Indochrome C heterologous expression engineered bacteria based on cold-stressed NRPS gene cluster and its construction method

Abstract

This invention relates to an engineered bacterium that heterologously expresses Indochrome C based on the cold-stressed NRPS gene cluster. The engineered bacterium is... Escherichia coli The pET41a-Indochrome C-Cluster, with accession number GDMCC No. 66715, is disclosed in this invention. The invention also discloses a method for constructing this engineered bacterium. By reconstructing the Indochrome C NRPS gene cluster using synthetic biology techniques, this invention solves the problems of long fermentation cycles and insufficient yields in the original strain, significantly improving pigment production efficiency and industrial application potential.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and microbial metabolic engineering, and in particular to Indochrome C heterologous expression engineered bacteria based on cold-stressed NRPS gene clusters and their construction methods. Background Technology

[0002] In recent years, the demand for naturally derived dyes, especially blue pigments, has increased significantly, but commercially available natural blue pigments are extremely scarce. Studies have shown that indochrome-like blue pigments possess good water solubility, remain stable below 60°C in neutral to alkaline environments (pH = 6–10), and are essentially non-toxic to organisms. These properties make them promising candidates for natural pigments with potential applications in both industrial and food sectors.

[0003] Antioxidant Arthritis Bacterium QL17 T This strain, isolated from soil from the Rongbuk Glacier on Mount Everest in the Himalayas, produces an extracellular, water-soluble blue pigment under low-temperature conditions. Genomic analysis revealed that the strain carries multiple genes related to cold adaptation and antioxidant activity, enabling it to adapt to extreme low-temperature environments. The blue pigment induced by this bacterium under low-temperature conditions has been named Indochrome C (…). Figure 1 (Disclosed in patent application number 202410028670.8), the transcription level of its core gene is about 17.5 times higher at 15℃ than at 25℃, indicating that its biosynthesis is temperature-regulated. However, the original strain has a long fermentation cycle (about 4 days), low color production temperature requirement (15℃), and a yield of only about 0.874 g / L, which is not conducive to the large-scale industrial production of the natural product Indochrome C. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an Indochrome C heterologous expression engineered bacterium based on the cold-shocked NRPS gene cluster with high production efficiency and short fermentation cycle.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the Indochrome C heterologous expression engineered bacteria based on the cold-stressed NRPS gene cluster.

[0006] To address the aforementioned problems, the Indochrome C heterologous expression engineered bacterium based on the cold-shocked NRPS gene cluster described in this invention is characterized in that: the engineered bacterium is... Escherichia coliThe strain pET41a-Indochrome C-Cluster, with accession number GDMCC No. 66715, was deposited on July 21, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, 510070, China.

[0007] The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-stressed NRPS gene cluster, as described above, includes the following steps:

[0008] (1) Using the whole gene synthesis method, the NRPS gene cluster fragment of Indochrome C was cloned into the pET41a expression vector to construct a recombinant plasmid, which was named pET41a-indochrome C cluster.

[0009] (2) Using the conventional heat shock transformation method, a DNA solution containing the pET41a-Indochrome CCluster recombinant plasmid at a concentration of 100 ng / μL was added to commercially available E. coli BL21 (… Escherichia coli BL21) competent cells were incubated on ice for approximately 30 minutes; the mixture was then heat-shocked at 42 °C for 45 seconds and immediately placed on ice for 5 minutes; then, LB broth without antibiotics was added to the mixture, and the cells were shaken at 37 °C and 200 rpm for 1 hour to recover; after recovery, the bacterial culture was evenly spread on LB agar plates containing kanamycin and incubated at 37 °C for 16–18 hours; resistant single clones were selected to obtain engineered bacteria that stably express the synthetic blue pigment Indochrome C. Escherichia coli pET41a-IndochromeC-Cluster.

[0010] In step (1), the NRPS gene cluster fragment is 8538 bp in length and has a GC content of 61%.

