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. 2026 May 11;110:202. doi: 10.1007/s00253-026-13862-6

Expression and identification of a novel high-activity recombinant humanized type I collagen SynthCol1 in Pichia pastoris GS115

Zhiwu Zhang 1,2, Jinyu Li 2, Zhengfeng Chen 2, Haiming Du 2, Wei Xia 2, Yana Wang 2, Huimin Yu 1,✉
PMCID: PMC13333565  PMID: 42115497

Abstract

Abstract

The limitations associated with animal-derived collagen, such as the risk of zoonotic pathogen transmission and batch variability, have expedited the development of recombinant alternatives. Nonetheless, achieving an optimal balance between the bioactivity of recombinant collagen and production efficiency to ensure superior techno-economic performance remains a significant challenge in the field. In this study, we engineered a novel recombinant humanized collagen, designated as SynthCol1, by incorporating a 9-mer repeat sequence from the human type I collagen α1 chain (G674–A736) that includes integrin-binding motifs (GFPGER/GMPGER). This design strategy effectively addressed the critical challenges of soluble expression and production yield, resulting in a high-producing strain. SynthCol1 was expressed at high titers (15.3 g/L) in a 500 L bioreactor using Pichia pastoris GS115 and was purified to greater than 95% homogeneity. Furthermore, functional assays demonstrated its capability to enhance cell adhesion. In a model of full-thickness human skin damaged by UVA exposure, SynthCol1 demonstrated significant efficacy in promoting tissue repair through structural reconstitution of the basement membrane, barrier regeneration and modulation of the inflammatory microenvironment. These results substantiate a strategic approach in the design of potent recombinant collagens, positioning SynthCol1 as a versatile and scalable biomaterial platform with substantial potential for therapeutic and cosmetic applications.

Key points

  • The study engineered a novel recombinant humanized type I collagen with high yield

  • SynthCol1 was designed with enhanced bioactivity via rational design

  • SynthCol1 was demonstrated to be effective in skin repair and photoprotection

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13862-6.

Keywords: Type I collagen, Recombinant humanized collagen, Scaled manufacture, Biomaterial, Skin care

Introduction

Collagen, a major structural protein and the primary constituent of the extracellular matrix (ECM), provides structural support and regulates essential biological processes, such as cell adhesion, migration, differentiation, and signal transduction (Naba 2024; Zhao et al. 2025). It is indispensable for tissue formation, maintaining structural integrity and facilitating injury repair (Hu et al. 2025). Type I collagen constitutes approximately 85% of the total collagen in the human body and is widely employed in dermatological, cosmetic, and biomedical applications due to its critical role in maintaining tissue biomechanics (Amirrah et al. 2022; Devos et al. 2023). Type I collagen is a heterotrimer composed of two α1 chains and one α2 chain. To date, 28 distinct collagen types have been identified, including fibrillar collagens, network-forming collagens, and fibril-associated collagens (Ricard-Blum 2010). Owing to its biocompatibility, biodegradability, and low immunogenicity, collagen is widely used as a biomaterial in medical devices, pharmaceuticals, health supplements, and cosmetics (Rezvani Ghomi et al. 2021; Wang et al. 2023; Wosicka-Frąckowiak et al. 2024).

Animal-derived collagen, traditionally sourced from animal tissues (e.g., porcine, bovine, and equine skin and tendon), presents several limitations, including potential viral contamination, challenges in achieving high purity, and inter-batch variability (Amirrah et al. 2022). Research into recombinant collagen production began about three decades ago with mammalian and bacterial expression systems; subsequent work has extended to yeast, insect cells, and transgenic plants (Cai et al. 2021; Fertala 2020; Geddis and Prockop 1993; Rutschmann et al. 2014; Shoseyov et al. 2013). Compared to traditional recombinant human collagen and recombinant collagen-like proteins, humanized collagen demonstrates superior properties in terms of biocompatibility, immunogenicity, and functional diversity. Its broad applications in antitumor therapy (Zeng et al. 2024), tissue regeneration (Hao et al. 2024), wound healing (Shuai et al. 2024), and biomaterial development (Qu et al. 2024) underscore its significant potential and growing importance in modern biomedical research.

