Modularized reconstructed recombinant escherichia coli for synthesizing indigo from de novo, fermentation method and application

Abstract

The invention relates to modular reconstructed recombinant Escherichia coli for synthesizing indigo from de novo, a fermentation method and application, and the recombinant Escherichia coli is characterized in that dispersedly distributed genes such as aroB, aroD, aroE, aroL, aroA, aroC and the like are integrated into an artificial operon at a host chromosome yjgX site, so that centralized expression of a tryptophan precursor synthesis module is realized, precursor supply and product yield are remarkably improved, and the recombinant Escherichia coli is suitable for industrial production. And a tryptophan metabolism module, an indigo synthesis module and a feedback inhibition removal and synthesis enhancement module are combined to jointly realize efficient synthesis of indigo in escherichia coli MG1655. In addition, four different naphthalene dioxygenase CNDO, PNDO, BNDO and NDO-ABC are introduced, so that precursor supply and product yield can be remarkably improved in indigo biosynthesis, and the highest CNDO yield can reach 10.24 g / L. The invention provides a new engineering strategy for efficient synthesis of indigo and other aromatic compounds.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to recombinant Escherichia coli for modular reconstructed de novo synthesis of indigo, fermentation methods, and applications. Background Technology

[0002] Indigo is a natural indole dye with a deep blue color. Originally obtained from the leaves of indigo plants such as woad and knotweed, it is one of the earliest organic dyes used by humans, with a history spanning thousands of years. Due to its unique color, good lightfastness, and biodegradability, indigo still holds significant economic value and application prospects in textile dyes, cosmetics, functional materials, and the polymer industry. Furthermore, indigo exhibits certain antibacterial, anti-inflammatory, and antioxidant activities, and has attracted attention in the development of pharmaceuticals and skincare products in recent years. Simultaneously, in the fields of organic electronics and flexible semiconductor materials, indigo molecules are considered a promising renewable semiconductor material due to their excellent charge transport properties and environmental friendliness.

[0003] Traditionally, indigo is obtained primarily through two methods: chemical synthesis and plant extraction. Chemical synthesis methods, which mostly use petrochemical compounds as raw materials, offer relatively high yields but often require high temperatures, high pressures, and strong redox conditions. This process generates various harmful byproducts such as cyanide, formaldehyde, and strong alkalis, leading to serious environmental and safety problems. While plant extraction can yield natural indigo, it is limited by long cultivation cycles, low extraction efficiency, and high costs, making it unsuitable for large-scale industrial production. Both of these methods fail to meet current requirements for green and sustainable production.

[0004] With the rapid development of synthetic biology and metabolic engineering, the production of natural pigments using microbial cell factories has become a new research direction. Existing research has modified hosts such as *E. coli* to synthesize indigo using carbon sources such as glucose or glycerol, with the addition of tryptophan precursors, through oxygenase or monooxygenase systems. This method significantly improves the environmental friendliness of production, but still suffers from problems such as dependence on exogenous precursors, high production costs, and unstable metabolic efficiency.

[0005] In contrast, de novo synthesis of indigo can directly synthesize indigo molecules from a carbon source via the host's own aromatic amino acid biosynthesis pathway, without the need for precursors such as tryptophan or indole. This method can effectively reduce costs, simplify the production process, and improve the overall metabolic efficiency of the system. However, reports on de novo biosynthesis of indigo are still limited, and mature metabolic pathway design and key enzyme system optimization schemes are lacking. Therefore, establishing an efficient, stable, and economically feasible de novo indigo synthesis system is of great significance for promoting the green production and industrialization of indigo. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art and to provide a modularly reconstructed recombinant Escherichia coli for de novo synthesis of indigo, a fermentation method, and its application. This invention enables efficient production of indigo using glucose as the sole carbon source, significantly improving the rate and yield of indigo synthesis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A modularly reconstructed recombinant Escherichia coli strain capable of de novo indigo synthesis, using Escherichia coli as a host, modularly expresses genes related to the indigo synthesis pathway; wherein the modular expression of genes related to the indigo synthesis pathway includes any one of modifications I-IV:

[0009] I. Reconstruction of the tryptophan precursor supply module: Knock out at least two of the following genes in the host: aroB, aroD, aroE, aroL, aroA, and aroC; and tandemly overexpress the knocked-out genes.

