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Abstract

The application discloses a Rhodococcus double oxygenase deletion library and a construction method and application thereof. Based on strict bioinformatics screening, the Rhodococcus double oxygenase gene is screened, different double oxygenase genes are knocked out, the Rhodococcus double oxygenase deletion mutant library is constructed, and the library is used for systematic degradation function screening and genotype-phenotype correlation analysis of aromatic compounds, so that key double oxygenase genes necessary or redundant for degradation of specific aromatic compounds can be rapidly identified, the substrate utilization spectrum and functional division spectrum of the double oxygenase family can be drawn, and the application prospect in multiple fields such as high value of lignin, synthesis of fine chemicals, and environmental biological remediation is potential.

Description

Technical Field

[0001] This invention belongs to the field of biodegradation technology, and relates to a Rhodococcus dioxygenase-deficient library, its construction method, and its application. Background Technology

[0002] Rhodococcus is a genus of Gram-positive, aerobic nocardioid actinomycetes. As a non-model bacterium, Rhodococcus can adapt to harsh environments and possesses diverse catabolic activities, capable of degrading various organic pollutants such as alkanes, cycloalkanes, aromatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), nitroaromatic hydrocarbons, and chlorinated hydrocarbons. Rhodococcus exhibits significant potential in biocatalysis, bioremediation, biosynthesis, and lignin utilization, and is receiving increasing attention. The powerful metabolic capacity of Rhodococcus is largely attributed to the large number of genes encoding oxidases in its genome, particularly the dioxygenase family.

[0003] Dioxygenases, as an important class of oxidoreductases, catalyze reactions that simultaneously introduce two oxygen atoms from a molecular oxygen atom into the substrate. Based on their mechanism of action, they are mainly divided into aromatic ring hydroxylation dioxygenases and ring-opening cleavage dioxygenases. These two types of enzymes work synergistically and are the rate-limiting step and core driving force in the degradation of aromatic compounds (such as benzoates, biphenyls, and naphthalenes) by Rhodococcus. Furthermore, the regioselective oxidation reactions catalyzed by dioxygenases also have significant industrial value in the synthesis of pharmaceutical intermediates.

[0004] Current research on the function of dioxygenases in Rhodococcus is mostly limited to the knockout and characterization of single or a few genes, resulting in low throughput and difficulty in revealing the relationship between genotype and phenotype from a global perspective. Furthermore, the Rhodococcus genome is large and contains multiple plasmids, including several highly homologous and functionally similar dioxygenase genes, leading to functional redundancy and overlapping substrate profiles. In the degradation of complex substrates (such as high-molecular-weight polycyclic aromatic hydrocarbons), multiple dioxygenases and their downstream pathways participate jointly, and the synergistic effects, substrate pathways, and metabolic flux allocation mechanisms among these enzymes remain unclear. Traditional gene knockout techniques based on homologous recombination are inefficient and time-consuming in Rhodococcus. Summary of the Invention

[0005] The purpose of this invention is to provide a Rhodococcus dioxygenase-deficient library, its construction method, and its applications. This invention utilizes molecular cloning technology to construct a mutant library of Rhodococcus bacteria lacking different dioxygenase genes. Using this library, by comparing and analyzing the differences in substrate utilization ability between wild-type and mutant strains, the dioxygenases involved in substrate degradation can be rapidly identified, and substrate specificity can be elucidated. Furthermore, the role and mechanism of dioxygenases in the degradation of aromatic compounds can be systematically analyzed, particularly how to utilize these enzyme systems to degrade different types of substrates. This not only provides new ideas for environmental pollution control but also supports applications in green catalysis and the bio-industry.

[0006] The technical solution to achieve the purpose of this invention is as follows:

[0007] The Rhodococcus dioxygenase deletion mutant library consists of Rhodococcus mutants with specific knockouts of genes from different members of the dioxygenase family.

[0008] Furthermore, the number of Rhodococcus mutants is more than 24.

[0009] Furthermore, Rhodococcus includes, but is not limited to, Rhodococcus PD630, Rhodococcus P14, Rhodococcus AJ270, Rhodococcus RHA1, and Rhodococcus J1.

