Engineering bacterium for high yield of L-tryptophan as well as construction method and application of engineering bacterium

Abstract

The invention belongs to the technical field of biology, and particularly relates to an engineering bacterium with high yield of L-tryptophan as well as a construction method and application of the engineering bacterium. The engineering bacterium provided by the invention is constructed by taking escherichia coli as a starting strain and carrying out gene modification, can realize high yield of L-tryptophan, has the advantages of high conversion rate, short fermentation period and stable hereditary character, does not need to add an inducer or antibiotic in the fermentation production process, simplifies the fermentation process, and is more suitable for industrial production of L-tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered bacterium that produces high levels of L-tryptophan, its construction method, and its applications. Background Technology

[0002] Tryptophan is an amino acid containing a chiral carbon atom, and its natural form is mainly L-tryptophan. L-tryptophan is widely used in medicine, food, feed and other fields.

[0003] The main methods for producing L-tryptophan are chemical synthesis, enzymatic conversion, and microbial fermentation. Microbial fermentation, using inexpensive raw materials such as glucose as carbon sources, is dominant due to its low cost and high sustainability. Commonly used strains include *Escherichia coli* and *Corynebacterium glutamicum*. Metabolic engineering optimization, such as removing feedback inhibition (aroG, trpE), overexpressing key enzymes (ppsA, tktA), blocking competitive pathways (pheA, tyrA), and modifying the transport system (yddG overexpression), can increase L-tryptophan yield. However, the L-tryptophan synthesis pathway is lengthy, involves numerous key enzymes, and has a complex feedback mechanism, resulting in low yields and conversion rates, as well as long fermentation cycles, for L-tryptophan production using microbial fermentation. According to literature reports, engineered strains of *E. coli* yielded approximately 5.1 g / L with a glucose conversion rate of about 0.180 g / g in a 48-hour shake-flask fermentation; the total yield in a 3 L bioreactor was approximately 43.0 g / L (conversion rate 0.180 g / g). *Corynebacterium glutamicum* achieved a yield of up to 58 g / L (conversion rate 0.17 g / g) in an 80-hour fed-batch fermentation. Furthermore, additional antibiotics and inducers were required during the fermentation process. Summary of the Invention

[0004] To address the above problems, this invention provides an engineered bacterium that produces high levels of L-tryptophan, its construction method, and its applications. This engineered bacterium can produce high levels of L-tryptophan, has a high glucose conversion rate, a short fermentation cycle, and stable genetic traits. No inducers or antibiotics are required during the fermentation process, making it suitable for the industrial production of L-tryptophan.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of this invention provides an engineered bacterium that produces high levels of L-tryptophan. This engineered bacterium is constructed from *Escherichia coli* strain through genetic modification. The genetic modification includes: not expressing LacI (lactose metabolism regulatory protein), TrpR (tryptophan operon repressor protein), TnaA (tryptophanase), TnaB (tryptophan permease), TnaC (tryptophan permease), PoxB (pyruvate oxidase), LdhA (L-lactate dehydrogenase), TdcD (propionate kinase / acetate kinase), PflB (pyruvate formate lyase), and TrpE (anthaminobenzoic acid synthase); not expressing the gene trpL (tryptophan attenuator gene); and integrating AroG... fbr (3-Deoxy-D-arabinohepylose-7-phosphate synthase relieved of negative feedback inhibition), SerA (D-3-phosphoglycerate dehydrogenase), TktA (transketolase A), PpsA (phosphoenolpyruvate synthase), TrpE fbr The genes encoding anthranilate synthase (which has been relieved of negative feedback inhibition), TrpD (glutamine aminotransferase), TalB (aldolase B), Gap (glyceraldehyde-3-phosphate dehydrogenase), YddG (aromatic amino acid transporter), and PntAB (binding transmembrane NAD(P)H dehydrogenase).

[0006] This invention optimizes the metabolic flux of L-tryptophan by modifying the genome of *E. coli*, enabling the engineered bacterium to produce high levels of L-tryptophan with high conversion rates and short fermentation cycles. This engineered bacterium does not contain exogenous plasmids, exhibits stable genetic traits, and requires no inducers or antibiotics during fermentation, simplifying the fermentation process and making it more suitable for industrial applications.

[0007] Preferably, the Escherichia coli is Escherichia coli W3110.

[0008] Preferably, the AroG fbr The encoding gene is aroG fbr Its nucleotide sequence is shown in SEQ ID NO.1.

[0009] Preferably, the TrpE fbr The encoding gene is trpE fbr Its nucleotide sequence is shown in SEQ ID NO.2.

[0010] Preferably, the gene encoding the gap is gap, which is derived from *Acetobutyricum* (a type of bacteria). Clostridium acetobutylic acid The nucleotide sequence is shown in SEQ ID NO.3.

[0011] Preferably, the gene encoding YddG is yddGpa, which originates from Burkholderia spp. (…). Paraburkholderia acidisoli The nucleotide sequence is shown in SEQ ID NO.4.

