An engineered bacterium with high yield of l-tryptophan, a construction method and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to an engineered bacterium for high-yield L-tryptophan and a construction method and application thereof. The engineered bacterium provided by the application is constructed by gene modification based on an Escherichia coli as a starting strain, can produce L-tryptophan at a high yield, has a high conversion rate, a short fermentation cycle, and stable genetic traits, and does not need to add an inducer and an antibiotic in a fermentation production process, so that the fermentation process is simplified, and the engineered bacterium is more suitable for industrialized production of L-tryptophan.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an engineered bacterium for high-yield production of L-tryptophan as well as a construction method and application thereof. BACKGROUND

[0002] Tryptophan is an amino acid containing a chiral carbon atom, and the naturally occurring form is mainly L-tryptophan. L-tryptophan is widely used in the fields of medicine, food, feed and the like.

[0003] The production methods of L-tryptophan mainly include chemical synthesis, enzymatic conversion and microbial fermentation. Among them, the microbial fermentation method is dominant due to low cost and strong sustainability, and the commonly used strains are Escherichia coli and Corynebacterium glutamicum, which uses glucose and the like as a carbon source in a microbial fermentation manner. Through metabolic engineering optimization, such as relieving feedback inhibition (aroG, trpE), overexpressing key enzymes (ppsA, tktA), blocking competitive pathways (pheA, tyrA) and modifying transport systems (yddG overexpression), the yield of L-tryptophan can be improved. However, the synthesis pathway of L-tryptophan is long, the key enzymes are more, and the feedback mechanism is complex, resulting in low yield and conversion rate of L-tryptophan produced by microbial fermentation method at present, and long fermentation period. According to the literature, the yield of the E. coli engineering strain in 48 hours of fermentation in a shake flask is about 5.1 g / L, and the conversion rate is about 0.180 g / g of glucose; in a 3 L bioreactor, the total yield is about 43.0 g / L (conversion rate 0.180 g / g). The yield of C. glutamicum in 80 hours of fed-batch fermentation can reach 58 g / L (conversion rate 0.17 g / g). And additional antibiotics and inducers need to be added during the fermentation process. SUMMARY

[0004] In view of the above problems, the present application provides an engineered bacterium for high-yield production of L-tryptophan as well as a construction method and application thereof. The engineered bacterium can produce high-yield L-tryptophan, has high glucose conversion rate, short fermentation period, stable genetic traits, does not need to add inducers and antibiotics during the fermentation production process, and is suitable for industrial production of L-tryptophan.

[0005] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0006] 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).

[0007] 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.

[0008] Preferably, the Escherichia coli is Escherichia coli W3110.

[0009] Preferably, the AroG fbr The encoding gene is aroG fbr Its nucleotide sequence is shown in SEQ ID NO.1.

[0010] Preferably, the TrpE fbr The encoding gene is trpE fbr Its nucleotide sequence is shown in SEQ ID NO.2.

[0011] Preferably, the gene encoding the gap is gap, which originates from *Acetobutyrobacter* (…). Clostridium acetobutylicum The nucleotide sequence is shown in SEQ ID NO.3.

[0012] Preferably, the gene encoding YddG is yddGpa, which originates from Burkholderia spp. (…).Paraburkholderia acidisoli ), the nucleotide sequence of SEQ ID NO. 4.

[0013] Preferably, the method of not expressing the LacI and TrpR is knocking out the corresponding coding genes and integrating the coding gene of the SerA in situ.

[0014] Preferably, the method of not expressing the TnaA, TnaB, TnaC, PoxB, LdhA, TdcD is knocking out the corresponding coding genes.

[0015] Preferably, the method of not expressing the PflB is knocking out the corresponding coding gene and integrating the coding gene of the YddG in situ.

