CN112877270A - Genetic engineering bacterium for producing hydroxyl tetrahydropyrimidine and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to construction and application of genetic engineering bacteria for producing hydroxyl tetrahydropyrimidine. The method strengthens key genes in a hydroxyl tetrahydropyrimidine synthesis path on a genome, weakens a hydroxyl tetrahydropyrimidine competitive inhibition path, constructs the hydroxyl tetrahydropyrimidine synthesis path, overexpresses an isocitrate dehydrogenase coding gene icd to increase the supply of a precursor alpha-ketoglutaric acid, dynamically regulates the supply of the precursor alpha-ketoglutaric acid through an esaI/esaR quorum sensing circuit strategy, and automatically regulates the metabolism of the alpha-ketoglutaric acid in a tricarboxylic acid cycle according to cell density to obtain the hydroxyl tetrahydropyrimidine high-yielding strain. The constructed strain can realize the high-efficiency synthesis of hydroxyl tetrahydropyrimidine under the fermentation condition without adding alpha-ketoglutaric acid, does not accumulate byproduct tetrahydropyrimidine, and has important industrial application value.

Description

Genetic engineering bacterium for producing hydroxyl tetrahydropyrimidine and application thereof

The technical field is as follows:

the invention belongs to the technical field of genetic engineering, and particularly relates to construction and application of genetic engineering bacteria for producing hydroxyl tetrahydropyrimidine.

Background art:

hydroxytetrahydropyrimidine ((4S,5S) -5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a hydroxylated derivative of tetrahydropyrimidine and is considered to be one of the most widely distributed compatible solutes. Due to their special biological properties, they are distinguished from other compatible solutes, such as their suitability for increasing the thermostability of proteins and cells, making them economically interesting targets.

The hydroxy tetrahydropyrimidines are used to stabilize macromolecules and whole cells, stabilize in vitro enzyme activity, promote in vivo protein folding, protect molecules and cells against freeze-thaw cycles, and promote resistance to desiccation, oxidation, and temperature stress protectants. In addition, the hydroxyl tetrahydropyrimidine is an excellent drying protective agent and has good vitrification performance. In addition, the hydroxy tetrahydropyrimidine-stabilized retroviral vectors for gene therapy, which allow long-term storage of the retroviral vectors, are potential gene therapy tools. The properties of hydroxy tetrahydropyrimidines, which enhance resistance to damage by cells and DNA from ionizing radiation and ultraviolet light and prevent UV-induced damage to skin cells, have prompted the widespread development of hydroxy tetrahydropyrimidines in skin care products.

At present, the production method of the hydroxyl tetrahydropyrimidine is mainly realized by a 'bacterial milking' process, namely Halomonas elongata is cultured under high salt concentration, and the large-scale synthesis of the hydroxyl tetrahydropyrimidine under high salt concentration and the quick release of the hydroxyl tetrahydropyrimidine under low salt concentration are realized by continuously increasing and reducing the salt concentration of fermentation liquor. The process has high requirements on equipment and energy consumption, in addition, the high-salt environment is not favorable for the growth of thalli, the production period is long, the synthesis efficiency of a target product is low, the product is usually a mixture of hydroxyl tetrahydropyrimidine and tetrahydropyrimidine, and the product needs to be further purified by adopting a chromatographic separation technology. Therefore, the yield of the hydroxyl tetrahydropyrimidine is low, the cost is high, and the market price is very expensive. The problems of high byproduct content, high cost and low yield seriously restrict the industrial production and application of the hydroxyl tetrahydropyrimidine, and the construction of the genetic engineering strain for efficiently synthesizing a single product has urgent need.

In order to obtain a high-efficiency production strain of the hydroxyl tetrahydropyrimidine, the invention takes Escherichia coli W3110 as a cell chassis and constructs a high-efficiency synthetic route of the hydroxyl tetrahydropyrimidine through rational design. Meanwhile, aiming at the problems of insufficient supply of alpha-ketoglutaric acid, uncoordinated TCA circulation and product synthesis and the like in the synthesis of the hydroxyl tetrahydropyrimidine, the invention constructs an escherichia coli genetic engineering bacterium for automatically carrying out dynamic regulation and control on the synthesis of the hydroxyl tetrahydropyrimidine based on cell density by utilizing an esaI/esaR quorum sensing system. The constructed strain can realize the high-efficiency synthesis of hydroxyl tetrahydropyrimidine under the fermentation condition without adding alpha-ketoglutaric acid, does not accumulate byproduct tetrahydropyrimidine, and has important industrial application value.

The invention content is as follows:

aiming at the problems, the invention aims to provide a genetic engineering bacterium for producing hydroxyl tetrahydropyrimidine, construction and application thereof, and a corresponding fermentation process control scheme is formulated, so that the invention has a good industrial application prospect.

The technical scheme of the invention is summarized as follows:

the invention provides an escherichia coli genetic engineering bacterium for producing hydroxyl tetrahydropyrimidine, which integrates a promoter P with xylose inducible on a host genome_xylFRNA polymerase of controlled T7 phage; integrating single copies from P_T7Ectoine synthase gene cluster ectABC of Halomonas elongata controlled by promoter; single copy by P_T7Promoter-controlled mutant gene lysC of aspartokinase from Corynebacterium glutamicum_cgl(G1A, C932T); double copy by P_T7A promoter-controlled tetrahydropyrimidine hydroxylase gene ectD from Halomonas elongata; knocking out a hydroxyl tetrahydropyrimidine competitive branch metabolism related gene thrA; replacing phosphoenolpyruvate carboxylase coding gene ppc promoter with P_trcA promoter to increase the supply of oxaloacetate; introducing a glucose metabolism transcription repression factor mutation mlc reported in a literature to replace the original mlc to relieve the glucose repression, and constructing a hydroxyl tetrahydropyrimidine synthesis pathway; increasing the copy number of the isocitrate dehydrogenase encoding gene icd to increase the supply of alpha-ketoglutarate, a precursor required for hydroxytetrahydropyrimidine; integration of constitutive promoter P from BioFAB library_BIOFABPromoter-controlled transcriptional regulator esaR170V replacing the original promoter of the key gene of the alpha-ketoglutarate catabolic pathway, the alpha-ketoglutarate dehydrogenase gene sucA, with P_esaSThe promoter is integrated with a promoter with proper strength to control a signal molecule acyl homoserine lactone synthase gene esaI, and further increases the accumulation of precursor substance alpha-ketoglutaric acid.

Preferably, the promoter controlling the easI gene is P_bs1、P_bs2、P_bs3Or P_bs4A promoter;

more preferably, the promoter controlling the easI gene is P_bs3A promoter;

the coding gene P_bs3The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 12;

the nucleotide sequence of the T7RNA polymerase gene is shown as a sequence table SEQ ID NO. 1;

the nucleotide sequence of the sugar metabolism transcription repression factor mutation mlc is shown in a sequence table SEQ ID NO. 2;

the ectoine synthase gene cluster ectABC is derived from Halomonas elongata (Halomonas elongata) DSM 2581, and the nucleotide sequence is shown as a sequence table SEQ ID NO. 3;

the aspartokinase mutant gene lysC_cglThe nucleotide sequence is shown in a sequence table SEQ ID NO. 4;

the ectoine hydroxylase gene ectD is derived from Halomonas elongata (H.elongata) DSM 2581, and the nucleotide sequence is shown as a sequence table SEQ ID NO. 5;

the isocitrate dehydrogenase coding gene icd is derived from escherichia coli (E.coli) W3110, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 6;

the coding gene P_BIOFABThe nucleotide sequence of the promoter is shown as a sequence table SEQ ID NO. 7;

the transcription regulator esaR170V is derived from Pantoea stewartii (Pantoea stewartia) DC 283, and the nucleotide sequence is shown as the sequence table SEQ ID NO. 8;

the coding gene P_esaSThe promoter is derived from Pantoea stewartii (P.stewartia) DC 283, and the nucleotide sequence is shown as the sequence table SEQ ID NO. 9;

the coding gene P_bs1The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 10;

the coding gene P_bs2The nucleotide sequence of the promoter is shown in a sequence table SEQ ID NO. 11;

the coding gene P_bs4The nucleotide sequence of the promoter is shown as a sequence table SEQ ID NO. 13;

the acyl homoserine lactone synthase gene esaI is derived from Pantoea stewartii (P.stewartia) DC 283, and the nucleotide sequence is shown as the sequence table SEQ ID NO. 14.

