase (NAD required) gene maeA of the recombinant strain GC03 to form a recombinant strain GC04; and (3) knocking out the malic acid dehydrogenase gene maeB of the recombinant strain GC04 to form a recombinant strain GC05.

3) Preparing competent cells from recombinant strain GC05, and introducing the plasmid pLB1k-cgAT (Cqu) in the step 1) into the GC05 to form recombinant strain GC06; competent cells were prepared from strain GC06, and pSB1a-mtk (Mca) -mcl (Rsp) -aceAK from step 1) was introduced into GC06 to construct recombinant Escherichia coli GC07 which synthesizes glycine from glucose.

The plasmid pLB1k-cgAT (Cqu) of the glutamate glyoxylate aminotransferase II gene cgAT is derived from quinoa Chenopodium quinoa; the malate thiokinase gene mtk is derived from Methylococcus capsulatus, and the malyl-CoA lyase gene mcl is derived from Rhodobacter sphaeroides; the recombinant plasmid pSB1a-mtk (Mca) -mcl (Rsp) -aceAK of isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase is derived from Escherichia coli.

Further, the calcium chloride method is mainly used for competent cell transformation.

Another purpose of the invention is to provide a method for preparing glycine by using the recombinant Escherichia coli and glucose as a raw material, which comprises the following steps:

1) Culturing of the cells and induction of enzymes: culturing recombinant Escherichia coli GC07 strain at 37 deg.C overnight, inoculating into shake flask containing 50mL ZYM self-induction culture medium with 1% inoculum size, adding kanamycin and ampicillin to final concentration of 50 μ g/mL and 100 μ g/mL respectively, and performing induction culture at 30 deg.C for 16 hr;

2) Whole-cell catalytic conversion: measuring the bacteria concentration of the culture solution after induction with spectrophotometer, and collecting 1.5 × 10 ¹¹ Centrifuging thallus at 4 deg.C and 5000rpm for 10min, collecting precipitate, washing with 0.85% physiological saline twice, discarding supernatant, re-suspending thallus with 5mL of transformation solution, transferring to test tube to final bacteria concentration of 3 × 10 ¹⁰ In the case of glycine production by whole-cell transformation, the whole-cell transformation system contained glucose at a final concentration of 50mM and 1 XPBS at 37 ℃ and 200rpm.

The invention has the beneficial effects that

The technology of the invention changes the metabolic pathway of recombinant Escherichia coli by carrying out gene modification on the recombinant Escherichia coli, the recombinant Escherichia coli takes glucose as a raw material and synthesizes glycine through glycolysis, glyoxylate pathway and reverse glyoxylate pathway in sequence, and the step of carbon fixation is arranged in the middle, so that the conversion rate is high, and the conversion cost is low.

Drawings

FIG. 1 shows the way of synthesizing glycine by recombinant Escherichia coli bacteria using glucose as raw material.

FIG. 2 shows the yield of glycine from recombinant E.coli engineered strain using glucose as raw material.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, E.coli BW25113 (Datsenko KA, wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-1 using PCR products. Proc. Natl. Acad. Sci. U.S.A.97 (12): 6640-6645.) is a non-pathogenic bacterium with clear genetic background, short generation time, easy culture and low cost of culture medium raw materials. Coli BW25113 is available from the institute of microbiology, national academy of sciences. Escherichia coli XY24 was obtained by genetic engineering from Escherichia coli BW25113 (Piao XY, wang L, lin BX, chen H, liu WF, tao Y. Metabolic engineering of Escherichia coli for production of L-aspartic acid and itsederivative β -alkane with high specificity and yield engineering.2019; 54.

Example 1 construction of recombinant engineered Escherichia coli strain GC07

The construction of the recombinant escherichia coli engineering strain GC07 comprises the following specific steps:

1. a plasmid pLB1k-cgAT (Cqu) of a glutamic glyoxylic acid transaminase II gene cgAT derived from Chenopodium quinoa was constructed.

1) PCR amplification of the cgAT gene. The nucleotide sequence of the modified Chenopodium quinoa (Chenopodium quinoa) exogenous glutamic glyoxylate transaminase II gene cgAT (Cqu) is shown as SEQ ID No. 1. The sequence was synthesized by a whole gene and ligated to a pUC57 vector to obtain a vector pUC57-cgaT (Cqu). The cgAT (Cqu) gene fragment was PCR-amplified with the cGAT (Cqu) -F and cGAT (Cqu) -R primers, the pUC57-cgAT (Cqu) plasmid vector as template, and the high fidelity TransStart Fastpfu DNA polymerase (Beijing Quantikin Biotechnology Co., ltd., product catalog AP 221).

2) A recombinant expression vector containing the cgAT (Cqu) gene was constructed. Carrying out agarose gel electrophoresis on the PCR amplification fragment obtained in the step 1) and recovering the target fragment. Meanwhile, the vector pLB1k is digested by NcoI and XhoI, the nucleotide sequence of the vector pLB1k is shown in SEQ ID No.2, and the large fragment LB1k-NX of the vector is recovered. The cgAT (Cqu) fragment was ligated to the LB1k-NX fragment using the Gibson assembly method (Gibson DG, young L, et al. Enzymatic assembly of DNA molecules up to a viral cloned primers. Nat. Methods.2009;6 (5): 343-345). With CaCl ₂ Escherichia coli DH5a competent cells were transformed by the method, purchased from Beijing Quanji gold Biotechnology Ltd, catalog CD201. Spread on LB plates containing 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃. Selecting clones, identifying clones capable of amplifying target fragments by using a primer 105-F/cgaT (Cqu) -R, sequencing, selecting positive clones, extracting plasmids, and obtaining positive plasmids which are named as pLB1k-cgaT (Cqu).