[0016] The second aspect of the present application provides an engineered bacterium SW-12, which is obtained by genetic modification of the E. coli W3110 as a starting strain; the genetic modification comprises:

[0017] knocking out the genes lacI and trpR and integrating the gene serA at the site of the gene trpR, which is activated by the trc promoter; knocking out the genes tnaA, tnaB, tnaC, poxB, ldhA and tdcD; knocking out the gene pflB and integrating the gene yddGpa at the site, which is activated by the Pj23110 promoter; knocking out the genes trpL and trpE and integrating the gene trpE at the original site fbr , which is replaced by the Pj23102 promoter; the nucleotide sequence of the gene yddGpa is shown in SEQ ID NO. 4;

[0018] integrating the gene aroG fbr , which is activated by the trc promoter; integrating the gene tktA, which is activated by the pj23114 promoter; integrating the gene ppsA, which is activated by the PbolA promoter; integrating the gene trpE fbr and the gene trpD, which is activated by the trc promoter; integrating the gene talB, which is activated by the pj23115 promoter; integrating the gene gap, which is activated by the PgapA promoter; integrating the gene pntAB, which is activated by the Ptrc promoter; the nucleotide sequence of the gene aroG fbr is shown in SEQ ID NO. 1, the nucleotide sequence of the gene trpE fbr is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene gap is shown in SEQ ID NO. 3.

[0019] wherein the “original promoter” is the wild-type promoter that the corresponding gene carries by itself.

[0020] The engineering bacteria SW-12 overexpresses the enzymes beneficial to the synthesis of L-tryptophan through the above gene modification, and cuts off the competition and decomposition pathway, thereby improving the yield and conversion rate of tryptophan. For example, the genes tnaA, tnaB and tnaC are knocked out to prevent L-tryptophan from being transported into cells and further decomposed into indole; the glycerol-3-phosphate dehydrogenase gene gap from the source of P. acetobutylicum is introduced to enhance the glycolysis pathway and improve the growth and metabolism ability of the strain; the pbolA promoter is used to dynamically regulate the expression of the ppsA gene, so that the ppsA gene is basically not expressed in the logarithmic growth phase, after entering the stable phase, the activity of the pbolA promoter rapidly increases, the expression level of the ppsA gene is increased by about 10-20 times, the metabolic pressure of the engineering bacteria in the growth process is reduced, the supply of the precursor PEP in the stable phase is improved, and the yield and conversion rate of L-tryptophan are improved; meanwhile, the aromatic amino acid transporter gene yddGpa from the source of Paraburkholderia acidisoli The aromatic amino acid transporter gene yddGpa from the source of

[0021] The third aspect of the present application provides a construction method of the above engineering bacteria SW-12, which specifically comprises the following operations:

[0022] The genes lacI and trpR are knocked out, and the gene serA is integrated at the site of the gene trpR and started by the trc promoter;

[0023] The gene aroG is integrated fbr and started by the trc promoter;

[0024] The genes tnaA, tnaB and tnaC are knocked out;

[0025] The genes trpL and trpE are knocked out, and the gene trpE is integrated at the original position fbr and the original promoter is replaced by the Pj23102 promoter;

[0026] The genes poxB, ldhA and tdcD are knocked out;

[0027] The gene gap is integrated and started by the PgapA promoter;

[0028] The genes trpE fbr and trpD are integrated and started by the trc promoter;

[0029] The gene ppsA is integrated and started by the PbolA promoter;

[0030] The gene tktA is integrated and started by the pj23114 promoter;

[0031] integrating the gene talB, which is initiated by the pj23115 promoter;

[0032] knocking out the gene pflB and integrating the gene yddGpa at the site, which is initiated by the Pj23110 promoter;

[0033] integrating the gene pntAB, which is initiated by the Ptrc promoter.

[0034] The present application does not limit the order of the above-mentioned knocking out and integrating, and the above-mentioned knocking out or integrating of the genome of E. coli W3110 in any order can achieve the purpose of the present application and are within the protection scope of the present application.

[0035] Further preferably, the gene aroG is integrated at the pseudogene site ygaY fbr .

[0036] Further preferably, the gene gap is integrated at the pseudogene site ydfE.

[0037] Further preferably, the gene trpE fbr and the gene trpD are integrated at the pseudogene site yedN.

[0038] Further preferably, the gene ppsA is integrated at the pseudogene site yjbI.

[0039] Further preferably, the gene tktA is integrated at the pseudogene site yehH.

[0040] Further preferably, the gene talB is integrated at the pseudogene site ilvG.

[0041] Further preferably, the gene pntAB is integrated at the pseudogene site ycjV.

[0042] The fourth aspect of the present application provides the use of the above-mentioned high-yield L-tryptophan engineering bacteria or the above-mentioned engineering bacteria SW-12 in the fermentation production of L-tryptophan.