Preferably, the genetically engineered bacterium takes escherichia coli e.

The invention also provides a construction method of the genetic engineering bacteria for producing the hydroxyl tetrahydropyrimidine, which comprises the following steps:

the E.coli W3110 is directionally transformed by using a CRISPR/Cas9 mediated gene editing technology, and the method specifically comprises the following steps:

(1) integration of xylose promoter P at the lacIZ Gene site_xylFControlled T7RNA polymerase (the nucleotide sequence is SEQ ID NO.1 of the sequence table).

(2) Constructing a hydroxyl tetrahydropyrimidine synthetic pathway. Firstly, the aspartokinase Gene thrA (Gene ID:945803) is knocked out to weaken the competitive inhibition of hydroxyl tetrahydropyrimidine, and the phosphoenolpyruvate carboxylase coding Gene ppc (Gene ID:948457) promoter is replaced by P_trcThe promoter was introduced into the literature to increase the supply of oxaloacetate as an aspartate precursor (Nakashima N, Tamura T.A new carbon carbohydrate expression mutation of Escherichia coli, mlc, and its use for producing isocutanol. journal of Bioscience&Bioengineering,2012,114(1):38-44.) reported sugar metabolism transcription repression factor mutation mlc (nucleotide sequence of SEQ ID NO.2) replaces original mlc to relieve glucose repression. While integrating at the pseudogenic site ybeM (Gene ID:4056041) with P_T7Ectoine synthetase gene cluster ectABC (nucleotide sequence is shown in sequence table SEQ ID NO.3) controlled by promoter; integration by P at the pseudogene site yghX (Gene ID:2847694)_T7Promoter-controlled mutant aspartate kinase gene lysC reported in the literature (Becker J, Zelder O, Hfner S, et al, from zero to homo-design-based systems for metabolic Engineering of Corynebacterium glutamicum for L-lysine production, 2011,13(2):159-168.)_cgl(the nucleotide sequence is SEQ ID NO.4 of the sequence table); at the Gene site trpR (Gene I)D:948917) and the genetic locus aceA (Gene ID:948517) by P_T7A tetrahydropyrimidine hydroxylase gene ectD (the nucleotide sequence is shown in a sequence table SEQ ID NO.5) controlled by a promoter.

(3) Enhance the accumulation of the precursor substance alpha-ketoglutaric acid. The isocitrate dehydrogenase encoding Gene icd (nucleotide sequence SEQ ID NO.6 of the sequence Listing) controlled by its own promoter was integrated at the pseudogene site yeEL (Gene ID: 2847764).

(4) Dynamically regulating the activity of alpha-ketoglutarate dehydrogenase. The pseudo Gene locus yjiP (Gene ID:38094982) integrated with the Gene encoded by P_BIOFABThe transcription regulator esaR170V (the nucleotide sequence is SEQ ID NO.8) controlled by a promoter (the nucleotide sequence is SEQ ID NO.7 of the sequence table, the number is apFAB104) replaces the promoter of an alpha-ketoglutarate dehydrogenase Gene sucA (Gene ID:945303) which is a key Gene of an alpha-ketoglutarate catabolism pathway by P_esaSThe promoter (nucleotide sequence is SEQ ID NO.9 of the sequence table) integrates different strengths (P) at the pseudogene site yjiT (Gene ID:945056)_bs1＜P_bs2＜P_bs3＜P_bs4) P of_bsThe accumulation of precursor substance alpha-ketoglutaric acid is further increased by a signal molecule acyl homoserine lactone synthase gene esaI (the nucleotide sequence is SEQ ID NO.14) controlled by a promoter (the nucleotide sequence is SEQ ID NO.10, 11, 12 and 13).

The invention also provides a method for producing the hydroxyl tetrahydropyrimidine by using the genetic engineering bacteria through shake flask fermentation:

(1) strain activation: taking the dipped bacterial liquid by an inoculating loop in a sterile operation room, densely marking in a solid inclined plane activation culture medium, culturing in an incubator at 37 ℃ for 10-12h, and marking on an activation inclined plane for subculturing for 10-12 h.

(2) Seed bottle culture: scraping a ring of thallus by using an inoculating ring in a sterile operation room, inoculating the thallus into a 500mL triangular flask filled with 30mL seed culture medium, sealing the flask by using twelve layers of gauze, and carrying out shake culture for 10-12h under the conditions of 35-37 ℃ and 240r/min under 180-;

(3) fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 10-15%, carrying out shake culture at 35-37 ℃ under the condition of 180-240r/min, maintaining the pH at 6.8-7.2 by supplementing ammonia water in the fermentation process, supplementing 0.5-2mL of 60% (m/v) glucose solution when the pH is not slowly reduced or even increased through a phenol red indicator, indicating that the thalli are lack of sugar, and fermenting for 36-48 h;

after 36-48h of shake flask fermentation, the yield of the hydroxyl tetrahydropyrimidine reaches 13-15g/L, the production intensity reaches 0.3-0.45g/(L multiplied by h), and the tetrahydropyrimidine can not be completely detected in the fermentation liquid.

Preferably, the slant culture medium comprises the following components: 3-10g/L of yeast powder, 5-10g/L, NaCl 3-5g/L of peptone, 5-10g/L of beef extract, 0.5-2g/L of sucrose, 15-30g/L of agar powder and the balance of water.

Preferably, the seed culture medium comprises the following components: 15-25g/L glucose, 5-10g/L yeast powder, 4-6g/L tryptone and KH₂PO₄ 1.2-2.4g/L，MgSO₄·7H₂O 0.5-2.0g/L，FeSO₄·7H₂O 5-10mg/L，MnSO₄·7H₂O 5-10mg/L，V_H 0.2-2mg/L，V_B10.1-2mg/L, 1-2 drops of defoaming agent and the balance of water, and the pH value is 6.8-7.2.

Preferably, the fermentation medium comprises the following components: 15-30g/L of glucose, 5-10g/L of xylose, 2-4g/L of yeast powder, 4-6g/L of tryptone and KH₂PO₄ 1.2-2.4g/L，MgSO₄·7H₂O 0.5-2g/L，FeSO₄·7H₂O 50-100mg/L，MnSO₄·7H₂O 50-100mg/L，V_H 0.2-2mg/L，V_B10.1-1mg/L, 1-3% phenol red indicator, 1-2 drops of defoaming agent and the balance of water, and the pH value is 6.8-7.2.

