Method for improving yield of aromatic or heterocyclic primary amine and reducing accumulation of toxic intermediate aldehyde by dynamic regulation

Abstract

The invention discloses a method for improving the yield of aromatic or heterocyclic primary amine and reducing accumulation of toxic intermediate aldehyde by utilizing dynamic regulation and control, and belongs to the technical field of gene engineering and fermentation engineering. In order to solve the problem that toxic intermediates are easily accumulated due to metabolism imbalance of carboxylic acid reductase ncCAR and transaminase OATA in the biosynthesis process of cinnamyl amine, promoters P4 and Pd which respond to accumulation of cinnamyl aldehyde are used for respectively regulating the expression levels of the carboxylic acid reductase ncCAR and transaminase OATA in the synthesis route of cinnamyl amine; the accumulation level of a toxic intermediate cinnamyl aldehyde is reduced, so that the inhibition of cinnamyl aldehyde on the activity of engineering bacteria is reduced, and the yield of cinnamyl amine is further improved.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and fermentation engineering, and particularly relates to a method for increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes by dynamic regulation. Background Art

[0002] Aromatic ring and aromatic heterocyclic methylamines (MAHs) are important pharmaceutical intermediates and organic synthesis intermediates, and have wide applications in the manufacture of pharmaceuticals, pesticides, polymers, dyes and detergents (Selective Hydrogenation of Nitriles to Primary Amines by using a Cobalt Phosphine Catalyst[J]. Chemsuschem 2017, 10, 842-846.). At present, the industrial synthesis method of MAHs is mainly chemical synthesis. The characteristics of such reactions are the use of noble metal catalysts (Ru, Pd, etc.), organic solvents (hydrazine hydrate, methanol, etc.) or the need for harsh reaction conditions (high temperature or high pressure) and expensive safety protection equipment (Efficient and Mild Reductive Amination of Carbonyl Compounds Catalyzed by Dual-Function Palladium Nanoparticles[J]. ACS Sustainable Chemistry And Engineering, 2020, 8(3): 1618-1626.). This results in high production risks, high production costs, and serious environmental pollution. The environmentally friendly and mild reaction condition biosynthesis method is a good supplement to the chemical synthesis method.

[0003] At present, there is little research on the biosynthesis method of MAHs. Our laboratory previously reported a new biosynthetic pathway for the biosynthesis of MAHs such as 3-(aminomethyl)pyridine, benzylamine, and cinnamylamine using carboxylic acids as substrates and catalyzed by CAR (ncCAR) from Neurospora crassa and ω-TA (OATA) from Ochrobactrum anthropi (Self-sufficient whole-cell biocatalysis for 3-(aminomethyl)pyridine synthesis. Biochemical Engineering Journal 2022, 183.). However, this method has some drawbacks. For example, during the biocatalytic synthesis of MAHs, the metabolic imbalance of the two enzymes easily leads to the accumulation of toxic intermediate aldehydes, which affects cell growth. Moreover, aldehydes are easily reduced by endogenous enzymes to form alcohols or oxidized to form acids, resulting in a decrease in the yield and production of MAHs.

[0004] Dynamic regulation refers to specific biological recognition elements that regulate the expression level of downstream proteins by responding to changes in the intracellular and extracellular environments, causing the expression level of the proteins to change with the environment. Dynamic regulation is divided into three categories: promoters responsive to metabolites, transcriptional regulators responsive to metabolites, and quorum sensing responsive to cell density. Among them, there are many reports on using promoters responsive to metabolites to regulate intracellular metabolite levels. For example, using a promoter responsive to vanillin (an intermediate in the catechol synthesis process) to dynamically regulate the expression levels of the enzymes required in the catechol synthesis pathway and the vanillin channel protein, resulting in a 40% increase in the yield of catechol (Toward engineering E. coli with an autoregulatory system for lignin valorization. PNAS 2018, 115, 2970-2975.); using a promoter responsive to farnesyl pyrophosphate (FPP, an intermediate in the ergosterol synthesis process) to dynamically regulate the expression levels of the enzymes required in the amorpha-4,11-diene synthesis pathway, doubling the yield of the product amorpha-4,11-diene (Engineering dynamic pathway regulation using stress-response promoters. Nat Biotechnol 2013, 31, 1039-46.). These reports indicate that dynamic regulation is a good method to solve the accumulation of intracellular metabolites and improve the product yield.

