CN116837015A - Genetically engineered bacterium for high-yield of L-cysteine as well as construction method and application thereof - Google Patents

Genetically engineered bacterium for high-yield of L-cysteine as well as construction method and application thereof Download PDF

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CN116837015A
CN116837015A CN202310748566.1A CN202310748566A CN116837015A CN 116837015 A CN116837015 A CN 116837015A CN 202310748566 A CN202310748566 A CN 202310748566A CN 116837015 A CN116837015 A CN 116837015A
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cyse
nrdh
cysteine
linker
gbd
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柳志强
陈立峰
张博
修晓玲
郑裕国
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Zhejiang University of Technology ZJUT
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Abstract

The invention relates to the technical field of microbial metabolism engineering, in particular to a genetically engineered bacterium for high-yield L-cysteine, a construction method and application thereof. The bacterial strain E.coli W3110EYC is hflC-cysM is ybbK-nrdH is hflK-cysE is chassis bacteria, key enzymes cysM, nrdH and cysE in the L-cysteine synthesis pathway are positioned on cysteine outer transport protein ydeD by using CRISPR-Cas9 gene editing technology, and the accumulation of important intermediate O-acetylserine of L-cysteine carbon metabolism is promoted by using the advantage of a compartment structure, the synthesis pathway of L-cysteine thiosulfate of escherichia coli is enhanced, the transport efficiency is improved, and the escherichia coli genetic engineering bacterial strain capable of producing L-cysteine at high yield is obtained.

Description

Genetically engineered bacterium for high-yield of L-cysteine as well as construction method and application thereof
Technical Field
The invention relates to the technical field of microbial metabolism engineering, in particular to a genetically engineered bacterium for high-yield L-cysteine, a construction method and application thereof.
Background
L-cysteine is one of the essential amino acids of great importance in organisms. L-cysteine is the only amino acid with an active thiol group compared to other essential amino acids, a property which makes L-cysteine closely related to many physiological processes in the organism. L-cysteine is also a common industrial raw material and has important applications in many industries, for example in the food industry, L-cysteine is often an excellent food flavor and primary flour treatment agent; in the pharmaceutical industry, L-cysteine can also be used for the treatment of radiopharmaceuticals poisoning, hepatitis, serosis, leukopenia and other diseases, and for the treatment and prevention of radiation-induced injury; in the cosmetic industry, the thiol in the L-cysteine structure has reducibility, can be used for regulating the generation of melanin, and has the effect of whitening skin.
With the continuous development of biotechnology, microbial fermentation methods have become a very promising production technology. At present, various amino acids have been industrially produced by microbial fermentation, such as lysine, threonine, etc., all over the world. In most bacteria and plants, the precursor of cysteine is L-serine, which is 3-phosphoglycerate from the glycolytic pathway, and the L-serine undergoes an acetylation reaction under the action of acetyl-CoA to give the key intermediate O-acetylserine, which can accept sulfur sources from different sulfur assimilation pathways to produce L-cysteine.
The microbial fermentation method is a promising industrial production method, has the advantages of environmental friendliness, high economic benefit and the like, but the microbial fermentation production of the L-cysteine still has a plurality of problems at present. Methods for microbial synthesis of excess L-cysteine are limited by many factors, such as the low level of accumulation of original L-cysteine in wild-type E.coli and the strong deleterious effects of excess L-cysteine accumulation on the cells themselves. Thus, the production of L-cysteine by microbial fermentation has a general problem of low yield. This may be related to the growth rate of the microorganism itself, metabolic pathways, environmental factors during fermentation, etc.
The L-cysteine production strain is modified by biotechnology such as genetic engineering, metabolic engineering and the like, the metabolic pathway of the strain can be regulated on the molecular level, the synthesis and accumulation of the cysteine are promoted, the toxicity of the L-cysteine to cells is reduced, the flux of the L-cysteine metabolic pathway is improved, and the method is an effective strategy for obtaining the L-cysteine high-yield strain. However, there are many uncertainty factors in achieving effective control of yield and quality by modifying the genome of the strain, regulating the expression of key enzymes, etc., and continuous attempts and improvements are required to achieve optimization and improvement of the production efficiency of L-cysteine. Therefore, constructing the genetic engineering bacteria capable of realizing high-yield L-cysteine is a research content worthy of continuous exploration.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a genetically engineered bacterium for producing L-cysteine at high yield and a construction method thereof, and is applied to fermentation production of L-cysteine so as to solve the problem that the metabolic pathway and transport channel of L-cysteine of an L-cysteine production strain in the prior art are unfavorable for synthesis and transport of L-cysteine, and the yield of the strain is lower in the fermentation production process of L-cysteine.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
the first object of the present invention is to provide a method for constructing a genetically engineered bacterium for producing L-cysteine at a high yield, comprising the steps of:
(1) Construction of plasmid pTrc99a-cysE Using pTrc99a plasmid as template f -ydeD-GBD-SH3-PDZ；
(2) The strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE was used as the chassis strain, and the base was usedThe nrdH gene on the genome is replaced by fusion protein gene nrdH-linker-GBD lig to obtain engineering bacterium E.coil W3110EYC:: hfl C-cysM:: ybbK-nrdH::: hfl K-cysE:: nrdH-linker-GBD lig, and the plasmid pTrc99a-cysE is obtained f -ydeD-GBD-SH3-PDZ is introduced into an engineering bacterium, thereby obtaining the genetically engineered bacterium for high-yield of L-cysteine.