In the sequence of SEQ ID No.2, the araC gene coding sequence: positions 86-964; p _BAD Promoter sequence: positions 1238-1266; RBS sequence: 1295-1299 bits; ncoI site: a 1308 th bit; xhoI site: 1367 th bit; t is a unit of _rrnB Terminator sequence: 1501 to 1658; P15A replication initiation site RepA gene coding sequence: 1742-2128 th; kanamycin resistance gene coding sequence: 2275-3090 th position.

2. A recombinant plasmid pSB1a-mtk (Mca) -mcl (R) -aceAK is constructed which can synergistically express Methycoccus capsulatus-derived malyl-CoA lyase, rhodobacter sphaeroides-derived malyl-CoA lyase, escherichia coli-derived isocitrate lyase, and isocitrate dehydrogenase kinase/phosphatase.

Artificially synthesizing an Escherichia coli codon-optimized malate thiokinase gene mtk derived from Methycoccus capsulatus and a Rhodobacter sphaeroides-derived malyl-CoA lyase gene mcl, wherein the sequences of RBSs are pre-primed before the mcl gene, and amplifying mtk (Mca) -mcl (Rsp) -NX fragments by using primers mtk (Mca) -F and mcl (Rsp) -R according to the method for amplifying a glutamic glyoxylate transaminase II gene cgAT (Cqu) derived from Chenopodium quinoa, wherein the nucleotide sequence of mtk (Mca) -mcl (Rsp) containing RBS is shown as SEQ ID No. 3. Gibson assembly was performed with the vector fragment pSB1a-NX, the nucleotide sequence of the vector pSB1a being shown in SEQ ID No.4, to give a plasmid pSB1a-mtk (Mca) -mcl (Rsp). The plasmid pSB1a-mtk (Mca) -mcl (Rsp) was digested with EcoRI and PstI to obtain a large fragment pSB1a-mtk (Mca) -mcl (Rsp) -EP.

In the sequence of SEQ ID No.3, the mtk (Mca) coding sequence: bits 1-2088; RBS sequence: 2089-2104 th bit; mcl (Rsp) coding sequence: 2105-3061.

In the sequence of SEQ ID No.4, the sequence of the araC gene: positions 86 to 964; p _BAD Promoter sequence: positions 1238-1266; RBS sequence: 1295-1299 bits; ncoI site: a 1308 th bit; xhoI site: 1367 th bit; t is _rrnB Terminator sequence: 1501 to 1658; P15A replication initiation site RepA gene coding sequence: 2260-3210 bit; ampicillin resistance gene coding sequence: 3832-4692.

Genomic DNA was extracted from E.coli, and aceAK gene fragment was amplified with primers aceA-F/aceK-R while RBS sequence was introduced in the primers. The aceAK fragment was ligated with the pSB1a-mtk (Mca) -mcl (Rsp) -EP fragment. Coli DH5a was transformed, clones capable of amplifying the desired fragment were identified by using primers 108-F/124-R and sequenced, positive clones were selected, and the resulting plasmid was named pSB1a-mtk (Mca) -mcl (Rsp) -aceAK. The nucleotide sequence of aceAK is shown as SEQ ID No. 5.

In the sequence of SEQ ID No.5, the coding sequence of aceA gene: bits 1-1305; aceK gene coding sequence: 1488-3224.

3. Construction of host bacteria

(1) Knockout of malate synthase a gene aceB.

a. Firstly, preparing P1 bacteriophage containing colibacillus gene fragment, wherein the contained gene fragment has aceB knockout character.

An E.coli gene fragment containing the aceB knockout trait is derived from E.coli strain JW3974, which is a W3110 series strain containing the aceB knockout trait, purchased from national institute of genetics (NIG, japan), in which the gene aceB encoding malate synthase A is replaced with a kanamycin resistance gene having FRT sites at both ends by about 1300bp, thereby knocking out the aceB gene (Baba T, ara T, et al. Construction of Escherichia coli K-12in-frame, single-gene knock-out variants: the Keio collection. Mol. Syst. Biol.2006; 2.2006.0008.. The P1 phage was prepared as follows: the JW3974 strain was cultured overnight at 37 ℃ and transferred to a medium containing 5mmol/LCaCl ₂ And 0.1% glucose in LB medium, culturing at 37 deg.C for 1h, adding wild type P1 bacteriophage, and culturing for 1-3h. Adding 200 mu L chloroform for culturing for 10min, centrifuging and taking supernatant to obtain the phage P1 virdeaceB containing the fragment.

b. The P1 phage transduction technology is utilized to construct an escherichia coli strain GC01-Kan, and the specific steps are as follows: after centrifugation of 1.5mL of the overnight-cultured recipient strain XY24 at 8000rpm for 3min, XY24 cells were resuspended in 0.75mL of P1 salt solution, 100mL of phage P1 virdeaceB was mixed with 100mL of XY24 cell suspension, and incubated at 37 ℃ for 30min. Then, adding 1mL of LB culture medium and 200mL of 1mol/L sodium citrate, continuing to culture for 1h at 37 ℃, centrifugally collecting thalli, resuspending the thalli by using 100mL of LB culture medium, coating the thalli on an LB flat plate containing 50mg/mL kanamycin, culturing overnight at 37 ℃, selecting clones, performing PCR amplification identification by using aceB-F/aceB-R primers, and selecting positive clones which are positive in the amplified 1900bp target band and named as GC01-Kan. The solute of the P1 salt solution is 10mM CaCl ₂ And 5mM MgSO ₄ The solvent is water.