[0012] Preferably, the method of not expressing LacI and TrpR is to knock out their corresponding coding genes and integrate the coding gene of SerA in situ.

[0013] Preferably, the method for not expressing TnaA, TnaB, TnaC, PoxB, LdhA, and TdcD is to knock out their corresponding coding genes.

[0014] Preferably, the method for not expressing the PflB is: knocking out its corresponding coding gene and integrating the coding gene of the YddG in situ.

[0015] A second aspect of this invention provides an engineered bacterium, SW-12, which is constructed from Escherichia coli W3110 as the starting strain through genetic modification; the genetic modification includes: Knock out genes lacI and trpR, and integrate gene serA at the site of gene trpR, using the trc promoter; knock out genes tnaA, tnaB, tnaC, poxB, ldhA, and tdcD; knock out gene pflB, and integrate gene yddGpa at the site, using the Pj23110 promoter; knock out genes trpL and trpE, and integrate gene trpE at the original site. fbr The original promoter was replaced with the Pj23102 promoter; the nucleotide sequence of the gene yddGpa is shown in SEQ ID NO.4; Integrating gene aroG fbr The trc promoter is used to start the integrative gene; the pj23114 promoter is used to start the integrative gene tktA; the pj23114 promoter is used to start the integrative gene ppsA; and the pbolA promoter is used to start the integrative gene trpE. fbr The gene trpD is started using the trc promoter; the integrated gene talB is started using the pj23115 promoter; the integrated gene gap is started using the PgapA promoter; the integrated gene pntAB is started using the Ptrc promoter; and the gene aroG... fbr The nucleotide sequence is shown in SEQ ID NO.1, and the gene trpE is... fbr The nucleotide sequence of the gene gap is shown in SEQ ID NO.2, and the nucleotide sequence of the gene gap is shown in SEQ ID NO.3.

[0016] Among them, the "original promoter" is the wild-type promoter that is naturally present in the corresponding gene.

[0017] The engineered bacterium SW-12, through the aforementioned gene modification, overexpresses enzymes favorable for L-tryptophan synthesis and disrupts competitive and decomposition pathways, thereby increasing tryptophan yield and conversion rate. For example, knocking out genes tnaA, tnaB, and tnaC prevents L-tryptophan from being transported into the cell and further decomposed into indole; introducing the glyceraldehyde-3-phosphate dehydrogenase gene gap from *Bacillus acetobutyricum* enhances the glycolysis pathway and improves the strain's growth and metabolic capacity; using the pbolA promoter to dynamically regulate ppsA gene expression, resulting in minimal expression of ppsA during the logarithmic growth phase, followed by a rapid increase in pbolA promoter activity and a 10-20 fold increase in ppsA gene expression level after entering the stationary phase, reducing the metabolic stress on the engineered bacteria during growth, increasing the supply of the precursor PEP during the stationary phase, and improving L-tryptophan yield and conversion rate; simultaneously introducing genes from... Paraburkholderia acid salts The aromatic amino acid transporter gene yddGpa was modified to reduce intracellular feedback inhibition, thereby further improving the L-tryptophan production and conversion rate of the engineered bacteria.

[0018] The third aspect of this invention provides a method for constructing the engineered bacterium SW-12, specifically including the following operations: Knock out the genes lacI and trpR, and integrate the gene serA at the site of the gene trpR, and start with the trc promoter; Integrating the gene aroG fbr Boot using the aforementioned trc bootloader; Knock out the genes tnaA, tnaB, and tnaC; Knock out the genes trpL and trpE, and integrate the gene trpE in its original position. fbr The original promoter is replaced with the Pj23102 promoter. Knock out the genes poxB, ldhA, and tdcD; Integrate the gene gap and start using the PgapA promoter; Integrating the trpE gene fbr And the trpD gene, started using the trc promoter; Integrate the ppsA gene and start it with the PbolA promoter; Integrate the tktA gene and start it using the pj23114 promoter; The gene talB is integrated and started using the pj23115 promoter; Knock out the gene pflB and integrate the gene yddGpa at that site, and start with the Pj23110 promoter; The pntAB gene is integrated and started using the Ptrc promoter.

[0019] The present invention does not limit the order of the above-mentioned knockout and integration. The purpose of the present invention can be achieved by knocking out or integrating the above-mentioned genes into the genome of Escherichia coli W3110 in any order, and all such actions are within the scope of protection of the present invention.

[0020] More preferably, the gene aroG is integrated at the pseudogene site ygaY. fbr .

[0021] More preferably, the gene gap is integrated at the pseudogene site ydfE.

[0022] More preferably, the trpE is integrated at the pseudogene site yedN. fbr And the trpD gene.

[0023] More preferably, the gene ppsA is integrated at the pseudogene site yjbI.

[0024] More preferably, the gene tktA is integrated at the pseudogene site yehH.

[0025] More preferably, the gene talB is integrated at the pseudogene site ilvG.

[0026] More preferably, the gene pntAB is integrated at the pseudogene site ycjV.