[0043] The fifth aspect of the present application provides a method for the fermentation production of L-tryptophan, which uses the above-mentioned high-yield L-tryptophan engineering bacteria or the above-mentioned engineering bacteria SW-12 as the fermentation bacteria.

[0044] Preferably, the method comprises the following operations:

[0045] The high L-tryptophan yield engineering bacteria or the engineering bacteria SW-12 are activated and cultured, and the bacteria grown to logarithmic phase are inoculated into a seed culture medium for culture, with the pH of the fermentation liquor controlled at 6.9-7.1, the dissolved oxygen >20%, and the temperature at 35-38℃; after 9-12 h of culture, the bacteria are inoculated into a fermentation culture medium for fermentation, with the pH controlled at 6.7-7.2 by adding ammonia water and glucose during the fermentation, the residual sugar concentration less than 5 g / L, and the fermentation time 42-50 h.

[0046] Optionally, the activation and culture are performed by inoculating the bacteria into an LB slant medium for activation, and the activated bacteria are inoculated into a flask for culture at 37℃ for 10-12 h.

[0047] Preferably, the seed culture medium comprises 30 g / L glucose, 7 g / L yeast powder, 3 g / L diammonium 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, with water as the solvent and sterilization at 121℃ before use.

[0048] Preferably, the fermentation culture medium comprises 20 g / L glucose, 5 g / L yeast powder, 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, with water as the solvent and sterilization at 121℃ before use. The addition of L-methionine and L-isoleucine in the fermentation culture medium can reduce the lag phase of the bacteria SW-12 in the fermentation tank, further shorten the fermentation cycle, and improve the conversion rate.

[0049] Further preferably, the trace element mixture in the seed culture medium and the fermentation culture medium comprises 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, and 4.5 g / L manganese sulfate, with 5 mmol / L H2SO4 as the solvent.

[0050] The application has the beneficial effects that the application provides an engineered bacterium with high yield of L-tryptophan, and specifically provides an engineered bacterium SW-12. The engineered bacterium provided by the application is constructed by modifying the genome of an Escherichia coli as a starting strain, and all operations are performed on the genome without plasmid in the construction process, so that the genetic traits of the constructed engineered bacterium are stable, and no antibiotics and inducers need to be added in the fermentation process. Experiments prove that the production of L-tryptophan by the engineered bacterium provided by the application has high yield and high conversion rate, and the fermentation cycle is short. The application also provides a method for fermenting and producing L-tryptophan by the engineered bacterium, and the method can produce L-tryptophan with higher yield and conversion rate, and further shorten the fermentation cycle. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The fermentation process of the engineered bacterium SW-12 in Example 2 in a 50L fermenter. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described in detail below in combination with specific examples. It should be understood that the specific examples described herein are only used to explain the application and not to limit the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0053] At present, the production of L-tryptophan is mainly based on microbial fermentation, and Escherichia coli is a commonly used strain. In Escherichia coli, the synthesis of L-tryptophan mainly involves three modules: central carbon metabolic pathway, shikimic acid pathway and chorismate pathway. Due to the long synthesis pathway of L-tryptophan, the large number of key enzymes and the complex feedback mechanism, and the fact that the engineered bacterium usually depends on exogenous plasmid, most of the existing L-tryptophan production strains on the market have the disadvantages of low yield, low conversion rate, the need for additional addition of antibiotics and inducers, and long fermentation cycle.

[0054] The application provides an engineered bacterium with high yield of L-tryptophan, which is constructed by genetic modification of Escherichia coli as a starting strain; the genetic modification includes: not expressing LacI, TrpR, TnaA, TnaB, TnaC, PoxB, LdhA, TdcD, PflB and TrpE; not expressing the gene trpL; integrating expression of AroG fbr , SerA, TktA, PpsA, TrpE fbrThe genes of TrpD, TalB, Gap, YddG and PntAB are deleted from the genome of the E. coli W3110, and the genes of TrpD, TalB, Gap, YddG and PntAB are inserted into the genome of the E. coli W3110, so that the engineered bacterium can produce L-tryptophan at a high yield, has a high glucose conversion rate, a short fermentation cycle, and stable genetic traits, and does not need to add an inducer and an antibiotic during the fermentation production process, and has an application potential in the industrial production of L-tryptophan.