[0005] Therefore, taking the biosynthesis of cinnamylamine as an example, the promoter responsive to cinnamaldehyde obtained by identification is used to regulate the expression levels of two enzymes required in the cinnamylamine synthesis pathway in real time, and realizing the dynamic regulation of cinnamylamine biosynthesis will be of great significance. Summary of the Invention

[0006] In order to solve the problem that the imbalance of carboxylic acid reductase ncCAR and transaminase OATA in the biosynthesis of cinnamylamine easily leads to the accumulation of toxic intermediates, the present invention uses a promoter responsive to the accumulation of cinnamaldehyde to regulate the expression levels of enzymes in the cinnamylamine synthesis pathway, reduce the accumulation level of the toxic intermediate cinnamaldehyde, so as to reduce the inhibition of the activity of engineering bacteria by cinnamaldehyde, and further improve the yield of cinnamylamine.

[0007] To achieve the above technical effects, the present invention provides the following technical solutions:

[0008] The first object of the present invention is to provide a method for increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes by dynamic regulation. The method uses Escherichia coli RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-oata as a template, replaces the promoter P of the oata gene with the promoter P4 T7 , to obtain strain S004, and then uses strain S004 as a template to replace the promoter P of the nccar gene with the promoter P d , to obtain strain S006, and perform shake-flask fermentation on strain S006 to produce MAHs; the promoter P4 is the first 300 bp upstream of the gene corresponding to the antibiotic resistance protein marR, and the nucleotide sequence is as shown in SEQ ID NO.1, and the promoter P T7 is the first 300 bp upstream of the gene corresponding to the DNA-binding transcriptional activator gadE, and the nucleotide sequence is as shown in SEQ ID NO.2. d

[0009] In one embodiment of the present invention, the construction method of the Escherichia coli RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-oata can be found in the article published by this laboratory before (Yuan S, Jin M, Xu C, et al. Self-sufficient whole-cell biocatalysis for 3-(aminomethyl)pyridine synthesis[J]. Biochemical Engineering Journal, 2022(183-):183.).

[0010] In one embodiment of the present invention, the method of replacing the promoter P of the oata gene with the promoter P4 T7 is to replace the P of oata in the plasmid pACYCDuet1-oata with the responsive promoter P4 T7 , LacO and RBS sequences, and the restriction enzyme sites are SmaI and NcoI.

[0011] In one embodiment of the present invention, the method of replacing the promoter P d of the nccar gene with the promoter P T7 is to replace the P of nccar in pET28a-nccar-pptase with the responsive promoter P d , LacO and RBS sequences, and the restriction enzyme sites are BglII and NcoI. T7

[0012] In one embodiment of the present invention, the nccar is a carboxylic acid reductase gene derived from Neurospora crassa; the pptase is a phosphopantetheine transferase gene derived from Escherichia coli; the oata is a transaminase gene derived from Ochrobactrum anthropi.

[0013] In one embodiment of the present invention, the GeneBank ID of the carboxylic acid reductase gene nccar is XM_950727.2; the GeneBank ID of the phosphopantetheine transferase gene pptase is CP024090.1; the GeneBank ID of the transaminase gene oata is CP000758.1.

[0014] In one embodiment of the present invention, the S004 is RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-P4-oata, and the S006 is RARE(DE3) / pET28a-P d -nccar-pptase / pACYCDuet1-P4-oata.