[0010] II. Tryptophan metabolism module: exogenous overexpression of indoleglycerol phosphate synthase gene BX1 and / or tryptophanase gene naphthalene dioxygenase gene tnaA;

[0011] III. Indigo synthesis module: exogenous overexpression of at least one of the genes encoding naphthalene dioxygenase, namely CNDO, NDO-ABC, BNDO, and PNDO;

[0012] IV. Feedback Inhibition Relief and Synthesis Enhancement Module: Exogenous overexpression of indigo hydrolase gene IH and gene aroG mut trpE gene mut trpC gene mut At least one of them.

[0013] Preferably, in item I, genes aroB, aroD, aroE, aroL, aroA, and aroC are integrated into the yjgX site of the host chromosome.

[0014] Preferably, in item II, the indoleglycerol phosphate synthase gene BX1 and / or tryptophanase gene naphthalene dioxygenase gene tnaA are overexpressed using the pCDFDuet vector.

[0015] Preferably, in item III, at least one of the following genes for naphthalene dioxygenase, namely CNDO, NDO-ABC, BNDO, and PNDO, is overexpressed using the pETDuet vector.

[0016] Preferably, in item IV, the indigo hydrolase gene IH and gene aroG are overexpressed using the pTrc99a vector.mut trpE gene mut trpC gene mut At least one of them.

[0017] Preferably, in items I-IV, the different genes in each module are linked together via ribosome binding sites (RBS).

[0018] Preferably, a modularly reconstructed recombinant *E. coli* strain capable of de novo indigo synthesis, using *Escherichia coli* as a host, modularly expresses genes related to the indigo synthesis pathway; wherein the modular expression of these genes includes any one of modifications I-IV:

[0019] I. Reconstruction of the tryptophan precursor supply module: Knock out the host genes aroB, aroD, aroE, aroL, aroA, and aroC, and then overexpress the knocked-out aroB, aroD, aroE, aroL, aroA, and aroC in tandem in sequence (more preferably, integrate the knocked-out aroB, aroD, aroE, aroL, aroA, and aroC into the yjgX site on the host chromosome in sequence to form an artificial operon);

[0020] II. Tryptophan metabolism module: Indoleglycerol phosphate synthase gene BX1 and / or tryptophanase gene tnaA are overexpressed using pCDFDuet vector (more preferably, tryptophanase gene tnaA is constructed between the NcoI and BamHI recognition sites of pCDFDuet vector, and codon-optimized indoleglycerol phosphate synthase gene BX1 is constructed between the NdeI and BglII recognition sites).

[0021] III. Indigo synthesis module: At least one of the following genes for naphthalene dioxygenase, namely CNDO, NDO-ABC, BNDO, and PNDO, is overexpressed using the pETDuet vector (more preferably, the codon-optimized naphthalene dioxygenase genes CNDO, NDO-ABC, BNDO, and PNDO are constructed between the NcoI and BamHI recognition sites of the pETDuet vector, respectively).

[0022] IV. Feedback Inhibition Relief and Synthesis Enhancement Module: The indigo hydrolase gene IH and gene aroG were overexpressed using the vector pTrc99a. mut trpE gene mut trpC gene mut (More preferably, the gene containing aroG) mut trpE gene mut trpC genemut The fragment was constructed between the NcoI and NotI sites at position 1 of the multiple cloning site of the expression vector pTrc99a, and the codon-optimized gene IH was constructed between the NdeI and MfeI sites at position 2 of the multiple cloning site of the expression vector pTrc99a.

[0023] Preferred gene aroG mut The nucleotide sequence is shown in SEQ ID No. 8; the gene trpE mut The nucleotide sequence is shown in SEQ ID No. 9; the gene trpC mut The nucleotide sequence is shown in SEQ ID No. 10.

[0024] Preferred gene aroG mut The encoded amino acid sequence was obtained by mutating aspartic acid (Asp) at position 146 of the amino acid sequence encoded by the gene aroG to asparagine (Asn).

[0025] Preferred gene trpE mut The encoded amino acid sequence was obtained by mutating alanine (Ala) at position 63 of the amino acid sequence encoded by the gene trpE to valine (Val).