[0055] The embodiment of the present application further provides an engineered bacterium SW-12 and a construction method thereof.

[0056] The embodiment of the present application further provides a method for producing L-tryptophan by fermentation using the engineered bacterium.

[0057] The scheme of the present application is described below through specific embodiments.

[0058] The reagents or instruments used in the following embodiments are all conventional commercially available products obtained from a commercial channel, unless otherwise specified. The methods used in the following embodiments are all conventional methods in the art, unless otherwise specified.

[0059] Embodiment 1

[0060] The embodiment provides an engineered bacterium SW-12 and a construction method thereof.

[0061] The primer sequences used in the construction process are shown in Table 1.

[0062] Table 1 Primers involved in the construction process of the strain

[0063]

[0064] The specific construction process of the engineered bacterium SW-12 is as follows:

[0065] 1. The genes lacI and trpR are knocked out from the genome of the E. coli W3110 as a starting strain, and the gene serA encoding D-3-phosphoglycerate dehydrogenase is integrated at the trpR site, and the trc promoter is used to obtain the strain SW-01:

[0066] Deletion or integration of chromosome was performed by CRISPR-Cas9 system according to the method described in the reference (Li Q i, Sun B, Chen J, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli [J]. Acta Biochimica et Biophysica Sinica, 2021, 53(5): 620-627.).

[0067] 1.1 Knockout gene lacI

[0068] A pair of primers with 20 bp complementary sequences (sg-lacI-F and sg-lacI-R listed in Table 1) were designed using 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 deletion of the target gene, two pairs of primers (lacI-1 / lacI-2, lacI-3 / lacI-4) were used to amplify the upstream and downstream homologous arms of lacI (~500 bp for each group) by PCR. 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 electrotransformation competent cells carrying plasmid pEcCas9. L-arabinose was used to induce the expression of lambda-Red recombinase. After electrotransformation, the bacteria were spread on LB plates containing kanamycin sulfate and tetracycline hydrochloride resistance and incubated at 37°C overnight. Positive colonies were verified by colony PCR, and then the knockout of the target gene lacI was confirmed by DNA sequencing.

[0069] 1.2 Knockout gene trpR and integrate gene serA at trpR site

[0070] The plasmid expressing sgRNA (pEcgRNA-trpR) was constructed using reverse PCR with primers sg-trpR-F and sg-trpR-R. The upstream and downstream homologous arms of serA (~500 bp each) were amplified by PCR using two pairs of primers (trpR-serA-1 / trpR-serA-2, trpR-serA-5 / trpR-serA-6) and serA was amplified by PCR using primers trpR-serA-3 and trpR-serA-4. The donor DNA fragment (DNA-serA) was constructed by overlap PCR of the two homologous arms and the target gene serA, which was integrated into the target site trpR. pEcgRNA-trpR and DNA-serA were co-transformed into E. coli electrocompetent cells carrying plasmid pEcCas9. Expression of lambda-Red recombinase was induced by L-arabinose. After electroporation, the bacteria were spread on LB plates containing kanamycin and tetracycline hydrochloride resistance and incubated at 37°C overnight. Positive colonies were verified by colony PCR and then confirmed by DNA sequencing whether the target gene was integrated.

[0071] 1.3 Elimination of pEcgRNA plasmid

[0072] The positive recombinants were inoculated into LB medium containing rhamnose and kanamycin and incubated at 37°C, and subcultured three times in succession, then diluted and spread on plates containing kanamycin and incubated at 37°C. LB plates containing tetracycline and kanamycin were spotted, and single colonies that grew on the kanamycin plates but not on the tetracycline plates were selected. Strain SW-01 was obtained.