Has the advantages that:

coli W3110 was used as the starting strain in the present invention, and xylose promoter (P) was used_xylF) Induction of RNA polymerase gene from T7 phage, binding of T7 Strong promoter System to Key genes in the Hydroxytetrahydropyrimidine Synthesis pathway (ectABC, ectD, lysC)_cglEtc.) and weakening the competitive inhibition pathway of hydroxyl tetrahydropyrimidine, constructing the synthesis pathway of hydroxyl tetrahydropyrimidine and over-expressing the isocitrate dehydrogenase coding gene icd to increase the supply of precursor alpha-ketoglutaric acidAnd simultaneously, dynamically regulating and controlling the activity of precursor alpha-ketoglutaric acid required by synthesizing the hydroxyl tetrahydropyrimidine through an esaI/esaR quorum sensing circuit strategy, and supplementing the alpha-ketoglutaric acid to obtain the hydroxyl tetrahydropyrimidine high-yielding strain. The yield of the hydroxyl tetrahydropyrimidine reaches 13-15g/L after 36-48h of shake flask fermentation, the production intensity reaches 0.3-0.45g/(L multiplied by h), and tetrahydropyrimidine can not be completely detected in fermentation liquor. The constructed strain can realize the high-efficiency synthesis of hydroxyl tetrahydropyrimidine under the fermentation condition without adding alpha-ketoglutaric acid, does not accumulate byproduct tetrahydropyrimidine, and has important industrial application value.

Description of the drawings:

FIG. 1: constructing a sucA gene knockout fragment and verifying an electrophoresis chart. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: overlapping segments; 4: original bacteria control; 5: and (4) identifying fragments of positive bacteria.

FIG. 2: an integration fragment of esaR170V gene, a knockout fragment of yjiP gene and an electrophoresis verification picture. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: p_BIOFAB-an esaR170V gene fragment; 4: overlapping segments; 5: original bacteria control; 6: and (4) identifying fragments of positive bacteria.

FIG. 3: shake flask fermentation yield map of genetically engineered bacteria.

Wherein: the production of hydroxytetrahydropyrimidine by fermentation with or without the addition of alpha-ketoglutaric acid (KG) to the control bacteria in example 2, and the different strengths (P) respectively_bs1、P_bs2、P_bs3、P_bs4) P of_bsAnd (3) carrying out bacteria-free addition of alpha-ketoglutaric acid fermentation on the esaI/esaR quorum sensing circuit engineering under the control of the promoter to produce the hydroxyl tetrahydropyrimidine.

The specific implementation mode is as follows:

the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

Example 1: construction of E.coli W3110 hydroxy tetrahydropyrimidine gene engineering bacteria

And performing directional modification on the gene by using a CRISPR/Cas9 gene editing technology. The gene editing methods employed in the present invention were performed with reference to literature (Li Y, Lin Z, Huang C, et al. metabolic engineering of Escherichia coli using CRISPR-Cas 9 dimensional genomic engineering,2015,31: 13-21.).

The method comprises the following specific steps:

(1) construction of pGRB plasmid:

CRISPR RGEN Tools are used to design specific target sequences ((PAM:5 '-NGG-3') used for cutting target genes, 10 mu L of each primer is taken to be arranged in a PCR tube after a forward primer and a reverse complementary primer are synthesized, after the primers are uniformly mixed, a double-chain fragment is formed by single-chain DNA through PCR annealing, the reaction conditions are that the pre-denaturation is carried out at 95 ℃ for 5min, the annealing is carried out at 50 ℃ for 1min, the obtained DNA fragment is connected with a linearized pGRB vector obtained through the reverse PCR through homologous recombination to obtain pGRB plasmid, and the kit used for the homologous recombination is

II One Step Cloning Kit series.

(2) Construction of overlapping DNA fragments for replacement:

the recombinant DNA segment required by knocking out the target gene is formed by overlapping two segments of an upstream homologous arm and a downstream homologous arm of the target gene. The recombination segment required by integrating the target gene is formed by overlapping the upstream and downstream homologous arms of the integration site gene and the target gene. The upstream and downstream sequences of the gene to be knocked out or the site to be integrated of the integrated target gene are taken as templates, upstream and downstream homology arm primers are designed, the length of a homology arm is usually about 500bp, and the gene to be integrated is taken as a template, and an amplification primer of the integrated gene is designed. Respectively amplifying upstream and downstream homologous arms and target gene fragments by a PCR method, and preparing recombinant fragments by overlapping PCR.

(3) Preparation of competent cells:

the cells were cultured to OD at 37 ℃ and 220rpm in a Erlenmeyer flask containing 100mL of 2 XYT medium₆₀₀Competent preparations were performed at 0.4-0.6. When pREDCas9 plasmid was carried in the cells, the culture temperature was adjusted to 32 ℃ and when the bacterial OD was₆₀₀When the concentration is 0.1-0.2, 0.1M IPTG is added. The preparation process refers to the conventional standard operation.

(4) Transformation of pGRB plasmid and recombinant DNA:

the pGRB plasmid and overlapping DNA fragment were simultaneously electrotransformed into electrotransformation competent cells containing predca 9. The electrically transformed bacteria are recovered and cultured for 2h, and then spread on LB plate containing ampicillin and spectinomycin, and cultured overnight at 32 ℃. And (3) carrying out colony PCR verification by using an upstream primer of the upstream homology arm and a downstream primer of the downstream homology arm or designing a special identifying primer, and screening positive recombinants.

(5) Elimination of plasmid:

the obtained positive recombinants were inoculated into LB shake tubes containing 0.2% L-arabinose and spectinomycin resistance and cultured at 32 ℃ for about 12 hours. And streaking the dipped bacterial liquid in the three zones of the spectinomycin resistant LB plate, and continuing to culture. Single colonies in the three-zone plate were picked with toothpicks, spotted on LB plates containing spectinomycin resistance and ampicillin resistance, respectively, and cultured continuously. And selecting a colony which grows in a spectinomycin resistant plate and does not grow in an ampicillin plate, wherein the colony is the recombinant strain for eliminating the pGRB plasmid. Transferring the positive recombinants into an LB liquid culture medium without resistance, and culturing at 42 ℃ for about 12 h. And (4) streaking the dipped bacterial liquid in a three-zone of the non-antibiotic plate, and continuing culturing. Single colonies in the three-zone plate are picked by toothpicks, and are respectively spotted into LB plates containing spectinomycin resistance and non-resistance, and the culture is continued. The single colonies that grew on the non-resistant plates and did not grow on the spectinomycin plates were selected as recombinant strains that had eliminated the pREDas9 plasmid.

The specific method for constructing the strain comprises the following steps:

(1) construction of a Hydroxytetrahydropyrimidine Synthesis pathway

Coli W3110 genome as template, upstream homology arm primers thrA-UF and thrA-UR and downstream homology arm primers thrA-DF and thrA-DR are designed according to upstream and downstream sequences of thrA gene, upstream and downstream homology arms are obtained by PCR, and overlapping fragments (thrA-U-thrA-D) are obtained by overlapping PCR. Searching a proper gRNA sequence through a gRNA searching tool (http:// www.rgenome.net/cas-designer), synthesizing gRNA-thrA-S and gRNA-thrA sequences, complementarily pairing two single-stranded primers through PCR annealing to obtain a double-stranded gRNA-thrA, and carrying out homologous recombination on the double-stranded gRNA-thrA and a pGRB linearized vector to obtain pGRB-thrA. And (3) electrically transforming the overlapped fragment and pGRB-thrA into an E.coli W3110 competent cell containing a pREDCas9 vector, coating thalli subjected to recovery culture after the electric transformation on an LB plate containing ampicillin and spectinomycin, performing overnight culture at 32 ℃, verifying a positive recon by colony PCR, eliminating pGRB-thrA for gene editing, and finally obtaining a positive strain for successfully knocking out thrA.