Preferably, the plasmid pTrc99a-cysE f The sequence of the-ydeD-GBD-SH 3-PDZ is shown as SEQ ID NO.10, and the sequence of the fusion protein gene nrdH-linker-GBD lig is shown as SEQ ID NO. 1.
The second object of the present invention is to provide a method for constructing a genetically engineered bacterium for producing L-cysteine at a high yield, comprising the steps of:
(1) Construction of plasmid pTrc99a-cysE Using pTrc99a plasmid as vector f -ydeD-GBD-SH3-PDZ；
(2) The strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE is used as a chassis strain, nrdH gene on the genome is replaced by fusion protein gene nrdH-linker-GBD lig, cysE gene is replaced by fusion protein gene cysE-linker-PDZ lig, engineering bacterium E.coil W3110EYC:: hflC-cysM::: ybbK-nrdH::: hflK-cysE:::: nrdH-linker-GBD lig:: cysE-linker-PDZ lig is obtained, and the plasmid pTrc99a-cysE is obtained f The ydeD-GBD-SH3-PDZ is imported into engineering bacteria E.coil W3110EYC:: hflC-cysM::: ybbK-nrdH::: hflK-cysE:::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig, thereby obtaining the genetic engineering bacteria of the high-yield L-cysteine.
Preferably, the plasmid pTrc99a-cysE f The sequence of the-ydeD-GBD-SH 3-PDZ is shown as SEQ ID NO.10, and the sequence of the fusion protein gene cysE-linker-PDZ lig is shown as SEQ ID NO. 2.
The third object of the present invention is to provide a method for constructing a genetically engineered bacterium for producing L-cysteine at high yield, comprising the steps of:
(1) Construction of plasmid pTrc99a-cysE Using pTrc99a plasmid as vector f -ydeD-GBD-SH3-PDZ；
(2) The strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE was used as the chassis strain, and the nrdH gene on its genome was replaced with the fusion protein gene nrdH-linker-GBThe expression D lig, cysE gene replaced with fusion protein gene cysE-linker-PDZ lig, cysM gene replaced with fusion protein gene cysM-linker-SH3lig, engineering bacterium E.coil W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE::: nrdH-linker-GBD lig:: cysE-linker-PDZ lig:: cysM-linker-SH3lig, and plasmid pTrc99a-cysE were obtained f The ydeD-GBD-SH3-PDZ is introduced into engineering bacteria E.coil W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig:: cysM-li linker-SH3lig, thereby obtaining the genetically engineered bacteria of high-yield L-cysteine.
Preferably, the plasmid pTrc99a-cysE f The sequence of the-ydeD-GBD-SH 3-PDZ is shown as SEQ ID NO.10, and the sequence of the fusion protein gene cysM-linker-SH3lig is shown as SEQ ID NO. 3.
Eukaryotic cells often utilize a number of organelles that are surrounded by a single or double layer of phospholipids, and such cellular compartments formed by the action of membranes may facilitate better cell performance of different metabolic activities. Further formation of the compartmental structure by assembly with a spatial scaffold is one of the common ways to improve cellular metabolism. The free enzyme in the cell can be assembled with the space bracket to form a compartment structure similar to a membraneless cell device, and the compartment structure can better control the metabolism and improve the conversion efficiency of metabolic pathways.
Common spatial scaffolds include protein scaffolds, RNA scaffolds, DNA scaffolds. By combining the characteristics of the space bracket structure, the efflux protein is used as a space bracket to be assembled with key enzymes in the L-cysteine synthesis path, the key enzymes are co-located on the efflux protein, and simultaneously, the expression of an output pump of the L-cysteine is improved, so that a complete L-cysteine synthesized compartment structure can be formed. The efflux protein-enzyme complex can fully exert the spatial advantage of a compartment structure, accelerate the aggregation of L-cysteine near the efflux protein, improve the transportation efficiency of the L-cysteine to the outside of cells, effectively improve the anabolic flux of the L-cysteine and reduce the toxicity of the accumulation of toxic compounds to the cells.
The chassis strain E.coli W3110EYC of the invention is hflC-cysM of the invention is ybbK-nrdH of the invention is disclosed in patent 202210078039.X, and cysM, nrdH, cysE is co-located on L-cysteine efflux protein ydeD to form a compartment structure, strengthen the synthesis pathway of L-cysteine thiosulfate of the escherichia coli, strengthen the sulfur source assimilation capability of the escherichia coli and the flux of the L-cysteine carbon metabolism pathway, promote the conversion of serine to O-acetylserine (OAS), exert the characteristics of the compartment structure, strengthen the flux of carbon flow and improve the supply of a carbon source; by co-locating cysteine production and efflux proteins, the capacity of cell production and outward transport of L-cysteine is improved, the advantage of a compartment structure is further enhanced, the L-cysteine transport efficiency is improved, the L-cysteine gathered near a cell membrane is transported out of the cell in time, and the toxic influence of excessive accumulation of the L-cysteine on the cell is reduced.