[0027] The fourth aspect of this invention relates to the application of the above-mentioned engineered bacteria that produce high levels of L-tryptophan or the above-mentioned engineered bacteria SW-12 in the fermentation production of L-tryptophan.

[0028] The fifth aspect of the present invention provides a method for producing L-tryptophan by fermentation, using the above-mentioned high-L-tryptophan engineered bacteria or the above-mentioned engineered bacteria SW-12 as the fermentation bacteria.

[0029] Preferably, the method includes the following operations: The engineered bacteria producing high levels of L-tryptophan or the engineered bacteria SW-12 were activated and cultured. The strain that had grown to the logarithmic phase was inoculated into a seed culture medium and cultured. The pH of the fermentation broth was controlled at 6.9-7.1, dissolved oxygen >20%, and temperature at 35-38℃. After culturing for 9-12 h, the culture was transferred to a fermentation medium for fermentation. During the fermentation process, the pH was controlled at 6.7-7.2 by adding ammonia and glucose, the residual sugar concentration was less than 5 g / L, and the fermentation time was 42-50 h.

[0030] Optionally, the activation and culture method is as follows: the strain is inoculated into LB slant medium for activation, and the activated strain is transferred into a flask and cultured at 37°C for 10-12 h.

[0031] Preferably, the seed culture medium consists of: 30 g / L glucose, 7 g / L yeast extract, 3 g / L diammonium hydrogen phosphate, 2 g / L potassium chloride, 3 g / L ammonium sulfate, 5 g / L citric acid, 2 g / L magnesium sulfate, 0.5 g / L isoleucine, and 1 mL / L trace element mixture; the solvent is water, and it is sterilized at 121°C before use.

[0032] Preferably, the fermentation medium comprises: 20 g / L glucose, 5 g / L yeast extract, 3.5 g / L magnesium sulfate, 2 g / L citric acid, 3 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 0.05 g / L isoleucine, 0.1 g / L ferrous sulfate, 0.5 g / L methionine, and 1 mL / L of trace element mixture; the solvent is water; and it is sterilized at 121°C before use. The addition of L-methionine and L-isoleucine to this fermentation medium reduces the lag phase of strain SW-12 in the fermenter, further shortening the fermentation cycle and improving the conversion rate.

[0033] More preferably, in the above-mentioned seed culture medium and fermentation culture medium, the components of the trace element mixture are: 4.0 g / L cobalt chloride hexahydrate, 0.6 g / L copper sulfate, 6.4 g / L zinc sulfate heptahydrate, 20 g / L sodium sulfate, 4.5 g / L manganese sulfate, and the solvent is 5 mmol / L H2SO4.

[0034] The beneficial effects of this invention are as follows: This invention provides an engineered bacterium that produces high levels of L-tryptophan, specifically an engineered bacterium SW-12. The engineered bacterium provided by this invention is constructed by modifying the genome of *Escherichia coli*, without the need for plasmids during construction; all operations are performed on the genome. Therefore, the constructed engineered bacterium has stable genetic traits and requires no antibiotics or inducers during fermentation. Experiments have shown that the engineered bacterium provided by this invention produces L-tryptophan with high yield and high conversion rate, and a short fermentation cycle. This invention also provides a method for producing L-tryptophan using this engineered bacterium through fermentation. This method can achieve even higher L-tryptophan yield and conversion rate, and further shorten the fermentation cycle. Attached Figure Description

[0035] Figure 1 This describes the fermentation process of engineered bacteria SW-12 in a 50L fermenter in Example 2. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0037] Currently, L-tryptophan production mainly relies on microbial fermentation, with *E. coli* being a commonly used strain. In *E. coli*, L-tryptophan synthesis primarily involves three modules: the central carbon metabolism pathway, the shikimic acid pathway, and the branched acid pathway. Due to the lengthy L-tryptophan synthesis pathway, numerous key enzymes, complex feedback mechanisms, and the fact that engineered strains typically depend on exogenous plasmids, most commercially available L-tryptophan production strains suffer from low yields, low conversion rates, the need for additional antibiotics and inducers, and long fermentation cycles.

[0038] This invention provides an engineered bacterium that produces high levels of L-tryptophan. This bacterium is constructed from *Escherichia coli* strain through genetic modification. The genetic modification includes: not expressing LacI, TrpR, TnaA, TnaB, TnaC, PoxB, LdhA, TdcD, PflB, and TrpE; not expressing the gene trpL; and integrating the expression of AroG. fbr SerA, TktA, PpsA, TrpE fbr The engineered bacteria contain the genes for TrpD, TalB, Gap, YddG, and PntAB. This strain can produce high levels of L-tryptophan, exhibits high glucose conversion, a short fermentation cycle, and stable genetic traits. No inducers or antibiotics are required during fermentation, making it a promising candidate for industrial-scale L-tryptophan production.

[0039] This invention also provides an engineered bacterium SW-12 and its construction method.