[0073] 2. Integration of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene aroG, which is relieved of negative feedback inhibition, at the pseudogene site ygaY of strain SW-01 fbr with the trc promoter to obtain strain SW-02:

[0074] The plasmid expressing sgRNA (pEcgRNA-ygaY) was constructed using primers sg-ygaY-F and sg-ygaY-R, and the upstream and downstream homologous arms of aroG were amplified using two pairs of primers (ygay-aroG-1 / ygay-aroG-2, ygay-aroG-5 / ygay-aroG-6) and aroG was amplified by PCR using primers ygay-aroG-3 and ygay-aroG-4 according to the method of “1.2”, and the gene aroG was integrated. fbr fbr fbr

[0075] ​​​3. Knock out the tryptophanase gene tnaA, tryptophan permease gene tnaB, tnaC of strain SW-02 to prevent L-tryptophan from being transported into the cell and further decomposed into indole, and obtain strain SW-03:

[0076] Construct an sgRNA expression plasmid using primers sg-tnaCAB-F and sg-tnaCAB-R, amplify the upstream and downstream homologous arms of tnaA, tnaB, and tnaC using two pairs of primers (△tnaCAB-1 / △tnaCAB-2, △tnaCAB-3 / △tnaCAB-4), and knock out the genes tna, tnaB, and tnaC according to the method of “1.1”.

[0077] Eliminate the pEcgRNA plasmid according to the method of “1.3”.

[0078] 4. Knock out the tryptophan attenuator gene trpL and anthranilate synthase gene trpE of strain SW-03, and integrate the trpE that has been relieved of negative feedback inhibition at the original site fbr , obtain strain SW-04 by replacing the original promoter with the Pj23102 promoter:

[0079] Construct an sgRNA expression plasmid (pEcgRNA-trpE) using primers sg-trpE-F and sg-trpE-R, amplify the upstream and downstream homologous arms of trpE using two pairs of primers (trpo-1 / trpo23102-2, trpo-5 / trpo-6), and amplify the target fragment trpE using two pairs of primers (trpo-3 / trpo-4) fbr . Integrate the gene trpE according to the method of “1.2” fbr .

[0080] Eliminate the pEcgRNA plasmid according to the method of “1.3”.

[0081] 5. Knock out the pyruvate oxidase gene poxB, L-lactate dehydrogenase gene ldhA, and propionic acid kinase / acetic acid kinase gene tdcD of strain SW-04, and obtain strain SW-05:

[0082] Construct an sgRNA expression plasmid using primers sg-poxB-F and sg-poxB-R, amplify the upstream and downstream homologous arms of poxB using two pairs of primers (△poxB-1 / △poxB-2, △poxB-3 / △poxB-4), and knock out the gene poxB according to the method of “1.1”.

[0083] The 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), and the gene ldhA was knocked out according to the method of "1.1".

[0084] The 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), and the gene tdcD was knocked out according to the method of "1.1".

[0085] The pEcgRNA plasmid was eliminated according to the method of "1.3".

[0086] 6. Integrating the glycerolaldehyde-3-phosphate dehydrogenase gene gap from Clostridium acetobutylicum Escherichia coli K-12 MG1655 at the pseudogene site ydfE of strain SW-05, which is driven by the gapA promoter, to obtain strain SW-06:

[0087] The plasmid expressing sgRNA (pEcgRNA-ydfE) was constructed using primers sg-ydfE-F and sg-ydfE-R, the upstream and downstream homologous arms of gap were amplified using two pairs of primers (ydfE-gap-1 / ydfE-gap-2, ydfE-gap-5 / ydfE-gap-6), and the gene gap was amplified by PCR using primers ydfE-gap-3 and ydfE-gap-4, and then integrated according to the method of "1.2".

[0088] The pEcgRNA plasmid was eliminated according to the method of "1.3".

[0089] 7. Integrating the tryptophan aminotransferase and glutamine amide transferase genes trpE fbr and trpD, which are relieved of negative feedback inhibition, at the pseudogene site yedN of strain SW-06, which is driven by the trc promoter, to obtain strain SW-07:

[0090] The plasmid expressing sgRNA (pEcgRNA-yedN) was constructed using primers sg-yedN-F and sg-yedN-R, the upstream and downstream homologous arms of trpE fbr and trpD were amplified using two pairs of primers (yedN-trpED-1 / yedN-trpED-2, yedN-trpED-5 / yedN-trpED-6), and the genes trpE fbr and trpD were amplified by PCR using primers yedN-trpED-3 and yedN-trpED-4, and then integrated according to the method of "1.2". fbrand trpD.

[0091] Eliminate pEcgRNA plasmid by the method of "1.3".

