Engineered corynebacterium glutamicum for synthesizing indigoidine and method for improving yield thereof

Abstract

The present application relates to the field of synthetic biology, metabolic engineering and microbial fermentation technology, and particularly relates to Corynebacterium glutamicum engineering bacteria for synthesizing natural blue pigment Indigoidine and a method for improving the yield thereof. pntAB The membrane-bound transhydrogenase coding gene is used in the natural blue pigment Indigoidine synthesis system of Corynebacterium glutamicum, the NADPH supply capacity is enhanced, the natural blue pigment synthesis is optimized from the overall reduction power supply and metabolic balance of cells, the problems of insufficient precursor supply and cell metabolic burden can be alleviated, the yield of natural blue pigment is significantly improved, and the application potential in industry is good, and a new technical path is provided for green biological manufacturing of natural blue pigment.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology, metabolic engineering, and microbial fermentation technology, specifically to an engineered strain of Corynebacterium glutamicum that synthesizes the natural blue pigment Indigoidine, and a method for increasing the yield of the natural blue pigment. Background Technology

[0002] Indigoidine is a natural blue pigment produced by non-ribosomal peptide synthase. It is characterized by its stable color, natural origin, and environmental friendliness, and has potential applications in food coloring, textile dyes, cosmetics, biomaterials, and sustainable manufacturing. Traditional chemically synthesized pigments often suffer from significant environmental pollution during production and non-renewable substrate sources. Therefore, utilizing microbial cell factories to produce natural blue pigments has become a research hotspot in recent years.

[0003] Indigoidine is typically synthesized from glutamine substrates by the catalysis of natural indigo synthase. Its biosynthesis depends not only on the expression and activation of the natural indigo synthase itself, but is also influenced by multiple factors including substrate glutamine supply, ATP supply, cellular reducing power balance, redox state, cell growth state, and product stress. Corynebacterium glutamicum (… Corynebacterium glutamicum Corynebacterium glutamicum is a traditional industrial microorganism with good amino acid synthesis capabilities, strong industrial fermentation adaptability, and a mature genetic manipulation system, making it suitable as a platform for the biosynthesis of natural blue pigments. Although there have been some studies on the synthesis of indigoidine using Corynebacterium glutamicum, further improvements in indigoidine yield require the development of new metabolic engineering targets that can promote indigoidine synthesis. Summary of the Invention

[0004] This invention provides an engineered strain of Corynebacterium glutamicum for synthesizing the natural blue pigment Indigoidine, and a method for increasing the yield of the natural blue pigment.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides the application of membrane-bound transhydrogenase, its encoding gene, or biological material containing said encoding gene in increasing the yield of natural blue pigment synthesized by Corynebacterium glutamicum or in constructing engineered Corynebacterium glutamicum for synthesizing natural blue pigment. The natural blue pigment is Indigoidine.

[0007] Preferably, the application includes: introducing and overexpressing a membrane-bound transhydrogenase encoding gene in Corynebacterium glutamicum, which has the ability to synthesize natural blue pigment.

[0008] Membrane-bound transhydrogenase (PntAB) catalyzes the reaction of NADH and NADP. + The reduction force transfer between NADH and NADPH converts the reducing force in NADH into NADPH, thereby increasing the intracellular NADPH supply capacity. In existing technologies, pntAB While NADPH / NADH balance has been used to regulate the synthesis of some amino acids or reduction-dependent products, its application in the *Corynebacterium glutamicum* natural blue pigment synthesis system to increase natural blue pigment yield has not yet been reported. This invention reveals that NADPH supply has a significant impact on indigoidine synthesis in *Corynebacterium glutamicum*. In *Corynebacterium glutamicum* strains capable of synthesizing indigoidine, expression of a membrane-bound transhydrogenase enhances NADPH supply, significantly improves indigoidine synthesis, and increases indigoidine yield.

[0009] Preferably, the membrane-bound transhydrogenase is derived from Escherichia coli.

[0010] Preferably, the amino acid sequence of the membrane-bound transhydrogenase is shown in SEQ ID NO.4 and 6.