C. Elimination of resistance: the pCP20 plasmid was purchased from Clontech, transformed into GC01-Kan by calcium chloride transformation, cultured overnight at 30 ℃ on LB plate containing ampicillin, and then cloned to obtain recombinant Escherichia coli GC01-Kan/pCP20 containing the plasmid pCP20. After being cultured in LB culture medium containing ampicillin resistance at 30 ℃, the mixture is coated on an LB plate without resistance and cultured at 42 ℃ overnight, clones are selected, aceB-F/aceB-R primers are used for PCR amplification and identification, the amplified 600bp target band is positive, and the selected positive clone is named as GC01.

(2) Knock-out of the glyoxylate pathway transcription repressor gene iclR.

Starting from recombinant bacterium GC01, knocking out glyoxylate pathway transcription repressing factor gene iclR by using the knocking-out method in the step (1) to obtain recombinant Escherichia coli GC02. Wherein the differences from the strains and primers used in the step (1) are as follows: coli strain containing the iclR knockout trait was changed to JW3978, and aceB was changed to iclR in the name of the corresponding primer.

(3) Knock-out of the G gene glcB of malate synthase.

And (2) knocking out the glcB gene of the malate synthase G by using the knocking-out method in the step (1) from the recombinant strain GC02 to obtain the Escherichia coli GC03. Wherein the differences from the strain used in the step (1) and the primer names are as follows: coli strain containing glcB knockout trait was changed to JW2943, and aceB was changed to glcB in the name of the corresponding primer.

(4) Knock-out of malate dehydrogenase (requiring NAD) gene maeA.

Starting from recombinant strain GC03, using the knockout method in the step (1) to knock out the gene maeA of malate dehydrogenase (NAD is required) to obtain Escherichia coli GC04. Wherein the differences from the strain used in the step (1) and the primer names are as follows: coli strain containing maeA knockout trait was changed to JW5238, and aceB was changed to maeA in the name of the corresponding primer.

(5) Knock-out of malate dehydrogenase gene maeB.

Starting from recombinant strain GC04, the malic acid dehydrogenase gene maeB is knocked out by using the knocking-out method in the step (1), and Escherichia coli GC05 is obtained. Wherein the difference of the strain and the primer name from the strain used in the step (1) is as follows: coli strain containing maeB knockout trait was changed to JW2447, and aceB was changed to maeB in the name of the corresponding primer.

4. Construction of genetically engineered bacteria

1) Construction of recombinant E.coli GC06.

Preparing strain GC05 into competent cells, and adding CaCl to plasmid pLB1k-cgAT (Cqu) ₂ GC05 was transformed, spread on LB plates containing 50. Mu.g/mL of kanamycin, cultured overnight at 37 ℃ and selected to obtain a clone designated as GC06.

2) Construction of recombinant E.coli GC07.

Strain GC06 was used to prepare competent cells, plasmid pSB1a-mtk-mcl-aceAK was applied with CaCl ₂ GC06 was transformed, plated on LB plates containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin, and cultured overnight at 37 ℃ to select a clone designated as GC07.

3) Construction of recombinant E.coli GC00.

The strain GC05 was used to prepare competent cells, and the plasmids pLB1K and pSB1a were ligated with CaCl ₂ GC05 was converted. The resulting suspension was plated on LB plates containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin, and cultured overnight at 37 ℃ to select a clone designated GC00.

The primers used in the above examples are shown in Table 1, and the host bacteria and their properties in the above examples are shown in Table 2.

TABLE 1 primer sequence List

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TABLE 2 host bacteria and their trait List

Note: * : the construction process is shown in Piao XY, wang L, lin BX, chen H, liu WF, tao Y. Metabolic engineering of Escherichia coli for the production of L-aspartic and its derivative beta-alkane with high specificity. 54:244-254.

The invention also provides a method for synthesizing glycine by using the engineering strain and taking glucose as a raw material. The method can use glucose as raw material to ferment and convert in industrial production, and simultaneously the introduction of the carbon fixation path and the reverse glyoxylate path leads the glucose to have higher conversion rate and leads the yield of the glycine to be improved.

Example 2 glycine was produced from glucose using recombinant escherichia coli strains GC00 and GC07.

The recombinant Escherichia coli takes glucose as a raw material, synthesizes glycine through glycolysis, a glyoxylate path and a reverse glyoxylate path in sequence, has a step of 'carbon fixation' in the middle, has the conversion rate as high as 1.67g/g, and has a metabolic path shown in figure 1. The general reaction formula is as follows: 1Glc +2CO ₂ +4NADPH+2ATP→2NADH+2FADH+4Glycine

The specific production steps are as follows:

(1) Culture of cells and Induction of enzymes

The engineered strains GC00 and GC07 cultured overnight were inoculated in an amount of 1% into shake flasks containing 50mL of ZYM self-induction medium, arabinose was added to a final concentration of 0.2%, kanamycin and ampicillin were further added to the resulting final concentrations of 50. Mu.g/mL and 100. Mu.g/mL, respectively, and O.D was measured by induction culture at 30 ℃ for 16 hours. ₆₀₀ Light absorption value, and 1.5X 10 was collected ¹¹ Cells according to O.D. ₆₀₀ The bacteria concentration is 1 × 10 when =1 ⁹ Calculating the volume of the solution per mL.