[0015] The second object of the present invention is to provide a method for constructing an engineered bacterium capable of increasing the production of MAHs and reducing the accumulation of toxic intermediate aldehydes. The construction method uses Escherichia coli RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-oata as a template, replaces the promoter P of the oata gene with the promoter P4 T7 , lacO and RBS to obtain the strain S004, and then uses the strain S004 as a template to use the promoter P d ​Replace the promoter P of the nccar gene T7 , lacO and RBS to obtain the engineered bacterium S006; the promoter P4 is the first 300 bp upstream of the gene corresponding to the antibiotic resistance protein marR, and its nucleotide sequence is as shown in SEQ ID NO.1. The promoter P d is the first 300 bp upstream of the gene corresponding to the DNA-binding transcriptional activator gadE, and its nucleotide sequence is as shown in SEQ ID NO.2.

[0016] In one embodiment of the present invention, the construction method of the Escherichia coli RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-oata can be referred to the article previously published by this laboratory (Yuan S, Jin M, Xu C, et al. Self-sufficient whole-cell biocatalysis for 3-(aminomethyl)pyridine synthesis[J]. Biochemical Engineering Journal, 2022(183-):183.).

[0017] In one embodiment of the present invention, the method of replacing the promoter P of the oata gene with the promoter P4 T7 is to replace the P T7 , LacO and RBS sequences of oata in the plasmid pACYCDuet1-oata with the responsive promoter P4, and the restriction enzyme sites are SmaI and NcoI.

[0018] In one embodiment of the present invention, the method of replacing the promoter P of the nccar gene with the promoter P d is to replace the P T7 , LacO and RBS sequences of nccar in pET28a-nccar-pptase with the responsive promoter P d , and the restriction enzyme sites are BglII and NcoI. T7

[0019] In one embodiment of the present invention, the nccar is the carboxylic acid reductase gene, derived from Neurospora crassa; the pptase is the phosphopantetheine transferase gene, derived from Escherichia coli; the oata is the transaminase gene, derived from Ochrobactrum anthropi.

[0020] ​In one embodiment of the present invention, the GeneBank ID of the carboxylic acid reductase gene nccar is XM_950727.2; the GeneBank ID of the phosphopantetheine transferase gene pptase is CP024090.1; the GeneBank ID of the transaminase gene oata is CP000758.1.

[0021] In one embodiment of the present invention, the S004 is RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-P4-oata, and the S006 is RARE(DE3) / pET28a-P d -nccar-pptase / pACYCDuet1-P4-oata.

[0022] The third object of the present invention is to provide an engineered bacterium obtained by the above construction method.

[0023] The fourth object of the present invention is to provide the application of the above-mentioned engineered bacterium in increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes.

[0024] Advantages of the present invention:

[0025] For the first time, the common problem of the accumulation of toxic intermediate aldehydes is solved by dynamic regulation, and the yield of MAHs is increased.

[0026] Taking the biosynthesis of cinnamylamine as an example, the present invention identifies the promoter responsive to cinnamaldehyde, and adjusts the expression levels of two enzymes (carboxylic acid reductase ncCAR and transaminase OATA) required in the cinnamylamine synthesis pathway in real time. Using the P4 and P d promoters, the dynamic regulation of cinnamylamine biosynthesis is achieved. For the first time, the common problem of the accumulation of toxic intermediate aldehydes is solved by a universal dynamic regulation, and the yield of MAHs is increased, making the amine / aldehyde reach 5.8 times that of the wild-type strain (WT). Description of the drawings

[0027] Figure 1 It is the relationship between cinnamaldehyde concentration and the expression level of the target enzyme; among them, Figure 1 a in is the protein expression level of ncCAR induced by different concentrations (0 mM, 0.1 mM, 0.2 mM, 0.35 mM, 0.5 mM, 0.7 mM, 0.85 mM, 1 mM) of cinnamaldehyde, Figure 1 b in is the protein expression level of OATA induced by different concentrations (0 mM, 0.2 mM, 0.5 mM, 0.75 mM, 1 mM) of cinnamaldehyde, Figure 1 c in is calculated by software Figure 1The proportion of ancCAR in a in Figure 1 d in is calculated by software Figure 1 The proportion of OATA in b in ;

