Polyketides are a diverse class of natural products with broad pharmacological activities and substantial industrial relevance; however, their scalable biosynthesis is often constrained by inefficient heterologous expression platforms. In this study, a systematic evaluation of multiple microbial hosts identified Aspergillus niger HL-1 as an efficient fungal chassis for polyketide production. Subcellular compartmentalization analysis revealed that cytosolic biosynthesis significantly outperformed peroxisomal localization, leading to a 5.88-fold increase in triacetic acid lactone (TAL) production. To further enhance production, intracellular acetyl-CoA and malonyl-CoA supplies were engineered and integrated with compartmentalized metabolic rewiring to redirect carbon flux toward polyketide biosynthesis. In addition, increasing the copy number of polyketide synthase genes further improved titers. As a result, monacolin J and TAL reached 1.68 g/L and 7.50 g/L in shake-flask cultures, respectively, and were further elevated to 2.61 g/L and 25.08 g/L in a 1.5-L bioreactor. Finally, a versatile type III polyketide synthase (PKS) expression platform was established, enabling the functional expression of ten heterologous PKSs. By further disrupting the endogenous katA gene to decelerate p-coumaric acid consumption, the competitive catabolic pathway was successfully blocked, yielding a naringenin shake-flask titer of 384.91 mg/L from p-coumaric acid. This work establishes a robust and scalable fungal platform for the efficient production of both complex polyketides and flavonoids, providing a promising strategy for biotechnological applications.

Enzyme-constrained metabolic modeling (ecModels) extends classical flux balance analysis (FBA) by incorporating enzyme catalytic and protein allocation constraints, enabling more quantitatively constrained simulations of cellular metabolism. However, existing ecModel toolkits rely on interpreted programming environments such as MATLAB or Python, which couples them to specific runtime ecosystems and limits their deployability as reusable computational backends. Here we present MeNetModel, a cross-platform computational engine for enzyme-constrained metabolic modeling implemented in the compiled .NET ecosystem (F#) and distributed as a language-agnostic dynamic link library (DLL). The framework adopts a modular architecture separating model construction, constraint formulation, and optimization, allowing the engine to be invoked natively from Python, MATLAB, C#, and F# without recompilation. MeNetModel further integrates automated enzyme kinetic parameter estimation via DLKcat, proteome allocation analysis, and the Expression-Efficiency Pressure (EEP or EEP+) metrics for systematic identification and prioritization of metabolic bottlenecks. We applied MeNetModel to construct an enzyme-constrained model of Aspergillus niger, which reproduces proteome-limited growth saturation and supports pathway-level bottleneck prioritization in the mevalonate biosynthesis pathway.

Ergothioneine (EGT) is a potent natural antioxidant, making it a promising candidate for potential applications in cosmetics, pharmaceuticals, and food industries. This study represents the successful heterologous synthesis of EGT in Aspergillus niger strain SH-2. By screening and co-expressing egt1 and egt2 genes from various sources, the combination of egt1 from Trichoderma reesei and egt2 from Neurospora crassa was the optimal EGT synthetic pathway. The synergistic effect of supplementing precursor resulted in a 34.70% increase in EGT production. However, the enhancement of precursor amino acid biosynthesis pathway played a negative role in the transcriptional level of EGT biosynthetic genes. Based on single-factor fermentation optimization, the maximum yield of EGT reached 3.12g/L in 5-L bioreactor (144h), the highest EGT titer heterologously synthesized in fungi. This study contributes to the field of heterologous EGT synthesis and offers an engineered A. niger strain OE4-T1N2 with potentially industrial application.

AIMS:Resumption of proliferation by quiescent hepatocytes in response to liver injury is a hallmark event in live regeneration. We have previously reported that the zinc finger transcription factor GATA4 contributes to liver regeneration by promoting hepatocyte proliferation. In the present study we investigated the underlying mechanism. METHODS AND MATERIALS:Cellular transcriptome was evaluated by RNA-seq and ChIP-seq. Liver regeneration was evaluated in a murine model of partial hepatectomy. KEY FINDINGS:Integrated transcriptomic analysis uncovered acetyl coenzyme A acetyltransferase 2 (Acat2) as one of the potential GATA4 targets in hepatocytes. Quantitative PCR and Western blotting confirmed that GATA4 knockdown down-regulated Acat2 expression whereas ChIP assay detected direct binding of GATA4 to the Acat2 promoter. Supplementation with a wild type, but not a catalytically inactive, Acat2 rescued the deficiency of proliferation in hepatocytes depleted of GATA4. Consistently, liver specific Acat2 knockdown dampened liver regeneration and exacerbated liver injury in mice. Importantly, a correlation between ACAT2 expression, hepatocyte proliferation, and liver injury was observed in patients with liver failure. SIGNIFICANCE:Our data suggest that Acat2, as a novel target for GATA4, may play a key role in liver regeneration.