(2) Whole cell catalyzed transformation

Collecting the above 1.5X 10 ¹¹ Centrifuging thallus at 4 deg.C and 5000rpm for 10min, collecting precipitate, washing with 0.85% normal saline twice, discarding supernatant, re-suspending thallus with 5mL of transformation solution, transferring to test tube to final bacteria concentration of 3 × 10 ¹⁰ and/mL, converting the whole cells into glycine by a whole cell conversion system containing 50mM of glucose and 1 XPBS at the final concentration at 37 ℃ and 200rpm, and sampling and testing after converting for 4h, 6h and 8h respectively.

(3) Glycine detection by high performance liquid chromatography

The sample was centrifuged at 13000rpm for 10min at 4 ℃, the supernatant was taken, amino acid derivatization was performed using 2, 4-Dinitrofluorobenzene (DNFB), 100. Mu.L of the centrifuged supernatant was aspirated, and 0.5M NaHCO was added ₃ 1% of DNFB and acetonitrile, 100. Mu.L of each of the DNFB and the acetonitrile was mixed, subjected to derivatization treatment in a water bath at 60 ℃ for 1 hour under a dark condition, and 700. Mu.L of 0.01M KH was added thereto after the derivatization reaction was completed ₂ PO ₄ (pH 7.0), the samples were filtered through a 0.22 μm microporous membrane and subjected to HPLC amino acid detection, and the experimental setup was repeated three times, and the results were averaged. The high performance liquid chromatography detection instrument and the detection conditions are as follows:

qualitative analysis of HPLC amino acids: the instrument used Shimadzu LC-20AT 220V (SHIMADZU, japan), the column used Agilent extended-C18 (4.6 mm. Times.250mm, 5 μ M), the mobile phase was A, pH7.0, 0.01M KH ₂ PO ₄ (ii) a B, an acetonitrile-methanol-water mixed solution (volume ratio of 9; the mobile phase was filtered using a 0.45 μm aqueous/organic membrane and degassed by ultrasound for 20min until no significant bubbles were observed. The flow rate is 1mL/min, the column temperature is 40 ℃, the sample injection amount is 1 mu L, the detection wavelength of the PDA is 360nm, the reference wavelength is 600nm, and the data acquisition time is 35min. Gradient elution, procedure (B pump): 1-2.5min,12 percent; 2.5-13min,16-36%;13-28min,38-100%;28-35, 10%.

And (4) making a standard curve according to the peak appearance time and the peak area of the standard product, and calculating the content of the glycine in the sample according to the peak area of the sample. As a result, as shown in FIG. 2, after 4 hours of transformation, the GC00 strain did not accumulate glycine, and the glycine yield of GC07 was 0.35g/L.

(5) The recombinant escherichia coli strain takes glucose as a raw material to produce glycine, and the culture medium is as follows:

a) LB medium formula:

yeast powder: 5g/L

Peptone: 10g/L

NaCl：10g/L

b) ZYM self-induction culture medium formula:

100mL A +2mL B +200 μ L D +100 μ L E (all following are mass percentage concentrations);

a, ZY:1% tryptone, 0.5% yeast powder;

B.50×M：1.25M Na ₂ HPO ₄ ，1.25M KH ₂ PO4，2.5M NH ₄ cl and 0.25M Na ₂ SO ₄ ；

C.50 × 5052:25% glycerol, 2.5% glucose, 10% l-arabinose;

D.500×MgSO ₄ ：1M MgSO ₄

e.1000 × microelements: 50mM FeCl ₃ ，20mM CaCl ₂ ，10mM MnCl ₂ ，10mM ZnSO ₄ ，CoCl ₂ 、NiCl ₂ 、Na ₂ Mo ₄ 、Na ₂ SeO ₃ And H ₃ BO ₃ 2mM each.

c) The formula of the conversion solution is as follows:

glucose 50mM, NH ₄ HCO ₃ 100mM、KH ₂ PO ₄ /K ₂ HPO ₄ buffer 100mM。

Sequence listing

<110> Nanjing Shengde Biotechnology research institute Co., ltd

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gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540

gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600

ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660

cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720

agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780

gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840

ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900

ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960

ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020

attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080

ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140

tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200

ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260

ctactgtttc tccatacccg ttttttgggc taacaggagg aattaaccat gggtacctct 1320

catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagcctcga gggtagatct 1380

ggtactagtg gtgaattcgg tgagctcggt ctgcagctgg tgccgcgcgg cagccaccac 1440

caccaccacc actaatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1500

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1560

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1620

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtcgacgcga gcggtatcag 1680

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 1740

tccatgtcag ccgttaagtg ttcctgtgtc actgaaaatt gctttgagag gctctaaggg 1800

cttctcagtg cgttacatcc ctggcttgtt gtccacaacc gttaaacctt aaaagcttta 1860

aaagccttat atattctttt ttttcttata aaacttaaaa ccttagaggc tatttaagtt 1920

gctgatttat attaatttta ttgttcaaac atgagagctt agtacgtgaa acatgagagc 1980

ttagtacgtt agccatgaga gcttagtacg ttagccatga gggtttagtt cgttaaacat 2040

gagagcttag tacgttaaac atgagagctt agtacgtgaa acatgagagc ttagtacgta 2100

ctatcaacag gttgaactgc ggatcttgct gacgctcagt ggaacgaaaa ctcacgttaa 2160

gggattttgg gcggccgccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 2220

ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagc 2280

catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat 2340

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 2400

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 2460

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 2520

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 2580

gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 2640

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 2700

agcgaccgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 2760

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 2820

cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 2880

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 2940

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 3000

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 3060

tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga 3120

atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccact 3180

tgcggagacc cggtcgtcag cttgtcgtcg gttcagggca gggtcgttaa atagcgcatg 3240

c 3241

<210> 3

<211> 3061

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atgaacattc acgagtacca ggcgaaggaa ctgctgaaaa cctatggcgt gccggttccg 60