[0028] Figure 2 is P4 / P d Results graph of the effects on the yields of cinnamylamine, aldehyde, and alcohol; among them, Figure 2 a in is P4 / P d Graph of the effects on the yield of cinnamylamine and the accumulation of cinnamaldehyde, Figure 2 b in is Figure 2 Graph of the relationship between the accumulation of cinnamaldehyde in a in and the OD600 nm of the strain;

[0029] Figure 3 Results graph of the effects of promoter strength changes on the yields of cinnamylamine and the accumulation of cinnamaldehyde; among them, Figure 3 a in is the results graph of screening constitutive promoters similar in strength to P4, Figure 3 b in is screening for P d Results graph of screening constitutive promoters similar in strength, Figure 3 c in is the results graph of the effects of constitutive promoters similar in strength to Pd or P4 on the yields of cinnamylamine and the accumulation of cinnamaldehyde. Detailed implementation manners

[0030] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with specific implementation manners and the accompanying drawings of the specification. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in this field, and those skilled in the art can obtain them through commercial channels without special instructions.

[0031] The abbreviations involved in the present invention are as follows:

[0032] Aromatic or heterocyclic primary amines: MAHs;

[0033] Carboxylic acid reductase from Neurospora crassa: ncCAR;

[0034] Transaminase from Ochrobactrum anthropi: OATA;

[0035] Phosphopantetheine transferase from Escherichia coli: PPTase;

[0036] Green fluorescent protein gene: vgfp;

[0037] Isopropyl β-D-thiogalactoside: IPTG;

[0038] Chloramphenicol: Cm;

[0039] Kanamycin: Kan;

[0040] Escherichia coli: E. coli.

[0041] The definitions involved in the present invention are as follows:

[0042] "Overexpression" means that after a specific gene in a cell is regulated by various signals, its expression level in an organism exceeds the original level, which can be achieved by enhancing endogenous expression or introducing exogenous genes.

[0043] E. coli RARE(DE3)(S000) used in the present invention: MG1655(DE3)ΔdkgBΔyeaEΔ(yqhC - dkgA)ΔyahKΔyjgB, purchased from Addgene, USA.

[0044] pACYCDuet1 - P4 - vgfp, pET28a - P d - vgfp, pACYCDuet1 - P4 - oata, pET28a - P d - nccar - pptase sequence information can be found in the sequence listing.

[0045] The plasmid extraction kit used in the present invention was purchased from OMEGA, USA, and the operation steps were carried out according to the product instruction manual. The PBS buffer solution used in the present invention was purchased from Nanjing Novoprotein Scientific Co., Ltd.

[0046] All media in the present invention were prepared with deionized water unless otherwise specified.

[0047] LB medium formula: yeast powder 5 g / L, NaCl 10 g / L, peptone 10 g / L, supplemented with chloramphenicol 50 μg / mL during inoculation.

[0048] The recombinant plasmids pACYCDuet1 - oata and pET28a - nccar - pptase used in the present invention are disclosed in the following literature: Yuan S, Jin M, Xu C, et al. Self - sufficient whole - cell biocatalysis for 3 - (aminomethyl)pyridine synthesis[J]. Biochemical Engineering Journal, 2022(183 -):183.

[0049] Example 1: Method for increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes by dynamic regulation

[0050] (1) Use the up-regulated promoter P4 responsive to cinnamaldehyde as the promoter of the oata gene to construct the recombinant plasmid pACYCDuet1-P4-oata, and transfer it into the RARE(DE3) competent cells (strain S000) to obtain strain S001. Use the down-regulated promoter P d , as the promoter of the nccar gene, to construct the recombinant plasmid pET28a-P d -nccar-pptase, and transfer it into the RARE(DE3) competent cells (strain S000) to obtain strain S002.