[0010] Furthermore, the Rhodococcus mutants include the Δ5610 mutant (knockout of dioxygenase gene K2Z90_005610), the Δ878 mutant (knockout of dioxygenase gene K2Z90_000878), the Δ946 mutant (knockout of dioxygenase gene K2Z90_000946), the Δ5533 mutant (knockout of dioxygenase gene K2Z90_005533), the Δ4578 mutant (knockout of dioxygenase gene K2Z90_004578), and the Δ5610 mutant (knockout of dioxygenase gene K2Z90_006305). The following mutants were identified: Δ6305 (knockout K2Z90_006318), Δ6318 (knockout K2Z90_006771), Δ6771 (knockout K2Z90_003455), Δ3455 (knockout K2Z90_000007), Δ007 (knockout K2Z90_001140), and Δ3455 (knockout K2Z90_000353). 53 mutant, Δ904 mutant with knockout of dioxygenase gene K2Z90_000904, Δ3511 mutant with knockout of dioxygenase gene K2Z90_003511, Δ5608 mutant with knockout of dioxygenase gene K2Z90_005608, Δ4785 mutant with knockout of dioxygenase gene K2Z90_004785, Δ648 mutant with knockout of dioxygenase gene K2Z90_000648, Δ5840 mutant with knockout of dioxygenase gene K2Z90_005840 The mutants include: Δ6111 (knockout of dioxygenase gene K2Z90_006111), Δ1381 (knockout of dioxygenase gene K2Z90_001381), Δ1519 (knockout of dioxygenase gene K2Z90_001519), Δ5045 (knockout of dioxygenase gene K2Z90_005045), Δ1796 (knockout of dioxygenase gene K2Z90_001796), and Δ5885 (knockout of dioxygenase gene K2Z90_005885).

[0011] The method for constructing the above-mentioned Rhodococcus dioxygenase-deficient mutant library includes the following steps:

[0012] (1) Target gene selection: Different members of the dioxygenase family were screened from the wild-type Rhodococcus genome as knockout targets;

[0013] (2) Construction of knockout vectors: Construct gene knockout vectors targeting each gene;

[0014] (3) Mutant screening: The constructed knockout vector was transformed into wild-type Rhodococcus, and the target gene was knocked out by homologous recombination technology. Mutants with different members of the dioxygenase family were obtained by screening, and a library of Rhodococcus dioxygenase deletion mutants was constructed.

[0015] Furthermore, in step (2), the gene knockout vector is a vector carrying PheS ※ Tagged suicide plasmid.

[0016] Furthermore, in step (3), the screening is carried out using an resistant screening method.

[0017] This invention also provides the application of the above-mentioned Rhodococcus dioxygenase-deficient mutant library, the specific application method being:

[0018] (1) All mutants in the Rhodococcus dioxygenase-deficient mutant library were inoculated into a culture medium with the target aromatic compound as the sole carbon source, and the utilization ability of each mutant was observed.

[0019] (2) Based on the utilization ability of each mutant, key dioxygenase genes in the metabolic pathway using target aromatic compounds as substrates were screened;

[0020] (3) Analyze the metabolic pathway of target aromatic compounds in Rhodococcus by key dioxygenase genes;

[0021] (4) Based on the analysis results, the target high-value chemical can be obtained by knocking out or overexpressing the relevant dioxygenase gene.

[0022] Furthermore, the target aromatic compounds include, but are not limited to, hydroquinone, 2-methylresorcinol, 4-methylcatechol, 2,5-dihydroxybenzoic acid, p-hydroxybenzaldehyde, catechol, 3,5-dihydroxytoluene, 3-methylcatechol, 4-ethylphenol, 3-hydroxybenzoic acid, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, m-hydroxybenzaldehyde, etc.

[0023] Furthermore, high-value chemicals include, but are not limited to, protocatechuic acid, mucilage acid, p-coumaric acid, ferulic acid, gallic acid, etc.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention follows a completely identical and reproducible genetic operation and molecular verification process for knocking out the target dioxygenase gene, thereby minimizing the systematic errors introduced by the differences in operation.

[0026] (2) This invention achieves precise regulation of the metabolic flux of aromatic compounds by blocking specific dioxygenase nodes, providing an ideal platform strain for blocking competitive pathways and accumulating target products.