gatggtgcgg tggcgtacag cgatgcgcag gcggcgagcg ttgcggagga aattggtggc 120

agccgttggg tggttaaggc gcaaattcat gcgggtggcc gtggcaaggc gggtggcgtg 180

aaagttgcgc acagcatcga ggaagtgcgt cagtatgcgg acgcgatgct gggcagccac 240

ctggttaccc atcaaaccgg tccgggtggc agcctggtgc agcgtctgtg ggttgagcaa 300

gcgagccaca ttaagaaaga atactatctg ggcttcgtga tcgatcgtgg taaccaacgt 360

atcaccctga ttgcgagcag cgagggtggc atggaaatcg aggaagtggc gaaggagacc 420

ccggaaaaga ttgttaaaga ggtggttgac ccggcgatcg gcctgctgga ttttcagtgc 480

cgtaaagtgg cgaccgcgat tggcctgaag ggtaaactga tgccgcaagc ggttcgtctg 540

atgaaggcga tctaccgttg catgcgtgac aaagatgcgc tgcaagcgga gattaacccg 600

ctggcgatcg tgggcgagag cgacgaaagc ctgatggttc tggatgcgaa gttcaacttt 660

gacgataacg cgctgtaccg tcaacgtacc attaccgaaa tgcgtgacct ggcggaggaa 720

gatccgaaag aggtggaagc gagcggccac ggtctgaact atattgcgct ggacggcaac 780

atcggttgca ttgttaacgg tgcgggtctg gcgatggcga gcctggacgc gatcaccctg 840

cacggtggcc gtccggcgaa cttcctggat gtgggtggcg gtgcgagccc ggagaaggtg 900

accaacgcgt gccgtatcgt tctggaagac ccgaacgtgc gttgcatcct ggttaacatt 960

tttgcgggca tcaaccgttg cgactggatt gcgaagggtc tgatccaggc gtgcgatagc 1020

ctgcaaatta aagtgccgct gatcgttcgt ctggcgggca ccaacgttga tgagggtcgt 1080

aaaattctgg cggaaagcgg cctgagcttc atcaccgcgg aaaacctgga cgatgctgcg 1140

gcgaaggcgg tggcgatcgt taaaggttaa cagtcaggag atataatgag cgtgttcgtt 1200

aacaagcaca gcaaagttat cttccagggt tttaccggcg agcacgcgac ctttcacgcg 1260

aaggacgcga tgcgtatggg cacccgtgtg gttggtggcg tgaccccggg taaaggtggc 1320

acccgtcacc cggacccgga gctggcgcac ctgccggtgt tcgataccgt tgcggaagcg 1380

gtggcggcga ccggtgcgga tgttagcgcg gtgtttgtgc cgccgccgtt taacgcggat 1440

gcgctgatgg aggcgatcga tgcgggtatt cgtgtggcgg ttaccatcgc ggacggcatt 1500

ccggttcacg atatgatccg tctgcaacgt taccgtgttg gcaaggacag catcgtgatt 1560

ggtccgaaca ccccgggcat cattaccccg ggtgaatgca aagtgggcat catgccgagc 1620

cacatttaca agaaaggtaa cgttggcatc gttagccgta gcggcaccct gaactatgag 1680

gcgaccgaac agatggcggc gctgggtctg ggcattacca ccagcgttgg tatcggtggc 1740

gacccgatta acggcaccga tttcgtgacc gttctgcgtg cgtttgaggc ggacccggag 1800

accgaaatcg tggttatgat cggcgagatt ggtggcccgc aagaagtggc tgcggcgcgt 1860

tgggcgaagg aaaacatgac caaaccggtt attggttttg tggcgggtct ggcggcgccg 1920

accggccgtc gtatgggtca cgcgggcgcg atcattagca gcgaggcgga caccgcgggt 1980

gcgaagatgg atgcgatgga agcgctgggc ctgtatgttg cgcgtaaccc ggcgcagatc 2040

ggtcaaaccg tgctgcgtgc ggcgcaggaa catggcattc gtttttaaag aggagaaagg 2100

taccatgagc tttcgtctgc aaccggctcc gccggcgcgt ccgaaccgtt gccaactgtt 2160

tggtccgggc agccgtccgg cgctgtttga gaaaatggcg gcgagcgcgg cggacgtgat 2220

caacctggac ctggaagata gcgttgcgcc ggatgataaa gcgcaggcgc gtgcgaacat 2280

cattgaggcg atcaacggtc tggactgggg ccgtaagtac ctgagcgtgc gtattaacgg 2340

tctggatacc ccgttttggt atcgtgacgt ggttgatctg ctggagcagg cgggtgaccg 2400

tctggatcaa atcatgattc cgaaagtggg ttgcgcggcg gacgtgtatg cggttgatgc 2460

gctggttacc gcgatcgaac gtgcgaaggg tcgtaccaaa ccgctgagct tcgaggtgat 2520

cattgaaagc gcggcgggca tcgcgcacgt tgaggaaatt gcggcgagca gcccgcgtct 2580

gcaagcgatg agcctgggtg cggcggattt tgcggcgagc atgggtatgc agaccaccgg 2640

cattggtggc acccaagaga actactatat gctgcacgac ggccagaaac actggagcga 2700

tccgtggcac tgggcgcaag cggcgattgt ggcggcgtgc cgtacccacg gtattctgcc 2760

ggttgacggt ccgttcggcg attttagcga cgatgaaggt tttcgtgcgc aggcgcgtcg 2820

tagcgcgacc ctgggtatgg tgggcaagtg ggcgatccac ccgaaacaag tggcgctggc 2880

gaacgaggtt tttaccccga gcgaaaccgc ggttaccgag gcgcgtgaaa ttctggcggc 2940

gatggacgcg gcgaaggcgc gtggtgaagg tgcgaccgtg tataaaggtc gtctggttga 3000

tatcgcgagc attaagcagg cggaagtgat cgttcgtcaa gcggaaatga ttagcgcgta 3060

a 3061

<210> 4

<211> 4792

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc 60

cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt 120

cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 180

agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 240

tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 300

taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 360

gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 420

cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 480

gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540

gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600

ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660

cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720

agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780

gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840

ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900

ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960

ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020

attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080

ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140

tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200

ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260

ctactgtttc tccatacccg ttttttgggc taacaggagg aattaaccat gggtacctct 1320

catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagcctcga gggtagatct 1380

ggtactagtg gtgaattcgg tgagctcggt ctgcagctgg tgccgcgcgg cagccaccac 1440

caccaccacc actaatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1500

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1560

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1620

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtcgaccaga cccgccataa 1680

aacgccctga gaagcccgtg acgggctttt cttgtattat gggtagtttc cttgcatgaa 1740

tccataaaag gcgcctgtag tgccatttac ccccattcac tgccagagcc gtgagcgcag 1800

cgaactgaat gtcacgaaaa agacagcgac tcaggtgcct gatggtcgga gacaaaagga 1860

atattcagcg atttgcccga gcttgcgagg gtgctactta agcctttagg gttttaaggt 1920

ctgttttgta gaggagcaaa cagcgtttgc gacatccttt tgtaatactg cggaactgac 1980

taaagtagtg agttatacac agggctggga tctattcttt ttatcttttt ttattctttc 2040

tttattctat aaattataac cacttgaata taaacaaaaa aaacacacaa aggtctagcg 2100

gaatttacag agggtctagc agaatttaca agttttccag caaaggtcta gcagaattta 2160

cagataccca caactcaaag gaaaaggtct agtaattatc attgactagc ccatctcaat 2220

tggtatagtg attaaaatca cctagaccaa ttgagatgta tgtctgaatt agttgttttc 2280

aaagcaaatg aactagcgat tagtcgctat gacttaacgg agcatgaaac caagctaatt 2340

ttatgctgtg tggcactact caaccccacg attgaaaacc ctacaaggaa agaacggacg 2400

gtatcgttca cttataacca atacgctcag atgatgaaca tcagtaggga aaatgcttat 2460

ggtgtattag ctaaagcaac cagagagctg atgacgagaa ctgtggaaat caggaatcct 2520

ttggttaaag gctttgagat tttccagtgg acaaactatg ccaagttctc aagcgaaaaa 2580

ttagaattag tttttagtga agagatattg ccttatcttt tccagttaaa aaaattcata 2640

aaatataatc tggaacatgt taagtctttt gaaaacaaat actctatgag gatttatgag 2700

tggttattaa aagaactaac acaaaagaaa actcacaagg caaatataga gattagcctt 2760

gatgaattta agttcatgtt aatgcttgaa aataactacc atgagtttaa aaggcttaac 2820

caatgggttt tgaaaccaat aagtaaagat ttaaacactt acagcaatat gaaattggtg 2880

gttgataagc gaggccgccc gactgatacg ttgattttcc aagttgaact agatagacaa 2940

atggatctcg taaccgaact tgagaacaac cagataaaaa tgaatggtga caaaatacca 3000

acaaccatta catcagattc ctacctacgt aacggactaa gaaaaacact acacgatgct 3060

ttaactgcaa aaattcagct caccagtttt gaggcaaaat ttttgagtga catgcaaagt 3120

aagcatgatc tcaatggttc gttctcatgg ctcacgcaaa aacaacgaac cacactagag 3180

aacatactgg ctaaatacgg aaggatctga ggttcttatg gctcttgtat ctatcagtga 3240

agcatcaaga ctaacaaaca aaagtagaac aactgttcac cgttagatat caaagggaaa 3300

actgtcgata tgcacagatg aaaacggtgt aaaaaagata gatacatcag agcttttacg 3360

agtttttggt gcatttaaag ctgttcacca tgaacagatc gacaatgtaa cagatgaaca 3420

gcatgtaaca cctaatagaa caggtgaaac cagtaaaaca aagcaactag aacatgaaat 3480

tgaacacctg agacaacttg ttacagctca acagtcacac atagacagcc tgaaacaggc 3540

gatgctgctt atcgaatcaa agctgccgac aacacgggag ccagtgacgc ctcccgtggg 3600

gaaaaaatca tggcaattct ggaagaaata gcgctttcag ccggcaaacc tgaagccgga 3660

tctgcgattc tgataacaaa ctagcaacac cagaacagcc cgtttgcggg cagcaaaacc 3720

cgcggccgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 3780

atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 3840

caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 3900

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 3960

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4020

tttccaatga tgagcacttt taaagttctg ctatgtgata cactattatc ccgtattgac 4080

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4140

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4200

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4260

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4320

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4380

atgccaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4440

caattaatag actgaatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4500

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4560

attgcagcac tggggccaga tggtaagcgc tcccgtatcg tagttatcta caccacgggg 4620

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 4680

aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4740

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatcgcat gc 4792

<210> 5

<211> 3224

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atgaaaaccc gtacacaaca aattgaagaa ttacagaaag agtggactca accgcgttgg 60