[0051] The method for constructing the recombinant plasmid pACYCDuet1-P4-oata is to first construct the recombinant plasmid pACYCDuet1-oata; then replace the P T7 , LacO and RBS sequences of oata in the plasmid pACYCDuet1-oata with the responsive promoter P4, and the restriction enzyme sites are SmaI and NcoI to obtain the plasmid pACYCDuet1-P4-oata.

[0052] The recombinant plasmid pET28a-P d -nccar-pptase is constructed by first constructing the recombinant plasmid pET28a-nccar-pptase; then replacing the P d of nccar in pET28a-nccar-pptase with the responsive promoter P T7 , LacO and RBS sequences, and the restriction enzyme sites are BglII and NcoI to obtain the plasmid pET28a-P d -nccar-pptase.

[0053] The above-mentioned nccar is the carboxylic acid reductase gene, derived from Neurospora crassa, and the GeneBank ID is XM_950727.2; the above-mentioned pptase is the phosphopantetheine transferase gene, derived from Escherichia coli, and the GeneBank ID is CP024090.1; the above-mentioned oata is the transaminase gene, derived from Ochrobactrum anthropi, and the GeneBank ID is CP000758.1.

[0054] The above-mentioned S001 is the strain RARE(DE3) / pACYCDuet1-P4-oata, and the above-mentioned S002 is the strain RARE(DE3) / pET28a-P d -nccar-pptase.

[0055] The above-upregulated promoter P4 is the first 300 bp upstream of the gene corresponding to the antibiotic resistance protein marR, and its nucleotide sequence is shown in SEQ ID NO.1. The above-downregulated promoter P d is the first 300 bp upstream of the gene corresponding to the DNA-binding transcriptional activator gadE, and its nucleotide sequence is shown in SEQ ID NO.2. The NCBI accession Nos. of marR and gadE are 945825 and 948023, respectively.

[0056] The nucleotide sequence of pACYCDuet1-P4-oata is shown in SEQ ID NO.3;

[0057] pET28a-P d -nccar-pptase has a nucleotide sequence shown in SEQ ID NO.4.

[0058] (2) SDS-PAGE analysis of the relationship between cinnamaldehyde concentration and OATA expression level in S001 and the relationship between cinnamaldehyde concentration and ncCAR expression level in S002. The specific analysis method is as follows:

[0059] ① Activation: Separate single colonies of the strains S001 and S002 obtained in step (1) and inoculate them into LB medium. Incubate at 37 °C and 220 rpm overnight for activation;

[0060] ② Inoculation: Take 0.2 mL of the seed solution obtained in ① and inoculate it into 20 mL of LB medium (containing 50 μg / mL Kan or 34 μg / mL Cm). Incubate at 37 °C and 220 rpm;

[0061] ③ Induction: When the OD 600nm reaches 0.5 - 0.7, add cinnamaldehyde at different concentrations for induction. The induction temperature is 37 °C;

[0062] ④ Sampling: After 2.5 h of induction, collect the bacterial cells and wash them once with PBS buffer. Lyse the cells by high-pressure homogenization, centrifuge at 10000 rpm and 4 °C for 20 min to obtain the lysate supernatant.

[0063] ⑤ Detect the expression levels of ncCAR or OATA in the two supernatants by SDS-PAGE, and analyze whether the cinnamaldehyde concentration is proportional to the ncCAR or OATA expression level.

[0064] The SDS-PAGE results are shown in Figure 1 , when the cinnamaldehyde concentration is 0 - 1 mM, the expression level of ncCAR is proportional to the cinnamaldehyde concentration ( Figure 1 a in); when the cinnamaldehyde concentration is 0 - 0.75 mM, the expression level of OATA is proportional to the cinnamaldehyde concentration (Figure 1 in b). This indicates that when the intracellular cinnamaldehyde concentration is 0 - 0.75 mM, P4 / P d can linearly regulate the expression levels of ncCAR and OATA according to the cinnamaldehyde concentration. The software Image lab can calculate the proportion of the target protein in the total protein based on the gray value of the SDS-PAGE gel (see Figure 1 in c and Figure 1 in d).