Coli (E.coli W3110) genome as template, upstream homology arm primers ybeM-UF and ybeM-UR and downstream homology arm primers ybeM-DF and ybeM-DR are designed according to upstream and downstream sequences of ybeM gene, Halomonas elongata (Halomonas elongata) genome as template, primer is designed according to sequence of ectABC gene, and promoter P_T7Then, the downstream primer of the upstream homology arm of the ybeM gene and the upstream primer of the ectABC gene are designed, and the upstream and downstream homology arms (ybeM-U, ybeM-D) and the target gene (P) are obtained by PCR_T7ectoABC), and then an overlapping fragment (ybeM-U-P) is obtained by the method of overlapping PCR_T7-ectABC-ybeM-D). Searching a proper gRNA sequence through a gRNA searching tool (http:// www.rgenome.net/cas-designer), synthesizing gRNA-ybeM-S and gRNA-ybeM-A sequences, complementarily pairing two single-stranded primers through PCR annealing to obtain double-stranded gRNA-ybeM, and carrying out homologous recombination on the double-stranded gRNA-ybeM and a pGRB linearized vector to obtain pGRB-ybeM. Electrically transforming the overlapped fragment and pGRB-ybeM into a thrA-successfully-knocked-out positive transformant competent cell containing a pREDCas9 vector, coating thalli obtained through recovery culture after the electric transformation on an LB plate containing ampicillin and spectinomycin, performing overnight culture at 32 ℃, verifying a positive recon by colony PCR, eliminating pGRB-ybeM used for gene editing, and finally obtaining a successfully-knocked-out ybeM gene and integrating P_T7Positive strain of ectABC.

To be combined withLike method for integrating P on gene locus lacIZ_xylFT7RNAP, integration of P at the pseudogene locus yghX_T7-lysC_cglIntegration of P at the gene loci trpR and aceA_T7ectoD, integration of P after ppc knockout at the gene site ppc_trcPpc, replacement of the mlc gene with a literature-reported mutant gene mlc. Constructing and strengthening a hydroxyl tetrahydropyrimidine synthesis path to obtain a positive transformant.

(2) Enhancing the accumulation of the precursor substance alpha-ketoglutarate

An upstream homology arm primer yeeL-UF, yeeL-UR, a downstream homology arm primer yeeL-DF and yeeL-DR are designed according to upstream and downstream sequences of yeeL genes by taking an escherichia coli (E.coli W3110) genome as a template, a primer is designed according to a sequence of an icd gene by taking the escherichia coli (E.coli W3110) genome as a template, an upstream homology arm primer icd-F and a downstream homology arm primer icd-R are designed, an upstream and downstream homology arm (yeeL-U, yeeL-D) and a target gene (icd) are obtained by PCR, and an overlapping fragment (yeeL-U-icd-yeeL-D) is obtained by an overlapping PCR method. Searching a proper gRNA sequence through a gRNA searching tool (http:// www.rgenome.net/cas-designer), synthesizing gRNA-yeeL-S and gRNA-yeeL-A sequences, complementarily pairing two single-stranded primers through PCR annealing to obtain double-stranded gRNA-yeeL, and carrying out homologous recombination on the double-stranded gRNA-yeeL and a pGRB linearized vector to obtain pGRB-yeeL. And (3) electrically transforming the overlapped fragment and pGRB-yeL into a positive transformant competent cell obtained in the step (1) containing a pREDCas9 vector, coating thalli subjected to recovery culture after the electric transformation on an LB plate containing ampicillin and spectinomycin, performing overnight culture at 32 ℃, verifying a positive recon by colony PCR, eliminating pGRB-yeL used for gene editing, and finally obtaining a positive strain for successfully knocking out yeL gene and integrating icd.

(3) Dynamic control of alpha-ketoglutarate dehydrogenase activity

Coli (E.coli W3110) genome as template, upstream homology arm primers sucA-UF-1, sucA-UR-1, downstream homology arm primers sucA-DF-1, sucA-DR-1 are designed according to the upstream and downstream sequences of sucA gene, upstream and downstream homology arms are obtained by PCR, and overlapping fragments (sucA-U-sucA-D) are obtained by overlapping PCR. Searching a proper gRNA sequence through a gRNA searching tool (http:// www.rgenome.net/cas-designer), synthesizing gRNA-sucA-S and gRNA-sucA-A sequences, complementarily pairing two single-stranded primers through PCR annealing to obtain double-stranded gRNA-sucA, and carrying out homologous recombination on the double-stranded gRNA-sucA and a pGRB linearized vector to obtain pGRB-sucA. And (3) electrically transforming the overlapped fragment and pGRB-sucA into a positive transformant competent cell obtained in the step (2) containing the pREDCas9 vector, coating thalli subjected to recovery culture after the electric transformation on an LB plate containing ampicillin and spectinomycin, performing overnight culture at 32 ℃, verifying a positive recon by colony PCR, and eliminating pGRB-sucA used for gene editing to obtain a positive strain for successfully knocking out sucA.

The electrophoresis chart of the construction of the sucA knockout overlapping fragment and the PCR verification of the positive strain is shown in FIG. 1. Wherein: m: 1kb DNA marker; 1: upstream homology arm 637 bp; 2: a downstream homology arm of 525 bp; 3: the overlapped fragment is 1119 bp; 4: original bacteria contrast 2531 bp; 5: positive bacteria identification fragment 1119 bp.

Escherichia coli (E.coli W3110) genome is used as a template, upstream homology arm primers yjiP-UF and yjiP-UR and downstream homology arm primers yjiP-DF and yjiP-DR are designed according to the upstream and downstream sequences of yjiP gene, Escherichia coli (E.coli W3110) genome is used as a template, primers are designed according to the sequence of esaR170V gene, and a constitutive promoter P from a BioFAB library (apFAB104) is used_BioFABThen, the downstream primer of the upstream homology arm of the yjiP gene and the upstream primer of the esaR170V gene are designed, and the upstream and downstream homology arms (yjiP-U, yjiP-D) and the target gene (P) are obtained by PCR_BioFABesaR170V), and then obtaining an overlapping fragment (yjiP-U-P) by an overlapping PCR method_BioFAB-esaR 170V-yjiP-D). Searching ｃA proper gRNA sequence through ｃA gRNA searching tool (http:// www.rgenome.net/cas-designer), synthesizing gRNA-yjiP-S and gRNA-yjiP-A sequences, complementarily pairing two single-chain primers through PCR annealing to obtain double-chain gRNA-yjiP, and homologously recombining the double-chain gRNA-yjiP and ｃA pGRB linearized vector to obtain pGRB-yjiP. Electrically transforming the overlapped fragment and pGRB-yjiP into a sucA positive transformant competent cell containing pREDCas9 vector, which is knocked out and constructed in the previous step, coating thalli obtained through recovery culture after the electric transformation on an LB plate containing ampicillin and spectinomycin, performing overnight culture at 32 ℃, and then performing colony PCR assayConfirming positive recombinants, then eliminating pGRB-yjiP used for gene editing, obtaining successful knockout of yjiP gene and integrating P_BioFAB-a positive strain of esaR 170V.

Integration of P at the genetic locus sucA in the same manner_esaS-sucA; integration of different intensities (P.Acs Synthetic Biology,2017:287) reported in the literature (Yang S, Liu Q, Zhang Y, et al. construction and Characterization of Broad-Spectrum precursors for Synthetic Biology) at the pseudogenic site yjiT (Gene ID:945056)_bs1＜P_bs2＜P_bs3＜P_bs4) P of_bsAnd (3) dynamically regulating the activity of alpha-ketoglutarate dehydrogenase by using a signal molecule acyl homoserine lactone synthase gene esaI controlled by a promoter, and finally obtaining a positive strain of an esaI/esaR quorum sensing circuit.

The construction of the yjiP locus esaR170V gene integration overlapping fragment and the electrophoresis chart of the PCR verification of the positive strain are shown in figure 2. Wherein: m: 1kb DNA marker; 1: an upstream homology arm of 530 bp; 2: 482bp of downstream homology arm; 3: target gene 839 bp; 4: an overlapping fragment 1780 bp; 5: the original strain is compared with 2055 bp; 6: positive bacteria identification fragment 1780 bp.