[0011] Membrane-bound transhydrogenases from E. coli are produced by pntAB Gene encoding, the pntAB Including the encoding of the α subunit of membrane-bound transhydrogenase (SEQ ID NO.4) pntA And encoding the β subunit of membrane-bound transhydrogenase (SEQ ID NO.6) pntB .

[0012] Preferably, the pntAB This is the gene encoding a membrane-bound transhydrogenase derived from *E. coli*, optimized according to the codon usage preferences of *Corynebacterium glutamicum*.

[0013] Preferably, the gene sequence optimized by Corynebacterium glutamicum codon usage preference is as follows: pntA The nucleotide sequence of the gene is shown in SEQ ID NO.3. pntB The nucleotide sequence of the gene is shown in SEQ ID NO.5.

[0014] In this invention, the biomaterial includes any one of the following: (1) An expression cassette containing a gene encoding a membrane-bound transhydrogenase; (2) A vector containing a membrane-bound transhydrogenase encoding gene or an expression cassette as described in (1); (3) Microorganisms containing a membrane-bound transhydrogenase encoding gene, the expression cassette described in (1) or the vector described in (2).

[0015] The expression cassette can be obtained by operatively linking the coding gene with transcriptional and / or translational regulatory elements (e.g., promoters, terminators, enhancers, etc.).

[0016] The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages, viral vectors, artificial chromosomes, etc.

[0017] The microorganisms may be bacteria, fungi, or viruses. Among them, the bacteria include Escherichia coli or Corynebacterium glutamicum.

[0018] In this invention, the introduction and overexpression of the membrane-bound transhydrogenase encoding gene can be achieved through conventional genetic engineering techniques, specifically including at least one of the following (1) and (2): (1) Integrate at least one copy of the membrane-bound transhydrogenase encoding gene into the genome of Corynebacterium glutamicum; (2) Introduce an expression plasmid containing a gene encoding a membrane-bound transhydrogenase into Corynebacterium glutamicum.

[0019] Wild-type strains of Corynebacterium glutamicum do not have the ability to synthesize indigoidine. Therefore, in this invention, Corynebacterium glutamicum with the ability to synthesize natural blue pigment expresses the gene encoding the natural blue pigment synthase. bpsA Or its functional variants.

[0020] Preferably, the Corynebacterium glutamicum with the ability to synthesize natural blue pigment also expresses a gene encoding an activation coenzyme for the natural blue pigment synthase. sfp .

[0021] In this invention, the natural blue pigment synthase encoding gene bpsA Derived from Streptomyces lilacinus ( Streptomyces lavendulae ), Streptomyces brownii ( Streptomyces chromofuscus ), Pseudomonas putida ( Pseudomonas putida ) or other microorganisms capable of catalyzing the synthesis of indigoidine. Preferably derived from *Streptomyces lilacinus* ( Streptomyces lavendulae ).

[0022] Preferably, the amino acid sequence of the natural blue pigment synthase is shown in SEQ ID NO.2.

[0023] Preferably, the natural blue pigment synthase encoding gene bpsA The nucleotide sequence is shown in SEQ ID NO.1.

[0024] Preferably, the amino acid sequence of the activating coenzyme of the natural blue pigment synthase is shown in SEQ ID NO.8.

[0025] Preferably, the sfpThe nucleotide sequence is shown in SEQ ID NO.7.

[0026] In a second aspect, the present invention provides a method for increasing the production of natural blue pigment by Corynebacterium glutamicum, the method comprising: introducing and overexpressing a membrane-bound transhydrogenase encoding gene in Corynebacterium glutamicum, which has the ability to synthesize natural blue pigment; The natural blue pigment is Indigoidine.

[0027] Preferably, the introduction and overexpression of the membrane-bound transhydrogenase encoding gene is achieved by integrating at least one copy of the membrane-bound transhydrogenase encoding gene into the genome or by introducing an expression plasmid containing the membrane-bound transhydrogenase encoding gene.