[0026] Preferred gene trpC mut The encoded amino acid sequence was obtained by mutating serine (Ser) at position 60 of the amino acid sequence encoded by the gene trpC to alanine (Ala).

[0027] Preferably, the Escherichia coli includes Escherichia coli MG1655 or a derivative thereof.

[0028] A method for synthesizing indigo includes: using glucose or glycerol as a carbon source, and subjecting the recombinant Escherichia coli to shake-flask fermentation culture or industrial fed-batch fermentation culture to obtain it.

[0029] Preferably, both shake-flask fermentation and industrial fed-batch fermentation use MR medium, which includes:

[0030] MR medium: 10 g / L glucose, 6.67 g / L potassium dihydrogen phosphate, 4 g / L diammonium hydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 0.8 g / L citric acid, and 10 mL / L trace metal elements. Other components include 10 g / L glucose, 5 g / L yeast extract, 7.2 g / L potassium dihydrogen phosphate, 2.5 g / L ammonium sulfate, 2 g / L citric acid, 1.5 g / L magnesium sulfate heptahydrate, 5 mL / L trace elements, and 1 mL / L polyether defoamer.

[0031] Trace elements: 10 g / L ferrous sulfate heptahydrate, 1.35 g / L anhydrous calcium chloride, 2.2 g / L zinc sulfate heptahydrate, 0.58 g / L manganese sulfate tetrahydrate, 1 g / L copper sulfate pentahydrate, 0.2 g / L sodium borate decahydrate, 0.1 g / L ammonium molybdate tetrahydrate;

[0032] The initial fermentation conditions were set as follows: temperature 30℃, aeration rate 2vvm, and stirring speed 300rpm.

[0033] During fermentation, dissolved oxygen is controlled at 20-40%, and online feeding is used to maintain glucose concentration below 1 g / L.

[0034] The application of the above-mentioned recombinant Escherichia coli in the production of indigo.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1. This invention systematically improves the efficiency of tryptophan precursor supply by integrating and overexpressing the tryptophan precursor supply gene, thereby significantly increasing the rate and yield of indigo synthesis.

[0037] 2. By constructing a tryptophan metabolism module for tandem expression and synergistic effect with the indigo synthesis module, the efficiency of the indole oxidation step is effectively promoted, and the accumulation of intermediate products is reduced.

[0038] 3. Modular metabolic regulation is achieved by integrating genome metabolism overexpression with multi-plasmid expression systems, balancing metabolic balance and host growth stability.

[0039] 4. The recombinant Escherichia coli constructed in this invention can achieve the self-synthesis of indigo using glucose as a carbon source without the addition of tryptophan. It has the advantages of simple process, high conversion rate and low production cost. After 60 hours of fermentation, the indigo yield reaches 10.24g, which is suitable for the biomanufacturing and industrial application of indigo and its derivatives.

[0040] In summary, this invention modularizes the initial strain of *E. coli*. Each module is functionally assembled through modular reconstruction of metabolic pathways and a multi-plasmid co-expression strategy. Specifically, the tryptophan metabolism module uses a medium copy number replication initiator; the indigo synthesis module uses a high copy number replication initiator; the feedback inhibition relief and synthesis enhancement module uses a low or medium copy number replication initiator; and the tryptophan precursor supply module is integrated and overexpressed on the host genome, thereby achieving de novo indigo synthesis. This process uses glucose as a carbon source and does not require the addition of tryptophan precursors, thus achieving efficient indigo production. Furthermore, the multi-plasmid system constructed by the above modifications can be built with different resistance markers and compatible replication initiators to ensure stable modular co-expression. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the indigo synthesis route of recombinant Escherichia coli according to the present invention;

[0042] Figure 2 This is a schematic diagram of the modular construction of the tryptophan precursor supply gene in Example 1;

[0043] Figure 3 This is a graph showing the yield of indigo produced by the recombinant bacteria in shake flasks in Example 2;

[0044] Figure 4 The synthesis of indigo and cell concentration OD of recombinant strains MG-Trp-C and MG-PTrp-C through fermentation. 600 The time-varying curve.

[0045] exist Figure 4 In the text, A represents indigo; B represents bacterial cell concentration (OD). 600 . Detailed Implementation

[0046] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. The described embodiments are only some embodiments of the present invention and not all embodiments.