[0065] (III) Effect of P4 / P d on cinnamylamine production

[0066] (1) Effects of P d and P4 on cinnamylamine production and cinnamaldehyde accumulation

[0067] Co-transformed pACYCDuet1-oata and pET28a-nccar-pptase constructed in step (I) into S000 competent cells to obtain strain S003 (RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-oata). Co-transformed pACYCDuet1-P4-oata and pET28a-nccar-pptase constructed in step (I) into S000 competent cells to obtain strain S004 (RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-P4-oata).

[0068] Detected the cinnamylamine production and cinnamaldehyde accumulation of strains S000 and S004 after 6 h of shake-flask culture. Specific shake-flask culture process: First, picked a single colony of the strain into LB medium containing 50 μg / mL Kan and 34 μg / mL Cm, cultured overnight at 37 °C with 200 rpm; transferred the seed culture into a 250 mL shake flask containing 50 mL LB medium (containing 50 μg / mL Kan and 34 μg / mL Cm), cultured at 37 °C with 200 rpm; added 0.2 mM IPTG when OD 600nm grew to 0.5 - 0.7, and continued to culture at 37 °C with 200 rpm for 6 h. The results showed that the cinnamylamine production of S004 was 1.8 times that of WT (0.58 mM), the aldehyde accumulation hardly changed, and the amine / aldehyde was 2.2 times that of WT, indicating that P4 played a role in promoting the conversion of aldehyde to amine ( Figure 2 in a). However, since the promoter of ncCAR was still P T7 , the conversion of acid to aldehyde did not decrease, resulting in no significant change in the cinnamaldehyde accumulation. In addition, since the aldehyde accumulation did not decrease, the OD 600nm of S004 after 4 h of reaction was as low as the OD 600nm of S003.

[0069] Subsequently, using strain S003 as a template, with P d replacing the P T7 of the nccar gene, strain S005 (RARE(DE3) / pET28a-P d -nccar-pptase / pACYCDuet1-oata) was obtained. The cinnamylamine production and cinnamaldehyde accumulation of S003 and S005 after 6 h of shake-flask culture were detected. The results showed that the cinnamylamine production of S005 was 1.8 times that of S003, the aldehyde accumulation decreased by 75%, and the amine / aldehyde was 9 times that of WT ([[]] Figure 2 a) in [[[]]]. This indicates that P d plays a role in reducing the conversion of acid to aldehyde. In addition, due to the reduced aldehyde accumulation, the OD 600nm of strain S005 reached 2.4, significantly higher than that of WT (1.4), which enabled the cinnamylamine production of S005 to reach 1.8 times that of WT.

[0070] Finally, using strain S004 as a template, with P d simultaneously replacing the P T7 of the nccar gene, strain S006 (RARE(DE3) / pET28a-P d -nccar-pptase / pACYCDuet1-P4-oata) was obtained. The cinnamylamine production and cinnamaldehyde accumulation of strains S003 and S006 after 6 h of shake-flask culture were detected. The results showed that the cinnamylamine production was 2.9 times that of S003 (0.75 mM), the aldehyde accumulation decreased by 50%, and the amine / aldehyde was 5.8 times that of S003 ([[]] Figure 2 a) in [[[]]]. Compared with S003, P4 and P d in S006 play a role in promoting the conversion of aldehyde to amine and reducing the conversion of acid to aldehyde, respectively; in addition, since the aldehyde accumulation of S006 is only 50% of that of S003, the OD 600nm of S006 reaches 2.0, which is beneficial to the conversion of cinnamic acid to cinnamylamine.