The fourth object of the present invention is to provide a genetically engineered bacterium which produces L-cysteine at a high yield, which is constructed by the above-described construction method.
The fifth object of the present invention is to provide the genetically engineered bacterium for high-yield L-cysteine constructed by the above construction method or the application of the genetically engineered bacterium for high-yield L-cysteine in microbial fermentation preparation of L-cysteine.
Preferably, the genetically engineered strain is inoculated into a fermentation medium, and fermentation culture is carried out at a temperature of between 32.5 and 37 ℃ and at a speed of between 200 and 800rpm, and OD is carried out 600 When the fermentation liquid is 10-30, adding IPTG with the final concentration of 0.1mM, continuously culturing for 48 hours, and taking the supernatant of the fermentation liquid after the fermentation is finished, and separating and purifying to obtain the L-cysteine.
Preferably, the composition of the fermentation medium is as follows: 25-50 g/L of dextrose monohydrate, (NH) 4 ) 2 SO 4 5～20g/L、KH 2 PO 4 0.5～2g/L、Na 2 S 2 O 3 5-20 g/L, yeast extract 1-10 g/L, na 2 HPO 4 0.5-2 g/L, peptone 0.5-2 g/L, trace element solution 0.5-2 ml/L, deionized water as solvent, and natural pH value.
The microelement solution comprises the following components: 5g/L MnSO 4 ·8H 2 O，0.7g/L CoCl 2 ·6H 2 O，5g/L ZnSO 4 ·7H 2 O，5g/L FeSO 4 ·7H 2 The O solvent is deionized water.
Further preferably, the fermentation medium is composed of: glucose monohydrate 46g/L, (NH) 4 ) 2 SO 4 9g/L、KH 2 PO 4 1g/L、Na 2 S 2 O 3 7g/L, yeast extract 8g/L, na 2 HPO 4 1g/L, 1g/L peptone, 1ml/L microelement solution, deionized water as solvent, and natural pH value.
Usually, before the genetically engineered bacteria ferment, the genetically engineered bacteria are inoculated into 10mL LB culture medium test tubes, cultured for 12 hours on a shaking table at 37 ℃ and a rotating speed of 180rpm, inoculated with 1mL of preculture into a 500mL shaking flask filled with 20mL of SM culture medium, cultured for 4 hours at 30 ℃ and 200rpm, added with IPTG with a final concentration of 0.1mM, and further cultured for 48 hours.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, an L-cysteine metabolism pathway and a transport channel of escherichia coli are modified, key enzymes cysM, nrdH and cysE in the L-cysteine synthesis pathway are positioned on cysteine outer transport protein ydeD by utilizing a CRISPR-Cas9 gene editing technology, the accumulation of an important intermediate product O-acetylserine of L-cysteine carbon metabolism is promoted by utilizing the advantage of a compartment structure, the escherichia coli L-cysteine thiosulfate synthesis pathway is strengthened, the transport efficiency is improved, and the escherichia coli genetic engineering strain with high yield of L-cysteine is obtained, wherein the yield of L-cysteine is increased from 1.43g/L to 2.63g/L.
Drawings
FIG. 1 is a schematic diagram of the modification site of the L-cysteine metabolic pathway of the present invention.
FIG. 2 shows the OD of the engineering bacteria constructed in example 2 600 And the content of L-cysteine in the supernatant of the fermentation broth.
FIG. 3 shows the OD of the engineering bacteria constructed in example 3 600 And the content of L-cysteine in the supernatant of the fermentation broth.
FIG. 4 shows the engineering bacteria constructed in example 4OD 600 And the content of L-cysteine in the supernatant of the fermentation broth.
Detailed Description
The invention is further described below with reference to the drawings and specific examples. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
The parent strain E.coli W3110EYC: : hflC-cysM: : ybbK-nrdH: : hflK-cysE is disclosed in patent 202210078039. X.
Strain e.coli W3110 is from the university of jerusalem CGSC collection (Coli Genetic Stock Center), 8 th month 5 th day of the collection date 1975, deposit number cgsc#4474, which is disclosed in patent US 2009/0298135a1, US2010/0248311 A1.
In an example, the final concentration of kanamycin in the medium is 0.05mg/L, the final concentration of spectinomycin in the medium is 0.05mg/L, and the final concentration of ampicillin in the medium is 0.10mg/L.
LB medium composition: 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride, and the solvent is deionized water, and the pH value is natural. LB plates were prepared by adding agar to LB liquid medium at a final concentration of 2 g/L.
The L-cysteine metabolic pathway modification site in the present invention is shown in FIG. 1.
TABLE 1 Gene editing-related genes and corresponding pathways
ydeD Export of L-cysteine
nrdH L-cysteine thiosulfate synthesis pathway
cysE L-cysteine carbon metabolic pathway
cysM L-cysteine thiosulfate synthesis pathway
TABLE 2 primer sequences
Example 1: determination of L-cysteine content
And (3) fermentation liquid treatment: 1mL of the bacterial liquid was centrifuged at 12000 Xg for 1min in a 2mL EP tube, and the supernatant and the pellet were separated. The supernatant was used for detection of L-cysteine and other metabolites.
