A genetically engineered Bacillus subtilis strain for producing Porphyra-334, its construction method, and its application.

Abstract

This invention discloses a genetically engineered Bacillus subtilis strain for producing the spore-like amino acid Porphyra-334, its construction method, and its applications, belonging to the fields of microbial metabolic engineering and synthetic biology. Using Bacillus subtilis WB600 as a chassis, this strain: 1) knocks out the competing transaldolase gene ywjH, blocking precursor competition and byproduct synthesis pathways; 2) systematically introduces the key gene cluster NlmysABCD from Nostoc lindy NIES-25 for Porphyra-334 synthesis; 3) knocks out the key regulatory genes spo0A and spoIIIE controlling spore formation, thereby blocking the cell differentiation process and redistributing metabolic resources to the biosynthesis of the target product. The finally constructed engineered strain achieved a Porphyra-334 yield of 2.71 g / L under shake-flask fermentation conditions and a yield of 10.07 g / L under top-fermentation conditions.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and metabolic engineering, specifically to a genetically engineered Bacillus subtilis strain for producing Porphyra-334, its construction method, and its application. Background Technology

[0002] Mycosporine-like amino acids (MAAs) are a class of natural ultraviolet-absorbing compounds synthesized by microorganisms such as cyanobacteria, microalgae, and some fungi. Among them, Porphyra-334, as a high-value MAA derivative with a complex structure and excellent functions, has become a star ingredient attracting much attention in the fields of high-end sunscreen skincare products, functional cosmetics, and biomedical materials due to its extremely high molar extinction coefficient in the 310-365 nm ultraviolet band (especially UVA), as well as its antioxidant, anti-inflammatory, and cell repair-promoting activities.

[0003] In recent years, the construction of microbial "cell factories" using synthetic biology and metabolic engineering techniques to achieve heterologous biosynthesis of MAAs has been regarded as the most promising direction for breaking through traditional production models. Among many microbial hosts, Bacillus subtilis is widely regarded as an ideal chassis cell for producing high-value-added natural products due to its non-pathogenicity, GRAS (Generally Recognized As Safe) certification by the US FDA, clear genetic background, low culture cost, rapid growth, strong protein secretion capacity, and mature genetic manipulation tools. However, constructing an efficient Porphyra-334 heterologous biosynthetic pathway in Bacillus subtilis still faces significant challenges: on the one hand, the expression level of exogenous gene clusters often does not match the host's endogenous metabolic network, and single copies or weak expression lead to insufficient enzyme levels, limiting metabolic flux; on the other hand, the host cell's own competitive metabolic pathways (such as the pentose phosphate pathway branch) divert precursor substances, and the inherent sporulation and development program is activated under certain conditions, resulting in a large amount of metabolic resources being used for cell differentiation rather than target product synthesis, which seriously restricts further yield increases. Therefore, developing a systematic modification strategy that can synergistically optimize gene expression dosage, eliminate metabolic competition, and reprogram cellular physiological states is key to constructing high-yielding Porphyra-334 Bacillus subtilis engineered strains. Summary of the Invention

[0004] This invention aims to address the lack of efficient and safe Porphyra-334-producing strains in existing technologies. By heterologously expressing the NlmysABCD gene cluster derived from Nostoc linckia NIES-25 in Bacillus subtilis WB600, a recombinant strain capable of efficiently synthesizing Porphyra-334 was constructed. Furthermore, this invention also co-expresses the key gene cluster mysABCD in the expression vector pP43NMK and the genome, and knocks out the key regulatory genes spo0A and spoIIIE for spore formation, achieving a significant improvement in Porphyra-334 synthesis capacity.

[0005] The first objective of this invention is to provide a genetically engineered Bacillus subtilis strain that produces Porphyra-334, wherein the genetically engineered Bacillus subtilis strain, in addition to knocking out the competing pathway gene ywjH and genes related to the synthesis of multicopy integration products, further knocks out the key regulatory genes spo0A and spoIIIE that control spore formation.

[0006] In one embodiment, the recombinant bacteria is Bacillus subtilis WB600 as the starting strain;

[0007] In one embodiment, the recombinant bacteria knocked out the competitive pathway transaldolase gene ywjH, whose encoded amino acid has the NCBI sequence number NP_391592.3;

[0008] In one embodiment, the recombinant bacteria integrates the key synthetic gene cluster NlmysABCD from Nostoclinckia NIES-25 into its genome, and this gene cluster is generated by an optimized strong promoter P. veg Driven expression.