[0027] (3) The 24 dioxygenase deletion mutants successfully constructed in this invention cover a variety of dioxygenase types, including those involving aromatic ring cleavage, substrate initial oxygenation, and those with unclear functions, forming a representative functional subset. This mutant set provides a direct and operable experimental basis for subsequently establishing the "substrate-key dioxygenase" correspondence through phenotypic and metabolic differences under different substrate conditions, and can be widely applied in multiple fields such as lignin high-value utilization, fine chemical synthesis, and environmental bioremediation. Attached Figure Description

[0028] Figure 1-24 Electrophoresis and sequencing results of 24 knockout mutants of dioxygenase genes, corresponding to Δ5610, Δ878, Δ946, Δ5533, Δ4578, Δ6305, Δ6318, Δ6771, Δ3455, Δ007, Δ1140, Δ353, Δ904, Δ3511, Δ5608, Δ4785, Δ648, Δ5840, Δ6111, Δ1381, Δ1519, Δ5045, Δ1796, and Δ5885, respectively.

[0029] Figure 25 The figure shows the results of the validation of the substrate utilization ability of the protocatechuic acid 3,4-dioxygenase deletion mutant.

[0030] Figure 26 The figure shows the results of the utilization test of 18 aromatic compounds by wild-type Rhodococcus PD630.

[0031] Figure 27 The figure shows the utilization test results of 12 aromatic compounds by four mutants, Δ4785, Δ353, Δ5608, and Δ5885.

[0032] Figure 28 The figure shows the utilization test results of 12 aromatic compounds by four mutants, Δ3455, Δ5533, Δ6305, and Δ007.

[0033] Figure 29 The figure shows the utilization test results of 12 aromatic compounds by three mutants, Δ904, Δ6771, Δ1140, and Δ3511.

[0034] Figure 30 The figure shows the utilization test results of 12 aromatic compounds by four mutants: Δ6318, Δ1381, Δ1796, and Δ648.

[0035] Figure 31 The figure shows the utilization test results of 12 aromatic compounds by four mutants, Δ1519, Δ5840, Δ878, and Δ6111.

[0036] Figure 32 The figure shows the utilization test results of 12 aromatic compounds by three mutants, Δ946, Δ5045, and Δ4578.

[0037] Figure 33 Figure 1 shows the results of secondary validation of the utilization of wild-type Rhodococcus PD630(a) and Δ904(b) with m-hydroxybenzaldehyde as the sole carbon source.

[0038] Figure 34 The figure shows the utilization test results of wild-type Rhodococcus PD630(a) and Δ904(b) using 3-hydroxybenzyl alcohol as the sole carbon source. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0040] Example 1

[0041] Screening process for target dioxygenase genes:

[0042] For Rhodococcus PD630 (NCBI Genome Accession Number GCF_020542785.1), all genes encoding dioxygenases in its genome were systematically screened and identified using a standardized bioinformatics workflow. First, based on publicly available genome annotations for this strain, a broad search was conducted in the NCBI database using keywords such as "R. opacus PD630", "dioxygenase", and "Rieske" to obtain candidate protein sequences, as shown in Table 1. Twenty-four dioxygenase-encoding genes were identified, covering multiple functional families and substrate types, including oxygenases that may be involved in aromatic hydroxylation and ring cleavage. After removing five genes located on plasmids, primers were designed using Primer Primer 5 for the remaining genes. Due to the high GC content (up to about 70%) and the presence of highly homologous paralogs in Rhodococcus PD630, the GC ratio of primers can be appropriately increased when designing primers. The Tm value can be calculated using Oligo software. At the same time, whole-genome alignment can be used to ensure that the primers are located in gene-specific regions to avoid non-specific amplification caused by homologous gene interference.