[0092] 8. Integrate phosphoenolpyruvate synthase gene ppsA with pbloA promoter in the pseudogene site yjbI of strain SW-07 to obtain strain SW-08:

[0093] Construct sgRNA expression plasmid (pEcgRNA-yjbI) using primers sg-yjbI-F and sg-yjbI-R, use two pairs of primers (yjbI-ppsA-1 / yjbI-ppsA-2, yjbI-ppsA-5 / yjbI-ppsA-6) to amplify the upstream and downstream homologous arms of ppsA, use primers yjbI-ppsA-3 and yjbI-ppsA-4 to amplify ppsA by PCR, and integrate gene ppsA by the method of "1.2".

[0094] Eliminate pEcgRNA plasmid by the method of "1.3".

[0095] 9. Integrate transketolase A gene tktA with pj23114 promoter in the pseudogene site yehH of strain SW-08 to obtain strain SW-09:

[0096] Construct sgRNA expression plasmid (pEcgRNA-yehH) using primers sg-yehH-F and sg-yehH-R, use two pairs of primers (yehH-tktA-1 / yehH-tktA-2, yehH-tktA-5 / yehH-tktA-6) to amplify the upstream and downstream homologous arms of tktA, use primers yehH-tktA-3 and yehH-tktA-4 to amplify tktA by PCR, and integrate gene tktA by the method of "1.2".

[0097] Eliminate pEcgRNA plasmid by the method of "1.3".

[0098] 10. Integrate transaldolase B gene talB with pj23115 promoter in the pseudogene site ilvG of strain SW-09 to obtain strain SW-10:

[0099] 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".

[0100] Eliminate the pEcgRNA plasmid according to method "1.3".

[0101] 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.

[0102] 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".

[0103] Eliminate the pEcgRNA plasmid according to method "1.3".

[0104] 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: strain SW-12 was obtained.

[0105] 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".

[0106] Eliminate the pEcgRNA plasmid according to method "1.3".

[0107] 13. Elimination of pEcCas9 plasmid

[0108] The positive recombinants eliminating pEcgRNA plasmid were inoculated into LB medium containing 50 g / L sucrose and cultured at 37°C, subcultured twice, diluted appropriately and then plated on LB plates containing 50 g / L sucrose and cultured at 37°C. Single colonies growing on the LB plates containing 50 g / L sucrose but not growing on the LB plates containing kanamycin were selected, which were the engineering bacteria SW-12.

[0109] Example 2

[0110] This example provides a method for producing L-tryptophan by fermentation of the engineering bacteria SW-12 of Example 1 in a 50 L fermenter, and the specific steps are as follows:

[0111] (1) The engineering bacteria SW-12 strain was inoculated in LB slant medium for activation, and the activated bacteria were transferred to a flask and cultured at 37°C for 12 h.

[0112] (2) The bacteria grown to the logarithmic phase were washed from the flask with sterile normal saline and inoculated into the seed medium in a seed tank, the pH of the fermentation broth was controlled at 6.9-7.1, the dissolved oxygen was >20%, and the temperature was 37°C. The composition of the seed medium was: 30 g / L glucose, 7 g / L yeast powder, 3 g / L diammonium 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 was water. The seed medium was sterilized at 121°C for 20 min before use. The composition of the trace element solution was: 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 was 5 mmol / L H2SO4.

[0113] (3) The engineering bacteria SW-12 is inoculated into the fermentation medium in the fermenter after 10 hours (OD>25) of seed tank culture, and the inoculation amount is 20% of the constant volume of the fermenter. The pH is controlled at 6.7-7.2 by adding ammonia water and 80% glucose solution, and the residual sugar concentration is less than 5 g / L. The fermentation time is 45 hours. The composition of the fermentation medium is: 20 g / L glucose, 7 g / L yeast powder, 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 mixed solution; the solvent is water. The fermentation medium is sterilized at 121°C for 20 minutes before use. The composition of the trace element solution is: 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.

[0114] The OD600 and L-tryptophan yield of the fermentation process are shown in Figure 1 The L-tryptophan yield of strain SW-12 reaches 57.2 g / L in a 50 L fermenter, the conversion rate is 21.2%, and the fermentation period is 45 hours.