Aspergillus niger, a generally recognized as safe (GRAS) filamentous fungus, is a promising chassis for edible mycelia but lacks meat-like sensory properties. Here, modular metabolic engineering including relieving feedback inhibition (ΔHRM), overexpressing rate-limiting enzymes, and enhancing iron uptake (ΔsreA) was employed to enhance heme biosynthesis, endowing mycelia with a meat-red color. Untargeted metabolomics uncovered a heme tolerance mechanism via precursor efflux mediated by ABC transporters. Heterologous expression of hemoglobins and a P450 enzyme further improved heme utilization, and genome-wide variations analysis uncovered adaptive responses to oxidative and endoplasmic reticulum (ER) folding stress under high-heme pressure. Notably, this enhanced heme supply substantially boosted functional metabolite production, increasing monacolin J yield by 9.99-fold and ergothioneine yield by 1.11-fold. The engineered mycelium closely resembled commercial plant-based meat in color. This study provides a feasible approach to developing A. niger as a sustainable chassis for functional meat alternatives.

To address the low efficiency of genetic manipulation and poor hyphal morphology control in Aspergillus oryzae, this study developed a synthetic biology toolkit and identified a key genetic target for morphological engineering. The toolkit features an RNP-mediated rapid knockout system, serine integrase-based gene integration, and a pipeline for screening high-activity neutral genomic sites. Systematic deletion of seven cell wall integrity-related genes revealed that disruption of the chitin synthase gene chsY most effectively enhanced protein secretion. The Delta chsY mutant exhibited a 34.8 % increase in hyphal diameter and a 30.6 % reduction in culture viscosity, coupled with upregulated secretory pathways and an activated unfolded protein response (UPR). Applying this discovery, we engineered a strain expressing a heterologous lipase (TLL), achieving a 52 % increase in extracellular activity in flasks. This benefit scaled to bioreactors, with a 42 % higher enzyme titer and similar to 50 % lower viscosity. Our work provides both a genetic toolkit and a scalable engineering strategy (chsY deletion) to enhance A. oryzae as a cell factory for industrial enzyme production.

Filamentous fungi such as Aspergillus oryzae and Aspergillus niger serve as valuable microbial cell factories with diverse applications. However, genetic manipulation in these fungi is often hindered by the lack of efficient resistance-based transformation systems. To address this challenge, we developed a novel transformation system for both A. oryzae and A. niger based on zhongshengmycin, a cost-effective streptothricin-class antibiotic, used in combination with the resistance gene encoding nourseothricin acetyltransferase (NAT). Our tests revealed that sensitization with SDS and EDTA-Na2 disrupts cell membrane integrity, thereby enhancing the susceptibility of Aspergillus to zhongshengmycin. Through an attB/attP-mediated integration system, we achieved heterologous expression of β-glucuronidase in A. oryzae with a selection efficiency of 85.71 % (6/7). Additionally, recombination of megfp-sed5 in A. niger achieved a transformation efficiency of 83.33 % (10/12), comparable to that of the PyrG system, thus establishing a versatile platform for fungal biotechnology. This system enables precise genomic integration for metabolic rewiring, offering new strategies for the synthetic biology-driven improvement of industrial filamentous fungi.

BACKGROUND:Thermotolerance is a critical trait for yeasts employed in industrial settings, and the utilization of unconventional yeasts has gained notable attention in recent years. However, the mechanisms underlying thermotolerance in unconventional yeasts, particularly Pichia spp., remain insufficiently elucidated. RESULTS:This study focuses on the thermotolerance of a non-traditional yeast strain Pichia kudriavzevii LC375240, renowned for its remarkable thermotolerance. Through transcriptomic analysis of both short-term and long-term heat shock exposures, we uncovered an intricate regulatory response in P. kudriavzevii. During long-term heat treatment, the yeast exhibited elevated expression of genes involved in the tricarboxylic acid (TCA) cycle and suppressed expression of genes in the pentose phosphate pathway (PPP). Additionally, long-term heat treatment led to an upregulation of heat shock proteins (HSPs) and an increase in trehalose, glutathione (GSH), and superoxide dismutase (SOD) levels, along with a reduction in the intracellular NADPH/NADP+ ratio and pyruvate content. These changes collectively contribute to the thermotolerance of P. kudriavzevii. CRISPR-Cas9-mediated knockout experiments further highlighted the critical roles of HSPs, antioxidases, and the trehalose metabolic pathway in the yeast's response to high temperatures. CONCLUSIONS:Taken together, this study demonstrates that P. kudriavzevii adapts to thermal stress through a combination of enhanced TCA cycle, reduced PPP, increased HSPs, trehalose, GSH, and SOD levels. These findings provide a comprehensive understanding of the molecular mechanisms underlying thermotolerance in P. kudriavzevii.