[0043] Table 1. Information on dioxygenases screened from Rhodococcus genomes

[0044] K2Z90_000007 UDG97274.1 372 PDR / VanB family oxidoreductases K2Z90_000353 UDG97588.1 343 Aromatic cyclic hydroxylated dioxygenase α subunit K2Z90_000648 UDG97754.1 357 Aromatic cyclic hydroxylated dioxygenase α subunit K2Z90_000878 UDG98069.1 341 Aromatic cyclic hydroxylated dioxygenase α subunit K2Z90_000904 UDG98095.1 330 Isopentin N synthase family oxygenases K2Z90_000946 UDG98134.1 300 Iron-dependent exodiol dioxygenase K2Z90_001140 UDG98317.1 371 Nitroalkyl acid monooxygenase family proteins K2Z90_001381 UDG98537.1 215 α-Ketoglutarate-dependent dioxygenase AlkB K2Z90_001519 UDG98669.1 401 4-Hydroxyphenylpyruvate dioxygenase K2Z90_001796 UDG98923.1 277 Phytanoyl-CoA dioxygenase family proteins K2Z90_003455 UDH00400.1 326 PDR / VanB family oxidoreductases K2Z90_003511 UDH00446.1 367 PDR / VanB family oxidoreductases K2Z90_004578 UDG94514.1 329 Nitroalkyl acid monooxygenase K2Z90_004785 UDG94706.1 259 dioxygenase K2Z90_005045 UDG94947.1 312 Nitroalkyl acid monooxygenase K2Z90_005533 UDG95393.1 318 TauD / TfdA family dioxygenases K2Z90_005608 UDG95459.1 390 Aromatic cyclic hydroxylated dioxygenase α subunit K2Z90_005610 UDG95461.1 114 Bifunctional 3-phenylpropionic acid / cinnamic acid dioxygenase ferroreductin subunit K2Z90_005840 UDG95678.1 402 4-Hydroxyphenylpyruvate dioxygenase K2Z90_005885 UDG95721.1 315 TauD / TfdA family dioxygenases K2Z90_006111 UDG95930.1 398 urinary acetylcholine 1,2-dioxygenase K2Z90_006305 UDG96117.1 288 TauD / TfdA family dioxygenases K2Z90_006318 UDG96129.1 386 3-Ketosteroid-9α-hydroxylase A subunit K2Z90_006771 UDG96535.1 323 PDR / VanB family oxidoreductases

[0045] Example 2

[0046] Construction of the knockout vector:

[0047] (1) Homologous arm amplification

[0048] Purified wild-type Rhodococcus PD630 genomic DNA was used as a template. Primers were designed based on the target gene sequence using Snapgene, Primer Primer 5, and Oligo software. The 5' end of the primers needed to contain a homologous sequence or restriction enzyme site (15-20 bp) matching the linearized vector. PCR amplification was performed using the high-fidelity polymerase Phanta SuperFidelity DNAPolymeras (Nanjing Novizan Biotechnology Co., Ltd.) to ensure the accuracy and specificity of the amplified fragments. 5% DMSO was added to the reaction system to reduce the secondary structure stability of the template. The thermal cycling program was set to a 3-minute pre-denaturation time, and the annealing temperature gradient was optimized. The extension time was calculated at 30 seconds per kb to overcome the extension difficulties of high-GC sequences. After amplification, the product was verified by agarose gel electrophoresis to confirm that it was a single, clear band. The product was then excised, purified, and stored at 4°C for subsequent cloning and assembly.

[0049] (2) Enzyme digestion

[0050] Based on the vector map, a Fast Digest restriction endonuclease was selected. The reaction system was prepared on ice. To prevent plasmid religation, 1 μL of the novel Fast AP alkaline phosphatase was added to the reaction system. After preparation, the reaction system was incubated in a 37°C water bath for 30 min. After the reaction, the completeness of the digestion was verified by agarose gel electrophoresis. The obtained linearized fragment can be used for one-step cloning.

[0051] (3) One-step cloning and recombination transformation

[0052] The DNA fragment was ligated to the plasmid backbone using a kit (Nanjing Novizan Biotechnology Co., Ltd.). After the reaction, an appropriate amount of product was added directly to 50 μL of competent cells (E. coli DH5α), mixed well, and incubated on ice for 30 minutes. Then, a heat shock was performed at 42°C for 45-90 seconds, followed by rapid cooling on ice for 2 minutes. 900 μL of antibiotic-free liquid medium (LB) was added, and the cells were incubated at 37°C with shaking at 200 rpm for 45 minutes. Finally, an appropriate amount of bacterial culture was evenly spread onto a selection plate containing the appropriate antibiotic, and the plate was inverted and incubated overnight at 37°C for 12-16 hours to obtain transformants.

[0053] (4) Colony PCR verification and genome sequencing

[0054] After 12-16 hours of growth, single colonies on the plates were picked, and plasmids were extracted using Easytaq (Beijing TransGen Biotechnology). Positive clones were then screened by agarose gel electrophoresis. Plasmid extracts from suspected positive clones were sent to a sequencing company (Sangon Biotech (Shanghai) Co., Ltd.) for bidirectional sequencing to compare the sequencing results with the designed sequence. If the sequencing results were correct, the recombinant plasmid was successfully constructed and subsequent experiments could proceed.