[0115] Comparative Example 1

[0116] This example provides an engineering bacteria, and the construction method is as follows:

[0117] On the basis of the strain SW-03 constructed in Example 1, the tryptophan attenuator gene trpL and the anthranilate synthase gene trpE are knocked out, and the trpE that is relieved of negative feedback inhibition is integrated at the original position fbr , and the original promoter is replaced with the pj23106 promoter. The construction method is the same as step 4 of Example 1.

[0118] Comparative Example 2

[0119] This example provides an engineering bacteria, and the construction method is as follows:

[0120] On the basis of the strain SW-03 constructed in Example 1, the tryptophan attenuator gene trpL and the anthranilate synthase gene trpE are knocked out, and the trpE that is relieved of negative feedback inhibition is integrated at the original position fbr , and the original promoter is replaced with the Ptrc promoter. The construction method is the same as step 4 of Example 1.

[0121] Test Example 1

[0122] The strain SW-03 constructed in Example 1 and the engineering bacteria constructed in Comparative Example 1 and Comparative Example 2 were respectively fermented in a 50 L fermenter to produce L-tryptophan according to the method of Example 2. After 45 h of fermentation, the L-tryptophan yield in the fermenter inoculated with the strain SW-03 was 35.14 g / L, and the conversion rate was 14.84%; the L-tryptophan yield in the fermenter inoculated with the engineering bacteria of Comparative Example 1 was 23.26 g / L, and the conversion rate was 12.36%; and the L-tryptophan yield in the fermenter inoculated with the engineering bacteria of Comparative Example 2 was 15.64 g / L, and the conversion rate was 8.96%.

[0123] Comparative Example 3

[0124] The present example provides an engineering bacteria, and the construction method thereof is as follows:

[0125] On the basis of the strain SW-09 constructed in Example 1, the transaldolase B gene talB was integrated into the pseudogene site ilvG, and the pj23108 promoter was used for starting. The construction method was the same as step 10 of Example 1.

[0126] Comparative Example 4

[0127] The present example provides an engineering bacteria, and the construction method thereof is as follows:

[0128] On the basis of the strain SW-09 constructed in Example 1, the transaldolase B gene talB was integrated into the pseudogene site ilvG, and the pj23102 promoter was used for starting. The construction method was the same as step 10 of Example 1.

[0129] Test Example 2

[0130] The strain SW-09 constructed in Example 1 and the engineering bacteria constructed in Comparative Example 3 and Comparative Example 4 were respectively fermented in a 50 L fermenter to produce L-tryptophan according to the method of Example 2. After 45 h of fermentation, the L-tryptophan yield in the fermenter inoculated with the strain SW-09 was 46.36 g / L, and the conversion rate was 18.03%; the L-tryptophan yield in the fermenter inoculated with the engineering bacteria of Comparative Example 3 was 23.69 g / L, and the conversion rate was 15.64%; and the L-tryptophan yield in the fermenter inoculated with the engineering bacteria of Comparative Example 4 was 16.65 g / L, and the conversion rate was 11.36%.

[0131] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An engineered bacterium with high L-tryptophan production, characterized in that, The engineering bacteria are constructed by gene modification based on Escherichia coli as a starting strain, and the gene modification comprises: not expressing LacI, TrpR, TnaA, TnaB, TnaC, PoxB, LdhA, TdcD, PflB and TrpE; not expressing gene trpL; integrating and expressing coding genes of AroG fbr , SerA, TktA, PpsA, TrpE fbr , TrpD, TalB, Gap, YddG and PntAB. The E. coli is E. coli W3110; The AroG fbr The coding gene of AroG is aroG fbr The nucleotide sequence of which is SEQ ID NO.

1. The TrpE fbr encoding gene is trpE fbr the nucleotide sequence of which is represented by SEQ ID NO. 2; The coding gene of the Gap is gap, which is derived from Klebsiella pneumoniae (ATCC 13882) Clostridium acetobutylicum , and the nucleotide sequence is shown in SEQ ID NO.

3. The gene encoding YddG is yddGpa, which originates from Burkholderia spp. (…). Paraburkholderia acidisoli The nucleotide sequence is shown in SEQ ID NO.4; The method for not expressing the LacI and TrpR is knocking out the corresponding coding genes and integrating the coding gene of the SerA in situ; The method for not expressing the TnaA, TnaB, TnaC, PoxB, LdhA and TdcD is knocking out the corresponding coding genes; The method for not expressing the PflB is knocking out the corresponding coding gene and integrating the coding gene of the YddG in situ.