TABLE 1 primers involved in the construction of the strains

Example 2: method for producing hydroxyl tetrahydropyrimidine by shaking flask fermentation of hydroxyl tetrahydropyrimidine genetic engineering bacteria which are obtained in step (2) of example 1 and are used for knocking out yeeL genes and integrating icds

(1) Activated slant culture: inoculating 1-2 ring strains from a refrigerator bacteria-protecting tube at-80 deg.C with an inoculating ring, uniformly coating on a slant culture medium, culturing at 37 deg.C for 12h, transferring to the second generation slant culture medium, and culturing at 37 deg.C for 12 h;

(2) seed bottle culture: inoculating the thalli on the inclined plane into a 500mL triangular flask filled with 30mL seed culture medium by using an inoculating loop for preparing seed liquid, sealing the triangular flask by using twelve layers of gauze, and carrying out shake culture for 12h at 37 ℃ and 220 r/min;

(3) fermentation culture: inoculating the seed solution into a 500mL baffle bottle filled with a fermentation culture medium according to the inoculation amount of 10% to ensure that the final volume is 30mL, sealing the bottle by using twelve layers of gauze, carrying out shake culture at 37 ℃ under the condition of 180r/min, and adding 0.4mL of 55% (m/v) alpha-ketoglutaric acid after fermenting for 0 h. In the fermentation process, ammonia water is supplemented to maintain the pH at 6.8-7.2, when the pH is not slowly reduced or even increased through a phenol red indicator, the thallus is lack of sugar, and 1mL of 60% (m/v) glucose solution is supplemented. After 36h of shake flask fermentation, the yield of the hydroxyl tetrahydropyrimidine can reach 14.68g/L, the highest production intensity can reach 0.41g/(L multiplied by h), and the yield and DCW of the hydroxyl tetrahydropyrimidine are shown in figure 3(Control + KG).

The slant culture medium comprises the following components: 3g/L of yeast powder, 5g/L, NaCl 3g/L of peptone, 5g/L of beef extract, 0.5g/L of sucrose, 15g/L of agar powder and the balance of water.

The seed culture medium comprises the following components: 20g/L glucose, 10g/L yeast powder, 6g/L tryptone and KH₂PO₄ 1.2g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 10mg/L，MnSO₄·7H₂O 10mg/L，V_H 0.2mg/L，V_B11.3mg/L, 1-2 drops of defoaming agent and the balance of water, and the pH value is 7.0.

The fermentation medium comprises the following components: 20g/L glucose, 10g/L xylose (added after 0h fermentation), 7.3g/L alpha-ketoglutaric acid (added after 0h fermentation), 2g/L yeast powder, 4g/L tryptone, KH₂PO₄ 2g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 100mg/L，MnSO₄·7H₂O 100mg/L，V_H 0.2mg/L，V_B10.8mg/L, 1-3% phenol red indicator, 1-2 drops of defoaming agent and the balance of water, and the pH value is 7.0.

Meanwhile, a Control group without adding alpha-ketoglutaric acid is arranged, the other experimental conditions are the same as above, and the yield of the hydroxyl tetrahydropyrimidine can reach 13.06g/L after 36 hours of shake flask fermentation.

Example 3: method for producing hydroxyl tetrahydropyrimidine by using shake flask fermentation of esaI/esaR quorum sensing line engineering bacteria

(1) Activated slant culture: inoculating 1-2 ring strains from a refrigerator bacteria-protecting tube at-80 deg.C with an inoculating ring, uniformly coating on a slant culture medium, culturing at 37 deg.C for 12h, transferring to the second generation slant culture medium, and culturing at 37 deg.C for 12 h;

(2) seed bottle culture: inoculating the thalli on the inclined plane into a 500mL triangular flask filled with 30mL seed culture medium by using an inoculating loop for preparing seed liquid, sealing the triangular flask by using twelve layers of gauze, and carrying out shake culture for 12h at 37 ℃ and 220 r/min;

(3) fermentation culture: inoculating the seed solution into a 500mL baffle bottle filled with a fermentation culture medium according to the inoculation amount of 10 percent to ensure that the final volume is 30mL, sealing the bottle by using twelve layers of gauze, carrying out shake culture at 37 ℃ and 180r/min, maintaining the pH at 6.8-7.2 by supplementing ammonia water in the fermentation process, indicating that the thalli are lack of sugar when the pH is not slowly reduced or even increased by observing a phenol red indicator, and supplementing 1mL of 60 percent (m/v) glucose solution. As shown in FIG. 3, P_bs3The yield of the esaI/esaR quorum sensing line engineering bacteria controlled by the promoter after fermentation for 36h reaches 14.93g/L, and the production intensity reaches 0.41g/(L multiplied by h). Under the fermentation condition without adding alpha-ketoglutaric acid, the high-efficiency synthesis of hydroxyl tetrahydropyrimidine is realized, and no accumulation of byproduct tetrahydropyrimidine is generated.

By comparing the fermentation results of example 3 and example 2 (fig. 3), it is shown that the quorum sensing system can effectively regulate the downstream metabolism of intracellular alpha-ketoglutarate, so that the intracellular synthesized alpha-ketoglutarate can be effectively used for synthesizing hydroxytetrahydropyrimidine, thereby avoiding additional addition by a feeding manner, reducing the production cost and reducing the complexity of operation. Meanwhile, the yield of the hydroxyl tetrahydropyrimidine is higher, and no tetrahydropyrimidine byproduct is accumulated.

The slant culture medium comprises the following components: 3g/L of yeast powder, 5g/L, NaCl 3g/L of peptone, 5g/L of beef extract, 0.5g/L of sucrose, 15g/L of agar powder and the balance of water.

The seed culture medium comprises the following components: 20g/L glucose, 10g/L yeast powder, 6g/L tryptone and KH₂PO₄ 1.2g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 10mg/L，MnSO₄·7H₂O 10mg/L，V_H 0.2mg/L，V_B11.3mg/L, 1-2 drops of defoaming agent and the balance of water, and the pH value is 7.0.

The fermentation medium comprises the following components: 20g/L glucose, 10g/L xylose (added after 0h fermentation), 2g/L yeast powder, 4g/L tryptone and KH₂PO₄ 2g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 100mg/L，MnSO₄·7H₂O 100mg/L，V_H0.2mg/L，V_B10.8mg/L, 1-3% phenol red indicator, 1-2 drops of defoaming agent and the balance of water, and the pH value is 7.0.

Example 4: method for producing hydroxyl tetrahydropyrimidine by using shake flask fermentation of esaI/esaR quorum sensing line engineering bacteria

(1) Strain activation: taking the dipped bacterial liquid by an inoculating loop in a sterile operation room, densely marking in a solid inclined plane activation culture medium, culturing in an incubator at 37 ℃ for 10h, and marking on an activation inclined plane for subculturing for 12 h.

(2) Seed bottle culture: scraping a ring of thallus with an inoculating ring in a sterile operation room, inoculating into a 500mL triangular flask containing 30mL seed culture medium, sealing with twelve layers of gauze, and performing shake culture at 35 deg.C and 200r/min for 12 h;

(3) fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 10%, carrying out shake culture at 35 ℃ under the condition of 200r/min, maintaining the pH at 6.8-7.2 by supplementing ammonia water in the fermentation process, and supplementing 0.5mL of 60% (m/v) glucose solution when the pH is not slowly reduced or even increased through a phenol red indicator, indicating that the thalli are lack of sugar. P_bs3After the esaI/esaR quorum sensing line engineering bacteria controlled by the promoter are subjected to shake flask fermentation for 40 hours, the yield of the hydroxyl tetrahydropyrimidine reaches 14.2g/L, the production intensity reaches 0.355g/(L multiplied by h), and the tetrahydropyrimidine can not be completely detected in the fermentation liquor.