[0055] Example 3

[0056] Knockout of dioxygenase gene in Rhodococcus PD630:

[0057] (1) Preparation of Rhodococcus PD630 competent cells

[0058] A single colony of Rhodococcus PD630 was picked from LB solid medium and inoculated into 5 mL of LB liquid medium. The culture was incubated at 30°C with shaking at 250 rpm until the culture became turbid. Then, 1 mL of the inoculum was transferred to 50 mL of LB liquid medium supplemented with 0.85% (w / v) glycine and 1% (w / v) sucrose, and cultured under the same conditions for approximately 24 hours. The OD of the culture was then measured. 600 When the bacterial concentration reaches 0.6-0.8, transfer it to a pre-chilled 50 mL centrifuge tube and centrifuge at 4°C and 8000 rpm for 10 minutes to collect the bacterial cells. Wash and resuspend the bacterial cells successively with 10 mL of pre-chilled sterile water and 1 mL of pre-chilled 10% glycerol, and finally aliquot and store at -80°C for later use or use directly for transformation.

[0059] (2) Gene knockout principle

[0060] In Rhodococcus PD630, gene knockout was performed using the PheS* mutant as a reverse selection marker. First, approximately 1 kb homologous arms upstream and downstream of the target gene were amplified, fused by overlap PCR, and cloned into the suicide vector pK18mob-pheS* (reference: [Cai Chenggu. Metabolic pathway design and process enhancement for the synthesis of lignin from erythropoiesis-1,4-diol and gallic acid [M]. Jiangsu: Nanjing University of Science and Technology, 2022.]). The constructed knockout plasmid was electroporated into Rhodococcus PD630, and the first recombination-positive clones were obtained by screening on SOB plates containing 50 μg / mL kanamycin using homologous recombination. Positive clones were then selected and cultured to OD. 600 ≈0.5, take 200 μL and spread it on LB agar plates containing 15 mM p-chlorophenylalanine (p-Cl-Phe), and incubate at 30℃ for 2–3 days. During this period, a second homologous recombination occurs, and the plasmid backbone is excluded. Finally, the target gene deletion strain was obtained through two rounds of screening and verified by PCR and genome sequencing.

[0061] (3) First homologous recombination and sporogen screening

[0062] The sequenced and verified knockout plasmid was introduced into pre-prepared Rhodococcus PD630 competent cells via electroporation. Electroporation parameters were set as follows: voltage 12.5 kV / cm, capacitance 25 μF, and resistance 400 Ω. Immediately after electroporation, 900 μL of recovery medium (SOB medium containing 0.5 M glucose) was added, and the cells were incubated at 30°C and 250 rpm for 3 hours. The revived bacterial culture was then plated onto SOB agar plates containing kanamycin (50 μg / mL) and incubated upside down at 30°C for 2–3 days. Single colonies (at which point the first single crossover occurs, and the plasmid integrates into the genome) were picked and transferred to LB broth and incubated with shaking at 30°C for 24–36 hours.

[0063] (4) Second homologous recombination and mutant screening

[0064] After appropriate dilution, the above culture was spread onto solid plates containing p-Cl-Phe and incubated at 30°C for 2-3 days. Under these conditions, pheS was expressed. ※ Cells that retain their genetic makeup and plasmid backbone (i.e., cells that have not undergone a second crossover) die because they cannot synthesize proteins normally; simultaneously, using the positive selection pressure of p-chlorophenylalanine, cells that have undergone a second homologous recombination (double crossover) and correctly lost pheS are enriched. ※ -Kan-tagged gene knockout candidate strains.

[0065] (5) Molecular verification of mutants

[0066] Multiple single clones were randomly selected from screening plates and initially screened using colony PCR. A set of validation primers was used: one primer located on the genomic sequence outside the homologous arm, another primer located inside the knocked-out gene, and another set of primers spanning the homologous arm. By comparing the size differences of the PCR products of wild-type and candidate mutants, clones that might have correctly double-crossovered were preliminarily identified. For clones that were initially positive, their genomic DNA was further extracted as templates, and amplification and sequencing were performed using multiple pairs of primers covering the knockout region and flanking sequences. Finally, by comparing the sequencing results with the wild-type sequence, it was confirmed that the coding region of the target gene had been completely and precisely deleted or inactivated, without any additional unexpected mutations, thus obtaining the knockout mutant of the gene. The above process was repeated for each target gene, and 24 knockout mutants of dioxygenase genes were successfully constructed and archived.