Three primary strategies exist for the recombinant expression of collagen: (a) recombinant human collagen, containing both the full-length collagen peptide chain and triple-helix structure; (b) recombinant humanized collagen, derived from human collagen genes but fabricated into fragment peptides lacking the triple-helix; and (c) recombinant collagen-like protein, featuring collagen-like amino acid sequences (originating from human, animal, bacterial, or artificial designs) with low homology to human collagen (Liu et al. 2022). Full-length, triple-helical are necessary for the recombinant human collagen. The similar recombinant collagen has been successfully expressed in systems including plant (tobacco) (Stein et al. 2009), Escherichia coli and yeast (Nokelainen et al. 2001; Toman et al. 2000); the high molecular weight of natural collagen, the complexity of its hierarchical structure, extensive post-translational modifications, and unique sequence periodicity pose significant challenges for achieving industrial-scale production at high levels (Yan et al. 2025), and it is difficult to make it commercially viable because of the high costs.

In order to realize collagen’s potential beyond structural roles for advanced functional applications in tissue repair and regeneration, research must prioritize two critical dimensions: biochemical attributes governing regenerative bioactivity and scalable manufacturing processes enabling clinical translation. Currently, commercialized recombinant collagens are recombinant humanized collagen (RHC) which derive human collagen genes including partial collagen fragments. RHC primarily expressed in E. coli or yeast-typically consist of partial collagen fragments or multimeric repeats (Xiang et al. 2023). These derivatives exhibit enhanced hydrophilicity and stability but display dynamic triple-helical folding observable only under specific conditions (e.g., via circular dichroism spectroscopy at low temperatures), reflecting properties distinct from natural collagen fibers (Naba 2024; Wosicka-Frąckowiak et al. 2024). Although E. coli is a commonly used system for recombinant protein expression, it exhibits significant limitations when processing complex proteins like collagen (Ramshaw et al. 2019). Studies indicate that E. coli typically fails to perform essential post-translational modifications during recombinant collagen expression, such as hydroxylation-a process critical for collagen stability and biological function (Rutschmann et al. 2014; Zhu et al. 2024). Furthermore, recombinant proteins expressed in E. coli often exist as inclusion bodies, requiring complicated folding and purification procedures that further increase production complexity and costs (Baghban et al. 2021; Gopal and Kumar 2013). In contrast, Pichia pastoris GS115 demonstrates significant advantages in producing recombinant collagen. As a eukaryotic expression system, P. pastoris can perform complex post-translational modifications such as glycosylation and hydroxylation, which are crucial for the functional activity and stability of certain proteins (Ejike et al. 2021; Pokoj et al. 2010). Studies have shown that P. pastoris achieves higher yields and better protein quality when expressing complex proteins. For instance, when expressing non-specific lipid transfer proteins, P. pastoris produces approximately 270 times more protein than E. coli, while maintaining high solubility, proper folding, and biological activity (Pokoj et al. 2010). Furthermore, P. pastoris has demonstrated exceptional advantages in industrial applications. Through optimized expression conditions and genetic engineering techniques, this yeast can significantly enhance both the yield and quality of recombinant proteins. For instance, employing a multi-strategy approach to improve phytase expression in E. coli, within P. pastoris, researchers achieved up to 384% improvement in enzyme activity (Helian et al. 2020). These findings indicate that P. pastoris not only excels at the laboratory scale but also holds tremendous potential for industrial production (Eskandari et al. 2023; Unver and Dagci 2024; Vijayakumar and Venkataraman 2024).