[0009] In one embodiment, the methyl-4-deoxycodone synthase gene NlmysA is derived from Nostoclinckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 1, and its nucleotide sequence is shown in SEQ ID NO. 7;

[0010] In one embodiment, the O-methyltransferase NlmysB is derived from Nostoc linckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 2, and its nucleotide sequence is shown in SEQ ID NO. 8;

[0011] In one embodiment, the ATP-grasp ligase gene NlmysC is derived from Nostoclinckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 9;

[0012] In one embodiment, the D-alanine-D-alanine ligase gene NlmysD is derived from Nostoc linckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 4, and its nucleotide sequence is shown in SEQ ID NO. 10;

[0013] In one embodiment, the recombinant bacteria further knocked out the key regulatory gene spo0A, which controls spore formation and has the NCBI sequence number NP_390302.1, to block the cells from entering the spore development stage and concentrate metabolic resources on the synthesis of Porphyra-334.

[0014] In one embodiment, the recombinant bacteria further knocked out the key regulatory gene spoIIIE, which controls spore formation and has the NCBI sequence number NP_389562.3, to block the cells from entering the spore development stage and concentrate metabolic resources on the synthesis of Porphyra-334.

[0015] In one embodiment, the Bacillus subtilis includes, but is not limited to, Bacillus subtilis WB600.

[0016] A second objective of this invention is to provide plasmids for the synthesis of key enzymes mysA, mysB, mysC, and mysD, which are initiated by expression on the vector pP43NMK.

[0017] In one implementation, pP43NMK is used as the expression vector.

[0018] In one embodiment, NlmysA, NlmysB, NlmysC, and NlmysD are expressed in the vector plasmid.

[0019] In one embodiment, the methyl-4-deoxycodone synthase gene NlmysA is derived from Nostoclinckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 1, and its nucleotide sequence is shown in SEQ ID NO. 7;

[0020] In one embodiment, the O-methyltransferase NlmysB is derived from Nostoc linckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 2, and its nucleotide sequence is shown in SEQ ID NO. 8;

[0021] In one embodiment, the ATP-grasp ligase gene NlmysC is derived from Nostoclinckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 9;

[0022] In one embodiment, the D-alanine-D-alanine ligase gene NlmysD is derived from Nostoc linckia NIES-25, and its encoded amino acid sequence is shown in SEQ ID NO. 4, and its nucleotide sequence is shown in SEQ ID NO. 10;

[0023] A third objective of this invention is to provide a method for producing Porphyra-334 by fermentation using the genetically engineered Bacillus subtilis.

[0024] In one embodiment, the seed culture of the genetically engineered Bacillus subtilis is added to a fermentation system containing 30 g / L glucose and cultured at 35-38 ºC and 180-220 rpm for no less than 72 h.

[0025] In one embodiment, the fermentation system further contains 24 g / L yeast extract, 12 g / L peptone, 12.54 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, 6 g / L ammonium sulfate, 3 g / L magnesium sulfate heptahydrate, 3 g / L urea, 0.1 g / L calcium chloride, and 30 g / L glucose.

[0026] In one embodiment, the seed culture of the genetically engineered Bacillus subtilis is inoculated into a fermenter system containing 30 g / L glucose. The fermentation system is maintained at a temperature of 35-38 ºC, a stirring speed of 200-800 r / min, an aeration rate of 2-8 vvm, and a pH of 7.0±0.2. Fermentation continues until the OD600 reaches 30-40, then yeast extract is added, and the culture is continued for at least 50 h.

[0027] In one embodiment, the strain ferments and grows to an OD600 of 30-40, while yeast extract is added to provide a sufficient nitrogen source to promote cell growth and the synthesis of Porphyra-334. This process is carried out at a low flow rate.

[0028] In one embodiment, the fermentation system or fermenter system further contains 24 g / L yeast extract, 12 g / L peptone, 12.54 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, 6 g / L ammonium sulfate, 3 g / L magnesium sulfate heptahydrate, 3 g / L urea, 0.1 g / L calcium chloride, and 30 g / L glucose.

[0029] A fourth objective of this invention is to provide the use of the genetically engineered Bacillus subtilis in the preparation of products containing Porphyra-334.

[0030] The fifth objective of this invention is to provide the application of the genetically engineered Bacillus subtilis in the fields of medicine and cosmetics.