gaaggcatta ctcgcccata cagtgcggaa gatgtggtga aattacgcgg ttcagtcaat 120

cctgaatgca cgctggcgca actgggcgca gcgaaaatgt ggcgtctgct gcacggtgag 180

tcgaaaaaag gctacatcaa cagcctcggc gcactgactg gcggtcaggc gctgcaacag 240

gcgaaagcgg gtattgaagc agtctatctg tcgggatggc aggtagcggc ggacgctaac 300

ctggcggcca gcatgtatcc ggatcagtcg ctctatccgg caaactcggt gccagctgtg 360

gtggagcgga tcaacaacac cttccgtcgt gccgatcaga tccaatggtc cgcgggcatt 420

gagccgggcg atccgcgcta tgtcgattac ttcctgccga tcgttgccga tgcggaagcc 480

ggttttggcg gtgtcctgaa tgcctttgaa ctgatgaaag cgatgattga agccggtgca 540

gcggcagttc acttcgaaga tcagctggcg tcagtgaaga aatgcggtca catgggcggc 600

aaagttttag tgccaactca ggaagctatt cagaaactgg tcgcggcgcg tctggcagct 660

gacgtgacgg gcgttccaac cctgctggtt gcccgtaccg atgctgatgc ggcggatctg 720

atcacctccg attgcgaccc gtatgacagc gaatttatta ccggcgagcg taccagtgaa 780

ggcttcttcc gtactcatgc gggcattgag caagcgatca gccgtggcct ggcgtatgcg 840

ccatatgctg acctggtctg gtgtgaaacc tccacgccgg atctggaact ggcgcgtcgc 900

tttgcacaag ctatccacgc gaaatatccg ggcaaactgc tggcttataa ctgctcgccg 960

tcgttcaact ggcagaaaaa cctcgacgac aaaactattg ccagcttcca gcagcagctg 1020

tcggatatgg gctacaagtt ccagttcatc accctggcag gtatccacag catgtggttc 1080

aacatgtttg acctggcaaa cgcctatgcc cagggcgagg gtatgaagca ctacgttgag 1140

aaagtgcagc agccggaatt tgccgccgcg aaagatggct ataccttcgt atctcaccag 1200

caggaagtgg gtacaggtta cttcgataaa gtgacgacta ttattcaggg cggcacgtct 1260

tcagtcaccg cgctgaccgg ctccactgaa gaatcgcagt tctaagcaac aacaaccgtt 1320

gctgactgta ggccggataa ggcgttcacg ccgcatccgg caatcggtgc acgatgcctg 1380

atgcgacgct tgcgcgtctt atcatgccta cagccgttgc cgaacgtagg ctggataagg 1440

cgtttacgcc gcatccggca attctctgct cctgatgagg gcgctaaatg ccgcgtggcc 1500

tggaattatt gattgctcaa accattttgc aaggcttcga tgctcagtat ggtcgattcc 1560

tcgaagtgac ctccggtgcg cagcagcgtt tcgaacaggc cgactggcat gctgtccagc 1620

aggcgatgaa aaaccgtatc catctttacg atcatcacgt tggtctggtc gtggagcaac 1680

tgcgctgcat tactaacggc caaagtacgg acgcggcatt tttactacgt gttaaagagc 1740

attacacccg gctgttgccg gattacccgc gcttcgagat tgcggagagc ttttttaact 1800

ccgtgtactg tcggttattt gaccaccgct cgcttactcc cgagcggctt tttatcttta 1860

gctctcagcc agagcgccgc tttcgtacca ttccccgccc gctggcgaaa gactttcacc 1920

ccgatcacgg ctgggaatct ctactgatgc gcgttatcag cgacctaccg ctgcgcctgc 1980

gctggcagaa taaaagccgt gacatccatt acattattcg ccatctgacg gaaacgctgg 2040

ggacagacaa cctcgcggaa agtcatttac aggtggcgaa cgaactgttt taccgcaata 2100

aagccgcctg gctggtaggc aaactgatca caccttccgg cacattgcca tttttgctgc 2160

cgatccacca gacggacgac ggcgagttat ttattgatac ctgcctgacg acgaccgccg 2220

aagcgagcat tgtttttggc tttgcgcgtt cttattttat ggtttatgcg ccgctgcccg 2280

cagcgctggt cgagtggcta cgggaaattc tgccaggtaa aaccaccgct gaattgtata 2340

tggctatcgg ctgccagaag cacgccaaaa ccgaaagcta ccgcgaatat ctcgtttatc 2400

tacagggctg taatgagcag ttcattgaag cgccgggtat tcgtggaatg gtgatgttgg 2460

tgtttacgct gccgggcttt gatcgggtat tcaaagtcat caaagacagg ttcgcgccgc 2520

agaaagagat gtctgccgct cacgttcgtg cctgctatca actggtgaaa gagcacgatc 2580

gcgtgggccg aatggcggac acccaggagt ttgaaaactt tgtgctggag aagcggcata 2640

tttccccggc attaatggaa ttactgcttc aggaagcagc ggaaaaaatc accgatctcg 2700

gcgaacaaat tgtgattcgc catctttata ttgagcggcg gatggtgccg ctcaatatct 2760

ggctggaaca agtggaaggt cagcagttgc gcgacgccat tgaagaatac ggtaacgcta 2820

ttcgccagct tgccgctgct aacattttcc ctggcgacat gctgtttaaa aacttcggtg 2880

tcacccgtca cgggcgtgtg gttttttatg attacgatga aatttgctac atgacggaag 2940

tgaatttccg cgacatcccg ccgccgcgct atccggaaga cgaacttgcc agcgaaccgt 3000

ggtacagcgt ctcgccgggc gatgttttcc cggaagagtt tcgccactgg ctatgcgccg 3060

acccgcgtat tggtccgctg tttgaagaga tgcacgccga cctgttccgc gctgattact 3120

ggcgcgcact acaaaaccgc atacgtgaag ggcatgtgga agatgtttat gcgtatcggc 3180

gcaggcaaag atttagcgta cggtatgggg agatgctttt ttga 3224

Claims (5)

1. An escherichia coli recombinant bacterium, wherein the strain comprises the following modifications:

1) Introducing exogenous glutamic glyoxylate aminotransferase gene cgAT;

2) Introducing exogenous malate thiokinase gene mtk and exogenous malyl-CoA lyase gene mcl;

3) Introducing a glyoxylate pathway aceK gene, namely a gene of isocitrate dehydrogenase kinase/phosphatase;

4) Knocking out aceB gene, namely gene of malate synthase A;

5) Knocking out an iclR gene, namely a gene of a transcription inhibitor of a glyoxylate pathway;

6) Knock-out of the glcB gene, the gene for the protein malate synthase G;

7) Knocking out maeA gene, namely a gene of malate dehydrogenase;

8) The maeB gene, namely the gene of malate dehydrogenase, is knocked out.

2. A method for constructing the recombinant Escherichia coli strain of claim 1, comprising the steps of:

1) Constructing two plasmids, wherein one plasmid is pLB1k-cgAT of the glutamate glyoxylate aminotransferase II gene cgAT, and the other plasmid is a recombinant plasmid pSB1a-mtk-mcl-aceAK of malate thiokinase, malyl-CoA lyase isocitrate lyase and isocitrate dehydrogenase kinase;

2) Constructing a host bacterium, and knocking out malate synthase A gene aceB of a recipient bacterium XY24 to form a recombinant bacterium GC01; knocking out a glyoxylate pathway transcription inhibitor gene iclR of the recombinant strain GC01 to form a recombinant strain GC02; knocking out a malic acid synthase G gene glcB of the recombinant strain GC02 to form a recombinant strain GC03; knocking out a malic acid dehydrogenase gene maeA of the recombinant strain GC03 to form a recombinant strain GC04; knocking out a malic acid dehydrogenase gene maeB of the recombinant strain GC04 to form a recombinant strain GC05;

3) Preparing competent cells from recombinant strain GC05, and introducing the plasmid pLB1k-cgAT in the step 1) into GC05 to form recombinant strain GC06; preparing competent cells from a strain GC06, and introducing pSB1a-mtk-mcl-aceAK obtained in the step 1) into the strain GC06 to construct an Escherichia coli recombinant strain GC07 for synthesizing glycine by using glucose.

3. The method for constructing a recombinant Escherichia coli according to claim 2, wherein: the plasmid pLB1k-cgAT of the glutamate glyoxylate aminotransferase II gene cgAT is derived from quinoa Chenopodium quinoa; the malate thiokinase gene mtk is derived from Methylococcus capsulatus, and the malyl-CoA lyase gene mcl is derived from Rhodobacter sphaeroides; the recombinant plasmid pSB1a-mtk-mcl-aceAK of isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase is derived from Escherichia coli.

4. The method for constructing a recombinant Escherichia coli according to claim 2, wherein: the competent cell transformation mainly adopts a calcium chloride method.

5. A method for preparing glycine from glucose by using the recombinant Escherichia coli of any one of claims 1 to 4, comprising the steps of:

1) Culturing of the cells and induction of enzymes: the recombinant Escherichia coli GC07 strain was transferred from the glycerol stock to 5mL of LB medium at 37 ℃ and 220 rpm overnight and transferred to the medium containing50mL of ZYM was cultured in an auto-induction medium, kanamycin and ampicillin were added to the medium so that the final concentrations were 50. Mu.g/mL and 100. Mu.g/mL, respectively, and after 16 hours of induction culture at 30 ℃ O.D was measured. ₆₀₀ Light absorption value, and 1.5X 10 was collected ¹¹ Thalli;

2) Whole-cell catalytic conversion: centrifuging the collected thallus at 4 deg.C and 5000rpm for 10min, washing with 0.85% normal saline twice, discarding supernatant, resuspending thallus with 5mL of transformation solution, transferring to test tube to make final bacteria concentration be 3 × 10 ¹⁰ Glycine was synthesized by whole-cell transformation at 37 ℃ and 200rpm in a whole-cell transformation system containing glucose and 1 XPBS at a final concentration of 50 mM.

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