In this study, through the optimization of a series of synthetic biology enabling technologies we successfully expressed a synthetic humanized collagen protein SynthCol1 in P. pastoris GS115, through lab-scale and pilot-scale testing, large-scale fermentation was successfully achieved in a 500 L bioreactor with high productivity. Further, we evaluated the biofunctions on Reconstructed Human Skin (RHS), demonstrating its exceptional efficacy in skin regeneration and repair. The comprehensive experimental design is shown in Fig. 1.

Fig. 1.

Fig. 1

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Schematic overview of the experimental design for recombinant SynthCol1 production and functional validation. The recombinant plastid vector pPIC9K-SynthCol1 was electroporated into yeast cells for SynthCol1 expression. The purified SynthCol1 protein was subsequently applied to NIH/3T3 cells to assess cellular activity and to a skin model to evaluate UVA damage repair

Materials and methods

Construction of recombinant P. pastoris

The 567aa recombinant humanized type I collagen protein SynthCol1 was designed as nine consecutive repeats of the human type I collagen α1 chain fragment (residues G674–A736). The SynthCol1 DNA sequence (1701 bp) was codon-optimized for P. pastoris, synthesized chemically by GenScript Biotech Co., Ltd. (Nanjing, China), and cloned into the pPIC9K plasmid (Thermo Fisher Scientific) to generate the pPIC9K-SynthCol1 construct by Gibson Assembly. This plasmid was amplified in Escherichia coli TOP10 cells. The gene sequences are detailed in Supplementary Table S1.

The expression vector pPIC9K-SynthCol1 was linearized with SalI restriction endonuclease (Takara, Dalian, China) and transformed into P. pastoris GS115 (Thermo Fisher Scientific) competent cells via electroporation. Positive transformants were selected on minimal dextrose (MD) medium (20 g/L glucose, 13.4 g/L YNB, 4 × 10–5 g/L biotin, 20 g/L agar). Transformants with high gene copy numbers were subsequently screened on YPD solid medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 20 g/L agar) containing Geneticin G418 at concentrations of 0.25, 0.5, and 1 mg/mL. Genomic DNA from selected transformants was extracted using the TIANamp Yeast DNA Kit (TIANGEN, Beijing, China) and analyzed by PCR with primers 5′AOX1 (5′-CGACTGGTTCCAATTGACAAGCT-3′) and 3′AOX1 (5′-GCAAATGGCATTCTGACATCCTCT-3′).

Shake flask expression and SDS-PAGE analysis

Small-scale SynthCol1 production was performed in 250 mL flasks at 28 °C with shaking at 220 rpm. The strain was inoculated into BMGY medium [10 g/L yeast extract, 20 g/L peptone, 100 mM potassium phosphate (pH 6.0), 13.4 g/L YNB, 4 × 10–5 g/L biotin, 1% (v/v) glycerol] and cultured until the optical density at 600 nm (OD600) reached 2–6. Cells were harvested by centrifugation and resuspended in BMMY medium [10 g/L yeast extract, 20 g/L peptone, 100 mM potassium phosphate (pH 6.0), 13.4 g/L YNB, 4 × 10–5 g/L biotin, 0.5% (v/v) methanol] to an initial OD600 of 1.0. Fermentation was terminated after 120 h of cultivation, with 0.5% (v/v) methanol supplemented every 24 h.

Protein samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4%–12% gradient gels (GenScript, Nanjing, China). Following electrophoresis, proteins were visualized by Coomassie Brilliant Blue G-250 staining. Gel images were captured using a multifunctional imaging system (Tanon, Shanghai, China), and band intensities were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Amino acid sequencing

The purified recombinant protein was separated by SDS-PAGE, and the gel band corresponding to the protein of SynthCol1 was excised and destained for LC–MS/MS analysis. The gel piece was dehydrated and then proteolytic digestion with trypsin, Glu-C or chymotrypsin as described (Wiśniewski et al. 2009). The eluted peptides were introduced online into a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) operated in positive ion mode. The raw data files were processed using BioPharma Finder software (Thermo Fisher Scientific).