Derivatization reaction system: 0.27g of CNBF was weighed and dissolved in 10mL of acetonitrile as solution I; the mother liquor is 0.2M boric acid solution and 0.05M borax solution, and the standard buffer solution with pH=9.0 is prepared by mixing 4:1 volumes and is named as solution II. The sample was diluted to a concentration of 0 to 5g/L, and mixed in a ratio of 100. Mu.L of the sample, 300. Mu.L of the I solution and 500. Mu.L of the II solution, and reacted at 600rpm for 1 hour in a constant temperature shaker. And filling the sample into a liquid phase bottle through a film to be tested.
Liquid phase detection: the instrument is a Siemens flight UPLC ultra-high pressure liquid chromatograph. The chromatographic column was a C18 column (4.6X105 mm,5 μm); the ultraviolet detector detects the wavelength of 260nm; the sample injection amount is 10 mu L; column temperature is 30 ℃; the flow rate is 0.8mL/min; the mobile phase used was AC biphasic, phase A neat acetonitrile, phase C50 mM HAc-NaAc buffer: acetonitrile=83: 17, ph=4.9. The gradient elution procedure is shown in table 3.
TABLE 3 gradient elution procedure
Sequence number Time (min) Phase A (%) C phase (%)
1 0 18 82
2 2.5 20 80
3 4 35 65
4 8 35 65
5 10 50 50
6 12 50 50
7 15 70 30
8 18 18 82
9 23 18 82
。
Example 2: construction of an effective strain E.coil W3110EYC: : hflC-cysM: : ybbK-nrdH: : hflK-cysE: : nrdH-linker-GBD lig and its fermentation with strain e.coil W3110EYC: : hflC-cysM: : ybbK-nrdH: : htlK-cysE was used as the starting strain, and the nrdH gene on the genome was replaced with the fusion protein gene nrdH-linker-GBD lig using CRISPR-Cas9 mediated gene editing technology (Yu Jiang et a1.2015Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied Environmental microbiology.81:2506-2514). The Linker nucleotide sequence of the fusion protein is shown as SEQ ID NO. 11.
(1) Construction of pTarget plasmid: a pTarget-nrdH plasmid was constructed capable of expressing sgRNA targeting the nrdH sequence of the gene of interest. PCR amplification was performed using pTarget F Plasmid (Addgene Plasmid # 62226) as template and pT-TB-nrdH-F and pT-TB-nrdH-R as primers. The PCR product was digested with Dpn I, the digested product was chemically transferred to E.coli DH 5. Alpha. And plated on spectinomycin plates, single colonies were picked and sequenced with the verification primer pT-YZ-F to verify the selection of the mutated pTarget-nrdH plasmid. The circular plasmid pTarget-nrdH was linearized using the primers pT-line-F and pT-line-R using the pTarget-nrdH plasmid as template.
(2) Construction of pTD plasmid: the E.coli W3110 genome is used as a template, the Donor-nrdH-up-F and the Donor-nrdH-up-R are used as primers for amplification to obtain the upstream homologous arm part of the nrdH, the Donor-nrdH-down-F and the Donor-nrdH-down-R are used as primers for amplification to obtain the downstream homologous arm part of the nrdH, the Donor-nrdH-F and the Donor-nrdH-R are used as primers for amplification to obtain the nrdH fragment of Donor-nrdH, the Donor-GBD lig-F and the Donor-GBD lig-R are used as primers for amplification to obtain the Donor GBD lig fragment of Donor GBD lig, TYTH-hrdH-F and TYTH-hrdH-R synonymous codon substitution targeting gene nrdH sequence. The PCR products were detected by 1.0% agarose gel electrophoresis and the PCR fragments were purified. The recovered DNA fragment was fused into a complete Donor fragment using fusion PCR, and the purified fragment was detected by 1.0% agarose gel electrophoresis and excised for recovery. The nucleotide sequence of the fusion protein gene nrdH-linker-GBD lig is shown as SEQ ID NO. 1. The linker sequence has been inserted between the fragments Donor nrdH and Donor GBD lig in this gene band. The linearized pTarget-nrdH plasmid was ligated with the Donor fragment, transformed into E.coli DH 5. Alpha. And plated onto spectinomycin plates, and single colonies were picked and sequenced using the verification primers pTD-YZ-F and pTD-YZ-F to verify the cloning of the successfully cloned pTD-nrdH plasmid according to the one-step cloning kit (Onestep clone kit, vazyme Biotech, nanjing, china).
(3) Electric transformation competence preparation: the pCas Plasmid (Addgene Plasmid # 62225) was introduced into E.coli W3110 EYC. See (Molecular Cloning: A Laboratory Manual,3ed edition, 99-102) for details. Selecting a monoclonal to a 10ml LB test tube containing 0.05mg/L kanamycin, and culturing overnight at 30 ℃; inoculating into 250mL shake flask containing 50mL LB medium at 1% by volume, and adding 500Mu.l of 1 mol/L-arabinose, 150rpm, 30℃to OD 600 0.4 to 0.6; cells were harvested by centrifugation at 4000rpm at 4℃for 10min to prepare electrotransformation competence, as described in detail in (Molecular Cloning: A Laboratory Manual,3ed edition, 99-102).