[0071] During the process of synthesizing cinnamylamine by shake-flask culture of strains S003, S004, S005, and S006, the accumulated cinnamaldehyde concentration is proportional to the OD 600nm ([[]] Figure 2 b) in [[[]]], further proving that the low OD 600nm of strain WT is due to the excessive accumulation of toxic cinnamaldehyde.

[0072] The specific steps of the method for detecting the effects of P d , P4 on cinnamylamine production and cinnamaldehyde accumulation are as follows:

[0073] ① Activation: Strains S003, S004, S005, and S006 were separately picked and inoculated into LB medium (containing 50 μg / mL Kan and 34 μg / mL Cm), and activated overnight at 37 °C with 220 rpm;

[0074] ② Inoculation: 0.5 mL of the seed solution was separately inoculated into 50 mL of LB medium (containing 50 μg / mL Kan and 34 μg / mL Cm), and cultured at 37 °C with 220 rpm;

[0075] ③ Induction: When the strain grew to OD 600nm = 0.5 - 0.7, 0.2 mM IPTG was added, along with 3 mM cinnamic acid and 6 mM L-Ala, and induced at 37 °C;

[0076] ④ Sampling: Samples were taken 2, 4, and 6 h after induction, and the filter membrane was filtered.

[0077] ⑤ HPLC was used to detect the concentrations of cinnamylamine, aldehyde, and alcohol.

[0078] (2) Excluding the increase in yield due to the change in promoter strength

[0079] Since the change in promoter strength can sometimes also increase the product concentration, therefore, the present invention needs to prove that the increase in amine / aldehyde of strain S006 (RARE(DE3) / pET28a - P d -nccar-pptase / pACYCDuet1-P4-oata) is due to the response of P4 / P d to cinnamaldehyde, rather than due to the change in promoter strength.

[0080] First, constitutive promoters with similar strength to P d / P4 were detected. P4 in strain S007 (RARE(DE3) / pACYCDuet1-P4-vgfp) was replaced with 5 constitutive promoters (P cons#2 、P cons#4 、P cons#5 、P cons#6 and P cons#7 ) respectively to obtain strains S008, S009, S010, S011, and S012. P d in strain S013 (RARE(DE3) / pET28a-P d -vgfp) was replaced with 5 constitutive promoters (P cons#3 、P cons#4 、P cons#5 、P cons#6 and P cons#7 ) respectively to obtain strains S014, S015, S016, S017, and S018.

[0081] The vgfp gene sequence is shown in SEQ ID NO.5.

[0082] The complete sequence of the pACYCDuet1-P4-vgfp recombinant plasmid is shown in SEQ ID NO.6;

[0083] pET28a-P d -vgfp recombinant plasmid complete sequence is shown in SEQ ID NO.7.

[0084] The nucleotide sequences of the constitutive promoters P const#1 、P const#2 、P const#3 、P cons#4 、P cons#5 、P cons#6 and P cons#7 are shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 respectively.

[0085] Subsequently, the fluorescence intensities of strains S007 and S008-S012 were compared to find a constitutive promoter with a fluorescence intensity similar to that of P4. The results showed that P const#2 had a fluorescence intensity similar to that of induced P4 ( Figure 3 a)). The fluorescence intensities of strains S013 and S014-S018 were compared to find a constitutive promoter with a fluorescence intensity similar to that of P d . The results showed that P const#5 had a fluorescence intensity similar to that of induced P d ( Figure 3 b)). In summary, the constitutive promoter with a fluorescence intensity similar to that of induced P4 is P const#2 , and the constitutive promoter with a fluorescence intensity similar to that of induced P d is P const#5 .

[0086] Finally, P4 in strain S006 was replaced with P const#2 , and P d was replaced with P const#5 to obtain strain S019. The cinnamylamine production and cinnamaldehyde accumulation of strain S019 and strain S006 were compared. The results showed that the cinnamylamine production in S019 was much lower than that in S006, and the cinnamaldehyde accumulation was as high as 1.2 mM ( Figure 3 c), which ruled out that the increase in cinnamylamine production or the decrease in cinnamaldehyde accumulation was due to the change in promoter strength.