The slant culture medium comprises the following components: 3g/L of yeast powder, 5g/L, NaCl 3g/L of peptone, 5g/L of beef extract, 0.5g/L of sucrose, 15g/L of agar powder and the balance of water.

The seed culture medium comprises the following components: 15g/L glucose, 5g/L yeast powder, 4g/L tryptone and KH₂PO₄ 1.2g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 5mg/L，MnSO₄·7H₂O 5mg/L，V_H 1mg/L，V_B11mg/L, 1-2 drops of defoaming agent and the balance of water, and the pH value is 6.8.

The fermentation medium comprises the following components: 15g/L glucose, 10g/L xylose, 2g/L yeast powder, 4g/L tryptone and KH₂PO₄1.2g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 50mg/L，MnSO₄·7H₂O 50mg/L，V_H 0.2mg/L，V_B10.5mg/L, 1-3% phenol red indicator, 1-2 drops of defoaming agent and the balance of water, and the pH value is 7.0.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

SEQUENCE LISTING

<110> Tianjin science and technology university

<120> genetic engineering bacterium for producing hydroxyl tetrahydropyrimidine and application thereof

<130> 1

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 2652

<212> DNA

<213> Escherichia coli (E. coli) BL21

<400> 1

atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60

ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120

catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180

gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240

atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300

acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360

accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420

atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540

gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600

tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660

attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720

tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780

ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840

attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900

agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960

aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020

atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080

ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140

gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200

atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260

gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320

aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380

aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440

ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500

tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560

gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620

tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680

ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740

attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800

aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860

ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920

tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980

tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040

atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100

tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220

aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280

attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340

aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400

aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460

gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520

gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580

atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640

gcgttcgcgt aa 2652

<210> 2

<211> 1301

<212> DNA

<213> Artificial sequence

<400> 2

aaaaatttga cgacacgtat tgaagtgctt taccatagcc tacagattat ttcggagcgc 60

gaaaatatag ggagtatgcg gtggttgctg aaaaccagcc tgggcacatt gatcaaataa 120

agcagaccaa cgcgggcgcg gtttatcgcc tgattgatca gcttggtcca gtctcgcgta 180

tcgatctttc ccgtctggcg caactggctc ctgccagtat cactaaaatt gtccgtgaga 240

tgctcgaagc acacctggtg caagagctgg aaatcaaaga agcggggaac cgtggccgtc 300

cggcggtggg gctggtggtt gaaactgaag cctggcacta tctttctctg cgcattagtc 360

gcggggagat tttccttgct ctgcgcgatc tgagcagcaa actggtggtg gaagagtcgc 420

aggaactggc gttaaaagat gacttgccat tgctggatcg tattatttcc catatcgatc 480

agttttttat ccgccaccag aaaaaacttg agcgtctaac ttcgattgcc ataaccttgc 540

cgggaattat tgatacggaa aatggtattg tacatcgcat gccgttctac gaggatgtaa 600

aagagatgcc gctcggcgag gcgctggagc agcataccgg cgttccggtt tatattcagc 660

atgatatcag cgcatggacg atggcagagg ccttgtttgg tgcctcacgc ggggcgcgcg 720

atgtgattca ggtggttatc gatcacaacg tgggggcggg cgtcattacc gatggtcatc 780

tgctacacgc aggcagcagt agtctcgtgg aaataggcca cacacaggtc gacccgtatg 840

ggaaacgctg ttattgcggg aatcacggct gcctcgaaac catcgccagc gtggacagta 900

ttcttgagct ggcacagctg cgtcttaatc aatccatgag ctcgatgtta catggacaac 960

cgttaaccgt ggactcattg tgtcaggcgg cattgcgcgg cgatctactg gcaaaagaca 1020

tcattaccgg ggtgggcgcg catgtcgggc gcattcttgc catcatggtg aatttattta 1080

acccacaaaa aatactgatt ggctcaccgt taagtaaagc ggcagatatc ctcttcccgg 1140

tcatctcaga cagcatccgt cagcaggccc ttcctgcgta tagtcagcac atcagcgttg 1200

agagtactca gttttctaac cagggcacga tggcaggcgc tgcactggta aaagacgcga 1260

tgtataacgg ttctttgttg attcgtctgt tgcagggtta a 1301

<210> 3

<211> 2433

<212> DNA

<213> Halomonas elongata (Halomonas elongata) DSM 2581

<400> 3

atgaacgcaa ccacagagcc ctttacaccc tccgccgacc tggccaagcc cagcgtggcc 60

gatgccgtgg tcggccatga ggcctcaccg ctcttcatcc gcaagccaag ccccgatgac 120

ggctggggca tctacgagct ggtcaagtcc tgtccgcctc tcgacgtcaa ttccgcctac 180

gcctatctgt tgctggccac ccagttccgc gatagctgcg ccgtggcgac caacgaagag 240

ggcgagatcg tcggcttcgt ttccggctac gtgaagagca acgcccccga tacctatttc 300

ctctggcagg ttgccgtggg cgagaaggca cgtggcaccg gcctggcccg tcgtctggtg 360

gaagccgtga tgacacgccc ggaaatggcc gaggtccacc atctcgagac cactatcacg 420

cccgacaacc aggcgtcctg gggcttgttc cgccgtctcg ccgatcgctg gcaggcgccg 480

ttgaacagcc gcgaatactt ctccaccgat caactcggcg gtgagcatga cccggaaaac 540

ctcgttcgca tcggcccgtt ccagaccgac cagatctgag ccgggacgcc gcctggccgg 600

cccggtacgg gccggcaacc cgtcttttcg ttttatcact ttccccccac aggaggtcgc 660

aatgcagacc cagattctcg aacgcatgga gtccgacgtt cggacctact cccgctcctt 720

cccggtcgtc ttcaccaagg cgcgcaatgc ccgcctgacc gacgaggaag ggcgcgagta 780

catcgacttc ctggccggtg ccggcaccct gaactacggc cacaacaacc cgcacctcaa 840

gcaggcgctg ctcgactata tcgacagcga cggcatcgtc cacggcctgg acttctggac 900

tgcggccaag cgcgactatc tggaaaccct ggaagaggtg atcctcaagc cgcgcggtct 960

cgactacaag gtgcatctgc ccggaccgac tggcaccaac gccgtcgagg cggccattcg 1020