[0040] This invention also provides a method for producing L-tryptophan by fermentation using the aforementioned engineered bacteria.

[0041] The present invention will be described below through specific embodiments.

[0042] Unless otherwise specified, the reagents or instruments used in the following examples are all commercially available products. The methods used in the following examples are conventional methods in the art.

[0043] Example 1 This embodiment provides the engineered bacteria SW-12 and its construction method.

[0044] The primer sequences used in the construction process are shown in Table 1.

[0045] Table 1 Primers used in strain construction

[0046] The following is the specific construction process of engineered bacteria SW-12: 1. Starting with Escherichia coli W3110, the genes lacI and trpR were knocked out from its genome, and the gene serA encoding D-3-phosphoglycerate dehydrogenase was integrated into the trpR site. The strain was then started using the trc promoter to obtain strain SW-01. Chromosomal deletion or integration was performed using the CRISPR-Cas9 system, as described in the reference (Li Qi, Sun B, Chen J, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli[J]. ActaBiochimica et Biophysica Sinica, 2021, 53(5): 620-627.).

[0047] 1.1 Knockout of gene lacI A pair of primers with 20 bp complementary sequences (sg-lacI-F and sg-lacI-R listed in Table 1) were designed using the CRISPR RGEN tool (http: / / www.rgenome.net / cas-designer / ), and a plasmid expressing sgRNA (pEcgRNA-lacI) was constructed using inverse PCR. To prepare the donor DNA fragment for the target gene deletion, the upstream and downstream homologous arms of lacI (each ~500 bp) were amplified by PCR using two pairs of primers (lacI-1 / lacI-2, lacI-3 / lacI-4). The two homologous arms were ligated by overlapping PCR to generate the donor DNA fragment (DNA-lacI). pEcgRNA-lacI and DNA-lacI were co-transformed into *E. coli* electroporation competent cells carrying plasmid pEcCas9. L-arabinose induced the expression of λ-Red recombinase. After electroporation, the cells were plated on LB agar plates containing kanamycin sulfate and tetracycline hydrochloride resistance and incubated overnight at 37°C. Positive colonies were verified by colony PCR, and then DNA sequencing was used to confirm whether the target gene lacI had been knocked out.

[0048] 1.2 Knock out gene trpR and integrate gene serA at the trpR site. Using primers sg-trpR-F and sg-trpR-R, a plasmid expressing sgRNA (pEcgRNA-trpR) was constructed using inverse PCR. Two pairs of primers (trpR-serA-1 / trpR-serA-2, trpR-serA-5 / trpR-serA-6) were used to amplify the upstream and downstream homologous arms of serA (~500 bp each), and primers trpR-serA-3 and trpR-serA-4 were used to amplify serA again. A donor DNA fragment (DNA-serA) was constructed by overlapping PCR of the two homologous arms and the target gene serA, and the target gene serA was integrated into the target site trpR. pEcgRNA-trpR and DNA-serA were co-transformed into *E. coli* electroporation competent cells carrying plasmid pEcCas9. L-arabinose induced the expression of λ-Red recombinase. After electroporation, the bacterial cells were plated on LB agar plates containing kanamycin sulfate and tetracycline hydrochloride resistance and incubated overnight at 37°C. Positive colonies were verified by colony PCR, and DNA sequencing was then used to confirm whether the target gene had been integrated.

[0049] 1.3 pEcgRNA plasmid elimination The positive recombinant was placed in LB medium containing rhamnose and kanamycin resistance and cultured at 37°C. After three consecutive subcultures, the culture was diluted appropriately and plated onto plates containing kanamycin, and incubated at 37°C. Single colonies that grew on kanamycin plates but not on tetracycline plates were selected and preserved. This yielded strain SW-01.

[0050] 2. The 3-deoxy-D-arabinoheptanoose-7-phosphate synthase gene aroG, which relieved negative feedback inhibition, was integrated into the pseudogene locus ygaY in strain SW-01. fbr Using the trc promoter, strain SW-02 was obtained: A plasmid expressing sgRNA (pEcgRNA-ygaY) was constructed using primers sg-ygaY-F and sg-ygaY-R. AroG was amplified using two pairs of primers (ygay-aroG-1 / ygay-aroG-2, ygay-aroG-5 / ygay-aroG-6). fbr The upstream and downstream homologous arms were amplified by PCR using primers ygay-aroG-3 and ygay-aroG-4. fbr The aroG gene was integrated according to method "1.2". fbr .

[0051] 3. Knock out the tryptophanase gene tnaA, tryptophan permease genes tnaB and tnaC of strain SW-02 to prevent L-tryptophan from being transported into the cell and further degraded into indole, thus obtaining strain SW-03: A plasmid expressing sgRNA was constructed using primers sg-tnaCAB-F and sg-tnaCAB-R. The upstream and downstream homologous arms of tnaA, tnaB, and tnaC were amplified using two pairs of primers (△tnaCAB-1 / △tnaCAB-2, △tnaCAB-3 / △tnaCAB-4). Genes tna, tnaB, and tnaC were knocked out according to method "1.1".