[0028] In some embodiments of the present invention, the introduction and overexpression of the membrane-bound transhydrogenase encoding gene is achieved by introducing an expression plasmid containing the membrane-bound transhydrogenase encoding gene into Corynebacterium glutamicum, which has the ability to synthesize natural blue pigment. Preferably, the expression plasmid is pEC-XK99E. The expression plasmid further contains a promoter driving its transcription upstream of the membrane-bound transhydrogenase encoding gene.

[0029] In this invention, the pntAB Expression can be driven by constitutive or inducible promoters.

[0030] In some embodiments of the present invention, the pntAB Expression is driven by a strongly constitutive promoter. Optional promoters include... P tuf , P sod , P eftu , P ilvC , P tac , P trc , P lac , P gapA , P dapA Or its mutants, truncated forms, or functional variants.

[0031] Preferably, the membrane-bound transhydrogenase is derived from Escherichia coli.

[0032] Preferably, compared with a control strain that does not express the membrane-bound transhydrogenase, the engineered bacteria produce at least 10%, 20%, 30%, 50%, 80%, or 100% more natural blue pigment.

[0033] Preferably, the Corynebacterium glutamicum with the ability to synthesize natural blue pigment expresses a gene encoding a natural blue pigment synthase. bpsA Or its functional variants.

[0034] Preferably, the Corynebacterium glutamicum with the ability to synthesize natural blue pigment also expresses a gene encoding an activation coenzyme for the natural blue pigment synthase. sfp .

[0035] Preferably, the natural blue pigment synthase encoding gene bpsA and its activation coenzyme encoding gene sfp Located on an expression vector (preferably on the same expression vector, preferably pXMJ19). Transcription is preferably driven by an inducible promoter.

[0036] To further increase the yield of indigoidine, the method further includes: overexpressing the glutamate synthase encoding gene in the *Corynebacterium glutamicum*. gltBD .

[0037] This invention discovers that, in expressing the gene encoding membrane-bound transhydrogenase... pntAB At the same time, overexpression gltBD The genes, together with the bacteria, work synergistically to significantly enhance the ability of Corynebacterium glutamicum to synthesize indigoidine, with the increase in indigoidine production being significantly better than that of other strains. gltBD , pntAB The effect of individual modification is also significantly better than the commonly used strategy of enhancing glutamine supply.

[0038] in, gltB The amino acid sequence encoding the protein is shown in SEQ ID NO.9. gltD The amino acid sequence encoding the protein is shown in SEQ ID NO.10.

[0039] Preferably, the method further includes: modifying the *Corynebacterium glutamicum* as follows (1) and / or (2): (1) Overexpression ppgk Gene; (2) Overexpression iolT1 and iolT2 Gene.

[0040] in, ppgk The amino acid sequence encoding the protein is shown in SEQ ID NO.11. iolT1 and iolT2 The amino acid sequence encoding the protein is shown in SEQ ID NO.12 and 13.

[0041] In this invention, the overexpression can be implemented in any one or more of the following ways: (1) Increase the copy number of the gene; (2) Replace the transcriptional and / or translational regulatory elements of the gene with more active elements.

[0042] Preferably, increasing the copy number can be achieved by integrating at least one copy of the gene into the genome, or by introducing an expression plasmid containing the gene.

[0043] Preferably, the transcriptional and / or translational regulatory elements include promoters, enhancers, etc.

[0044] In some embodiments of the present invention, the overexpression is achieved by introducing an expression plasmid containing the gene. The overexpressed gene can be... pntAB The genes are expressed in tandem within the same expression plasmid.

[0045] Thirdly, the present invention provides a Corynebacterium glutamicum engineered strain that synthesizes natural blue pigment, wherein the engineered strain contains a gene encoding a natural blue pigment synthase. bpsA and membrane-bound transhydrogenase encoding genes pntAB ; The natural blue pigment is Indigoidine.