Targeted in vivo hypermutation mediated by base deaminase-T7 RNA polymerase (T7 RNAP) fusions promotes genetic diversification and accelerates continuous directed evolution. Due to the lack of a T7RNAP expression regulation system and functionally compatible linker for fusion protein expression, T7RNAP-guided continuous evolution has not been established in Bacillus subtilis, which limited long gene fragment continuous evolution targeted on genome. Here, we developed BS-MutaT7 system, which introduced mutations into specific genomic regions by leveraging chimeric fusions of base deaminases with T7RNAP in B. subtilis. We selected seven different sources of adenosine and cytosine deaminases, 14 fusion protein linkers to be fused with T7RNAP, constructing four libraries with the size of 5000, where deaminases were fused at either the N- or C-terminus of T7RNAP. Based on the efficiency of binding to T7 promoter and high mutagenesis activity, two optimal chimeric mutators, BS-MutaT7A (TadA8e-Linker0-T7RNAP) and BS-MutaT7C (PmCDA1-(GGGGS)3-T7RNAP co-expressed with UGI) were identified. The target mutation rates reached 1.2 × 10-5 per base per generation (s.p.b.) and 5.8 × 10-5 s.p.b., representing 7000-fold and 37,000-fold increases over the genomic mutation rate, respectively. Both exhibited high processivity, maintaining mutation rates of 5.8 × 10-6 s.p.b. and 2.9 × 10-5 s.p.b. within a 5 kb DNA region. Notably, BS-MutaT7C exhibited superior mutagenic activity, making it well-suited for applications requiring intensive and sustained genomic diversification. Application of BS-MutaT7 enabled a 16-fold increase in tigecycline resistance and enhanced β-lactoglobulin (β-Lg) expression by evolving the global transcriptional regulator codY, achieving a β-Lg titer of 3.92 g/L. These results highlight BS-MutaT7 as a powerful and versatile tool for genome-scale continuous evolution in B. subtilis.

Aspergillus niger is a powerful and efficient cell factory, with the potential to synthesize valuable products as chassis cells. The use of microbial cell factories to produce monacolin J, a precursor for statin synthesis, as an alternative to chemical synthesis could meet increasing market demand. However, the need for precise large fragment gene editing and the availability of suitable integration loci hinders the application of this strain. Herein, we identified neutral integration sites of A. niger based on the combination of ATAC-seq, H3K4me3 epigenetic datasets. Next, a landing pad system was developed for the one-step integration of the MJ biosynthesis gene cluster (BGC) in A. niger. Furthermore, we optimized the precursor module supply, the auxiliary factor supply module of NADPH, the module for eliminating oxidative stress pressure, and the transporter module to improve the production of MJ. Finally, a multi-copy integration strategy was applied to the rapid integration of MJ BGC, achieving MJ titer up to 1851.52 mg/L at the 500 mL shaker level.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes enormous economic losses in the food and feed industries. Simple, rapid, low-cost, and quantitative analysis of ZEN is particularly urgent in the fields of food safety and animal husbandry. Using the bioluminescent bacterium Photobacterium phosphoreum T3, we propose a bioluminescence inhibition assay to evaluate ZEN levels quickly. The limit of detection (LOD), limit of quantification (LOQ), and quantitative working range of this bioluminescence inhibition assay were 0.1 µg/mL, 5 µg/mL, and 5-100 µg/mL, respectively. The concentration-response curve of the bioluminescence inhibition rate and ZEN concentration was plotted within the range 5 to 100 μg/mL, as follows: y = 0.0069x2 - 0.0190x + 7.9907 (R2 = 0.9943, y is luminescence inhibition rate, x is ZEN concentration). First, we used the bioluminescence inhibition assay to detect the remaining ZEN in samples treated with purified lactonohydrolase ZHD101. The bioluminescence inhibition assay results showed a strong correlation with the HPLC analysis. Furthermore, we successfully evaluated the overall toxicity of samples treated with purified peroxidase Prx and H2O2 using the P. phosphoreum T3 bioluminescence inhibition assay. The results indicate that the degradation products of ZEN created by purified peroxidase Prx and H2O2 showed little toxicity to P. phosphoreum T3. In this study, a simple, rapid, and low-cost assay method of zearalenone by bioluminescent P. phosphoreum T3 was developed. The bioluminescence inhibition assay could be used to estimate the efficiency of enzymatic degradation of ZEN.