Fermentation, from 5 to 500 L

Fed-batch fermentation was performed in a 5 L bioreactor (Baoxing Bio-Engineering, Shanghai, China). The yeast strain was inoculated into 100 mL of BMGY medium and cultivated until OD600 exceeded 10. The culture was then transferred into 2 L of basal salt medium (BSM) containing (w/v): 1.34% H₃PO₄, 0.046% CaSO₄·2H₂O, 0.91% K₂SO₄, 0.75% MgSO₄·7H₂O, 0.206% KOH, 4% glycerol, and 0.435% PTM1 trace salts. Fermentation parameters were maintained at: temperature 28 °C; pH 5.0 (controlled with ammonium hydroxide); agitation speed 600 rpm; aeration rate 3 vvm (air volume per medium volume per minute). A glycerol fed-batch phase continued until OD600 reached > 100. After a 1-h glycerol depletion period, methanol induction commenced at a constant feed rate of 18 mL/h/L. Samples were collected for expression analysis 24 h after induction. Fermentation was terminated following a 120-h induction period. Further, amplified the bioreactor to 50 L and 500 L (Gaoji Bio-Engineering, Shanghai, China). The culture medium conditions were consistent with the 5 L fermentation process, with airflow rate and methanol feed rate proportionally scaled up.

Purification of recombinant collagen

The fermentation broth was centrifuged at 7000 × g for 20 min at 4 °C. The supernatant was diluted to a conductivity of 5–10 mS/cm and loaded onto an SP Big Beads™ cation-exchange chromatography column (Smart-Lifesciences, Changzhou, China) pre-equilibrated with equilibration buffer (20 mM sodium phosphate, pH 6.5). After loading at pH 6.0–7.0, bound proteins were eluted using a linear NaCl gradient (0–1 M) in the same buffer. Fractions containing the target protein were pooled and concentrated via ultrafiltration using a 5-kDa molecular weight cut-off (MWCO) membrane cassette (Cobetter, Hangzhou, China). Protein concentration was quantified by BCA assay before storage at − 80 °C.

Size-exclusion chromatography (SEC) analysis

Desalted protein samples were adjusted to 1 mg/mL. Molecular weights were determined using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an AdvanceBio SEC 300 Å column (7.8 × 300 mm, 2.7 μm; Agilent). Separation was performed isocratically with mobile phase (50 mM sodium phosphate, 150 mM NaCl, pH 7.0) at 0.5 mL/min and 25 °C. Proteins were detected by UV absorbance at 210 nm. A calibration curve was generated using AdvanceBio SEC Standards (1.35–670 kDa; Agilent) under identical conditions. Sample molecular weights were determined by correlating retention times with the calibration curve.

Cell adhesion assay

Purified and desalted protein samples were diluted to 0.5 mg/mL in sterile phosphate-buffered saline (PBS, pH 7.4). Aliquots (100 µL) of SynthCol1 and bovine type I collagen (prepared in-house) were used to coat 96-well plates via overnight incubation at 4 °C. Wells were then blocked with 1% (w/v) bovine serum albumin (BSA) in PBS for 1 h at 37 °C. NIH/3T3 cells were harvested at 80–90% confluency, resuspended in serum-free medium at 1 × 105 cells/mL, and seeded (100 µL/well) onto protein-coated surfaces. After 4 h of adhesion at 37 °C under 5% CO₂, non-adherent cells were removed by three washes with pre-warmed PBS. Then each well was filled with complete medium containing 10% Hoechst 33,342, covered with aluminum foil, and incubate for 1 h at 37℃ in a 5% CO₂ incubator. Images were captured at random fields using a fluorescence microscope. Adhesion strength was quantified indirectly through metabolic activity measurement using a CCK-8 assay kit (Solarbio Science & Technology, Beijing, China). Fresh medium containing 10% (v/v) CCK-8 reagent was added (110 µL/well), incubated at 37 °C for 1 h, and absorbance measured at 450 nm using a microplate reader (BioTek Synergy H1).