[0031] Beneficial effects:

[0032] The genetically engineered Bacillus subtilis strain constructed in this invention has several significant beneficial effects: First, by selecting the non-pathogenic and GRAS-certified Bacillus subtilis WB600 as the production host, the safety of downstream products in cosmetics, pharmaceuticals, and other fields is fundamentally guaranteed. Second, through the implementation of a systematic metabolic engineering strategy, not only was the competitive branch gene ywjH knocked out to guide carbon metabolism to the target pathway, but the heterologous gene cluster NlmysABCD was also integrated in multiple copies at several neutral sites in the genome, strongly driving the synthesis throughput of Porphyra-334 through gene dosage effects. Furthermore, by knocking out the key spore formation genes spo0A and spoIIIE, the growth-differentiation coupling of the host cell is effectively decoupled, allowing more metabolic resources and energy to be used for product accumulation. The synergistic effect of these strategies significantly increased the yield of shake-flask fermentation of engineered bacteria from the initial 0.77 g / L to 2.71 g / L, demonstrating excellent high-yield performance, greatly reducing production costs and simplifying process control, and laying a solid foundation for the large-scale, low-cost biomanufacturing of Porphyra-334. Attached Figure Description

[0033] Figure 1 A schematic diagram of the recombinant plasmid p43NMK-Pveg-UTR4-NlmysABCD. Detailed Implementation

[0034] The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were all commercially available products, and the specific operations were performed according to the kit instructions. Routine 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).

[0035] (a) Culture medium

[0036] (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0037] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0038] (3) Fermentation medium: yeast extract 24 g / L, peptone 12 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, ammonium sulfate 6 g / L, magnesium sulfate heptahydrate 3 g / L, urea 3 g / L, calcium chloride 0.1 g / L, glucose 30 g / L.

[0039] (5) Preparation of competent cells of Bacillus subtilis: The strain preserved in the laboratory was streaked on an agar plate and incubated at 37 °C for 10-12 h. Single colonies were picked and placed in 1 mL of LB medium into 50 mL centrifuge tubes and incubated at 37 °C for about 8 h. 4 mL of LB medium diluted five times was added, along with 300 μL of 50% xylose, and incubated for 2 h. Finally, 1.25 mL of 50% glycerol was added and the culture was frozen at -80 °C.

[0040] (II) Porphyra-334 shake-flask fermentation process: The constructed strain was inoculated into LB liquid medium and cultured overnight at 37 ºC and 220 rpm for 12 h to obtain seed liquid. 1 mL of seed liquid was inoculated into 25 mL of fermentation medium and cultured at 37 ºC and 220 rpm for 72 h.

[0041] (III) Porphyra-334 detection:

[0042] Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, collect the supernatant, and use it for HPLC determination.

[0043] HPLC detection conditions: High performance liquid chromatography (HPLC) system (Agilent); Column: ZORBAX Eclipse Plus C18; Detector: Agilent UV detector; Mobile phase: 0.25% formic acid in water; Flow rate: 0.65 mL / min; Column temperature: 30 ºC; Injection volume: 10 μL.

[0044] (iv) strains

[0045] The dual-plasmid gene editing system has been published in the literature: Wu Y, Liu Y, Lv X, Li J, Du G, Liu L. CAMERS‐B: CRISPR / Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering. 2020;117:1817–1825.