(4) Electric shock conversion: 150ng of pTD-nrdH plasmid is mixed with 200 μl of electrocompetent cells, transferred into a precooled 2mm electric shock cup, subjected to electric shock transformation by an electroporation device (MicroPluserTM, BIO-RAD) for about 1min, immediately added with 1mL of LB medium after electric shock is finished and immediately sucked out gently, transferred into a 1.5mL centrifuge tube, resuscitated at 30 ℃ for 3-4 h, plated with LB plate containing 0.05mg/L kanamycin and 0.05mg/L spectinomycin, inversely cultured at 30 ℃ for 18-20 h, and subjected to colony PCR verification by using nrdH-VF and nrdH-VR as primers, and if about 1400bp fragments can be successfully amplified, the fragment is proved to be E.coli W3110EYC: hflC-cysM: gbbbK-nrdH:: hflK-cysE: nrdH-line-linker-D positive colony.
(5) pTD and pCas plasmid elimination: positive single colonies were picked up and inoculated into LB tubes containing 1mM IPTG and 0.05mg/L kanamycin, incubated overnight at 30℃and the next day bacterial solution streaked on LB plates containing 0.05mg/L kanamycin, incubated at 30℃for 24 hours, and single colonies were picked up and streaked on LB plates containing 0.05mg/L spectinomycin, which were unable to successfully eliminate the pTD-nrdH plasmid on LB plates containing 0.05mg/L spectinomycin. Single colonies were picked up for successful elimination of pTD-nrdH plasmid in LB tubes, incubated overnight at 37℃and streaked with the next day bacterial solution in LB plates for 12h at 37℃and single colonies were picked up for streaking with LB plates containing 0.05mg/L kanamycin and were unable to be successfully eliminated with pCas plasmid in LB plates containing 0.05mg/L kanamycin, resulting in plasmid-free E.coli W3110EYCE.coil W3110EYC:: hflC-cysM:: ybbK-nrdH:: hfLK-cysE:: nrdH-linker-GBD lig.
(6) Introducing a fermentation plasmid: preparation E.coli W3110EYCE.coil W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig chemical conversion competence, see description of the detailed procedure (Molecular Cloning: A La boratory Manual,3ed edition, 99-102). The plasmid pTrc99a-cysE was fermented f Conversion of ydeD-GBD-SH3-PDZ (abbreviated as pEDGSP) to E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE:: nrdH-liIn the nker-GBD lig, a plasmid-containing strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE::: nrdH-linker-GBD lig/pEDGSP is obtained.
(7) Fermentation verification: e.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig/pEDGSP as the experimental group, E.coli W3110EYC:: hflC-cysM::: ybbK-nrdH:: hflK-cysE/pE DGSP as the control group were inoculated into 10mL of LB medium, respectively, and cultured overnight at 37℃and 200 rpm. 1mL of the preculture was inoculated into a 500mL shaking flask containing 20mL of SM medium, and the culture was continued at 30℃and 200rpm for 4 hours, and IPTG was added at a final concentration of 0.1mM, followed by further culturing for 48 hours. After fermentation, 1mL of fermentation broth is taken to determine OD 600 1mL of the fermentation broth was centrifuged at 12000rpm for 3min at room temperature, and the fermentation supernatant was assayed for OD as in example 1 600 And the L-cysteine content in the supernatant of the fermentation broth is shown in FIG. 2. ( In the drawing: e.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig/pEDGSP is abbreviated as: ECY:: NG/PEDGSP, E.coli W3110EYC:: hflC-cys M:: ybbK-nrdH:: hflK-cysE/pEDGSP is abbreviated as: ECY/PEDGSP )
As can be seen from fig. 2, after co-localized assembly of nrdH with ydeD, L-cysteine production was increased from 1.43g/L to 1.76g/L, while od600= 21.64 was raised to od600=23.96. Indicating that the co-localization assembly enhances the synthesis and export of the L-cysteine and reduces the toxicity of the L-cysteine to cells themselves.
Example 3: construction of an effective Strain E.coil W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE:: nrdH-GBD lig::: cysE-linker-PDZ lig and fermentation thereof
(1) Construction of pTarget plasmid: a pTarget-cysE plasmid was constructed which was able to express sgRNA targeting the cysE sequence of the gene of interest. PCR amplification was performed using pTarget F Plasmid (Addgene Plasmid # 62226) as template and pT-TB-cysE-F and pT-TB-cysE-R as primers. The PCR product was digested with Dpn I. The digestion products are transferred to E.coli DH5 alpha, coated on a spectinomycin plate, single colonies are picked up and sequenced with the verification primer pT-YZ-F to verify and screen the mutated pTarget-cysE plasmid. The circular plasmid pTarget-cysE was linearized using the primers pT-line-F and pT-line-R using the pTarget-cysE plasmid as template.