2. An engineered bacterium SW-12, characterized in that, The engineering bacteria SW-12 is constructed by genetic modification based on the E. coli W3110 as a starting strain; the genetic modification is as follows: knocking out the genes lacI and trpR and integrating the gene serA at the site of the gene trpR, activated with the trc promoter; knocking out the genes tnaA, tnaB, tnaC, poxB, ldhA and tdcD; knocking out the gene pflB and integrating the gene yddGpa at this site, activated with the Pj23110 promoter; knocking out the genes trpL and trpE and integrating the gene trpE in the original position fbr the nucleotide sequence of the gene yddGpa is represented in SEQ ID NO. 4; Integrated gene aroG fbr with trc promoter; integrated gene tktA with pj23114 promoter; integrated gene ppsA with PbolA promoter; integrated gene trpE with PgapA promoter fbr and gene trpD with trc promoter; integrated gene talB with pj23115 promoter; integrated gene gap with PgapA promoter; integrated gene pntAB with Ptrc promoter; the nucleotide sequence of the gene aroG fbr is shown in SEQ ID NO. 1, the nucleotide sequence of the gene trpE fbr is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene gap is shown in SEQ ID NO.

3.

3. The method for constructing the engineered bacteria SW-12 according to claim 2, characterized in that, Specifically comprising the following operations: Knocking out the genes lacI and trpR and integrating the gene serA at the site of the gene trpR, which is activated by the trc promoter; integration of the gene aroG fbr with the trc promoter; Knocking out the genes tnaA, tnaB and tnaC; knocking out the gene trpL and the gene trpE and integrating the gene trpE in its original position fbr replacing the original promoter by the Pj23102 promoter; Knocking out the genes poxB, ldhA and tdcD; Integrating the gene gap, which is activated by the PgapA promoter; integrate the gene trpE fbr and the gene trpD, with the trc promoter; Integrating the gene ppsA, which is activated by the PbolA promoter; Integrating the gene tktA, which is activated by the pj23114 promoter; Integrating the gene talB, which is activated by the pj23115 promoter; Knocking out the gene pflB and integrating the gene yddGpa at the site, which is activated by the Pj23110 promoter; Integrating the gene pntAB, which is activated by the Ptrc promoter.

4. The construction method according to claim 3, characterized in that, Integrate the gene aroG at the pseudogene site ygaY fbr ; Integrating the gene gap at the pseudogene site ydfE; In the pseudogene site yedN integrate the trpE fbr and the gene trpD; Integrating the gene ppsA at the pseudogene site yjbI; Integrating the gene tktA at the pseudogene site yehH; Integrating the gene talB at the pseudogene site ilvG; Integrating the gene pntAB at the pseudogene site ycjV.

5. The application of the engineering bacteria of claim 1 or the engineering bacteria SW-12 of claim 2 in the fermentation production of L-tryptophan.

6. A method for the fermentative production of L-tryptophan, characterized in that, The engineering bacteria of claim 1 or the engineering bacteria SW-12 of claim 2 is used as a fermentation bacteria.

7. The method of claim 6, wherein, The method comprises the following operations: activating the engineering bacteria with high yield of L-tryptophan or the engineering bacteria SW-12, culturing, inoculating the bacteria grown to the logarithmic phase into a seed culture medium for culture, controlling the pH of the fermentation liquor to be 6.9-7.1, the dissolved oxygen to be >20%, and the temperature to be 35-38℃; after 9-12 h of culture, inoculating into a fermentation medium for fermentation, and controlling the pH to be 6.7-7.2 by adding ammonia water and glucose in the fermentation process, the residual sugar concentration to be less than 5g / L, and the fermentation time to be 42-50 h.

8. The method of claim 7, wherein, The components of the seed culture medium are: 30 g / L glucose, 7 g / L yeast powder, 3 g / L diammonium 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, 1 mL / L trace element mixture; the solvent is water; sterilized at 121°C before use; and / or The components of the fermentation medium are: 20 g / L glucose, 5 g / L yeast powder, 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, 1 mL / L trace element mixture; the solvent is water; sterilized at 121°C before use.

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