[0011] The expression regulatory elements of the recombinant plasmid in step (1) include the T7 promoter, ribosome binding site, lactose operon, and T7 terminator.

[0012] In step (2), Escherichia coli BL21 ( Escherichia coli The concentration of competent cells in BL21 was 1×10⁻⁶. 8 The volume ratio of competent cells to recombinant plasmid DNA solution was 5:1 (CFU / mL).

[0013] In step (2), the LB liquid culture medium is prepared by adding 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride to deionized water, mixing thoroughly, adjusting the pH to 7.0 with 5 mol / L NaOH, bringing the volume to 1 L with deionized water, and then sterilizing by steaming at 15 psi for 21 min. The volume ratio of this LB liquid culture medium to the recombinant plasmid DNA solution is 45:1.

[0014] The LB plate containing the kanamycin resistance screening marker in step (2) is prepared by adding 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin to deionized water, mixing thoroughly, adjusting the pH to 7.0 with 5 mol / L NaOH, bringing the volume to 1 L with deionized water, and then sterilizing by steaming at 15 psi for 21 min.

[0015] A fermentation product prepared using an engineered bacterial strain with Indochrome C heterologous expression based on the cold-shocked NRPS gene cluster as described above.

[0016] The method for preparing fermentation products of engineered bacteria as described above is characterized by: using engineered bacteria... Escherichia coli A single colony of pET41a-Indochrome C-Cluster was inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37 ℃ with shaking at 200 rpm to obtain the seed culture. 1 mL of the seed culture was then inoculated into 100 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured until OD... 600 When the concentration of the culture medium is 0.6~0.8, IPTG with a final concentration of 200 μM is added for induction, and the culture temperature is adjusted to 18 ℃. The culture is then continued to be shaken at 200 rpm for 42 h to obtain the fermentation product of the engineered bacteria.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. This invention uses Escherichia coli (E. coli) Escherichia coli Using pET41a-Indochrome C-Cluster as the host, a non-ribosomal peptide synthase (NRPS) expression system was constructed through whole-genome synthesis, and an engineered strain heterologously expressed the natural product Indochrome C with antifreeze activity. The constructed engineered strain was confirmed by LC-MS to successfully synthesize the target product Indochrome C. The establishment of this engineered strain significantly improved the production efficiency of Indochrome C and shortened the fermentation cycle, demonstrating promising prospects for industrial application.

[0019] 2. The method of the present invention is simple and easy to implement, and effectively solves the problems of low Indochrome C yield and harsh cultivation conditions in the prior art. Attached Figure Description

[0020] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0021] Figure 1 The chemical structure of indochrome C in this invention is shown.

[0022] Figure 2 This is a map of the pET41a-indochrome C NRPS-like cluster recombinant plasmid of the present invention.

[0023] Figure 3 This is the biosynthetic gene cluster of Indochrome C as predicted in this invention.

[0024] Figure 4 This is the mass spectrometry analysis result of the indochrome C heterologous expression engineered bacteria of this invention.

[0025] Figure 5 This is a comparison of the yield and fermentation cycle of the engineered strain of this invention with the original strain Indochrome C. Detailed Implementation

[0026] Indochrome C heterologous expression engineered bacteria based on cold-shocked NRPS gene clusters; this engineered bacteria is Escherichia coli The strain pET41a-Indochrome C-Cluster, with accession number GDMCC No. 66715, was deposited on July 21, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, 510070, China.

[0027] The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-stressed NRPS gene cluster includes the following steps:

[0028] (1) Using a whole-genome synthesis method, the 8538 bp NRPS gene cluster fragment of Indochrome C with a GC content of 61% was cloned into the pET41a expression vector to construct a recombinant plasmid (e.g., Figure 2As shown in the figure, the recombinant plasmid is named pET41a-indochrome C cluster. Its expression regulatory elements include the T7 promoter, ribosome binding site (RBS), lactose operon (lacO), and T7 terminator, with the aim of ensuring efficient transcriptional expression of the target gene cluster in the host cell.