[0047] 1. Unless otherwise specified, all materials and reagents used in this invention are commercially available;

[0048] 2. Unless otherwise specified, the experimental methods of this invention are all conventional methods.

[0049] 3. The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in this invention were all commercially available products, and the specific operations were performed according to the kit instructions. Conventional procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to *Molecular Cloning: A Laboratory Manual (Fourth Edition)*. Sequencing of the plasmids and DNA products was performed by Genewiz (Suzhou).

[0050] 4. Culture medium

[0051] (1) LB liquid medium: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L.

[0052] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0053] (3) Primary seed culture medium LB: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L.

[0054] (4) Secondary seed culture medium LB: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L.

[0055] (5) MR medium composition (initial fermentation medium, shake flask fermentation medium): 10 g / L glucose, 6.67 g / L potassium dihydrogen phosphate, 4 g / L diammonium hydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 0.8 g / L citric acid and 10 mL / L trace metal elements, 1 mL / L polyether defoamer;

[0056] The trace elements are: 10 g / L ferrous sulfate heptahydrate, 1.35 g / L anhydrous calcium chloride, 2.2 g / L zinc sulfate heptahydrate, 0.58 g / L manganese sulfate tetrahydrate, 1 g / L copper sulfate pentahydrate, 0.2 g / L sodium borate decahydrate, and 0.1 g / L ammonium molybdate tetrahydrate.

[0057] 5. Primers: See Table 1;

[0058] Table 1

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] 6. Indigo shake-flask fermentation

[0065] (1) Indigo shake flask fermentation process: Freshly activated seed culture was inoculated into 4 mL LB liquid medium containing ampicillin, kanamycin and streptomycin, and cultured overnight at 37℃ and 200 rpm for 12 h to obtain seed liquid. 2500 μL of seed liquid was inoculated into a 250 mL flask containing 25 mL of fermentation medium, and cultured at 30℃ and 200 rpm for 24 h. Ammonia water was added to adjust the pH to 7.0, and fermentation continued for 48 h.

[0066] 7. Indigo detection

[0067] Mix the entire fermentation system thoroughly and take a sample (1 mL from each fermentation system). Centrifuge at 12000 rpm for 10 min and collect the precipitate. Wash the precipitate twice thoroughly with 100% methanol, centrifuge at 12000 rpm for 5 min, and after the methanol has fully evaporated, add 2-10 mL of DMSO and use ultrasound to break up and dissolve the precipitate (the precipitate will be colorless when extraction is complete). Centrifuge at 12000 rpm for 5 min and collect the supernatant. Detect the absorbance at 620 nm using an ELISA reader, substitute it into the standard curve of indigo standard, and calculate the yield of indigo in the fermentation system.

[0068] Example 1: Construction of a recombinant strain of indigo synthesized de novo

[0069] (I) Modular construction of tryptophan precursor supply gene in recombinant bacteria

[0070] To improve the synthesis throughput of aromatic amino acids and tryptophan precursors, the chromosome of *E. coli* MG1655 was modified at specific sites using the CRISPR / Cpf1 gene editing system, integrating and expressing genes such as aroB (Gene ID: 947927), aroD (Gene ID: 946210), aroE (Gene ID: 947776), aroL (Gene ID: 945031), aroA (Gene ID: 945528), and aroC (Gene ID: 946814) into a gene cluster. These genes, originally scattered across the genome, were arranged in tandem at specific sites (yjgX) on the host chromosome and placed under a unified regulatory element, forming an artificial operon structure, thereby achieving centralized and efficient expression of the precursor synthesis module. Figure 2 As shown, the operation steps are as follows:

[0071] (1) Target gene knockout, vector construction, and host pretreatment. To avoid duplicate expression and reduce metabolic burden, the aroB, aroD, aroE, aroL, aroA, and aroC genes, which are scattered on the E. coli MG1655 chromosome, were first knocked out one by one using the CRISPR / Cpf1 system; the specific steps are as follows:

[0072] a. Using the MG1655 genome as a template, 23bp target sequences corresponding to each target gene were designed and introduced into pcrEG plasmids to construct editing plasmids such as pcrEG-aroB, pcrEG-aroD, pcrEG-aroE, pcrEG-aroL, pcrEG-aroA, and pcrEG-aroC; at the same time, homologous arm fragments of about 500bp upstream and downstream of each target gene were amplified to mediate recombination repair.