[0046] Table 1. Strains involved in the following examples

[0047] Strain Plasmid Yield g / L Bacillus subtilis WB600 p43NMK-P43-NlmysABCD 0.32 Bacillus subtilis WB600 ΔywjH p43NMK-P43-NlmysABCD 0.51 Bacillus subtilis WB600 ΔywjH p43NMK-Pveg-NlmysABCD 0.64 Bacillus subtilis WB600 ΔywjH p43NMK-PspoVG-NlmysABCD 0.42 Bacillus subtilis WB600 ΔywjH p43NMK-Pyvyd-NlmysABCD 0.47 Bacillus subtilis WB600 ΔywjH p43NMK-Pveg-UTR3-NlmysABCD 0.42 Bacillus subtilis WB600 ΔywjH p43NMK-Pveg-UTR7-NlmysABCD 0.48 Bacillus subtilis WB600 ΔywjH p43NMK-Pveg-UTR8-NlmysABCD 0.54 Bacillus subtilis WB600 ΔywjH p43NMK-Pveg-UTR4-NlmysABCD 0.77 <![CDATA[Bacillus subtilis WB600 ΔywjH ΔsacB::P veg -NlmysABCD]]> p43NMK-Pveg-UTR4-NlmysABCD 0.92 <![CDATA[Bacillus subtilis WB600 ΔywjH ΔsacB::P veg -NlmysABCD Δctc::P veg -NlmysABCD]]> p43NMK-Pveg-UTR-four-NlmysABCD 1.75 <![CDATA[Bacillus subtilis WB600 ΔywjH ΔsacB::P veg -NlmysABCD Δctc::P veg -NlmysABCD ΔyckB::P veg -NlmysABCD]]> p43NMK-Pveg-UTR-four-NlmysABCD 2.31 <![CDATA[Bacillus subtilis WB600 ΔywjH ΔsacB::P veg -NlmysABCD Δctc::P veg -NlmysABCD ΔyckB::P veg -NlmysABCD ΔsacA::P veg -NlmysABCD]]> p43NMK-Pveg-UTR-four-NlmysABCD 2.64 <![CDATA[Bacillus subtilis WB600 ΔywjH ΔsacB::P veg -NlmysABCD Δctc::P veg -NlmysABCD ΔyckB::P veg -NlmysABCD ΔsacA::P veg -NlmysABCD Δspo0A]]> p43NMK-Pveg-UTR-four-NlmysABCD 2.68 <![CDATA[Bacillus subtilis WB600 ΔywjH ΔsacB::P veg -NlmysABCD Δctc::P veg -NlmysABCD ΔyckB::P veg -NlmysABCD ΔsacA::P veg -NlmysABCD Δspo0A ΔspoIIIE]]> p43NMK-Pveg-UTR-four-NlmysABCD 2.71