[0029] [Obtaining and Functional Annotation of the Target Gene]

[0030] To obtain the biosynthesis-related genes of Indochrome C, this invention uses antioxidant Arthrobacter QL17 ( Arthrobacter antioxidans QL17 T The entire genome of this strain (NCBI: https: / / www.ncbi.nlm.nih.gov / datasets / taxonomy / 2895818 / ) was used as the research object, and a systematic analysis of its secondary metabolite biosynthesis gene clusters was performed using the antiSMASH bioinformatics tool. The results showed that there are seven biosynthetic gene clusters (BGCs) related to natural product synthesis in the strain's genome. Among them, cluster 7 is a non-ribosomal peptide synthase-like (NRPS-like) gene cluster, which is presumed to be closely related to the synthesis of Indochrome C (see...). Figure 3 This gene cluster is approximately 8.5 kb in length and contains several key structural genes (see Table 1). The Indochrome C biosynthetic gene cluster includes IncA-IncG. Specifically: IncA has the nucleotide sequence shown in positions 1 to 243 of SEQ ID NO.1 in the sequence listing; IncB has the nucleotide sequence shown in positions 1 to 1264 of SEQ ID NO.2 in the sequence listing; IncC has the nucleotide sequence shown in positions 1 to 305 of SEQ ID NO.3 in the sequence listing; IncD has the nucleotide sequence shown in positions 1 to 228 of SEQ ID NO.4 in the sequence listing; IncE has the nucleotide sequence shown in positions 1 to 239 of SEQ ID NO.5 in the sequence listing; IncF has the nucleotide sequence shown in positions 1 to 320 of SEQ ID NO.6 in the sequence listing; and IncG has the nucleotide sequence shown in positions 1 to 183 of SEQ ID NO.7 in the sequence listing.

[0031] Table 1. Inferred Functions of Open Reading Frames in the Indochrome C Biosynthetic Gene Cluster

[0032]

[0033] With model strain Streptomyces chromofuscusA comparative analysis of the indigoidine synthesis pathways revealed that the two pathways share some similarities in the cyclization step using L-glutamine. However, the Indochrome C pathway has additional modifications such as glycosylation, indicating that its synthesis mechanism is unique in both conservation and difference.

[0034] The above results indicate that the Cluster 7 gene cluster used in this invention possesses all the structural and regulatory genes required for Indochrome C synthesis, providing a clear genetic basis for achieving efficient heterologous expression in Escherichia coli.

[0035] (2) Using the conventional heat shock transformation method, a DNA solution containing the pET41a-Indochrome CCluster recombinant plasmid at a concentration of 100 ng / μL was added to commercially available E. coli BL21 (… Escherichia coli In BL21 competent cells, the mixture was incubated on ice for approximately 30 minutes. After heat shock at 42 °C for 45 seconds, the mixture was immediately placed on ice for 5 minutes to promote plasmid entry into the host cells. Then, LB broth without antibiotics was added to the mixture, and the cells were shaken at 37 °C and 200 rpm for 1 hour to recover. After recovery, the bacterial culture was evenly spread on LB agar plates containing kanamycin and incubated at 37 °C for 16–18 hours. Resistant single clones were selected to obtain engineered bacteria stably expressing the synthetic blue pigment Indochrome C. Escherichia coli pET41a-Indochrome C-Cluster.

[0036] Among them: Escherichia coli BL21 ( Escherichia coli The concentration of competent cells in BL21 was 1×10⁻⁶. 8 The volume ratio (μL / μL) of the competent cells to the recombinant plasmid DNA solution was 5:1 (CFU / mL).