Aspergillus niger is a cell factory widely used in industries to produce proteases, organic acids, drugs, and other substances. The hyphal morphology of A. niger is a complex differentiated elongated tubular structure, which limits its basic research and application. In this study, the mpkA, bck1, steC, and Tpk2 genes were successfully deleted using a quick way to knock out genes based on the RNP (Ribonucleoprotein) complex. The study showed that the knockout of mpkA and bck1 kinase gene strains resulted in smaller, denser colonies, short rod-shaped hypha, and a significant increase in glucoamylase secretion. The mechanism of short hypha formation and high protein production for A. niger is the cell wall integrity signaling (CWIS) pathway. The CWIS pathway passed through the bck1-mkkA-mpkA tertiary kinase to deliver phosphorylation signals to the rlmA transcription factor, which regulated the expression of the cell wall synthesis gene agsA, thus regulating hyphal morphology. The mpkA kinase regulated the expression of the transcription factor amyR, which affected the expression of the genes glaA and amyA, thus enhancing the expression of proteins in A. niger. This study provides a strategy for the regulation of hyphal morphology and promotes the application of A. niger in industrial production.

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis , Bacillus amyloliquefaciens , and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. Key points • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.

BACKGROUND:Research on protein production holds significant importance in the advancement of food technology, agriculture, pharmaceuticals, and bioenergy. Aspergillus niger stands out as an ideal microbial cell factory for the production of food-grade proteins, owing to its robust protein secretion capacity and excellent safety profile. However, the extensive oxidative folding of proteins within the endoplasmic reticulum (ER) triggers ER stress, consequently leading to protein misfolding reactions. This stressful phenomenon results in the accelerated generation of reactive oxygen species (ROS), thereby inducing oxidative stress. The accumulation of ROS can adversely affect intracellular DNA, proteins, and lipids. RESULT:In this study, we enhanced the detoxification of ROS in A. niger (SH-1) by integrating multiple modules, including the NADPH regeneration engineering module, the glutaredoxin system, the GSH synthesis engineering module, and the transcription factor module. We assessed the intracellular ROS levels, growth under stress conditions, protein production levels, and intracellular GSH content. Our findings revealed that the overexpression of Glr1 in the glutaredoxin system exhibited significant efficacy across various parameters. Specifically, it reduced the intracellular ROS levels in A. niger by 50%, boosted glucoamylase enzyme activity by 243%, and increased total protein secretion by 88%. CONCLUSION:The results indicate that moderate modulation of intracellular redox conditions can enhance overall protein output. In conclusion, we present a strategy for augmenting protein production in A. niger and propose a potential approach for optimizing microbial protein production system.

Growth-advantageous microbial chassis cells are beneficial for shortening fermentation period and boosting biomolecule productivity. This study focused on enhancing recombinant proteins synthesis efficiency in Bacillus subtilis by CRISPRi-mediated metabolism regulation for improved cell growth and screening expression elements. Specifically, by repressing odhA gene expression to reallocate cellular resource and overexpressing atpC, atpD and atpG genes to reprogram energy metabolism, the growth-advantageous chassis cell with high specific growth rate of 0.63 h-1 and biomass yield of 0.41 g DCW/g glucose in minimum medium was developed, representing 61.54 % and 46.43 % increasements compared to B. subtilis 168. Subsequently, using screened optimal P566 promoter and (EAAAK)3 protein linker, secretory bovine alpha-lactalbumin (α-LA) titer reached 1.02 mg/L. Finally, to test protein synthesis capability of cells, intracellular GFP, secretory α-LA and α-amylase were expressed with P566 promoter, representing 43.76 %, 75.49 % and 82.98 % increasements. The growth-advantageous B. subtilis chassis cells exhibit their potential to boost bioproduction productivity.