[0067] Figure 1-24Electrophoresis and sequencing results of 24 knockout mutants of dioxygenase genes, corresponding to Δ5610, Δ878, Δ946, Δ5533, Δ4578, Δ6305, Δ6318, Δ6771, Δ3455, Δ007, Δ1140, Δ353, Δ904, Δ3511, Δ5608, Δ4785, Δ648, Δ5840, Δ6111, Δ1381, Δ1519, Δ5045, Δ1796, and Δ5885, respectively.

[0068] Example 4

[0069] Metabolic pathway analysis using m-hydroxybenzaldehyde as a substrate:

[0070] (1) Selecting the substrate

[0071] Eighteen aromatic compounds, including hydroquinone, 2-methylresorcinol, catechol, 3-methylcatechol, 4-methylcatechol, 3,5-dihydroxytoluene, 4-ethylphenol, p-hydroxyphenylpropionic acid, 3-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 4-methoxycinnamic acid, resveratrol, o-cresol, m-cresol, p-cresol, m-hydroxybenzaldehyde, and p-hydroxybenzaldehyde, were used as substrates for testing wild-type Rhodococcus PD630.

[0072] (2) Phenotypic screening

[0073] Wild-type Rhodococcus PD630 and mutant glycerol tubes, preserved at -80℃, were used. Under aseptic conditions in a laminar flow hood, 200 μL of the bacterial culture was added to 1 mL of LB broth and incubated at 250 rpm and 30℃ for 24 h to activate the strain. Subsequently, under aseptic conditions in a laminar flow hood, 1 mL of the bacterial culture was added to 50 mL of LB broth and incubated at 250 rpm and 30℃ for 24–36 h. The OD of the strain was then measured. 600 At approximately 2.0, the culture medium was centrifuged, and the bacterial cells were washed twice with 20 mL of sterile physiological saline. Finally, 1 mL of physiological saline was added to suspend all bacterial cells, forming a bacterial suspension. The OD of the bacterial suspension was measured. 600 Value, and with the initial OD 600 The bacterial culture medium was inoculated at a concentration of 0.25 μL into MSM medium and cultured at 250 rpm and 30°C. Samples were taken at 0 h, 12 h, and 24 h after inoculation to detect changes in substrate composition and OD. 600 .

[0074] (3) Path Analysis

[0075] Figure 25This study validates the substrate utilization ability of protocatechuic acid 3,4-dioxygenase-deficient mutants. Wild-type Rhodococcus PD630 can grow normally and effectively utilize the substrate, while the K2Z90_005610 deletion mutant (Δ5610) shows significant growth inhibition and cannot utilize vanillic acid, vanillin, p-hydroxybenzoic acid, ferulic acid, or p-coumaric acid as carbon sources. This result is consistent with previous research findings on the key cyclic cleavage step of protocatechuic acid 3,4-dioxygenase in the protocatechuic acid degradation pathway, indicating that the substrate screening and phenotypic analysis system established in this invention can accurately reflect the impact of specific dioxygenase deletion on substrate utilization ability, demonstrating the feasibility of this method in resolving the "substrate-key dioxygenase" correspondence.

[0076] Figure 26 The figure shows the utilization results of wild-type Rhodococcus PD630 on 18 aromatic compounds. It can be seen that wild-type Rhodococcus PD630 can effectively utilize 12 of these compounds, including hydroquinone, catechol, various methyl / hydroxy substituted phenols, and benzoic acid derivatives. The remaining 6 compounds failed to support significant growth under the experimental conditions and were excluded from subsequent mutant screening.

[0077] Depend on Figure 27 It can be seen that the four mutants Δ4785, Δ353, Δ5608, and Δ5885 can all effectively utilize 12 aromatic compounds, including catechol, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde, as the sole carbon source for growth.

[0078] Depend on Figure 28 It can be seen that the four mutants Δ3455, Δ5533, Δ6305, and Δ007 can all effectively utilize 12 aromatic compounds, including catechol, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde, as the sole carbon source for growth.

[0079] Depend on Figure 29 It can be seen that the three mutants Δ6771, Δ1140, and Δ3511 can effectively utilize 12 aromatic compounds, including catechol, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde, as the sole carbon source for growth. Δ904, however, shows growth inhibition when m-hydroxybenzaldehyde is used as the sole carbon source.

[0080] Depend on Figure 30 It can be seen that the four mutants Δ6318, Δ1381, Δ1796, and Δ648 can all effectively utilize 12 aromatic compounds, including catechol, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde, as the sole carbon source for growth.