[0037] LB liquid medium is prepared by adding 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride to deionized water, mixing thoroughly, adjusting the pH to 7.0 with 5 mol / L NaOH, and then bringing the volume to 1 L with deionized water. The medium is then autoclaved at 15 psi for 21 min. The volume ratio (μL / μL) of this LB liquid medium to the recombinant plasmid DNA solution is 45:1.

[0038] LB plates containing kanamycin resistance selection markers are prepared by adding 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin to deionized water, mixing thoroughly, adjusting the pH to 7.0 with 5 mol / L NaOH, bringing the volume to 1 L with deionized water, and then autoclaving at 15 psi for 21 min.

[0039] The yield of the Indochrome C heterologous expression engineered bacteria based on the cold-shocked NRPS gene cluster constructed by the above method was 2.82 g / L.

[0040] The strain of this engineered bacterium was analyzed by mass spectrometry (MS), and the results are as follows: Figure 4 As shown in the chromatogram, the characteristic ion peak of Indochrome C pigment was detected, confirming the successful expression of the target product.

[0041] A fermentation product prepared using an engineered bacterium with Indochrome C heterologous expression based on the cold-shock NRPS gene cluster is described by the following method: The engineered bacterium... Escherichia coli A single colony of pET41a-Indochrome C-Cluster was inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37 ℃ with shaking at 200 rpm to obtain the seed culture. 1 mL of the seed culture was then inoculated into 100 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured until OD... 600 When the concentration of the culture medium reaches 0.6–0.8, IPTG at a final concentration of 200 μM is added for induction, and the culture temperature is adjusted to 18 °C. The culture is then continued with shaking at 200 rpm for 42 h to obtain the fermentation product of the engineered bacteria. Samples are taken periodically during the culture process to monitor the cell growth curve (OD). 600 ) and Indochrome C production.

[0042] [Validation of Fermentation Cycle and Yield Improvement of Engineered Strains]

[0043] Indochrome C yield standard curve: Based on the known concentrations of Indochrome C yield (0.1 mg / mL to 1 mg / mL), the absorbance (Abs) of different concentrations of fermentation product at 590 nm was measured. 590 Plotting the absolute concentration of Indochrome C production on the x-axis and the corresponding absorbance on the y-axis, a standard curve of Indochrome C production concentration versus absorbance was obtained, yielding the formula: y = 0.1274x + 0.0052 R. 2=0.999. This standard curve was used to calculate the blue pigment yield of each sample at different time points. All experiments were performed in triplicate to ensure data reliability.

[0044] To address the shortcomings of the original strain, this invention significantly shortens the fermentation cycle and increases yield by heterologously expressing the Indochrome C gene cluster in Escherichia coli BL21. Figure 5 The fermentation curves and pigment yield changes of the original strain and the engineered strain are shown. The results indicate that the fermentation time of the engineered strain is shortened by 30 h: the original strain requires approximately 72 h of cultivation at low temperature (15℃) to reach peak color production, while the engineered strain constructed in this invention reaches peak color production in about 42 h under common conditions (18℃, IPTG induction), shortening the fermentation cycle by approximately 30 h compared to the original strain. Furthermore, compared to the original strain, the engineered strain increases the indochrome C yield by 3.32 times: the original strain's Indochrome C yield is only about 0.874 g / L, while the engineered strain of this invention increases the yield to approximately 2.82 g / L under the same conditions, an increase of approximately 3.23 times.

[0045] In summary, this invention reconstructs the Indochrome C NRPS gene cluster using synthetic biology techniques, solving the problems of long fermentation cycle and insufficient yield of the original strain, and significantly improving the production efficiency and industrial application potential of the pigment.

Claims

1. An engineered Indochrome C heterologous expression strain based on the cold-stressed NRPS gene cluster, characterized in that: This engineered bacteria is Escherichia coli pET41a-Indochrome C-Cluster, with accession number GDMCC No.66715.