cctggcccgg gtcgccaagg ggcgccacaa tatcgtctcc ttcaccaacg gctttcatgg 1080

cgtcaccatg ggcgcgctgg cgaccaccgg taaccgcaag ttccgcgagg ccaccggtgg 1140

cgtgccgacc caggctgctt ccttcatgcc gttcgatggc tacctcggca gcagcaccga 1200

caccctcgac tacttcgaga agctgctcgg cgacaagtcc ggcggcctgg acgtgcccgc 1260

ggcggtgatc gtcgagacag tgcagggcga gggcggtatc aatgtcgccg gcctggagtg 1320

gctcaagcgc ctcgagagca tctgccgcgc caatgacatc ctgctgatca tcgacgacat 1380

ccaggcgggc tgcggccgga ccggcaagtt cttcagcttc gagcatgccg gcatcacgcc 1440

ggatatcgtg accaactcca agtcgctgtc cggttacggc ctgccgttcg ctcacgtcct 1500

gatgcgcccc gagctcgaca agtggaagcc cggtcagtac aacggcacct tccgcggctt 1560

caacctggct ttcgccactg ctgctgccgc catgcgcaag tactggagcg acgacacctt 1620

cgagcgtgac gtgcagcgca aggctcgcat cgtcgaggaa cgcttcggca agatcgccgc 1680

ctggctgagc gagaacggca tcgaggcctc cgagcgcggc cgcgggctga tgcggggcat 1740

cgacgtgggt tccggcgata tcgccgacaa gatcacccac caagccttcg agaacgggtt 1800

gatcatcgaa accagcggtc aggacggcga agtggtcaag tgcctgtgcc cgctgaccat 1860

tcccgacgaa gacctggtcg agggactcga catcctcgag accagcacca agcaggcctt 1920

tagctgatcg cctgaggtgc gccatcgggc ctgtccatgg catcctgtat cggtcggccg 1980

tgcgcggccg gccagtcatt gattcactgg agaatcgaca tgatcgttcg caatctcgaa 2040

gaagcgcgcc agaccgaccg tctggtcacc gccgaaaacg gcaactggga cagcacccgc 2100

ctgtcgctgg ccgaagatgg tggcaactgc tccttccaca tcacccgcat cttcgagggt 2160

accgagaccc acatccacta taagcatcac ttcgaggctg tttattgcat cgaaggcgag 2220

ggcgaagtgg aaaccctggc cgatggcaag atctggccca tcaagccggg tgacatctac 2280

atcctcgacc agcacgacga gcacctgctg cgcgccagca agaccatgca cctggcctgc 2340

gtgttcacgc cgggcctgac cggcaacgaa gtgcaccgcg aagacggttc ctacgcacct 2400

gccgacgaag ccgacgacca gaagccgctg taa 2433

<210> 4

<211> 1266

<212> DNA

<213> Artificial sequence

<400> 4

atggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60

aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120

tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180

ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240

gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300

ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt 360

gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420

aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480

ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540

accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600

atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660

cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720

attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc 780

gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840

aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900

tcttctgtag aagacggcac caccgacatc atcttcacct gccctcgttc cgacggccgc 960

cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020

gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt 1080

accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc 1140

tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca 1200

ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga 1260

cgctaa 1266

<210> 5

<211> 999

<212> DNA

<213> Halomonas elongata (H. elongata) DSM 2581

<400> 5

atgtcagtgc agacatcgtc caaccgaccg ctgccacaag cgaacctgca tatcgccacg 60

gagacacccg aggccgacag ccggatccgt agcgcgccgc gtccggggca ggatccctat 120

ccgacccgac tgagcgagcc gctggatctt ccctggctca atcgccgcga gccggtggtc 180

aagggagagg aggccgatgg gccgctctcg gccgcgcagc tcgatacctt cgagcgccag 240

ggcttcatct tcgagccgga cttcctgaaa ggcgaggaac tcgaggcgtt gcgccacgaa 300

ctcaacgccc tgctggcccg ggatgacttc cgcggacgag acttcgccat caccgagccg 360

cagggcaacg agatccgctc gctgttcgcg gtgcactacc tgtcgcgagt cttcagccgc 420

ctggccaacg acgaacgcct gatgggtcgc gcccggcaga ttctcggcgg cgagccctat 480

gtccatcagt cgcgcatcaa ctacaagccc ggcttcgagg gcaagggctt caattggcat 540

tccgattttg aaacctggca cgccgaggat ggcatgcccg ccatgcatgc ggtgagtgcg 600

tccatcgtgc tgaccgacaa ccacaccttc aacgggccgc tgatgctggt gcccggctca 660

caccgggtat tcgtgccgtg cctgggtgaa acgccggagg atcatcaccg gcagtcgctc 720

aagacccagg aattcggcgt gccgagccgc caggcgctgc gcgagttgat cgaccgacat 780

ggtatcgaag cgcccaccgg cgcggcgggt ggcctgctgc tgttcgactg caataccctg 840

cacggctcca acgccaacat gtcgccggat ccgcgcagca acgccttttt cgtctacaac 900

cgtcgtgaca accgctgcgt cgaaccttat gcggcctcca agcgccgccc gcgcttcctg 960

gcccacgagc cggatgaggc gtggtcgccg gatggctga 999

<210> 6

<211> 1251

<212> DNA

<213> Escherichia coli (E. coli) W3110

<400> 6

atggaaagta aagtagttgt tccggcacaa ggcaagaaga tcaccctgca aaacggcaaa 60

ctcaacgttc ctgaaaatcc gattatccct tacattgaag gtgatggaat cggtgtagat 120

gtaaccccag ccatgctgaa agtggtcgac gctgcagtcg agaaagccta taaaggcgag 180

cgtaaaatct cctggatgga aatttacacc ggtgaaaaat ccacacaggt ttatggtcag 240

gacgtctggc tgcctgctga aactcttgat ctgattcgtg aatatcgcgt tgccattaaa 300

ggtccgctga ccactccggt tggtggcggt attcgctctc tgaacgttgc cctgcgccag 360

gaactggatc tctacatctg cctgcgtccg gtacgttact atcagggcac tccaagcccg 420

gttaaacacc ctgaactgac cgatatggtt atcttccgtg aaaactcgga agacatttat 480

gcgggtatcg aatggaaagc agactctgcc gacgccgaga aagtgattaa attcctgcgt 540

gaagagatgg gggtgaagaa aattcgcttc ccggaacatt gtggtatcgg tattaagccg 600

tgttcggaag aaggcaccaa acgtctggtt cgtgcagcga tcgaatacgc aattgctaac 660

gatcgtgact ctgtgactct ggtgcacaaa ggcaacatca tgaagttcac cgaaggagcg 720

tttaaagact ggggctacca gctggcgcgt gaagagtttg gcggtgaact gatcgacggt 780

ggcccgtggc tgaaagttaa aaacccgaac actggcaaag agatcgtcat taaagacgtg 840

attgctgatg cattcctgca acagatcctg ctgcgtccgg ctgaatatga tgttatcgcc 900

tgtatgaacc tgaacggtga ctacatttct gacgccctgg cagcgcaggt tggcggtatc 960

ggtatcgccc ctggtgcaaa catcggtgac gaatgcgccc tgtttgaagc cacccacggt 1020

actgcgccga aatatgccgg tcaggacaaa gtaaatcctg gctctattat tctctccgct 1080

gagatgatgc tgcgccacat gggttggacc gaagcggctg acttaattgt taaaggtatg 1140

gaaggcgcaa tcaacgcgaa aaccgtaacc tatgacttcg agcgtctgat ggatggcgct 1200

aaactgctga aatgttcaga gtttggtgac gcgatcatcg aaaacatgta a 1251

<210> 7

<211> 65

<212> DNA

<213> Artificial sequence

<400> 7

tcgacataaa gtctaaccta taggatactt acagccatca attcattaaa gaggagaaag 60

gatcc 65

<210> 8

<211> 753

<212> DNA

<213> Pantoea tile (Pantoea stewartia) DC 283

<400> 8

atgttttctt ttttccttga aaatcaaaca ataacggata cgcttcagac ttacatacag 60

agaaagttat ctccgctggg tagtccggat tacgcttaca ctgttgtgag caaaaaaaat 120

ccttcaaatg ttctgattat ttccagttat cctgacgaat ggattaggtt ataccgcgct 180

aacaactttc agctgaccga tccggttatt ctcacggcct ttaaacgcac ctcgccgttt 240

gcctgggatg agaatattac gctgatgtcc gacctgcggt tcaccaaaat tttctcttta 300

tccaagcaat acaacatcgt taacggcttt acctatgtcc tgcatgacca catgaacaac 360

cttgctctgt tgtccgtgat cattaaaggc aacgatcaga ctgcgctgga gcaacgcctt 420