[0052] Eliminate the pEcgRNA plasmid according to method "1.3".

[0053] 4. The tryptophan attenuator gene trpL and the anthranilate synthase gene trpE in strain SW-03 were knocked out, and the trpE gene, which relieved the negative feedback inhibition, was integrated into its original position. fbr By replacing the original promoter with the Pj23102 promoter, strain SW-04 was obtained. A plasmid expressing sgRNA (pEcgRNA-trpE) was constructed using primers sg-trpE-F and sg-trpE-R. The upstream and downstream homologous arms of trpE were amplified using two pairs of primers (trpo-1 / trpo23102-2, trpo-5 / trpo-6), and the target fragment trpE was amplified using two pairs of primers (trpo-3 / trpo-4). fbr The trpE gene was integrated according to method "1.2". fbr .

[0054] Eliminate the pEcgRNA plasmid according to method "1.3".

[0055] 5. By knocking out the pyruvate oxidase gene poxB, the L-lactate dehydrogenase gene ldhA, and the propionate kinase / acetate kinase gene tdcD of strain SW-04, strain SW-05 was obtained. A plasmid expressing sgRNA was constructed using primers sg-poxB-F and sg-poxB-R. The upstream and downstream homologous arms of poxB were amplified using two pairs of primers (△poxB-1 / △poxB-2, △poxB-3 / △poxB-4). The poxB gene was knocked out according to the method in "1.1".

[0056] A plasmid expressing sgRNA was constructed using primers sg-ldhA-F and sg-ldhA-R. The upstream and downstream homologous arms of ldhA were amplified using two pairs of primers (△ldhA-1 / △ldhA-2, △ldhA-3 / △ldhA-4). The ldhA gene was knocked out according to the method in "1.1".

[0057] A plasmid expressing sgRNA was constructed using primers sg-tdcD-F and sg-tdcD-R. The upstream and downstream homologous arms of tdcD were amplified using two pairs of primers (△tdcD-1 / △tdcD-2, △tdcD-3 / △tdcD-4). The tdcD gene was then knocked out according to the method in "1.1".

[0058] Eliminate the pEcgRNA plasmid according to method "1.3".

[0059] 6. Integration source of ydfE at the pseudogene locus in strain SW-05 Clostridium acetobutylicum The glyceraldehyde-3-phosphate dehydrogenase gene gap was extracted and promoted using the gapA promoter to obtain strain SW-06. A plasmid expressing sgRNA (pEcgRNA-ydfE) was constructed using primers sg-ydfE-F and sg-ydfE-R. The upstream and downstream homologous arms of the gap were amplified using two pairs of primers (ydfE-gap-1 / ydfE-gap-2, ydfE-gap-5 / ydfE-gap-6). The gap was amplified by PCR using primers ydfE-gap-3 and ydfE-gap-4. The gene gap was then integrated according to the method in "1.2".

[0060] Eliminate the pEcgRNA plasmid according to method "1.3".

[0061] 7. Integration of the pseudogene site yedN into strain SW-06 relieved the negative feedback inhibition of trpE. fbr The glutamine aminotransferase gene trpD was used as a promoter to obtain strain SW-07. A plasmid expressing sgRNA (pEcgRNA-yedN) was constructed using primers sg-yedN-F and sg-yedN-R. trpE was amplified using two pairs of primers (yedN-trpED-1 / yedN-trpED-2, yedN-trpED-5 / yedN-trpED-6). fbr The upstream and downstream homologous arms of trpD were amplified by PCR using primers yedN-trpED-3 and yedN-trpED-4. fbr And trpD, integrate the gene trpE according to method "1.2". fbr And trpD.

[0062] Eliminate the pEcgRNA plasmid according to method "1.3".

[0063] 8. The phosphoenolpyruvate synthase gene ppsA was integrated into the pseudogene site yjbI of strain SW-07 and promoted using the pbloA promoter to obtain strain SW-08: A plasmid expressing sgRNA (pEcgRNA-yjbI) was constructed using primers sg-yjbI-F and sg-yjbI-R. The upstream and downstream homologous arms of ppsA were amplified using two pairs of primers (yjbI-ppsA-1 / yjbI-ppsA-2, yjbI-ppsA-5 / yjbI-ppsA-6). ppsA was amplified by PCR using primers yjbI-ppsA-3 and yjbI-ppsA-4. The ppsA gene was then integrated according to the method described in "1.2".

[0064] Eliminate the pEcgRNA plasmid according to method "1.3".

[0065] 9. The transketolase A gene tktA was integrated into the pseudogene site yehH of strain SW-08, and the strain was promoted using the pj23114 promoter to obtain strain SW-09: A plasmid expressing sgRNA (pEcgRNA-yehH) was constructed using primers sg-yehH-F and sg-yehH-R. The upstream and downstream homologous arms of tktA were amplified using two pairs of primers (yehH-tktA-1 / yehH-tktA-2, yehH-tktA-5 / yehH-tktA-6). tktA was amplified by PCR using primers yehH-tktA-3 and yehH-tktA-4. The tktA gene was then integrated according to the method described in "1.2".