[0081] Depend on Figure 31 It can be seen that the four mutants Δ1519, Δ5840, Δ878, and Δ6111 can all effectively utilize 12 aromatic compounds, including catechol, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde, as the sole carbon source for growth.

[0082] Depend on Figure 32 It can be seen that the three mutants Δ946, Δ5045, and Δ4578 can all effectively utilize 12 aromatic compounds, including catechol, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde, as the sole carbon source for growth.

[0083] Depend on Figure 33 It was found that the utilization of m-hydroxybenzaldehyde as the sole carbon source by wild-type Rhodococcus PD630 and Δ904 was validated a second time. Under the same culture conditions, batch fermentation experiments of wild-type Rhodococcus PD630 and mutant Δ904 with m-hydroxybenzaldehyde as the sole carbon source were repeated, and the substrate consumption during the culture process was accurately quantified by high-performance liquid chromatography (HPLC). The repeated experimental results confirmed the initial observation: the wild-type strain exhibited rapid consumption of m-hydroxybenzaldehyde during the logarithmic growth phase; in stark contrast, the Δ904 mutant showed a significantly reduced OD during the 24-hour culture period. 600 The value has not increased significantly.

[0084] Figure 34 The figure shows the utilization results of wild-type Rhodococcus PD630 and Δ904 using 3-hydroxybenzyl alcohol as the sole carbon source. It can be seen that wild-type Rhodococcus PD630 grows well and rapidly consumes the substrate, while the growth of the Δ904 mutant is limited. Furthermore, under identical HPLC analysis conditions, the elution time of the 3-hydroxybenzyl alcohol standard is completely consistent with the first unknown main peak in the mutant fermentation broth.

[0085] In summary, after substrate screening using the constructed dioxygenase-deficient mutant library, Δ904 exhibited a complete growth defect towards a single substrate—m-hydroxybenzaldehyde. Based on this, it is hypothesized that the Δ904 gene is not a functionally redundant member, but likely dominates the initiation or key transformation step of an independent and specific metabolic pathway. To clearly identify the changes in metabolites in the Δ904 mutant when m-hydroxybenzaldehyde is the sole carbon source, the fermentation broth supernatant was analyzed by liquid chromatography-mass spectrometry (LC-MS). Through precise molecular weight determination and isotope distribution comparison using primary mass spectrometry, a significant characteristic peak was found in the mutant fermentation supernatant sample, with a quasi-molecular ion peak [MH]. - The precise mass-to-charge ratio (m / z) is 123.280, which is in high agreement with the calculated value (124.137) for the molecular formula C7H8O2.

[0086] The molecular formula of m-hydroxybenzaldehyde (C7H6O2) indicates that it contains six degrees of unsaturation. The detected metabolite molecule (C7H8O2) has two more hydrogen atoms (H2) than the substrate, and its degree of unsaturation is reduced by one. The most direct and reasonable chemical explanation for this change is that the aldehyde group (-CHO) of the substrate was reduced to a hydroxymethyl group (-CH2OH). Therefore, we infer that this metabolite is most likely m-hydroxybenzyl alcohol. A m-hydroxybenzyl alcohol standard was prepared. Under identical HPLC analytical conditions, the peak time of this standard was completely consistent with the first unknown main peak in the mutant fermentation broth.

[0087] The true first step in the metabolism of m-hydroxybenzaldehyde in Rhodococcus PD630: it is not a hydroxylation or oxidation reaction as initially assumed, but rather mediated by an unusual reduction reaction. Specifically, the protein encoded by the K2Z90_000904 gene in Rhodococcus PD630 likely does not directly attack the aromatic ring with its classic dioxygenase activity, but rather reduces m-hydroxybenzaldehyde to m-hydroxybenzyl alcohol through a novel reductase or dehydrogenase mechanism. The identification of this crucial step leads to a completely new direction for subsequent research: elucidating how m-hydroxybenzyl alcohol is reoxidized and further metabolized through ring-opening metabolism.

Claims

1. A library of Rhodococcus dioxygenase-deficient mutants, characterized in that, It consists of Rhodococcus mutants in which genes of different members of the dioxygenase family have been specifically knocked out.

2. The Rhodococcus dioxygenase deletion mutant library according to claim 1, characterized in that, There are more than 24 Rhodococcus mutants.