2. A method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-stressed NRPS gene cluster, comprising the following steps: (1) Using a whole-genome synthesis method, the NRPS gene cluster fragment of synthesized Indochrome C was cloned into the pET41a expression vector to construct a recombinant plasmid, which was named pET41a-indochrome C cluster. The NRPS gene cluster of synthesized Indochrome C includes IncA-IncG; wherein: IncA encodes the sequence shown in SEQ ID NO.1; IncB encodes the sequence shown in SEQ ID NO.2; IncC encodes the sequence shown in SEQ ID NO.3; IncD encodes the sequence shown in SEQ ID NO.4; IncE encodes the sequence shown in SEQ ID NO.5; IncF encodes the sequence shown in SEQ ID NO.6; and IncG encodes the sequence shown in SEQ ID NO.

7. (2) Using the conventional heat shock transformation method, a DNA solution containing the pET41a-Indochrome C Cluster recombinant plasmid at a concentration of 100 ng / μL was added to commercially available E. coli BL21 competent cells and incubated on ice for 30 minutes. The mixture was then heat-shocked at 42 °C for 45 seconds and immediately placed on ice for 5 minutes. Antibiotic-free LB broth was then added to the mixture, and the cells were shaken at 37 °C and 200 rpm for 1 hour to recover. After recovery, the bacterial culture was evenly spread on LB agar plates containing kanamycin and incubated at 37 °C for 16–18 hours. Resistant single clones were selected to obtain engineered bacteria stably expressing the synthetic blue pigment Indochrome C. Escherichia coli pET41a-Indochrome C-Cluster.

3. The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-shocked NRPS gene cluster as described in claim 2, characterized in that: In step (1), the NRPS gene cluster fragment is 8538 bp in length and has a GC content of 61%.

4. The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-shocked NRPS gene cluster as described in claim 2, characterized in that: The expression regulatory elements of the recombinant plasmid in step (1) include the T7 promoter, ribosome binding site, lactose operon, and T7 terminator.

5. The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-stressed NRPS gene cluster as described in claim 2, characterized in that: In step (2), Escherichia coli BL21 ( Escherichia coli The concentration of competent cells in BL21 was 1×10⁻⁶. 8 The volume ratio of competent cells to recombinant plasmid DNA solution was 5:1 (CFU / mL).

6. The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-shocked NRPS gene cluster as described in claim 2, characterized in that: In step (2), the LB liquid culture medium is prepared by adding 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride to deionized water, mixing thoroughly, adjusting the pH to 7.0 with 5 mol / L NaOH, bringing the volume to 1 L with deionized water, and then sterilizing by steaming at 15 psi for 21 min. The volume ratio of this LB liquid culture medium to the recombinant plasmid DNA solution is 45:

1.

7. The method for constructing Indochrome C heterologous expression engineered bacteria based on the cold-stressed NRPS gene cluster as described in claim 2, characterized in that: The LB plate containing the kanamycin resistance screening marker in step (2) is prepared by adding 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 50 μg / mL kanamycin to deionized water, mixing thoroughly, adjusting the pH to 7.0 with 5 mol / L NaOH, bringing the volume to 1 L with deionized water, and then sterilizing by steaming at 15 psi for 21 min.

8. A fermentation product containing Indochrome C prepared using the Indochrome C heterologous expression engineered bacteria based on the cold-shocked NRPS gene cluster as described in claim 1.

9. The method for preparing fermentation products containing Indochrome C by engineered bacteria as described in claim 8, characterized in that: engineered bacteria Escherichia coli A single colony of pET41a-Indochrome C-Cluster was inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37 ℃ with shaking at 200 rpm to obtain the seed culture. 1 mL of the seed culture was then inoculated into 100 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured until OD... 600 When the concentration of the culture medium is 0.6~0.8, IPTG with a final concentration of 200 μM is added for induction, and the culture temperature is adjusted to 18 ℃. The culture is then continued to be shaken at 200 rpm for 42 h to obtain the fermentation product of the engineered bacteria.

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