[0046] This invention introduces and overexpresses a membrane-bound transhydrogenase encoding gene in Corynebacterium glutamicum, which has the ability to synthesize natural blue pigment. pntAB It promotes the reduction conversion of NADH to NADPH in cells, enhances the NADPH supply capacity, and thus enhances the supply of precursors related to the synthesis of natural blue pigment and the cell's reducing power balance, thereby increasing the production of natural blue pigment.

[0047] Preferably, the engineered bacteria overexpress the gene encoding the natural blue pigment synthase. bpsA and membrane-bound transhydrogenase encoding genes pntAB .

[0048] Preferably, the engineered bacteria overexpress the gene encoding the activation coenzyme of the natural blue pigment synthase. sfp .

[0049] Preferably, the membrane-bound transhydrogenase is derived from Escherichia coli.

[0050] In the above-mentioned engineered bacteria, the bpsA and pntAB They can be located in the same expression vector, different expression vectors, or in the genome of Corynebacterium glutamicum.

[0051] In some embodiments of the present invention, the bpsA and pntAB They are located in different expression vectors.

[0052] To further increase the yield of natural blue pigment, the engineered bacteria also overexpressed the gene encoding glutamate synthase. gltBD .

[0053] This invention discovers that, in expressing the gene encoding membrane-bound transhydrogenase... pntAB At the same time, overexpression gltBD The genes, together with the bacteria, can work synergistically to significantly enhance the ability of Corynebacterium glutamicum to synthesize indigoidine, thus further increasing the yield of indigoidine.

[0054] Preferably, the engineered bacteria also overexpress the genes in (1) and / or (2) below: (1) ppgk Gene; (2) iolT1 and iolT2 Gene.

[0055] Some embodiments of the present invention provide an engineered strain of Corynebacterium glutamicum, which contains a carrier bpsA and sfp Gene expression plasmids and carriers pntAB Gene expression plasmids.

[0056] Some embodiments of the present invention provide an engineered strain of Corynebacterium glutamicum, which contains a carrier bpsA and sfp Gene expression plasmids and carriers pntAB and gltBD Gene expression plasmids.

[0057] Some embodiments of the present invention provide an engineered strain of Corynebacterium glutamicum, which contains a carrier bpsA and sfp Gene expression plasmids and carriers pntAB, gltBD and ppgk Gene expression plasmids.

[0058] Some embodiments of the present invention provide an engineered strain of Corynebacterium glutamicum, which contains a carrier bpsA and sfp Gene expression plasmids and carriers pntAB, gltBD, iolT1 and iolT2 Gene expression plasmids.

[0059] In this invention, the original starting strain of the engineered Corynebacterium glutamicum can be selected from ATCC 13032, ATCC13869, ATCC 14067, RES167, or their derived strains.

[0060] Fourthly, the present invention provides the application of the engineered bacteria described above in the preparation of natural blue pigment or in the construction of engineered bacteria for the production of natural blue pigment; The natural blue pigment is Indigoidine.

[0061] The Indigoidine prepared by the engineered bacteria of this invention can be used to prepare food colorings, textile dyes, cosmetic colorings, pharmaceutical intermediates or bio-based materials.

[0062] Fifthly, the present invention provides a method for preparing natural blue pigment, the method comprising: fermenting and culturing the engineered bacteria described above, and collecting the natural blue pigment in the culture; The natural blue pigment is Indigoidine.

[0063] Preferably, the carbon source used in the fermentation culture is one or more of glucose, sucrose, fructose, glycerol, molasses, starch hydrolysate, corn steep liquor hydrolysate, and lignocellulose hydrolysate; The nitrogen source used in the fermentation culture is one or more of the following: ammonium sulfate, ammonium chloride, urea, yeast powder, peptone, corn steep liquor, glutamic acid, and glutamine.

[0064] Preferably, the pH of the fermentation culture is controlled between 6.0 and 8.0. More preferably, the pH is between 6.8 and 7.4.

[0065] Preferably, the fermentation culture temperature is 28-34°C. The fermentation is carried out under aerobic conditions.