[0048] (v) Primers

[0049] Table 2 Primers required in the following examples

[0050] ywjH-V-F ATCAAAGGTGCAGATCGTCGAACGGCAGATC ywjH-V-R CTTATGTAGCGAATTCGGCCAAGCTAACTAAGTTTG ywjH-UH-F GGCCGAATTCGCTACATAAGGAGGACATTCGACATGC ywjH-UH-R GCCCCTTTCATTTCAAAAAGCCTCCCTGATTAAGAAATTTTCTG ywjH-DH-F CTTTTTGAAATGAAAGGGGCGGCAAACAGC ywjH-DH-R CGACGATCTGCACCTTTGATTTTGGCGCCC p43NMK-F1 ATGATGAAAGCTTGGCGTAATCATGGTCATAG p43NMK-R1 GTGTACATTCCTCTCTTACCTATAATGGTACC p43NMK-V-F It should be noted that there may be some inaccuracies in the translation of "p43NMK-Pveg-UTR-four-NlmysABCD" in the original text as it seems an incorrect or unclear expression. It might be better to double-check the original content for more accurate translation if possible. GTGATTGATGAAAGCTTGGCGTAATCATGGTCATAG p43NMK-V-R CGAAAACATACCACCTATCAGGATCCAGTTGCTC Pyvyd-F TGAGCAACTGGATCCGATCAATTGGTCTCTTTCTCT Pyvyd-R TTTTATCACCTCCTTTATTAAGGATATGTATCTATTTCTCTTTTACCC PspoVG-F GAGCAACTGGATCCTGCGGAAGTAAACGAAGTGTACGGAC PspoVG -R GTGTACATTCCTCTCTTACCTATATAAAAGCATTAGTGTATCAATTCCACG Pveg-F TTTTTTTGAGCAACTGGATCCTATGGGAAGTGCTCCGTAATACGC Pveg- R TTTTATCACCTCCTTTCACTACATTTATTGTACAACAC UTR3-F GTGATTAGAAAGGAGGAATGTACACATGTCAATAGTACAAGC UTR3-R GTGTACATTCCTCCTTTCTAATCACCACTACATTTATTGTACAACAC UTR4-F GTGATAGCGGTACCATTATAGGTAAAGGAGGAATGTACACATGTCAATAGTACAAGC UTR4-R GTGTACATTCCTCCTTTACCTATAATGGTACCGCTATCACCACTACATTTATTGTACAACAC UTR7-F GTGATAGCGGTACATTAGAAAGGAGGAATGTATAATGTCAATAGTACAAGC UTR7-R TATACATTCCTCCTTTCTAATGTACCGCTATCACCACTACATTTATTGTACAACAC UTR8-F GTGATAGCGGTATATTAGAAAGGAGGAATGTATAATGTCAATAGTACAAGC UTR8-R TATACATTCCTCCTTTCTAATATACCGCTATCACCACTACATTTATTGTACAACAC sacB-V-F AGAAAAGCGGCAGATCGTCGAACGGCAGATC sacB-V-R GATGTACAGCGAATTCGGCCAAGCTAACTAAGTT sacB-UH-F GGCCGAATTCGCTGTACATCCAGCCTTCATTC sacB-UH-R ACTTCCCATACGTTCATGTCTCCTTTTTTATGTACTGTGTTAG Pveg-F GACATGAACGTATGGGAAGTGCTCCGTAATACGC Pveg-R GTGTACATTCCTCTCTTACCACTACATTTTATTGTACAACACGAGCCC NlmysA-F GGTAAGAGGAATGTACACATGTCAATAGTACAAGCTAAATTTG NlmysA-R GTGTACATTCCTCTCTTACTTAGATTGCGCTACCCACCAGTTCGG NlmysB-F AGTAAGAGAGGAATGTACACATGATGTCAACTACAATAGCAAAACCCACG NlmysB-R GTGTACATTCCTCTCTTACTTAAGCCGTACGTCTAATG NlmysC-F GTAAGAGAGGAATGTACACATGGCTCAATCTATATCCGTAAGCAGTTC NlmysC-R GTGTACATTCCTCTCTTACTTAATCACCACCCAATTCAACTAATTTG NlmysD-F GTAAGAGAGGAATGTACACATGCCCGTACTAAGGATATTACACTTG NlmysD-R CAGGAAACAGCTATGACCATGATTACGCCAAGCTTTCATCATTAAATCATTTGGCTCAACTC cowB-DH-F TGTTAATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAAACGCAAAAGAAAATGCCG sacB-DH-R CGACGATCTGCCGCTTTTCTAATGGATCTGTGC ctc-N23-F TTACCCGTATCCTTCCCATATATAATTTCTACTGTTGTAGATCGTCTCTGGAACTGATTCAAGCAAGC ctc-N23-R GAAATTATATATGGGAAGGATACGGGTAAATCTACAACAGGAAAATTATTAAAGTTCTTAGACCTCGCGTCTCTACCTCTAC ctc-VF AGCGTTTTTCCAGATCGTCGAACGGCAGATC ctc-VR ATTCAAAGAAAAATTCGGCCAAGCTAACTAAGTT ctc-UH-F GGCCGAATTCTTCTTTGAATTCGAATCCAGAGCTTGC ctc-UH-R ACTTCCCATAATTCAGCACCATCCTCTTGTCGT Pveg-F GGTGCTGAATTATGGGAAGTGCTCCGTAATACGC Pveg-R GTGTACATTCCTCTCTTACCACTACATTTATTGTACAACACGAGCCC ctc-DH-F TGTTATCCGCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTATAGCTTAAGGCGTAACCCTCCC ctc-DH-R CGACGATCTGGAAAAACGCTTTGGTCTAGTGTCTACAC yckB-N23-F CACTCCATATAAAATTCATACGAATTTCTACTGTTGTAGATCGTCTCTGAACTGATTCAAGCAAGC yckB-N23-R CGTATGAATATTTATGGAGTGATCTACAACAGTAGAATTTTAAGTTCTTAGACCTCGCGTCTCTACCTCTAC yckB-VF AATCGTAACCCAGATCGTCGAACGGCAGATC yckB-VR CCGCTGAAAAGAATTCGGCCAAGCTAACTAAGTT yckB-UH-F GGCCGAATTCTTTTCAGCGGCCTCAGCTG yckB-UH-R ACTTCCCATACTATAGTTCCCCCAATCTGTTAATCAACTAAAAAG yckB-DH-F TGTTATCCGCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTATCGGACGCCATGATCAATAGC yckB-DH-R CGACGATCTGGGTTACGATTGAGCCTGTATCTTATTGG sacA-N23-F TTGGAAGGGCAGCTATCATGTATAATTTCTACTGTTGTAGATCGTCTCTGAACTGATTCAAGCAAGC sacA-N23-R ATACATGATAGCTGCCCTTCCAAATCTACAACAGTAGAAATTTTAAAGTTCTTAGACCTCGCGTCTCTACCTCTAC sacA-VF ACGGTTGGCACAGATCGTCGAACGGCAGATC sacA-VR CCCAGCAATCGAATTCGGCCAAGCTAACTAAGTT sacA-UH-F GGCCGAATTCGATTGCTGGGTATGATCAATGCGTT sacA-UH-R ACTTCCCATATTTTTCCTCTCCTCATCCTCATTAATCTTC sacA-DH-F TGTTATCCGCTCAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAATCCTTCTATTTTCTTATGTGAAATC sacA-DH-R CGACGATCTGTGCCAACCGTTGACATTATCGAG spo0A-N23-F AGATCTGATTCCCTCAGCCTCTCAATTTCTACTGTTGTAGATCGTCTCTGAACTGATTCAAGCAAGC spo0A-N23-R GAGAGGCTGAGGGAATCAGATCTATCTACAACAGTAGAAATTATTTAAAGTTCTTAGACCTCGCGTCTCTACCTCTAC spo0A-VF CGTTTTTCATCAGATCGTCGAACGGCAGATC spo0A-VR TTCTGTTTATGAATTCGGCCAAGCTAACTAAGTT spo0A-UH-F GGCCGAATTCATAAACAGAAAATCAAAACGAAGCTGATCC spo0A-UH-R AAGCTCATGTGTTTCTTCCTCCCCAAATGTAGTTAACAG spo0A-DH-F AGGAAGAAACACATGAGCTTATTAAGTGGTCATTAAATCAAAC spo0A-DH-R CGACGATCTGATGAAAAACGATTAGCCTTCCCGC spoIIIE-N23-F TGAAAATGCGAGAAAGCTTGAACAATTTCTACTGTTGTAGATCGTCTCTGAACTGATTCAAGCAAGC spoIIIE-N23-R GTTCAAGCTTTCTCGCATTTTCAATCTACAGTAGAAATTATTTAAAGTTCTTAGACCTCGCGTCTCTACCTCTAC spoIIIE-VF TGAGAGAGAACAGATCGTCGAACGGCAGATC spoIIIE-VR CTGTATTTTAGAATTCGGCCAAGCTAACTAAGTT spoIIIE-UH-F GGCCGAATTCTAAAATACAGCAGCTTGGTGAAACGAC spoIIIE-UH-R ACTCCCTTCACACCTTACTGCGTTAAAAAATGTAAGTTCATAC spoIIIE-DH-F CAGTAAGGTGTGAAGGGAGTTCCGCTTTCTATAGTTG spoIIIE-DH-R CGACGATCTGTTCTCTCTCAACATTTTCACCCATTCACT