3. The Rhodococcus dioxygenase deletion mutant library according to claim 1, characterized in that, Rhodococcus is Rhodococcus PD630, Rhodococcus P14, Rhodococcus AJ270, Rhodococcus RHA1 or Rhodococcus J1.

4. The Rhodococcus dioxygenase deletion mutant library according to claim 1, characterized in that, Rhodococcus mutants include the Δ5610 mutant (K2Z90_005610 knockout of dioxygenase gene), the Δ878 mutant (K2Z90_000878 knockout of dioxygenase gene), the Δ946 mutant (K2Z90_000946 knockout of dioxygenase gene), the Δ5533 mutant (K2Z90_005533 knockout of dioxygenase gene), the Δ4578 mutant (K2Z90_004578 knockout of dioxygenase gene), and the Δ63 mutant (K2Z90_006305 knockout of dioxygenase gene). 05 mutant, Δ6318 mutant with knockout of dioxygenase gene K2Z90_006318, Δ6771 mutant with knockout of dioxygenase gene K2Z90_006771, Δ3455 mutant with knockout of dioxygenase gene K2Z90_003455, Δ007 mutant with knockout of dioxygenase gene K2Z90_000007, Δ1140 mutant with knockout of dioxygenase gene K2Z90_001140, Δ353 mutant with knockout of dioxygenase gene K2Z90_000353 Variants, including the Δ904 mutant (K2Z90_000904 knockout of dioxygenase gene), the Δ3511 mutant (K2Z90_003511 knockout of dioxygenase gene), the Δ5608 mutant (K2Z90_005608 knockout of dioxygenase gene), the Δ4785 mutant (K2Z90_004785 knockout of dioxygenase gene), the Δ648 mutant (K2Z90_000648 knockout of dioxygenase gene), and the Δ5840 mutant (K2Z90_005840 knockout of dioxygenase gene). The following mutants were identified: Δ6111 (knockout of dioxygenase gene K2Z90_006111), Δ1381 (knockout of dioxygenase gene K2Z90_001381), Δ1519 (knockout of dioxygenase gene K2Z90_001519), Δ5045 (knockout of dioxygenase gene K2Z90_005045), Δ1796 (knockout of dioxygenase gene K2Z90_001796), and Δ5885 (knockout of dioxygenase gene K2Z90_005885).

5. The method for constructing a Rhodococcus dioxygenase-deficient mutant library according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Target gene selection: Different members of the dioxygenase family were screened from the wild-type Rhodococcus genome as knockout targets; (2) Construction of knockout vectors: Construct gene knockout vectors targeting each gene; (3) Mutant screening: The constructed knockout vector was transformed into wild-type Rhodococcus, and the target gene was knocked out by homologous recombination technology. Mutants with different members of the dioxygenase family were obtained by screening, and a library of Rhodococcus dioxygenase deletion mutants was constructed.

6. The construction method according to claim 5, characterized in that, In step (2), the gene knockout vector is a vector carrying PheS ※ Tagged suicide plasmid.

7. The construction method according to claim 5, characterized in that, In step (3), the screening is carried out using resistance screening.

8. The application of the Rhodococcus dioxygenase deletion mutant library according to any one of claims 1 to 4, characterized in that, The specific application method is as follows: (1) All mutants in the Rhodococcus dioxygenase-deficient mutant library were inoculated into a culture medium with the target aromatic compound as the sole carbon source, and the utilization ability of each mutant was observed. (2) Based on the utilization ability of each mutant, key dioxygenase genes in the metabolic pathway using target aromatic compounds as substrates were screened; (3) Analyze the metabolic pathway of target aromatic compounds in Rhodococcus by key dioxygenase genes; (4) Based on the analysis results, the target high-value chemical can be obtained by knocking out or overexpressing the relevant dioxygenase gene.

9. The application according to claim 8, characterized in that, The target aromatic compound is selected from one or more of hydroquinone, 2-methylresorcinol, 4-methylcatechol, 2,5-dihydroxybenzoic acid, p-hydroxybenzaldehyde, catechol, 3,5-dihydroxytoluene, 3-methylcatechol, 4-ethylphenol, 3-hydroxybenzoic acid, 4-methoxycinnamic acid, p-hydroxyphenylpropionic acid, o-cresol, m-cresol, p-cresol, and m-hydroxybenzaldehyde.

10. The application according to claim 8, characterized in that, High-value chemicals include protocatechuic acid, mucilage acid, p-coumaric acid, ferulic acid, or gallic acid.

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