Aspergillus niger is a primary cell factory for food-grade protein (enzyme) production due to its strong protein secretion capacity and unique safety characteristics. The bottleneck issue for the current A. niger expression system is the difference in expression yield of heterologous proteins of non-fungal origin compared to those of fungal origin, which is about three orders of magnitude. The sweet protein monellin, derived from West African plants, has the potential to become a food-grade sweetener due to its high sweetness and the benefit of not containing sugar itself, but it is extremely difficult to establish a research model for heterologous expression in A. niger, owing to extremely low expression, a small molecular weight, and being undetectable with conventional protein electrophoresis. HiBiT-Tag was fused with low-expressing monellin in this work to create a research model for heterologous protein expression in A. niger at ultra-low levels. We increased monellin expression by increasing the monellin copy number, fusing monellin with the endogenous highly expressed glycosylase glaA, and eliminating extracellular protease degradation, among other strategies. In addition, we investigated the effects of overexpression of molecular chaperones, inhibiting the ERAD pathway, and enhancing the synthesis of phosphatidylinositol, phosphatidylcholine, and diglycerides in the biomembrane system. Using medium optimization, we finally obtained 0.284 mg/L of monellin in the supernatant of the shake flask. This is the first time recombinant monellin has been expressed in A. niger, with the goal of investigating ways to improve the secretory expression of heterologous proteins at ultra-low levels, which can serve as a model for the expression of other heterologous proteins in A. niger.

The maltose α-amylase AmyM from Bacillus stearothermophilus can be used for flour modification, baked goods preservation, and maltose production. Here, we optimized the recombinant expression of AmyM in Bacillus subtilis WB800 via several strategies. By screening the optimal promoter, a double promoter combination (P43 and PamyL) could improve the expression level of AmyM by 61.25%, compared with the strong promoter P43. Then, we optimized the secretion efficiency of recombinant AmyM by over-expressing the molecular chaperone prsA gene. SDS-PAGE results suggested that over-expression of the prsA could improve the secretion efficiency of AmyM to the extracellular environment. The extracellular enzyme activity of AmyM was increased by 101.58% compared to the control strain. To further improve the expression of AmyM, we introduced the hemoglobin gene of Vitreoscilla (vgb) into the AmyM recombinant strain. The results revealed that the introduction of vgb could promote the transcription and translation of AmyM in B. subtilis. This may be due to the increasing level of intracellular NADPH and NADP+ caused by the expression of vgb. By this strategy, the expression level of AmyM was increased by 204.08%. Finally, we found the recombinant AmyM showed an optimal temperature of 65 °C and an optimal pH of 5.5. Our present results provided an effective strategy for increasing the heterologous expression level of AmyM in B. subtilis.

Efficient protein secretion is closely correlated with vesicle sorting and packaging, especially with cargo receptor-mediated selective transport for ER exit. Even though Aspergillus niger is considered an industrially natural host for protein production due to its exceptional secretion capacity, the trafficking mechanism in the early secretory pathway remains a black box for us to explore. Here, we identified and characterized all putative ER cargo receptors of the three families in A. niger. We successfully constructed overexpression and deletion strains of each receptor and compared the colony morphology and protein secretion status of each strain. Among them, the deletion of Erv14 severely inhibited mycelial growth and secretion of extracellular proteins such as glucoamylase. To gain a comprehensive understanding of the proteins associated with Erv14, we developed a high-throughput method by combining yeast two-hybrid (Y2H) with next-generation sequencing (NGS) technology. We found Erv14 specifically interacted with transporters. Following further validation of the quantitative membrane proteome, we determined that Erv14 was associated with the transport of proteins involved in processes such as cell wall synthesis, lipid metabolism, and organic substrate metabolism.

Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.

Collagen plays a vital role in the human body and is widely used in food, health products, and medical treatment. Codon optimization of the human type Ⅲ collagen gene was carried out according to the codon usage bias of Pichia pastoris. The single-tandem,two-tandem, and four-copy two-tandem expression vectors pPIC9K-COL3-S, pPIC9K-COL3-2, and pPIC9K-COL3-4, respectively, were constructed and transformed into P. pastoris GS115 to achieve integrated expression of human type Ⅲ collagen, thereby obtaining engineered strains of P. pastoris containing single-tandem collagen, two-tandem collagen, and four-copy two-tandem collagen. The pPIC9K-COL3-S and pPIC9K-COL3-2 recombinant strains were mixed and shaken with a 0.5% induction concentration of methanol to stimulate high-density fermentation. SDS-PAGE and western blot analysis demonstrated that the recombinant strains successfully expressed recombinant collagen,where the apparent molecular weight of the single-tandem protein was approximately 26.7 ku, and that of the two-tandem protein was approximately 52.3 ku. The yield of the high-density shake flask fermentation of the four-copy recombinant strain induced by 0.5% methanol was the highest, with an optimal induction time of 72 h and protein yield reaching approximately 0.45 g/L. A high-purity recombinant protein of this strain was obtained after purification using a nickel column. Antioxidant activity experiments showed that the DPPH free-radical scavenging rate of the recombinant collagen reached 51.49%, whereas the ABTS free-radical scavenging rate reached 41.24%, thus proving its antioxidant activity. This provides a theoretical basis for its application in the fields of food, health products, and medicine.