Rheological analysis of collagen

The viscosity behavior of the collagen solution was characterized using a rotational rheometer (HR10, TA Instruments, USA) equipped with a Peltier temperature control system. A parallel plate geometry (25 mm diameter, PP40) with a fixed gap of 1.0 mm was employed for all measurements. To prevent dehydration during the thermal scan, a solvent trap was utilized, and a thin layer of low-viscosity silicone oil was applied around the edge of the sample. Prior to measurement, the rheometer plate and base were pre-cooled to 4 °C. The collagen solution (5 mg/mL) was loaded onto the plate on ice to avoid premature thermal denaturation. After loading, the sample was allowed to equilibrate at 4 °C for 3 min to ensure thermal homogeneity and to relax the structure. Subsequently, a temperature ramp test was performed, heating the sample from 4  to 37 °C at a constant rate of 2 °C/min. During the process, a constant shear rate of 1 s⁻1 was applied, and the apparent viscosity (η) was recorded in real-time.

Reconstructed human skin model assay

A full-thickness reconstructed human skin model (T-Skin™) assessed ultraviolet radiation a (UVA)-protective effects (Bataillon et al. 2019). Models were allocated to four groups: (1) untreated control (NC): 22.5 µL PBS with visible light; (2) UVA-damaged (UVA): PBS + 2.5 mJ/cm2 UVA; (3) positive control (UVA + Vc): L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate + UVA; and (4) test group (UVA + SynthCol1): SynthCol1 (0.5 mg/mL in PBS) + UVA. After 30-min incubation, models were PBS-rinsed and cultured in fresh medium for 48 h (37 °C/5% CO₂).

Conditioned media underwent IL-1α quantification by enzyme-linked immunosorbent assay (ELISA). Tissues were bisected: one half flash-frozen in optimal cutting temperature (OCT) compound for immunofluorescence (IF), the other fixed in 4% paraformaldehyde (PFA) for paraffin sectioning. IF staining employed primary antibodies (4 °C, overnight) followed by Alexa Fluor-conjugated secondaries (37 °C, 1–2 h) with DAPI counterstaining. Paraffin sections were hematoxylin–eosin (H&E) stained. Ceramide NP (N-stearoyl phytosphingosine) was quantified via LC–MS/MS using isopropanol extracts and multiple reaction monitoring (MRM). Antibody specifications and reagent sources are detailed in Supplementary Table S2.

Statistical analysis

All experiments were performed in three independent biological replicates. Raw data were processed in Microsoft Excel and analyzed statistically using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Continuous variables are expressed as mean ± standard deviation (SD). Statistical comparisons were performed as follows: two-group comparisons via Student’s t-test or multi-group comparisons via one-way ANOVA with Tukey’s multiple comparison test. Statistical significance was defined as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

Construction and expression of SynthCol1

We designed a recombinant humanized collagen, named SynthCol1, based on a 9-mer repeat sequence (G674–A737) from the human type I collagen α1 chain. Its theoretical molecular weight is 51.04 kDa (Fig. 2 and Fig. S1). SynthCol1 contains specific integrin-binding motifs (GFPGER and GMPGER) for α2β1 and α1β1 integrins but has reduced glycosylation to improve functional specificity (Sweeney et al. 2008). Protein expression was confirmed in multiple clones (Fig. 3A, B). On SDS-PAGE, SynthCol1 migrated at ~ 74 kDa, about 40% larger than predicted. This discrepancy may result from P. pastoris post-translational modifications (e.g., glycosylation) and/or reduced SDS-binding capacity of the recombinant collagen, causing anomalous electrophoretic migration (Song et al. 2024). After amino acid sequencing, the obtained protein showed a 100% match with the designed sequence (Fig. 4).

Fig. 2.

Fig. 2

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Diagram of the sequence design for the collagen type I α1 peptide fragment. The segment G674-G736 (63 aa) of the collagen type I α1 chain was selected and tandemly repeated to form a nonamer

Fig. 3.