[0066] Eliminate the pEcgRNA plasmid according to method "1.3".

[0067] 10. The transaldolase B gene talB was integrated into the pseudogene site ilvG of strain SW-09, and the strain was started using the pj23115 promoter to obtain strain SW-10: A plasmid expressing sgRNA (pEcgRNA-ilvG) was constructed using primers sg-ilvG-F and sg-ilvG-R. The upstream and downstream homologous arms of talB were amplified using two pairs of primers (ilvG-talB-1 / 110talB-2, ilvG-talB-5 / ilvG-talB-6). talB was amplified by PCR using primers ilvG-talB-3 and ilvG-talB-4. The gene talB was then integrated according to the method described in "1.2".

[0068] Eliminate the pEcgRNA plasmid according to method "1.3".

[0069] 11. Knock out the pyruvate-formate lyase gene pflB in strain SW-10 and integrate the source at this site. Paraburkholderia acidisoli The aromatic amino acid transporter gene yddGpa was used as a promoter with the Pj23110 promoter to obtain strain SW-11. A plasmid expressing sgRNA (pEcgRNA-pflB) was constructed using primers sg-pflB-F and sg-pflB-R. The upstream and downstream homologous arms of yddGpa were amplified using two pairs of primers (pflB-yddGpa-1 / pflB-yddGpa-2, pflB-yddGpa-5 / pflB-yddGpa-6). yddGpa was amplified by PCR using primers pflB-yddGpa-3 and pflB-yddGpa-4. The gene yddGpa was then integrated according to the method described in "1.2".

[0070] Eliminate the pEcgRNA plasmid according to method "1.3".

[0071] 12. The pseudogene site ycjV of strain SW-11 was integrated into the membrane-binding transmembrane NAD(P)H dehydrogenase gene pntAB, and the strain was generated by using the trc promoter. A plasmid expressing sgRNA (pEcgRNA-ycjV) was constructed using primers sg-ycjv-F and sg-ycjv-R. The upstream and downstream homologous arms of pntAB were amplified using two pairs of primers (ycjv-pntAB-1 / ycjv-pntAB-2, ycjv-pntAB-5 / ycjv-pntAB-6). pntAB was amplified by PCR using primers ycjv-pntAB-3 and ycjv-pntAB-4. The pntAB gene was then integrated according to the method in "1.2".

[0072] Eliminate the pEcgRNA plasmid according to method "1.3".

[0073] 13. Elimination of pEcCas9 plasmid The positive recombinant with pEcgRNA plasmid eliminated was inoculated into LB medium containing 50 g / L sucrose and cultured at 37°C. After two consecutive subcultures, the culture was diluted appropriately and plated onto LB agar plates containing 50 g / L sucrose and cultured at 37°C. Single colonies that grew on LB agar plates containing 50 g / L sucrose but not on kanamycin agar plates were selected and preserved as engineered strain SW-12.

[0074] Example 2 This embodiment provides a method for producing L-tryptophan by fermentation of the engineered strain SW-12 from Example 1 in a 50 L fermenter. The specific steps are as follows: (1) The engineered strain SW-12 was inoculated into LB slant medium for activation. The activated strain was then transferred into a flask and cultured at 37°C for 12 h. (2) The inoculum grown to the logarithmic phase was washed off the flask using sterile physiological saline and inoculated into the seed culture medium in the seed tank. The fermentation broth was controlled at pH 6.9-7.1, dissolved oxygen >20%, and 37℃. The seed culture medium consisted of: 30 g / L glucose, 7 g / L yeast extract, 3 g / L diammonium hydrogen phosphate, 2 g / L potassium chloride, 3 g / L ammonium sulfate, 5 g / L citric acid, 2 g / L magnesium sulfate, 0.5 g / L isoleucine, and 1 mL / L of trace element solution; the solvent was water. The seed culture medium was sterilized at 121℃ for 20 min before use. The trace element solution consisted of: 4.0 g / L cobalt chloride hexahydrate, 0.6 g / L copper sulfate, 6.4 g / L zinc sulfate heptahydrate, 20 g / L sodium sulfate, 4.5 g / L manganese sulfate, and 5 mmol / L H2SO4 as the solvent.

[0075] (3) After the engineered strain SW-12 was cultured in a seed tank for 10 h (OD>25), it was transferred to the fermentation medium in the fermenter at an inoculation rate of 20% of the fermenter's fixed volume. The pH was controlled at 6.7-7.2 by adding ammonia and 80% glucose solution, and the residual sugar concentration was less than 5 g / L. The fermentation time was 45 h. The fermentation medium consisted of: 20 g / L glucose, 7 g / L yeast extract, 4 g / L magnesium sulfate, 4 g / L citric acid, 8 g / L ammonium sulfate, 8 g / L potassium dihydrogen phosphate, 0.2 g / L isoleucine, 0.08 g / L ferrous sulfate, 0.8 g / L methionine, and 1 mL / L of trace element mixture; the solvent was water. The fermentation medium was sterilized at 121℃ for 20 min before use. The trace element solution consists of: 4.0 g / L cobalt chloride hexahydrate, 0.6 g / L copper sulfate, 6.4 g / L zinc sulfate heptahydrate, 20 g / L sodium sulfate, 4.5 g / L manganese sulfate, and 5 mmol / L H2SO4 as the solvent.