[0066] The beneficial effects of the present invention include at least the following: (1) This invention is the first to encode a membrane-bound transhydrogenase gene. pntAB This technology, used in the Corynebacterium glutamicum natural blue pigment synthesis system, optimizes natural blue pigment synthesis from the perspective of overall cellular reducing power supply and metabolic balance by enhancing NADPH supply capacity. It can alleviate the problems of insufficient precursor supply and cellular metabolic burden, and is comparable to that of non-overexpressing strains. pntAB Compared with the control strain, it significantly increased the production of natural blue pigment.

[0067] (2) The engineered bacteria provided by the present invention can be used in combination with other engineering strategies and has good scalability.

[0068] (3) The engineered bacteria provided by the present invention can synthesize natural blue pigment in a culture system with glucose, sucrose, molasses, corn steep liquor hydrolysate or other fermentable carbon sources as substrates, and have good industrial application potential, providing a new technical path for the green bio-manufacturing of natural blue pigment. Attached Figure Description

[0069] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0070] Figure 1 These are color phenotypic diagrams of different engineered bacteria fermentation broths or cell precipitates in embodiments of the present invention.

[0071] Figure 2 This is a comparison chart of the indigoidine production of different engineered bacteria in the embodiments of the present invention. Detailed Implementation

[0072] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0073] Example 1: Construction of a basic strain for synthesizing the natural blue pigment indigoidine by Corynebacterium glutamicum ATCC 13032 was used as the starting strain to construct the basic strain for the synthesis of natural blue pigment.

[0074] Gene encoding the synthase of the natural blue pigment indigoidine bpsA and its activation coenzyme encoding gene sfp Cloned into the Corynebacterium glutamicum expression vector pXMJ19, using P... tac Promoter tandem expression bpsA and sfp Among them, the gene encoding the natural blue pigment synthase... bpsA Derived from Streptomyces lilacinus ( Streptomyces lavendulae Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence encoding the protein is shown in SEQ ID NO.2, activating coenzyme encoding gene. sfp Derived from Corynebacterium glutamicum ( Corynebacterium glutamicum Its nucleotide sequence is shown in SEQ ID NO.7, and the amino acid sequence encoding the protein is shown in SEQ ID NO.8.

[0075] Will contain bpsA and sfp The expression vector pXMJ19 was introduced. Corynebacterium glutamicumATCC13032 was used to screen for positive transformants, and the basic strain for natural blue pigment synthesis was obtained, denoted as CG-BS.

[0076] Example 2: Overexpression of Escherichia coli source pntAB Construction of engineered bacteria Based on the PntAB protein sequence from *E. coli*, the sequences encoding the α subunit of membrane-bound transhydrogenase were extracted. pntA and the encoding membrane-bound transhydrogenase β subunit pntB Corresponding amino acid sequence. Based on the codon preference of Corynebacterium glutamicum, for pntA and pntB The nucleotide sequence was codon-optimized to obtain an optimized sequence suitable for efficient expression by Corynebacterium glutamicum while maintaining its encoding amino acid sequence. Subsequently, the codon-optimized sequence was obtained through artificial gene synthesis. pntA (The gene sequence is shown in SEQ ID NO.3, and the protein encoding sequence is shown in SEQ ID NO.4) and pntB (The gene sequence is shown in SEQ ID NO.5, and the protein sequence is shown in SEQ ID NO.6.) The coding sequence was used as a membrane-bound transhydrogenase expression module for subsequent engineered bacteria construction.

[0077] Will pntAB The bacteria were cloned into the Corynebacterium glutamicum expression vector pEC-XK99E and placed in a strong constitutive promoter. P tuf Under control, the following was constructed. pntAB Expressive vehicle. pntAB The expression vector was introduced into the natural blue pigment synthesis strain CG-BS obtained in Example 1, and positive transformants were screened to obtain overexpressing strains. pntAB The engineered bacterium that synthesizes the natural blue pigment was designated CG-BS-PntAB. Verification was performed using colony PCR, plasmid extraction, sequencing, or RT-qPCR. pntAB The introduction and expression of .