Fig. 3

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Expression analysis of recombinant collagen in shake flasks, 5 L and 500 L bioreactor. A SDS-PAGE analysis of protein expression in 10 different transformants (Clone 1—10). BSA was used as a loading control. G, GS115 B SDS-PAGE analysis of protein expression at different time points post-induction in shake flasks. C Cell growth analysis at different time points post-induction. D Yield of recombinant collagen at different time points post-induction in a 5-L bioreactor. E SDS-PAGE analysis of protein expression during fermentation in a 5 L bioreactor. Lanes 3 and 4: 48 h post-induction; lanes 5 and 6: 72 h post-induction; lanes 7 and 8: 96 h post-induction; lanes 9 and 10: 120 h post-induction. Samples in lanes 3, 5, 7, and 9 represent 20-fold dilutions of the fermentation supernatant; samples in lanes 4, 6, 8, and 10 represent 40-fold dilutions. F Yield of recombinant collagen at different time points post-induction in a 500 L bioreactor

Fig. 4.

Fig. 4

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Amino acid sequence of SynthCol1 (1–567aa). Solid lines with arrows indicate the trypsin, Glu-C and chymotrypsin digest fragments analyzed in the study with a total sequence coverage of 100%

High density fermentation

Recombinant strain was selected for 5 L scale fermentation. Culture supernatants sampled at 48, 72, 96, and 120 h post-induction were analyzed by SDS-PAGE, revealing SynthCol1's expression profile (Fig. 3C-E). Densitometric quantification of Coomassie-stained gels showed SynthCol1 reached 7.0 g/L by 96 h (Fig. 3E). Notably, extending fermentation beyond 96 h yielded no significant increase in recombinant protein production, establishing the feasibility of large-scale SynthCol1 manufacturing. The fermentation process was successfully scaled up to a 500 L bioreactor level. As depicted in Fig. 3F, the expression profile of SynthCol1 from 48 to 120 h post-induction mirrored the trend observed in the 5 L fermentation. Specifically, the protein concentration reached a plateau phase at 72 h, achieving a yield of 15.3 g/L. These results demonstrate the potential of SynthCol1 for the large-scale production of recombinant collagen.

Purification of SynthCol1

To obtain high-purity recombinant collagen, fermentation broth was purified. Owing to SynthCol1’s theoretical pI (10.47), the clarified supernatant was diluted and loaded onto a cation-exchange column. Bound proteins were eluted via stepwise NaCl gradient. SDS-PAGE analysis of elution fractions (Fig. 5A) revealed most contaminants eluted at 0.25 M NaCl, while SynthCol1 was efficiently recovered at 0.5 M NaCl. Densitometric analysis of Coomassie-stained gels indicated ~ 95% purity for SynthCol1 in the 0.5 M NaCl fraction. These results demonstrate effective purification of SynthCol1 by cation-exchange chromatography.

Fig. 5.

Fig. 5

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Purification of recombinant collagen SynthCol1. A SDS-PAGE analysis illustrating the purification process. Lane 1: Crude fermentation supernatant. Lane 2: Flow-through fraction. Lanes 3—9: Elution fractions collected using step gradients of increasing NaCl concentration: lane 3 (0.05 M), lane 4 (0.1 M), lane 5 (0.2 M), lane 6 (0.25 M), lanes 7 and 8 (0.5 M), lane 9 (1 M NaCl). The target recombinant collagen band is indicated (arrow). B Size exclusion chromatography (SEC) analysis of purified recombinant collagen SynthCol1. SEC profile of molecular weight standards and purified SynthCol1

Purified, desalted protein underwent SEC analysis. The chromatographic profile (Fig. 5B) indicated ~ 95% purity, consistent with SDS-PAGE results. Notably, comparison with molecular mass standards revealed an apparent native molecular mass of ~ 316 kDa—significantly exceeding both the theoretical monomeric mass (51 kDa) and the SDS-PAGE-derived apparent mass (74 kDa). These findings suggest SynthCol1 may adopt a tetrameric conformation under native conditions.