[0076] OD600 and L-tryptophan production during fermentation process, as follows Figure 1 As shown, strain SW-12 achieved an L-tryptophan yield of 57.2 g / L in a 50 L fermenter, with a conversion rate of 21.2% and a fermentation period of 45 h.

[0077] Comparative Example 1 This embodiment provides an engineered bacterium, the construction method of which is as follows: Based on the strain SW-03 constructed in Example 1, the tryptophan attenuator gene trpL and the anthranilate synthase gene trpE were knocked out, and the trpE gene, which had been relieved of negative feedback inhibition, was integrated in its original position. fbr The original promoter is replaced with the pj23106 promoter. The construction method is the same as step 4 of Example 1.

[0078] Comparative Example 2 This embodiment provides an engineered bacterium, the construction method of which is as follows: Based on the strain SW-03 constructed in Example 1, the tryptophan attenuator gene trpL and the anthranilate synthase gene trpE were knocked out, and the trpE gene, which had been relieved of negative feedback inhibition, was integrated in its original position. fbr Replace the original promoter with the Ptrc promoter. The construction method is the same as step 4 of Example 1.

[0079] Test Example 1 Using the strain SW-03 constructed in Example 1 and the engineered strains constructed in Comparative Examples 1 and 2, L-tryptophan was produced by fermentation in a 50 L fermenter according to the method in Example 2. After 45 h of fermentation, the L-tryptophan yield in the fermenter inoculated with strain SW-03 was 35.14 g / L, with a conversion rate of 14.84%; the L-tryptophan yield in the fermenter inoculated with the engineered strain of Comparative Example 1 was 23.26 g / L, with a conversion rate of 12.36%; and the L-tryptophan yield in the fermenter inoculated with the engineered strain of Comparative Example 2 was 15.64 g / L, with a conversion rate of 8.96%.

[0080] Comparative Example 3 This embodiment provides an engineered bacterium, the construction method of which is as follows: Based on the strain SW-09 constructed in Example 1, the transaldolase B gene talB was integrated into the pseudogene site ilvG and started using the pj23108 promoter. The construction method is the same as step 10 of Example 1.

[0081] Comparative Example 4 This embodiment provides an engineered bacterium, the construction method of which is as follows: Based on the strain SW-09 constructed in Example 1, the transaldolase B gene talB was integrated into the pseudogene site ilvG and started using the pj23102 promoter. The construction method is the same as step 10 of Example 1.

[0082] Test Example 2 Using the strain SW-09 constructed in Example 1 and the engineered strains constructed in Comparative Examples 3 and 4, L-tryptophan was produced by fermentation in a 50 L fermenter according to the method in Example 2. After 45 h of fermentation, the L-tryptophan yield in the fermenter inoculated with strain SW-09 was 46.36 g / L, with a conversion rate of 18.03%; the L-tryptophan yield in the fermenter inoculated with the engineered strain of Comparative Example 3 was 23.69 g / L, with a conversion rate of 15.64%; and the L-tryptophan yield in the fermenter inoculated with the engineered strain of Comparative Example 4 was 16.65 g / L, with a conversion rate of 11.36%.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered bacterium that produces high levels of L-tryptophan, characterized in that, The engineered bacteria were constructed from *Escherichia coli* strains through genetic modification. The genetic modification included: not expressing LacI, TrpR, TnaA, TnaB, TnaC, PoxB, LdhA, TdcD, PflB, and TrpE; not expressing the trpL gene; and integrating AroG. fbr SerA, TktA, PpsA, TrpE fbr The genes encoding TrpD, TalB, Gap, YddG, and PntAB.

2. The engineered bacteria according to claim 1, characterized in that, The *E. coli* is *E. coli* W3110; and / or The AroG fbr The encoding gene is aroG fbr Its nucleotide sequence is shown in SEQ ID NO.1; and / or The TrpE fbr The encoding gene is trpE fbr Its nucleotide sequence is shown in SEQ ID NO.2; and / or The gene encoding the gap is named gap, and it originates from *Acetobutyricum* (…). Clostridium acetobutylicum ), the nucleotide sequence shown in SEQ ID NO.3; and / or The gene encoding YddG is yddGpa, which originates from Burkholderia spp. (…). Paraburkholderia acidisoli ), the nucleotide sequence shown in SEQ ID NO.4; and / or The method by which LacI and TrpR are not expressed is to knock out their corresponding coding genes and integrate them in situ into the coding gene of SerA; and / or The method for preventing the expression of TnaA, TnaB, TnaC, PoxB, LdhA, TdcD, and TnaC is: knocking out their corresponding coding genes; and / or The method for not expressing PflB is to knock out its corresponding coding gene and integrate the coding gene of YddG in situ.