[0073] b. The pcrEG-aroB, pcrEG-aroD, pcrEG-aroE, pcrEG-aroL, pcrEG-aroA, and pcrEG-aroC plasmids constructed in a, along with their corresponding donor DNA fragments, were electroporated into MG1655 competent cells carrying pEcCpf1 (denoted as MG-pEcCpf1). After transformation, the cells were plated on LB agar plates containing kanamycin and spectinomycin and incubated at 37°C for 24 h. Colonies were then picked for PCR identification.

[0074] c. Verified positive clones were further inoculated into LB medium containing rhamnose to induce the loss of the edit plasmid, obtaining intermediate strains with each gene knocked out; through multiple rounds of editing, all target genes were sequentially deleted, finally obtaining a basic chassis strain without aroB, aroD, aroE, aroL, aroA, and aroC, named MG-Δaro( Figure 2 ).

[0075] (2) Using the genome of Escherichia coli MG1655 as a template, the upstream and downstream homologous arm fragments of yjgX were amplified using primers yjgX-UP-F / R and yjgX-DH-F / R, respectively; at the same time, the target genes were amplified using primers aroB-F / R, aroD-F / R, aroE-F / R, aroL-F / R, aroA-F / R, and aroC-F / R. After gel recovery and purification of the fragments, the recovered fragments were sequentially ligated by overlapping PCR to form donor DNA fragments containing homologous arms and target genes.

[0076] (3) The N on plasmid pcrEG was amplified using PCR technology. 23 The sequence is replaced with N, which is complementary to a specific 23bp sequence on yjgX. 23 Using yjgX-N23-F / R as primers, the plasmid pcrEG-yjgX targeting yjgX was obtained. The PCR product was used to remove template DNA with DpnI enzyme, and then transformed into E. coli JM109 competent cells using a heat shock transformation method. The cells were then plated on LB agar plates containing spectinomycin and incubated overnight at 37°C. Single colonies were picked for expansion culture, plasmid extraction, and sequencing.

[0077] (4) The pEcCpf1 plasmid was transformed into the electrocompetent cells of Escherichia coli MG165-Δaro. The transformed bacterial solution was spread on LB plates containing kanamycin and cultured overnight at 37°C to obtain MG-Δaro-pEcCpf1. MG-Δaro-pEcCpf1 was then prepared as electrocompetent cells.

[0078] (5) The validated pcrEG-yjgX plasmid and the donor DNA fragment were electroporated together into MG-Δaro-pEcCpf1 electroporated competent cells. The transformation products were inoculated on LB plates containing kanamycin and spectinomycin. After culturing at 37°C for 24 h, single colonies were picked for PCR identification.

[0079] (II) Construction of recombinant plasmids

[0080] To achieve modular expression of indigo de novo synthesis, multi-plasmid systems were constructed, including a feedback inhibition relief and synthesis enhancement module, a tryptophan metabolism module, and an indigo synthesis module.

[0081] 1. Construction of feedback inhibition removal and synthesis enhancement module

[0082] The aroG, trpE, and trpC gene fragments were amplified from the genome of *E. coli* MG1655 using primers Trc-aroG-F / R, Trc-trpE-F / R, and Trc-trpC-F / R, respectively. Fragments 1 were obtained by ligating the three fragments using overlap PCR. Separately, the codon-optimized IH gene fragment from *P. putida* (synthesized by GenScript Biotech, Inc., the nucleotide sequence of the codon-optimized indigo hydrolase gene IH is shown in SEQ ID No. 7) was amplified using primer Trc-IH-F / R. Fragments 1 and IH were then constructed into the multiple cloning site of the expression vector pTrc99a: fragment 1 was constructed between the NcoI and NotI sites at position 1 of the multiple cloning site of pTrc99a, and IH was constructed between the NdeI and MfeI sites at position 2 of the multiple cloning site of pTrc99a. Subsequently, primer aroG-F / R was used to amplify the fragments. mut -F / R, trpE mut -F / R, trpC mut -F / R mutations were performed at specific sites in the corresponding gene to construct the recombinant plasmid pTrc99a-pJ23119-aroG. mut -trpE mut -trpC mut -pJ23119-IH;

[0083] The gene aroG obtained after mutation mut The nucleotide sequence is shown in SEQ ID No. 8;

[0084] The gene trpE obtained after mutation mut The nucleotide sequence is shown in SEQ ID No. 9;

[0085] The trpC gene obtained after mutation mutThe nucleotide sequence is shown in SEQ ID No. 10.