gctgccgaac agggcacgat gcagatgctg ctgattgatt ttaacgagca gatgtaccgc 480

ctggccggta ccgaaggcga gcgagccccg gcgttaaatc agagcgcgga caaaacgata 540

ttttcctcgc gtgaaaatga ggtgttgtac tgggcgagta tgggcaaaac ctatgctgag 600

attgccgcta ttacgggcat ttctgtgagt accgtgaagt ttcacatcaa gaatgtggtc 660

gtgaaactgg gcgtcagtaa cgcccgacag gctatcagac tgggtgtaga actggatctt 720

atcagaccgg cagcgtcagc ggccaggtag tga 753

<210> 9

<211> 184

<212> DNA

<213> Pantoea tile (P. stewartia) DC 283

<400> 9

gctcacaaca gtgtaagcgt atccgttatt gtttgatttt caaggaaaaa agaaaacatt 60

caggctccat gctgcttctt ttacttaacg tggacttaac ctgcactata gtacaggcaa 120

gatgatactt aagagtaact tacaatgaat cattcagagg ttacaatggc ttcagttgtt 180

tagc 184

<210> 10

<211> 115

<212> DNA

<213> Artificial sequence

<400> 10

ggcgcgccct aatttgacag tagaattagc atgtgatata ataaaataat ttttactacc 60

caagcttata aaagagcact gttgggcgtg agtggaggcg ccggaggagg aaaaa 115

<210> 11

<211> 114

<212> DNA

<213> Artificial sequence

<400> 11

ggcgcgccgt agattgacac cctctgtagc atgtgatata ataaatttta tattctaccc 60

aagcttataa aagagcactg ttgggcgtga gtggaggcgc cggaggagga aaaa 114

<210> 12

<211> 114

<212> DNA

<213> Artificial sequence

<400> 12

ggcgcgccgt tagttgacac ttagcctagc atgtgatata attatgttat ttatctaccc 60

aagcttataa aagagcactg ttgggcgtga gtggaggcgc cggaggagga aaaa 114

<210> 13

<211> 114

<212> DNA

<213> Artificial sequence

<400> 13

ggcgcgcccc tccttgacac tgaatttagc atgtgatata attaacttaa tattctaccc 60

aagcttataa aagagcactg ttgggcgtga gtggaggcgc cggaggagga aaaa 114

<210> 14

<211> 698

<212> DNA

<213> Pantoea tile (P. stewartia) DC 283

<400> 14

aagcttgtaa aatcagtgca ggataaccgc gagggccgca gtaactttta agaggaaatg 60

gaatgcttga actgtttgac gtcagttacg aagaactgca aaccacccgt tcagaagaac 120

tttataaact tcgcaagaaa acatttagcg atcgtctggg atgggaagtc atttgcagtc 180

agggaatgga gtccgatgaa tttgatgggc ccggtacacg ttatattctg ggaatctgcg 240

aaggacaatt agtgtgcagc gtacgtttta ccagcctcga tcgtcccaac atgatcacgc 300

acacttttca gcactgcttc agtgatgtca ccctgcccgc ctatggtacc gaatccagcc 360

gtttttttgt cgacaaagcc cgcgcacgtg cgctgttagg tgagcactac cctatcagcc 420

aggtcctgtt tttagcgatg gtgaactggg cgcaaaataa tgcctacggc aatatctata 480

cgattgtcag ccgcgcgatg ttgaaaattc tcactcgctc tggctggcaa atcaaagtca 540

ttaaagaggc tttcctgacc gaaaaggaac gtatctattt gctgacgctg ccagcaggtc 600

aggatgacaa gcagcaactc ggtggtgatg tggtgtcacg tacgggctgt ccgcccgtcg 660

cagtcactac ctggccgctg acgctgccgg tctgataa 698

Claims (7)

1. A strain of escherichia coli genetic engineering bacteria for producing hydroxyl tetrahydropyrimidine is characterized in that a promoter P with xylose inducible is integrated on a host genome_xylFRNA polymerase of controlled T7 phage; integrating single copies from P_T7Ectoine synthase gene cluster ectABC controlled by a promoter; single copy by P_T7Promoter-controlled aspartokinase gene lysC_cgl(ii) a Double copy by P_T7Tetrahydropyrimidine hydroxylase gene ecD controlled by promoter; knocking out a hydroxyl tetrahydropyrimidine competitive branch metabolism related gene thrA; replacing phosphoenolpyruvate carboxylase coding gene ppc promoter with P_trcA promoter; replacement of original by mutant mlc of transcription repressing factor of sugar metabolismmlc; integrating a single copy isocitrate dehydrogenase encoding gene icd; integration P_BIOFABThe transcriptional regulator esaR170V under the control of the promoter; replacement of the original promoter of the alpha-ketoglutarate dehydrogenase gene sucA with P_esaSA promoter; the signal molecule acyl homoserine lactone synthase gene esaI is integrated.

2. The genetically engineered Escherichia coli for producing hydroxytetrahydropyrimidine of claim 1, wherein the promoter of the easI gene is P_bs1、P_bs2、P_bs3Or P_bs4A promoter.

3. The genetically engineered Escherichia coli for producing hydroxytetrahydropyrimidine according to claim 1 or 2, wherein:

the nucleotide sequence of the T7RNA polymerase gene is shown as a sequence table SEQ ID NO. 1;

the nucleotide sequence of the sugar metabolism transcription repression factor mutation mlc is shown in a sequence table SEQ ID NO. 2;

the nucleotide sequence of the ectoine synthetase gene cluster ectABC is shown in a sequence table SEQ ID NO. 3;

the aspartokinase gene lysC_cglThe nucleotide sequence is shown in a sequence table SEQ ID NO. 4;

the nucleotide sequence of the tetrahydropyrimidine hydroxylase gene ectD is shown in a sequence table SEQ ID NO. 5;

the nucleotide sequence of the isocitrate dehydrogenase coding gene icd is shown in a sequence table SEQ ID NO. 6;

the P is_BIOFABA promoter, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 7;

the nucleotide sequence of the transcription regulator esaR170V is shown in a sequence table SEQ ID NO. 8;

the P is_esaSA promoter, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 9;

the P is_bs1A promoter, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 10;

the P is_bs2A promoter, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 11;

the P is_bs3A promoter, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 12;

the P is_bs4A promoter, the nucleotide sequence of which is shown in a sequence table SEQ ID NO. 13;

the nucleotide sequence of the acyl homoserine lactone synthase gene esaI is shown in a sequence table SEQ ID NO. 14.

4. The genetically engineered escherichia coli for producing hydroxytetrahydropyrimidine of claim 1, wherein the genetically engineered escherichia coli is a host strain escherichia coli e.

5. Use of the engineered bacterium of any one of claims 1 to 4 in the production of hydroxytetrahydropyrimidine.

6. Use according to claim 6, characterized in that the process for the fermentative production of hydroxytetrahydropyrimidines comprises the following steps:

inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 10-15%, carrying out shake culture at 35-37 ℃ under the condition of 180-240r/min, maintaining the pH at 6.8-7.2 by supplementing ammonia water in the fermentation process, and supplementing 0.5-2mL of 60% glucose solution for fermentation for 36-48h when the pH is not slowly reduced or even increased through a phenol red indicator, indicating that the thalli are lack of sugar.

7. The use according to claim 6, wherein the fermentation medium comprises: 15-30g/L of glucose, 5-10g/L of xylose, 2-4g/L of yeast powder, 4-6g/L of tryptone and KH₂PO₄ 1.2-2.4g/L，MgSO₄·7H₂O 0.5-2g/L，FeSO₄·7H₂O 50-100mg/L，MnSO₄·7H₂O 50-100mg/L，V_H 0.2-2mg/L，V_B10.1-1mg/L, 1-3% phenol red indicator, 1-2 drops of defoaming agent and the balance of water, and the pH value is 6.8-7.2.

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