3. An engineered bacterium SW-12, characterized in that, The engineered bacterium SW-12 was constructed from Escherichia coli W3110 as the starting strain through genetic modification; the genetic modification included: Knock out genes lacI and trpR, and integrate gene serA at the site of gene trpR, using the trc promoter; knock out genes tnaA, tnaB, tnaC, poxB, ldhA, and tdcD; knock out gene pflB, and integrate gene yddGpa at the site, using the Pj23110 promoter; knock out genes trpL and trpE, and integrate gene trpE at the original site. fbr The original promoter was replaced with the Pj23102 promoter; the nucleotide sequence of the gene yddGpa is shown in SEQ ID NO.4; Integrating gene aroG fbr The trc promoter is used to start the integrative gene; the pj23114 promoter is used to start the integrative gene tktA; the pj23114 promoter is used to start the integrative gene ppsA; and the pbolA promoter is used to start the integrative gene trpE. fbr The gene trpD is started using the trc promoter; the integrated gene talB is started using the pj23115 promoter; the integrated gene gap is started using the PgapA promoter; the integrated gene pntAB is started using the Ptrc promoter; and the gene aroG... fbr The nucleotide sequence is shown in SEQ ID NO.1, and the gene trpE is... fbr The nucleotide sequence of the gene gap is shown in SEQ ID NO.2, and the nucleotide sequence of the gene gap is shown in SEQ ID NO.

3.

4. The method for constructing the engineered bacteria SW-12 according to claim 3, characterized in that, Specifically, the following operations are included: Knock out the genes lacI and trpR, and integrate the gene serA at the site of the gene trpR, and start with the trc promoter; Integrating the gene aroG fbr Boot using the aforementioned trc bootloader; Knock out the genes tnaA, tnaB, and tnaC; Knock out the genes trpL and trpE, and integrate the gene trpE in its original position. fbr The original promoter is replaced with the Pj23102 promoter. Knock out the genes poxB, ldhA, and tdcD; Integrate the gene gap and start using the PgapA promoter; Integrating the trpE gene fbr And the trpD gene, started using the trc promoter; Integrate the ppsA gene and start it with the PbolA promoter; Integrate the tktA gene and start it using the pj23114 promoter; The gene talB is integrated and started using the pj23115 promoter; Knock out the gene pflB and integrate the gene yddGpa at that site, and start with the Pj23110 promoter; The pntAB gene is integrated and started using the Ptrc promoter.

5. The construction method according to claim 4, characterized in that, The gene aroG is integrated at the pseudogene locus ygaY. fbr ; and / or Integrate the gene gap at the pseudogene site ydfE; and / or The trpE is integrated at the pseudogene locus yedN. fbr and gene trpD; and / or The gene ppsA is integrated at the pseudogene site yjbI; and / or The gene tktA is integrated at the pseudogene site yehH; and / or The gene talB is integrated at the pseudogene site ilvG; and / or The pntAB gene is integrated at the pseudogene site ycjV.

6. The application of the engineered bacteria according to claim 1 or 2, or the engineered bacteria SW-12 according to claim 3, in the fermentation production of L-tryptophan.

7. A method for producing L-tryptophan by fermentation, characterized in that, The engineered bacteria described in claim 1 or 2, or the engineered bacteria SW-12 described in claim 3, are used as the fermentation bacteria.

8. The method according to claim 7, characterized in that, The method includes the following operations: activating and culturing the engineered bacteria that produce high levels of L-tryptophan or the engineered bacteria SW-12; inoculating the strain that has grown to the logarithmic phase into a seed culture medium for cultivation; controlling the pH of the fermentation broth to be 6.9-7.1, dissolved oxygen >20%, and temperature to be 35-38℃; after culturing for 9-12 h, transferring the strain to a fermentation medium for fermentation; during the fermentation process, controlling the pH to be 6.7-7.2 and the residual sugar concentration to be less than 5 g / L by adding ammonia and glucose; and fermenting for 42-50 h.

9. The method according to claim 8, characterized in that, The seed culture medium consists of: 30 g / L glucose, 7 g / L yeast extract, 3 g / L diammonium hydrogen phosphate, 2 g / L potassium chloride, 3 g / L ammonium sulfate, 5 g / L citric acid, 2 g / L magnesium sulfate, 0.5 g / L isoleucine, and 1 mL / L of trace element mixture; the solvent is water; sterilized at 121°C before use; and / or The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 3.5 g / L magnesium sulfate, 2 g / L citric acid, 3 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 0.05 g / L isoleucine, 0.1 g / L ferrous sulfate, 0.5 g / L methionine, and 1 mL / L of trace element mixture; the solvent is water; and it is sterilized at 121°C before use.

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