[0086] 2. Construction of tryptophan metabolism module

[0087] The tnaA gene cluster fragment was amplified from the genome of *E. coli* MG1655 using primers tnaA-F / R. The BX1 gene fragment derived from *Zea mays* with optimized codons was amplified using primers BX1-F / R (the nucleotide sequence of the codon-optimized indoleglycerol phosphate synthase gene BX1 is shown in SEQ ID No. 2). The obtained tnaA fragment (the nucleotide sequence of the tryptophanase gene tnaA is shown in SEQ ID No. 1) was constructed between the NcoI and BamHI recognition sites of the pCDFDuet vector, and the BX1 fragment was constructed between the NdeI and BglII recognition sites to obtain the recombinant vector pCDFDuet-pJ23119-tnaA-BX1.

[0088] 3. Construction of the Indigo Synthesis Module

[0089] The codon-optimized CNDO fragment derived from Commonas sp. MQ was amplified using primers CNDO-F / R, and the codon-optimized CNDO fragment (the nucleotide sequence of the codon-optimized naphthalene dioxygenase gene CNDO is shown in SEQ ID No. 3) was constructed between the NcoI and BamHI recognition sites of the pETDuet vector to obtain the recombinant plasmid pET-CNDO. Similarly, primers PNDO-F / R, BNDO-F / R, and NDO-ABC-F / R were used to amplify codon-optimized PNDO (derived from Pseudomonasputida, the nucleotide sequence of the codon-optimized naphthalene dioxygenase gene PNDO is shown in SEQ ID No. 6), BNDO (derived from P. balearica DSM 6083, the nucleotide sequence of the codon-optimized naphthalene dioxygenase gene BNDO is shown in SEQ ID No. 5), and NDO-ABC (derived from Pseudomonasputida, the nucleotide sequence of the codon-optimized naphthalene dioxygenase gene NDO-ABC is shown in SEQ ID No. 4), and these fragments were sequentially constructed between the NcoI and BamHI recognition sites of the pETDuet vector to obtain recombinant plasmids pET-PNDO, pET-BNDO, and pET-NDO-ABC.

[0090] (III) Obtaining engineered strains

[0091] The above-mentioned plasmids for different indigo production modules (pET-CNDO, pET-PNDO, pET-BNDO, or pET-NDO-ABC) were respectively coupled with pCDFDuet-pJ23119-tnaA-BX1 and pTrc99a-pJ23119-aroG mut -trpE mut -trpC mut -pJ23119-IH was co-transformed into the MG-Trp strain, resulting in four different engineered strains: MG-Trp-C, MG-Trp-P, MG-Trp-B, and MG-Trp-ABC.

[0092] Example 2: Construction of a non-modular enhanced aromatic amino acid precursor supply strain

[0093] To verify the effectiveness of the modular integrated expression system in improving the synthesis throughput of aromatic amino acids and tryptophan precursors, a non-modular enhanced strain (strain MG-PTrp) was constructed as a control strain for subsequent studies.

[0094] In strain MG-PTrp, the six genes aroB, aroD, aroE, aroL, aroA, and aroC are expressed separately by independent promoters, rather than being clustered and integrated as operons.

[0095] (I) Construction of strain MG-PTrp, the specific steps are as follows:

[0096] (1) Using Escherichia coli MG1655 as the host, the promoter pJ23119 was linked upstream of the aroB, aroD, aroE, aroL, aroA and aroC genes respectively by GibsonAssembly method to construct six separate expression cassettes.

[0097] (2) The six expression cassettes aroB to aroC were integrated into the chromosome at the same gene loci as the in situ genes to maintain the gene copy number consistent with the genomic background. Integration was performed using the CRISPR / Cpf1 site-directed editing system, following the method described in Example 1.