[0078] Example 3: pntAB Overexpression increases shake-flask fermentation yield of the natural blue pigment indigoidine The CG-BS obtained in Example 1 and the CG-BS-PntAB obtained in Example 2 were respectively inoculated into seed culture medium and then transferred to fermentation culture medium with the same inoculation amount. The fermentation medium used was CGXII medium, with the following composition: glucose 80 g / L, MgSO4·7H2O 0.25 g / L, (NH4)2SO4 20 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, urea 5 g / L, MOPS 42 g / L, biotin 20 μg / L, thiamine 100 μg / L, and trace elements (including MnSO4·5H2O 10 mg / L, FeSO4·7H2O 10 mg / L, ZnSO4·7H2O 10 mg / L, CuSO4·5H2O 2 mg / L, NiSO4·6H2O 20 mg / L, CaCl2 10 mg / L, CoCl2·6H2O 0.31 mg / L). The initial pH of the medium was adjusted to 7.0 with NaOH; for engineered bacteria carrying plasmids, appropriate antibiotics were added to maintain plasmid stability.

[0079] The engineered bacteria were inoculated into seed culture medium and cultured at 30°C with shaking for 16 h to obtain seed culture. Subsequently, at an inoculation rate of 4% (v / v), the seed culture was transferred to shake flasks containing CGXII medium and cultured at 30°C and 220 rpm for 72 h. When the OD of the culture medium... 600 When the concentration reached approximately 0.8, 0.1 mM IPTG was added to initiate the synthesis of the natural blue pigment. After fermentation, the fermentation broth was collected, and the yield of the natural blue pigment, indigoidine, was determined. The natural blue pigment was extracted by centrifugation and DMSO solvent extraction, and quantified using high-performance liquid chromatography.

[0080] Different engineered bacteria fermentation broth or cell precipitate color phenotypes, such as Figure 1 As shown, the specific yield results are as follows: Figure 2 As shown, the production of the natural blue pigment Indigoidine in CG-BS-PntAB was higher than that in the control strain CG-BS. The production of natural blue pigment Indigoidine in the control strain CG-BS was 2.0 g / L, indicating that overexpression... pntAB The engineered strain CG-BS-PntAB produced 3.1 g / L of the natural blue pigment indigoidine; compared with the control strain, the production of natural blue pigment indigoidine by CG-BS-PntAB was increased by 55%. This result indicates that overexpression of the membrane-bound transhydrogenase encoding gene... pntAB It can effectively enhance the ability of Corynebacterium glutamicum to synthesize the natural blue pigment Indigoidine.

[0081] Example 4: pntABCombination with glutamate / glutamine precursor supply enhancement strategies Indigoidine biosynthesis uses glutamine as a direct substrate, and its yield is influenced not only by the expression levels of indigoidine synthase and its activating enzyme, but also by the combined effects of the host cell's intracellular glutamate / glutamine precursor pool, reducing power supply, and nitrogen assimilation metabolic flux. Although overexpression of the glutamine synthase encoding gene... glnA This is a common strategy to enhance glutamine supply, but in the Indigoidine synthesis system, simply strengthening... glnA It may be difficult to fully increase the production of Indigoidine due to limitations in upstream glutamate supply, NADPH supply, and overall cellular nitrogen metabolism balance.

[0082] To further improve indigoidine production, this embodiment introduces a glutamate / glutamine precursor supply enhancement module based on CG-BS-PntAB. Specifically, the glutamate synthase encoding gene is... gltBD Cloning to the construct containing Example 2 pntAB On the pEC-XK99E, with pntAB In strongly constitutive promoters P tuf Under the control of tandem expression, that is, forming P tuf The expression cassette -pntAB-gltBD was used. The expression plasmid was introduced into the CG-BS strain to obtain simultaneous overexpression. pntAB and gltBD The engineered bacteria CG-BS-PntAB-GltBD.