[0051] Example 1: Knockout of competitive pathways in chassis strains

[0052] The dual-plasmid gene editing system used in this invention has been disclosed in the literature: Wu Y, Liu Y, Lv X, Li J, Du G, Liu L. CAMERS‐B: CRISPR / Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering. 2020;117:1817–1825. The specific operation is as follows:

[0053] The gene editing system of Bacillus subtilis consists of two plasmids, pHT-XCR2 and pcrF11. pHT-XCR2 is a Cpf1 expression vector, and pcrF11 is a crRNA expression vector, used to express crRNA and insert homology repair template.

[0054] (1) A 23 bp specific targeting sequence (N23) was designed targeting the spo0A knockout / integration site on the Bacillus subtilis genome. Using pcrF11 plasmid as a template, primers were designed to replace the original N23 sequence on the plasmid with the N23 sequence targeting spo0A through reverse PCR or site-directed mutagenesis. The PCR product was digested with DpnI enzyme to remove the template plasmid, purified, and transformed into Escherichia coli DH5α competent cells. After successful sequencing, the plasmid pcrF11-spo0A was obtained.

[0055] (2) Three pairs of primers were designed and synthesized to amplify the upstream homologous arm (UH, approximately 1000 bp) of the spo0A gene locus, the downstream homologous arm (DH, approximately 1000 bp) of the sacB gene locus, and the linearized backbone of the vector pcrF11-spo0A. The PCR products were subjected to gel electrophoresis and purified. Using a seamless cloning (or Gibson Assembly) kit, the fragments were mixed in proportion and homologous recombination was performed to obtain the plasmid pcrF11-spo0A-Δ.

[0056] (3) The plasmid pHT-XCR2 containing the Cpf1 protein was first transformed into Bacillus subtilis WB600 competent cells to obtain an intermediate host with CRISPR-Cpf1 editing capability. Electroporation competent cells were prepared from this intermediate host. The donor plasmid pcrF11-spo0A-Δ constructed in step (2) was electroporated into the competent cells. After electroporation, resuscitation medium was added and the cells were revived at 37°C for 2 h. The revived bacterial culture was plated on double antibiotic plates (such as chloramphenicol and kanamycin) and incubated upside down at 37°C for 24-48 h to screen for transformants that underwent homologous recombination.