[0098] (3) After verification by PCR and sequencing, the resulting engineered strain was named MG-PTrp. In this strain, each gene is expressed independently and no clustered regulatory structure is formed.

[0099] (II) Obtaining engineered strains

[0100] The different indigo production module plasmids (pET-CNDO, pET-PNDO, pET-BNDO, or pET-NDO-ABC) prepared in Example 1 were respectively coupled with pCDFDuet-pJ23119-tnaA-BX1 and pTrc99a-pJ23119-aroG mut -trpE mut -trpC mut -pJ23119-IH was co-transformed into the MG-PTrp strain to obtain engineered strains MG-PTrp-C, MG-PTrp-P, MG-PTrp-B, and MG-PTrp-ABC that did not undergo modular integration of the tryptophan precursor supply module.

[0101] Example 3: Production of indigo from engineered strains in shake flasks

[0102] The MG-Trp-C, MG-Trp-P, MG-Trp-B, and MG-Trp-ABC strains constructed in Example 1, and the corresponding engineered strains MG-PTrp-C, MG-PTrp-P, MG-PTrp-B, and MG-PTrp-ABC strains constructed in Example 2 (without modular integration of the tryptophan precursor supply module), were inoculated into 4 mL of LB liquid medium containing ampicillin, kanamycin, and streptomycin, respectively. The cultures were incubated overnight at 37°C and 200 rpm for 12 h to obtain seed culture. 2500 μL of the seed culture was inoculated into a 250 mL flask containing 25 mL of fermentation medium (MR medium). The cultures were incubated at 30°C and 200 rpm for 24 h. Ammonia was added to adjust the pH to 7.0, and fermentation continued for 48 h.

[0103] Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, discard the supernatant, wash the precipitate thoroughly with 100% methanol, centrifuge at 10,000 rpm for 5 min, and after the methanol has fully evaporated, add 2 mL of DMSO and use sonication to dissolve the precipitate. Centrifuge the resulting liquid at 10,000 rpm for 10 min, collect the supernatant, filter through a membrane for HPLC analysis.

[0104] Indigo yield was calculated, and the results are shown in [the original text]. Figure 3 ;

[0105] like Figure 3 As shown, all eight different hosts exhibited de novo indigo synthesis capability after fermentation. Among them, the modularly integrated strains MG-Trp-C, MG-Trp-P, MG-Trp-B, and MG-Trp-ABC generally showed increased yields compared to strains without modular integration of the tryptophan precursor, with MG-Trp-C exhibiting the highest yield (1.2 g / L). This invention achieved a certain yield by strengthening key genes in the tryptophan production pathway using plasmids, providing a reference for the construction of strains for industrial indigo production.

[0106] Example 4: Production of indigo from engineered strains using a fed-batch fermentation process

[0107] This embodiment provides a method for utilizing recombinant Escherichia coli MG-Trp-C (its indigo synthesis principle is described in [link to documentation]). Figure 1 A method for batch fed-batch fermentation production of indigo using MG-PTrp-C.

[0108] (1) Seed liquid preparation

[0109] Fresh single colonies of engineered strains MG-Trp-C and MG-PTrp-C were selected and inoculated into 4 mL of LB liquid medium containing ampicillin (100 μg / mL), kanamycin (50 μg / mL), and streptomycin (50 μg / mL). The cultures were then shaken at 37°C and 200 rpm for 12 h to obtain the primary seed culture.

[0110] (2) Preparation of secondary seed liquid

[0111] The primary seed culture was inoculated into the secondary seed culture medium at an inoculation rate of 2% (v / v) and cultured at 37℃ and 200 rpm for 6–8 h. When the bacterial cell concentration OD 600 When the pH reaches 2.0–2.5, it is used as the inoculum for fermentation.

[0112] (3) Fermentation by batch feeding

[0113] The secondary seed culture was inoculated into the fermentation medium in a 5L fermenter at an inoculation rate of 5% (v / v); the initial fermentation conditions were set as follows: temperature 30℃, aeration rate 2vvm, and stirring speed 300rpm.