(2) Construction of pTD plasmid: using the E.coli W3110 genome as a template, the primers Donor-cysE-up-F and Donor-cysE-up-R, donor-cysE-down-F and Donor-cysE-down-R, TYTH-cysE-F and TYTH-cysE-R, donor-ybbK-F and Donor-ybbK-R, donor-nrdH-F and Donor-nrdH-R, TYTH-cysE-F and TYTH-cysE-R were the same as in example 2 (2), pTD-cysE plasmid was obtained. The nucleotide sequence of the fusion protein gene cysE-linker-PDZ lig is shown as SEQ ID NO. 2. Escherichia coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: the cysE gene on the hflK-cysE genome has undergone site-directed mutagenesis (A237V) to relieve feedback inhibition.
(3) Electric transformation competence preparation: after the pCas Plasmid (Addgene Plasmid # 62225) was introduced into E.coli E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE:: nrdH-linker-GBD lig conversion competence, preparation of electric conversion competence was started, and the preparation method was the same as in example 2 (3).
(4) Electric shock conversion: the E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig is electrotransferred to competent E.coli W3110EYC, and E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE:: nrdH-linker-PDZ lig positive colonies were constructed in the same manner as in example 2 (4).
(5) pTD and pCas plasmid elimination: the procedure was as in example 2 (5), to give plasmid-free E.coli W3110EYC:: nrdH-linker-GBD lig:: cysE-linker-PDZ lig.
(6) Introducing a fermentation plasmid: the plasmid pTrc99a-cysE was fermented f The conversion of ydeD-GBD-SH3-PDZ to W3110EYC:: nrdH-linker-GBD lig:: cysE-linker-PDZ lig resulted in plasmid-containing strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig/pEDGSP.
(7) Fermentation verification: the E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBDlig:: cysE-linker-PDZlig/pEDSGP production strain was used as the experimental set, and the E.coli W3110EYC:: hflC-cysM: ybbK-nrdH::: hflK-cysE:: nrdH-linker-GBD lig/pEDSGP constructed in example 2 was used as the control set for fermentation test and detection as in example 2 (7). OD (optical density) 600 And the L-cysteine content in the supernatant of the fermentation broth is shown in FIG. 3. (in the drawing, E.coli W3110EYC:: hflC-cysM::: ybbK)-nrdH:: hfLK-cysE:: nrdH-linker-GBDlig:: cysE-linker-PDZlig/pEDSGP abbreviated as ECY::: NG:: EP/PEDGSP)
As can be seen from FIG. 3, L-cysteine yield increased from 1.76 to 2.19g/L after co-localized assembly of cysE with ydeD. Co-localization assembly of L-cysteine carbon metabolic pathway effectively improves the conversion efficiency of serine to L-cysteine, fully exerts the structural characteristics and further promotes the accumulation of L-cysteine.
Example 4: construction of an effective Strain E.coil W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE:: nrdH-linker-GBDlig::: cysE-linker-PDZlig::: cysM-linker-SH3lig and fermentation thereof
(1) Construction of pTarget plasmid: a pTarget-cysM plasmid was constructed which was able to express an sgRNA targeting the yeaS sequence of the gene of interest. PCR amplification was performed using pTarget F Plasmid (Addgene Plasmid # 62226) as template and pT-TB-cysM-F and pT-TB-cysM-R as primers. The PCR product was digested with Dpn I. The digestion products are transferred to E.coli DH5 alpha, coated on a spectinomycin plate, single colonies are picked up and sequenced with the verification primer pT-YZ-F to verify and screen the mutated pTarget-cysM plasmid. The circular plasmid pTarget-cysM was linearized using the primers pT-line-F and pT-line-R using the pTarget-cysM plasmid as template.
(2) Construction of pTD plasmid: the procedure of example 2 (2) was followed using E.coli W3110 genome as template, and the genome of Donor-cysM-up-F and Donor-cysM-up-R, donor-cysM-F and Donor-cysM-down-R, TYTH-cysM-F, TYTH-cysM-F as primers and Escherichia coli W3110EYC as template to obtain pTD-cysM plasmid. The nucleotide sequence of the fusion protein gene cysM-linker-SH3lig is shown as SEQ ID NO. 3.
(3) Electric transformation competence preparation: after the pCas Plasmid (Addgene Plasmid # 62225) was introduced into E.coil W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE:::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig conversion competence, the preparation of electric conversion competence was started, in the same manner as in example 2 (3).
(4) Electric shock conversion: the E.coli W3110EYC: hflC-cysM: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig:: cysE-linker-PDZ lig were screened after electrotransformation to construct E.coli W3110EYC: hflC-cysM:: ybbK-nrdH::: hflK-cysE::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig:: cysM-linker-SH3lig positive colonies, and the construction method was the same as in example 2 (4).
(5) pTD and pCas plasmid elimination: the procedure was as in example 2 (5), to give plasmid-free E.coli W3110EY C:: hflC-cysM:: ybbK-nrdH:: hflK-cysE::: nrdH-linker-GBDlig:: cysE-linker-PDZlig:: cysM-linker-SH3lig.