[0057] (4) The successfully screened single colonies were subjected to colony PCR to verify whether the target gene was successfully knocked out. To ensure that the target gene on the genome was knocked out, the remaining PCR products were sent to Sanger sequencing to obtain the correct recombinant strain.

[0058] Other knockouts, including ΔspoIIIE, follow the steps above.

[0059] Example 2: Integration and Modification of Key Genes

[0060] The dual-plasmid gene editing system used in this invention has been disclosed in the literature: Wu Y, Liu Y, Lv X, Li J, Du G, Liu L. CAMERS‐B: CRISPR / Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering. 2020;117:1817–1825. The specific operation is as follows:

[0061] The gene editing system of Bacillus subtilis consists of two plasmids, pHT-XCR2 and pcrF11. pHT-XCR2 is a Cpf1 expression vector, and pcrF11 is a crRNA expression vector, used to express crRNA and insert homology repair template.

[0062] (1) A 23 bp specific targeting sequence (N23) was designed targeting the sacB knockout / integration site on the Bacillus subtilis genome. Using pcrF11 plasmid as a template, primers were designed to replace the original N23 sequence on the plasmid with the N23 sequence targeting sacB through reverse PCR or site-directed mutagenesis. The PCR product was digested with DpnI enzyme to remove the template plasmid, purified, and transformed into Escherichia coli DH5α competent cells. After successful sequencing, the plasmid pcrF11-sacB was obtained.

[0063] (2) Three pairs of primers were designed and synthesized to amplify the upstream homologous arm (UH, approximately 1000 bp) of the sacB gene locus, the downstream homologous arm (DH, approximately 1000 bp) of the sacB gene locus, the key enzyme gene NlmysABCD, and the linearized backbone of the vector pcrF11-sacB. The PCR products were subjected to gel electrophoresis and purified. Using a seamless cloning (or Gibson Assembly) kit, the fragments were mixed in proportion and homologous recombination was performed to obtain the plasmid pcrF11-sacB-Pveg-NlmysABCD.

[0064] (3) The plasmid pHT-XCR2 containing the Cpf1 protein was first transformed into Bacillus subtilis WB600 competent cells to obtain an intermediate host with CRISPR-Cpf1 editing capability. Electroporation competent cells were prepared from this intermediate host. The donor plasmid pcrF11-sacB-Pveg-NlmysABCD constructed in step (2) was electroporated into competent cells. After electroporation, resuscitation medium was added and the cells were revived at 37°C for 2 h. The revived bacterial culture was plated on double antibiotic plates (such as chloramphenicol and kanamycin) and incubated upside down at 37°C for 24-48 h to screen for transformants that underwent homologous recombination.

[0065] (4) The successfully screened single colonies were subjected to colony PCR to verify whether the target gene was successfully knocked out. To ensure that the target gene on the genome was knocked out, the remaining PCR products were sent to Sanger sequencing to obtain the correct recombinant strain.

[0066] Other integrations, including ctc::P veg -NlmysABC、yckB::P veg -NlmysABC and sacA::P veg -NlmysABC refers to the steps above.

[0067] Example 3: Construction of the Porphyra-334 chassis strain

[0068] The engineered strain was obtained by transformation. The specific procedure is as follows: 1 μL of the constructed plasmid was injected into competent Bacillus subtilis cells, and the cells were shaken at 37ºC for 2 h. The mixture was then spread onto LB agar plates containing the corresponding antibiotic concentration. The plates were incubated at 37ºC for 10–12 h, and the engineered strain containing the expression plasmid was obtained by picking bacteria.

[0069] Example 4: Shake Flask Fermentation Production

[0070] The engineered bacteria were inoculated into LB liquid medium containing the corresponding antibiotic and cultured overnight at 37ºC and 220 rpm for 12 h to obtain a seed culture. 1 mL of the seed culture was inoculated into 25 mL of fermentation medium and cultured at 37ºC and 220 rpm for 72 h. 1 mL of the fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The highest yield achieved in shake flasks was 2.71 g / L.

[0071] Example 5: Fermentation tank fed-batch culture

[0072] A batch-feed fermentation experiment was conducted in a 50 L fermenter.