[0083] As a control, the gene encoding glutamine synthase was used. glnA Cloned into pEC-XK99E and placed in a strongly constitutive promoter. P tuf Under control, the following was constructed. glnA Expressive vehicle. glnA The expression vector was introduced into the natural blue pigment synthesis strain CG-BS obtained in Example 1, and positive transformants were screened to obtain overexpressing strains. glnA The engineered bacterium that synthesizes natural blue pigment is designated CG-BS-GlnA; the gene encoding glutamate synthase... gltBD Cloned into pEC-XK99E and placed in a strongly constitutive promoter. P tuf Under control, the following was constructed. gltBD Expressive vehicle. gltBD The expression vector was introduced into the natural blue pigment synthesis strain CG-BS obtained in Example 1, and positive transformants were screened to obtain overexpressing strains. gltBDThe engineered bacterium that synthesizes natural blue pigment is designated CG-BS-GltBD; the gene encoding glutamine synthase... glnA Cloning to the construct containing Example 2 pntAB On the pEC-XK99E, with pntAB In strongly constitutive promoters P tuf Under controlled tandem expression, simultaneous overexpression was achieved. pntAB and glnA The engineered strain CG-BS-GlnA-PntAB. The above control strain and the strain only overexpressing CG-BS-GlnA-PntAB were compared. pntAB Engineered bacteria CG-BS-PntAB, simultaneously overexpressing pntAB and gltBD The engineered strain CG-BS-PntAB-GltBD was subjected to shake-flask fermentation using the same method as in Example 3, and the indigoidine yield of each engineered strain was determined.

[0084] The results are as follows Figure 2 As shown, simultaneous overexpression pntAB and gltBD The engineered strain CG-BS-PntAB-GltBD produced 6.1 g / L of indigoidine, a 205% increase compared to the basic strain CG-BS. This was significantly higher than that produced by overexpression alone. pntAB (3.2 g / L), overexpression alone gltBD (2.8 g / L) and the conventional glutamine synthesis-enhancing strain CG-BS-GlnA (2.3 g / L). Meanwhile, the indigoidine yield of CG-BS-PntAB-GltBD was also higher than that of CG-BS-GlnA-PntAB (3.0 g / L), indicating that... pntAB and gltBD The combination is not a simple additive, but rather a further promotion of the formation of glutamate / glutamine precursor pools on the basis of enhancing NADPH supply, thereby producing a synergistic effect of increased production.

[0085] The above results indicate that pntAB Mediated NADPH supply enhancement and gltBD There is a synergistic effect between the mediated enhancement of glutamate / glutamine precursor supply. This combined strategy differs from simple overexpression. pntAB Or conventional overexpression glnA The modification method can more effectively improve the limiting of reducing power supply and precursor supply in the synthesis process of Indigoidine, thereby significantly increasing the yield of Indigoidine synthesized by Corynebacterium glutamicum.

[0086] Example 5: pntABA combination of strategies to enhance glutamate / glutamine precursor supply and ATP supply and glucose uptake. Since the synthesis of the natural blue pigment Indigoidine relies on non-ribosomal peptide synthase catalysis, energy supply has a significant impact on the accumulation of the natural blue pigment product. To further increase the yield of the natural blue pigment Indigoidine, an ATP supply enhancement module was introduced into the CG-BS-PntAB-GltBD model.

[0087] Will iolT1 and iolT2 Combination and ppgk Genes were seamlessly cloned into plasmids constructed in Example 4 containing... pntAB and gltBD On pEC-XK99E, with pntAB-gltBD in a strongly constitutive promoter P tuf Under the control of tandem expression, that is, forming respectively... P tuf -pntAB-gltBD - iolT1-iolT2 expression boxes and P tuf -pntAB-gltBD - The ppgk expression box will contain respectively P tuf -pntAB-gltBD - iolT1-iolT2 expression box, P tuf -pntAB-gltBD - The pEC-XK99E expression plasmid of the ppgk expression cassette was introduced into the CG-BS strain to obtain the NADPH and ATP-enhanced engineered strain CG-BS-PntAB-GltBD-IolT (IolT represents simultaneous overexpression). iolT1 and iolT2 ) and CG-BS-PntAB-GltBD-Ppgk.