(6) Introducing a fermentation plasmid: the plasmid pTrc99a-cysE was fermented f Transformation of the-ydeD-GBD-SH 3-PDZ into E.coli W3110EYC: hflC-cysM:: ybbK-nrdH:: hflK-cysE::: nrdH-linker-GBD lig:: cysE-linker-PDZ lig:: cysM-linker-SH3lig resulted in the plasmid-containing strain E.coli W3110 EYC::: hflC-cysM:: ybbK-nrdH::: hflK-cysE::::: nrdH-GBD lig cysE-linker-PDZ lig::::: cysM-SH 3 lig/pEDSGP. The procedure was as in example 2 (6).
(7) Fermentation verification: the E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig:: cysE-linker-PDZ lig:: cysM-linker-SH3 lig/pEDSGP production strain was used as experimental group, and the E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE::::: nrdH-GBZ lig/pEDGSP as control group, and fermentation test and detection were performed according to the method of example 2 (7). OD (optical density) 600 And the L-cysteine content in the supernatant of the fermentation broth is shown in FIG. 4. ( In the drawing: e.coli W3110EYC:: hflC-cysM:: yb BK-nrdH:: hflK-cysE:: nrdH-linker-GBD lig:: cysE-linker-PDZ lig:: cysM-linker-SH3 lig/pEDSG P is abbreviated as ECY::: NG:: EP:: MD/PEDGSP )
As can be seen from FIG. 4, after co-localization of cysM to the outer transferrin ydeD, the L-cysteine yield increased from 2.19g/L to 2.63g/L. The compartmental structure is said to enhance L-cysteine synthesis and to alleviate L-cysteine toxicity to the cell itself.
Example 5: plasmid pTrc99a-cysE comprising a scaffold protein domain gene f— Construction of ydeD-GBD-SH3-PDZ
(1) Construction of pTrc99a-cysE f -ydeD-GBD-SH3-PDZ plasmid: PCR amplification was performed using pTrc99a plasmid as a template and pTrc99a-line-F and pTrc99a-line-R as primers to obtain the linear vector pTrc99a-line. PCR products were subjected to Dpn I inAfter digestion for 3h at 37℃the DNA fragment was recovered using the Clean up kit. Performing PCR amplification by using the E.coli W3110 genome as a template and cysE-F and cysE-R as primers to obtain cysE fragments, and performing PCR amplification by using ydeD-F and ydeD-R as primers to obtain ydeD fragments. PCR amplification is carried out by taking plasmid clf-SH3 synthesized by Beijing engine biotechnology Co., ltd as a template, taking SH3-F and SH3-R as primers to obtain SH3 fragments (GenBank accession number: EDL 06069), taking plasmid clf-GBD synthesized by Beijing engine biotechnology Co., ltd as a template, taking GBD-F and GBD-R as primers to obtain GBD fragments (GenBank accession number: BAA 21534), taking plasmid clf-PDZ synthesized by Beijing engine biotechnology Co., ltd as a template, taking PDZ-F and PDZ-R as primers to obtain PDZ fragments (GenBank accession number: AAH 31149), and recovering DNA fragments by using a Clean up kit. The linearized pTrc99a-line plasmid, fragment cysE, fragment ydeD, fragment SH3, fragment GBD, fragment PDZ were ligated together according to the instructions of (One step clone kit, vazyme Biotech, nanjing, china) and the ligation product was transformed into DH 5. Alpha. Competence by chemical transformation. Positive clones were selected by colony PCR using primers pTrc99a-VF and pTrc99a-VR, and the pTrc99a-cysE-ydeD-GBD-SH3-PDZ plasmid was obtained by sequencing. The plasmid pTrc99a-cysE-ydeD-GBD-SH3-PDZ is used as template, cysE is used f -F and cysE f And (3) carrying out PCR amplification by taking R as a primer. The PCR product was digested with Dpn I at 37℃for 3h, and the DNA fragment was recovered using Clean up kit, and the product was converted into DH 5. Alpha. Competence by chemical conversion. Colony PCR selection of positive clones was performed with primers pTrc99a-VF and pTrc99a-VR, and pTrc99a-cysE was obtained by sequencing f -ydeD-GBD-SH3-PDZ plasmid. The nucleotide sequence of the fragment SH3 is shown as SEQ ID NO.4, the nucleotide sequence of the fragment GBD is shown as SEQ ID NO.5, the nucleotide sequence of the fragment PDZ is shown as SEQ ID NO.6, the nucleotide sequence of the fragment cysE is shown as SEQ ID NO.7, and the fragment cysE is shown as SEQ ID NO.5 f The nucleotide sequence of the fragment ydeD is shown as SEQ ID NO.8, the nucleotide sequence of the fragment ydeD is shown as SEQ ID NO.9, and the plasmid pTrc99a-cysE is shown as a plasmid f The nucleotide sequence of the-ydeD-GBD-SH 3-PDZ is shown in SEQ ID NO. 10.

Claims (10)

1. The construction method of the genetically engineered bacterium for producing the L-cysteine at high yield is characterized by comprising the following steps of:
(1) Construction of plasmid pTrc99a-cysE Using pTrc99a plasmid as vector f -ydeD-GBD-SH3-PDZ；
(2) The strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE is taken as a chassis strain, the nrdH gene on the genome is replaced by fusion protein gene nrdH-linker-GBD lig, engineering bacterium E.coil W3110 EYC::: hflC-cysM:: ybbK-nrdH::: hflK-cysE:: nrdH-linker-GBD lig is obtained, and the plasmid pTrc99a-cysE is obtained f -ydeD-GBD-SH3-PDZ is introduced into an engineering bacterium, thereby obtaining the genetically engineered bacterium for high-yield of L-cysteine.