The cell bioactivity properties of SynthCol1

As a critical ECM protein, collagen establishes microenvironments that support cell adhesion and proliferation essential for tissue growth. To evaluate SynthCol1’s bioactivity, NIH/3T3 cell adhesion assays were performed. As shown in Fig. 6, cell adhesion was minimal in the PBS control. Both SynthCol1 and bovine type I collagen markedly enhanced cell adhesion relative to PBS (p < 0.01), and SynthCol1 outperformed bovine collagen by 25.3% (p < 0.05), demonstrating its superior performance in supporting cell adhesion.

Fig. 6.

Fig. 6

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Cell adhesion assay of recombinant collagen SynthCol1 on NIH/3T3 fibroblasts. A Negative control: PBS-coated surface. B Surface coated with purified SynthCol1. C Positive control: Surface coated with bovine type I collagen. D Quantification of cell adhesion rate (%) (mean ± SD; n ≥ 3). Error bars represent SD (n = 3). One-way ANOVA p < 0.05, Tukey’s multiple comparison test. Scale bars: 50 μm (A–C)

Efficacy assessment in a reconstructed human skin model

Collagen, as the primary dermal structural protein, forms a fibrous network critical for maintaining skin elasticity, structural integrity, hydration retention, antioxidant activity, and barrier function (Amirrah et al. 2022). To evaluate SynthCol1’s tissue-repair efficacy, we employed a UVA-damaged reconstructed human skin model (Fig. 7 and Fig. S2). Compared to untreated controls (NC), UVA-damaged models showed significantly reduced fluorescence intensity for Collagen IV, VII, XVII, Loricrin, AQP3, and Keratin (p < 0.05). Positive control treatment significantly upregulated all biomarkers versus damaged models (p < 0.05), confirming successful damage induction. Critically, SynthCol1 also significantly increased all six biomarkers versus damaged models (p < 0.05) (Fig. 7 and Fig. S2).

Fig. 7.

Fig. 7

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Topical photoprotective effects of recombinant collagen SynthCol1 in a 3D full-thickness skin model (T-Skin™). Skin models received topical pre-treatment with: Untreated Control (NC): PBS + visible light exposure. Model Group (UVA): PBS + UVA irradiation (X J/cm2). Positive Control (UVA + Vc): 200 μM vitamin C derivative + UVA irradiation. Test Group (UVA + SynthCol1): SynthCol1 + UVA irradiation. A-G Relative fluorescence intensity of immunofluorescence staining of Collagen IV (A), Collagen VII (B), Collagen XVII (C), Loricrin (D), Aquaporin 3 (AQP3) (E), Cytokeratin (F), and reactive oxygen species (ROS) (G). H Interleukin-1α (IL-1α) content. I Ceramide NP content. Data = mean ± SD (n = 3). #p < 0.05 vs. NC group; *p < 0.05 vs. UVA group (t-test)

UVA damage significantly elevated IL-1α and ROS levels versus NC (p < 0.05). Positive control treatment reduced both markers (p < 0.05), further validating the model. SynthCol1 similarly decreased IL-1α and ROS versus damaged models (p < 0.05) (Fig. 7G, H). Ceramide NP content decreased post-UVA exposure but increased with both positive control and SynthCol1 treatments (Fig. 7I). In addition, histologically, UVA-damaged models exhibited stratum corneum disorganization, epidermal thickening, and dermo-epidermal junction abnormalities—all ameliorated by positive control and SynthCol1 treatments (Fig. 8). These results demonstrate SynthCol1 which could promote skin repair in the UVA-damaged skin model.

Fig. 8.