[0114] When dissolved oxygen (DO) drops below 30%, the stirring speed is gradually increased to maintain DO within the range of 20-40%. During fermentation, the glucose concentration in the culture medium is monitored every 2 hours, and an online feeding method is used to control its concentration to remain below 1 g / L. The glucose feeding rate is dynamically adjusted based on the cell growth rate and metabolic status. The total fermentation time is approximately 60 hours. After fermentation, samples are taken to analyze the indigo yield.

[0115] (4) Fermentation results

[0116] like Figure 4As shown, the engineered strain MG-Trp-C achieved a maximum indigo yield of 10.24 g / L in a 5L fermentation system, while MG-PTrp-C achieved a maximum indigo yield of 6.2 g / L, approximately 60% of that of the modularly integrated strain MG-Trp-C. These results indicate that modularizing aroBDELAC and placing it under a unified regulatory unit significantly improves precursor supply throughput and product synthesis efficiency. Furthermore, these results demonstrate that highly efficient de novo indigo biosynthesis was achieved by comprehensively optimizing the expression of aromatic amino acid and tryptophan synthesis pathways and the indigo synthesis module.

[0117] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A modularly reconstructed recombinant Escherichia coli for de novo synthesis of indigo, characterized in that, Using Escherichia coli as the host, indigo synthesis pathway genes were expressed in a modular manner; Modular expression of indigo biosynthesis pathway genes includes any one of the modifications from I to IV: Ⅰ: Reconstruction of the tryptophan precursor supply module: Knock out at least two of the following genes in the host: aroB, aroD, aroE, aroL, aroA, and aroC; and tandemly overexpress the knocked-out genes. II: Tryptophan metabolism module: exogenous overexpression of indoleglycerol phosphate synthase gene BX1 and / or tryptophanase gene naphthalene dioxygenase gene tnaA; III: Indigo synthesis module: exogenous overexpression of at least one of the genes encoding naphthalene dioxygenase, namely CNDO, NDO-ABC, BNDO, and PNDO; IV: Feedback Inhibition Relief and Synthesis Enhancement Module: Exogenous overexpression of indigo hydrolase gene IH and gene aroG mut trpE gene mut TrpC gene mut At least one of them.

2. The recombinant Escherichia coli as described in claim 1, characterized in that, In item I, genes aroB, aroD, aroE, aroL, aroA, and aroC are integrated into the yjgX site on the host chromosome.

3. The recombinant Escherichia coli as described in claim 1, characterized in that, In item II, the indoleglycerol phosphate synthase gene BX1 and / or tryptophanase gene naphthalene dioxygenase gene tnaA were overexpressed using the pCDFDuet vector.

4. The recombinant Escherichia coli as described in claim 1, characterized in that, In item III, at least one of the genes encoding naphthalene dioxygenase, namely CNDO, NDO-ABC, BNDO, and PNDO, is overexpressed using the pETDuet vector.

5. The recombinant Escherichia coli as described in claim 1, characterized in that, In section IV, the indigo hydrolase gene IH and gene aroG were overexpressed using the pTrc99a vector. mut trpE gene mut trpC gene mut At least one of the following; wherein the gene aroG mut The nucleotide sequence is shown in SEQ ID No. 8; the gene trpE mut The nucleotide sequence is shown in SEQ ID No. 9; the gene trpC mut The nucleotide sequence is shown in SEQ ID No.

10.

6. The recombinant Escherichia coli as described in claim 1, characterized in that, In items I-IV, different genes in each module are linked together through ribosome binding sites.

7. The recombinant Escherichia coli as described in claim 1, characterized in that, The Escherichia coli includes Escherichia coli MG1655 or its derivative strains.

8. A method for synthesizing indigo, characterized in that, include: The recombinant Escherichia coli described in any one of claims 1-7 is prepared by shaking flask fermentation or industrial fed-batch fermentation using glucose or glycerol as a carbon source.

9. The method as described in claim 8, characterized in that, Both shake-flask fermentation and industrial fed-batch fermentation cultures used MR medium. The initial fermentation conditions for industrial fed-batch fermentation culture were set as follows: temperature 30℃, aeration rate 2 vvm, and stirring speed 300 rpm. During fermentation, dissolved oxygen was controlled at 20-40%, and online feeding was used to maintain glucose concentration below 1 g / L.

10. The use of the recombinant Escherichia coli according to any one of claims 1-7 in the production of indigo.

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