[0073] The optimal engineered bacteria were inoculated into 8 mL of LB medium containing the corresponding antibiotic and cultured overnight at 37ºC and 220 rpm for 10–12 h to obtain primary seed culture. 8 mL of the primary seed culture was then inoculated into 800 mL of fermentation medium containing the corresponding antibiotic and fermentation medium and cultured at 37ºC and 220 rpm until OD reached [value missing]. 600 The OD value was 3-4, yielding a secondary seed culture. 200 mL of this secondary seed culture was inoculated into a 50 L fermenter containing 2 L of fermenter medium with the corresponding antibiotic for fermentation. The fermentation temperature was maintained at 37ºC. After the initial 30 g / L glucose in the medium was completely depleted, glucose was added continuously. When the OD... 600 When the pH reaches 30-40, yeast extract is added at a low flow rate, maintaining the pH at 7.0±0.2 throughout, and foaming is controlled by adding an antifoaming agent. Dissolved oxygen is controlled by adjusting the stirring speed (200-800 rpm) and aeration rate (2-8 vvm). In a 50 L fermenter, the yield of Porphyra-334 can reach 10.07 g / L over 60 h, with an OD of 600 With a maximum value of 73.2, it demonstrates significant production potential in large-scale industrial applications.

[0074] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered Bacillus subtilis strain for producing Porphyra-334, characterized in that, Using Bacillus subtilis WB600 as the origin strain, the gene cluster NlmysABCD was heterologously expressed using plasmid p43NMK as the vector, and the original promoter and untranslated region UTR of plasmid p43NMK were replaced with promoter P. veg UTR4; Knock out the transaldolase gene ywjH, the spore formation regulatory protein gene spo0A, and the spore septum DNA transport protein gene spoIIIE; integrate 1-4 copies of the gene cluster NlmysABCD into the genome of the originating strain. The gene cluster NlmysABCD consists of the methyl-4-deoxycodone alcohol synthase gene NlmysA, the O-methyltransferase gene NlmysB, the ATP-grasp ligase gene NlmysC, and the D-alanine-D-alanine ligase gene NlmysD, all derived from Nostoc linckia NIES-25.

2. A genetically engineered Bacillus subtilis strain for producing Porphyra-334, characterized in that, Using Bacillus subtilis WB600 as the starting strain, the modification method for the starting strain is as follows: 1) Knock out the transaldolase gene ywjH; 2) Knock out the fructan sucrase gene sacB and replace it with one generated by a strong constitutive promoter P. veg The gene cluster that initiates expression is NlmysABCD; 3) Knock out the BL25 ribosomal protein gene ctc that binds to 5S ribosomal RNA and replace it with one generated by the strong promoter P. veg The gene cluster that initiates expression is NlmysABCD; 4) Introduce the recombinant plasmid p43NMK-Pveg-UTR4-NlmysABCD; The gene cluster NlmysABCD consists of the methyl-4-deoxycodone alcohol synthase gene NlmysA, the O-methyltransferase gene NlmysB, the ATP-grasp ligase gene NlmysC, and the D-alanine-D-alanine ligase gene NlmysD, all derived from Nostoc linckia NIES-25.

3. The genetically engineered Bacillus subtilis for producing Porphyra-334 according to claim 2, characterized in that, The modification method also includes: Knock out the amino acid ABC transporter gene yckB and replace it with the strong promoter P veg The gene cluster NlmysABCD is initiated for expression.

4. The genetically engineered Bacillus subtilis for producing Porphyra-334 according to claim 3, characterized in that, The modification method also includes: Knock out the sucrose-6-phosphate hydrolase gene sacA and replace it with the strong promoter P veg The gene cluster NlmysABCD is initiated for expression.

5. The genetically engineered Bacillus subtilis for producing Porphyra-334 according to claim 4, characterized in that, The modification method also includes: Knock out the gene spo0A, which regulates spore formation.

6. The genetically engineered Bacillus subtilis for producing Porphyra-334 according to claim 5, characterized in that, The modification method also includes: Knock out the spoIIIE gene, a DNA transporter protein in spore septum.

7. A method for constructing a genetically engineered Bacillus subtilis strain for producing Porphyra-334, characterized in that, The construction method includes the modification method according to any one of claims 2-6.

8. A method for producing Porphyra-334 by fermentation, characterized in that, include: The genetically engineered Bacillus subtilis producing Porphyra-334 as described in any one of claims 1-6, or the genetically engineered Bacillus subtilis producing Porphyra-334 obtained by the construction method described in claim 7, is cultured in a culture medium.

9. The use of the genetically engineered Bacillus subtilis for producing Porphyra-334 as described in any one of claims 1-6, or the genetically engineered Bacillus subtilis for producing Porphyra-334 obtained by the construction method described in claim 7, in the production of Porphyra-334.

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