2. The method for constructing genetically engineered bacterium capable of producing L-cysteine as claimed in claim 1, wherein the plasmid pTr c99a-cysE f The sequence of the-ydeD-GBD-SH 3-PDZ is shown as SEQ ID NO.10, and the sequence of the fusion protein gene nrdH-link-GBD lig is shown as SEQ ID NO. 1.
3. The construction method of the genetically engineered bacterium for producing the L-cysteine at high yield is characterized by comprising the following steps of:
(1) Construction of plasmid pTrc99a-cysE Using pTrc99a plasmid as vector f -ydeD-GBD-SH3-PDZ；
(2) The strain E.coli W3110EYC:: hflC-cysM:: ybbK-nrdH:: hflK-cysE is used as a chassis strain, nrdH gene on the genome is replaced by fusion protein gene nrdH-linker-GBD lig, cysE gene is replaced by fusion protein gene cysE-linker-PDZ lig, engineering bacterium E.coil W3110EYC:: hflC-cysM::: ybbK-nrdH::: hflK-cysE:::: nrdH-linker-GBD lig:: cysE-linker-PDZ lig is obtained, and the plasmid pTrc99a-cysE is obtained f The ydeD-GBD-SH3-PDZ is imported into engineering bacteria E.coil W3110EYC:: hflC-cysM::: ybbK-nrdH::: hflK-cysE:::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig, thereby obtaining the genetic engineering bacteria of the high-yield L-cysteine.
4. The method for constructing genetically engineered bacterium capable of producing L-cysteine at high yield as claimed in claim 3, wherein said plasmid pTr c99a-cysE f The sequence of the-ydeD-GBD-SH 3-PDZ is shown as SEQ ID NO.10, and the sequence of the fusion protein gene cysE-link-PDZ lig is shown as SEQ ID NO. 2.
5. The construction method of the genetically engineered bacterium for producing the L-cysteine at high yield is characterized by comprising the following steps of:
(1) Construction of plasmid pTrc99a-cysE Using pTrc99a plasmid as vector f -ydeD-GBD-SH3-PDZ；
(2) The strain E.coli W3110EYC:: hflC-cysM: ybbK-nrdH:: hflK-cysE is used as a chassis strain, the nrdH gene on the genome is replaced by fusion protein gene nrdH-linker-GBD lig, the cysE gene is replaced by fusion protein gene cysE-linker-PDZ lig, the cysM gene is replaced by fusion protein gene cysM-linker-SH3lig, engineering bacteria E.coil W3110EYC: hflC-cysM:: ybbK-nrdH::::: hflK-cysE:: nrdH-linker-GBD lig:: cysE-PDZ lig:: cysM-linker-SH3lig are obtained, and the plasmid pTrc99a-cysE f The ydeD-GBD-SH3-PDZ is introduced into engineering bacteria E.coil W3110EYC:: hflC-cysM:: ybbK-nrdH::: hflK-cysE::: nrdH-linker-GBD lig::: cysE-linker-PDZ lig:: cysM-li linker-SH3lig, thereby obtaining the genetically engineered bacteria of high-yield L-cysteine.
6. The method for constructing a genetically engineered bacterium capable of producing L-cysteine as claimed in claim 5, wherein the plasmid pTr c99a-cysE f The sequence of the-ydeD-GBD-SH 3-PDZ is shown as SEQ ID NO.10, and the sequence of the fusion protein gene cysM-linker-SH3lig is shown as SEQ ID NO. 3.
7. A genetically engineered bacterium capable of producing L-cysteine at a high yield, which is constructed by the construction method according to any one of claims 1 to 6.
8. The use of the genetically engineered bacterium of high-yield L-cysteine constructed by the construction method of any one of claims 1 to 6 or the genetically engineered bacterium of high-yield L-cysteine of claim 7 in microbial fermentation preparation of L-cysteine.
9. The use according to claim 8, wherein the genetically engineered strain is inoculated into a fermentation medium and subjected to fermentation culture at a temperature of 32.5-37 ℃ and at a speed of 200-800 rpm, OD 600 When the fermentation liquid is 10-30, the culture is continued after adding IPTG, and the supernatant of the fermentation liquid is taken after the fermentation is finished, and the L-cysteine is obtained through separation and purification.
10. The use according to claim 9, wherein the composition of the fermentation medium is as follows: 25-50 g/L of dextrose monohydrate, (NH) 4 ) 2 SO 4 5～20g/L、KH 2 PO 4 0.5～2g/L、Na 2 S 2 O 3 5-20 g/L, yeast extract 1-10 g/L, na 2 HPO 4 0.5-2 g/L, peptone 0.5-2 g/L, trace element solution 0.5-2 ml/L, deionized water as solvent, and natural pH value.
CN202310748566.1A 2023-06-25 2023-06-25 Genetically engineered bacterium for high-yield of L-cysteine as well as construction method and application thereof Pending CN116837015A (en)

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