[0087] The data listed in this example are all average values of multiple repeated experiments.

[0088] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes by dynamic regulation, characterized in that, Using E. coli RARE(DE3) / pET28a-nccar-pptase / pACYCDuet1-oata as template, the promoter P of the oata gene was replaced by the promoter P4. T7 , strain S004 was obtained, and then strain S004 was used as a template and promoter P d Replace the promoter P of the nccar gene T7 , strain S006 is obtained, and the strain S006 is subjected to shake flask fermentation to produce aromatic or heterocyclic primary amines; the promoter P4 is the first 300 bp upstream of the gene corresponding to the antibiotic resistance protein marR, and the nucleotide sequence is shown in SEQ ID NO.1, and the promoter P d It is the first 300 bp upstream of the gene corresponding to the DNA binding transcription activator gadE, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, Replacing the promoter P of the oata gene with the promoter P4 T7 The method is to replace the P of oata in the plasmid pACYCDuet1-oata with the responsive promoter P4 T7 , LacO and RBS sequences, and the restriction enzyme sites are SmaI and NcoI.

3. The method according to claim 1, characterized in that, The promoter P d to replace the promoter P of the nccar gene T7 is a method using a responsive promoter P d to replace the P of nccar in pET28a - nccar - pptase T7 , LacO and RBS sequences, and the restriction enzyme sites are BglII and NcoI.

4. The method according to claim 1, characterized in that, The nccar is a carboxylic acid reductase gene, derived from Neurospora crassa, with GeneBank ID XM_950727.2; the pptase is a phosphopantetheinyl transferase gene, derived from Escherichia coli, with GeneBank ID CP024090.1; the oata is a transaminase gene, derived from Ochrobactrum anthropi, with GeneBank ID CP000758.

1.

5. A method for constructing an engineered bacterium capable of increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes, characterized in that, Using E. coli RARE (DE3) / pET28a-nccar-pptase / pACYCDuet-oata as template, the promoter P of the oata gene was replaced by the promoter P4. T7 , strain S004 was obtained, and then strain S004 was used as a template and promoter P d Replace the promoter P of the nccar gene T7 , and obtain the engineered bacteria S006; the promoter P4 is the first 300 bp upstream of the gene corresponding to the antibiotic resistance protein marR, and the nucleotide sequence is shown in SEQ ID NO.

1. d It is the first 300 bp upstream of the gene corresponding to the DNA binding transcription activator gadE, and the nucleotide sequence is shown in SEQ ID NO.

2.

6. The method according to claim 5, characterized in that, The method of replacing the promoter P of the oata gene with the promoter P4 T7 is to replace the P of oata in the plasmid pACYCDuet1-oata with the responsive promoter P4 T7 , LacO and RBS sequences, and the restriction enzyme sites are SmaI and NcoI.

7. The method according to claim 5, characterized in that, The promoter P d is replaced with the promoter P of the nccar gene T7 by a method that uses the responsive promoter P d to replace the P of nccar in pET28a - nccar - pptase T7 , LacO and the RBS sequence, and the restriction enzyme sites are BglII and NcoI.

8. The method according to claim 5, characterized in that, The nccar is a carboxylic acid reductase gene, derived from Neurospora crassa, with GeneBank ID XM_950727.2; the pptase is a phosphopantetheinyl transferase gene, derived from Escherichia coli, with GeneBank ID CP024090.1; the oata is a transaminase gene, derived from Ochrobactrum anthropi, with GeneBank ID CP000758.

1.

9. An engineered bacterium obtained by the construction method according to any one of claims 5 - 8.

10. Use of the engineered bacterium according to claim 9 in increasing the yield of aromatic or heterocyclic primary amines and reducing the accumulation of toxic intermediate aldehydes.

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