[0088] Shake-flask fermentation was carried out using the same method as in Example 3, and the results were as follows: Figure 2 As shown, the production of the natural blue pigment Indigoidine from the NADPH and ATP-enhanced engineered bacteria CG-BS-PntAB-GltBD-IolT and CG-BS-PntAB-GltBD-Ppgk was further increased to 6.2 g / L and 6.6 g / L, respectively, indicating that... pntAB The mediated enhancement of reducing power, together with enhanced energy supply and sugar uptake, can improve the biosynthetic efficiency of the natural blue pigment Indigoidine.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of membrane-bound transhydrogenase, its encoding gene, or biological material containing said encoding gene in increasing the yield of natural blue pigment synthesized by Corynebacterium glutamicum or in constructing engineered Corynebacterium glutamicum for synthesizing natural blue pigment; in, The natural blue pigment is Indigoidine.

2. The application according to claim 1, characterized in that, The application includes: introducing and overexpressing a membrane-bound transhydrogenase encoding gene in Corynebacterium glutamicum, which has the ability to synthesize natural blue pigment; Preferably, the membrane-bound transhydrogenase is derived from Escherichia coli.

3. A method for increasing the yield of natural blue pigment synthesized by Corynebacterium glutamicum, characterized in that, The method includes: introducing and overexpressing a membrane-bound transhydrogenase encoding gene in Corynebacterium glutamicum, which has the ability to synthesize natural blue pigment; The natural blue pigment is Indigoidine.

4. The method according to claim 3, characterized in that, The introduction and overexpression of the membrane-bound transhydrogenase encoding gene is achieved by integrating at least one copy of the membrane-bound transhydrogenase encoding gene into the genome or by introducing an expression plasmid containing the membrane-bound transhydrogenase encoding gene. Preferably, the membrane-bound transhydrogenase is derived from Escherichia coli; Preferably, compared with a control strain that does not express the membrane-bound transhydrogenase, the engineered bacteria exhibit an increase in the production of natural blue pigment of at least 10%, 20%, 30%, 50%, 80%, or 100%. Preferably, the Corynebacterium glutamicum with the ability to synthesize natural blue pigment expresses a gene encoding a natural blue pigment synthase. bpsA or its functional variations; Preferably, the Corynebacterium glutamicum with the ability to synthesize natural blue pigment also expresses a gene encoding an activation coenzyme for the natural blue pigment synthase. sfp .

5. The method according to claim 3 or 4, characterized in that, The method further includes: overexpressing a glutamate synthase-encoding gene in the *Corynebacterium glutamicum*. gltBD ; Preferably, the method further includes: modifying the *Corynebacterium glutamicum* as follows (1) and / or (2): (1) Overexpression ppgk Gene; (2) Overexpression iolT1 and iolT2 Gene.

6. A strain of Corynebacterium glutamicum that synthesizes natural blue pigment, characterized in that, The engineered bacteria contain a gene encoding a natural blue pigment synthase. bpsA and membrane-bound transhydrogenase encoding genes pntAB ; The natural blue pigment is Indigoidine.

7. The engineered bacteria according to claim 6, characterized in that, The engineered bacteria overexpress the activation coenzyme encoding gene of natural blue pigment synthase. sfp ; Preferably, the membrane-bound transhydrogenase is derived from Escherichia coli.

8. The engineered bacteria according to claim 6 or 7, characterized in that, The engineered bacteria also overexpress the gene encoding glutamate synthase. gltBD ; Preferably, the engineered bacteria also overexpress the genes in (1) and / or (2) below: (1) ppgk Gene; (2) iolT1 and iolT2 Gene.

9. The use of the engineered bacteria according to any one of claims 6 to 8 in the preparation of natural blue pigment or in the construction of engineered bacteria for the production of natural blue pigment; in, The natural blue pigment is Indigoidine.

10. A method for preparing natural blue pigment, characterized in that, The method includes: fermenting and culturing the engineered bacteria according to any one of claims 6 to 8, and collecting the natural blue pigment in the culture; The natural blue pigment is Indigoidine.

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