Fig. 8

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Histological assessment of photoprotective effects by recombinant collagen SynthCol1 in a 3D full-thickness skin model. Skin models received topical pre-treatment followed by light exposure: A Untreated control (NC): PBS + visible light exposure B Model group (UVA): PBS + UVA irradiation (X J/cm2). C Positive control (UVA + VC): 200 μM vitamin C derivative + UVA irradiation. D Test group (UVA + SynthCol1): SynthCol1 + UVA irradiation, Representative hematoxylin and eosin (H&E) stained sections

Discussion

Collagen is the most abundant protein in mammals. The amino acid sequences of collagen from different species (e.g., bovine, porcine, fish) share high homology with human collagen. Telopeptides are often the primary source of immunogenicity; thus, the recombinant humanized collagen which derived from the triple-helix region of human collagen possesses extremely low immunogenicity. Besides, the gene sequence is directly derived from humans, resulting in a protein structure that is nearly identical to endogenous human collagen. These factors collectively contribute to the low immunogenicity of recombinant collagen. This characteristic is the cornerstone of its widespread application within the human internal environment (from oral ingestion to injection and implantation) and represents its greatest advantage over other xenogeneic protein materials.

Here, we engineered a humanized type I collagen derivative SynthCol1 and established a high-yield expression system in P. pastoris GS115. Through optimized fermentation and purification, we obtained recombinant SynthCol1 with ~ 95% purity. As early as 2004, Yao et al. (2004) demonstrated that using a sequence derived from type I collagen α1 chain G950–V961 combined with GER (Gly-Glu-Arg) significantly enhanced cell adhesion. The protein design strategy of utilizing human-derived collagen sequence fragments with repeated tandem sequences is also a primary design approach for current commercial recombinant collagens. Ramshaw et al. (2025) cited numerous cases, with the most frequently used sequences being various repetitive fragments derived from human type III collagen. In this study, for the first time, a triple-helical fragment (G674–A736) was selected, which features a near-native isoelectric point (pI), high hydrophilicity, critical integrin-binding motifs (GFPGER/GMPGER for α2β1/α1β1 binding) (Knight et al. 1998), and reduced glycosylation sites. The designed sequence in this study contains 18 GER regions, which increase the domains for protein binding to integrin receptors. According to cell adhesion experiments (Fig. 6), this design shows a 25% improvement compared to commonly used bovine collagen.

Currently, the predominant expression systems for therapeutic proteins are E. coli and P. pastoris (Krishna et al. 2025). The yeast system offers distinct advantages for clinical applications: endotoxin-free expression and efficient secretory production that simplifies purification from culture supernatants (Werten et al. 2019). Moreover, due to moderate molecular weight, codon optimization and stable sequence design followed by systematic process optimization and scaled-up production, the final yield of SynthCol1 reached 15.3 g/L in large-scale fermentation, achieving a high level of expression. Pan et al. (2023) summarized the yields of recombinant collagen across different expression systems, reporting the maximum yields of 13.2 g/L in E. coli and 4.7 g/L in yeast. In comparison, Li et al. (2021) demonstrated that the maximum expression level in a similar yeast system could reach 8 g/L for recombinant type III collagen. Notably, the SynthCol1 yield of 15.3 g/L achieved in this study substantially surpasses these previously reported yields. This study not only establishes a practical framework for developing functional recombinant collagen-based materials and scalable manufacturing processes, but also provides foundational insights that support the expanded application of recombinant collagen in biomedicine and beyond. Thus, these engineered designs concurrently address the dual challenges of soluble expression and production yield in recombinant collagen platforms.

Current microbial expression systems for recombinant collagen face challenges due to the characteristic Gly-X–Y repeats and high molecular weight of collagen, which hinder the expression of full-length native collagen in E. coli and yeast (Yan et al. 2025). A key strategy involves producing recombinant humanized collagen with engineered repeats or functional domains derived from natural collagens. Successful production of functional recombinant collagen requires both rational selection of collagen fragments and scalable expression platforms, because individual collagen segments mediate distinct biological functions. For example, E. coli-expressed 18 × repeats of type III collagen (Gly300–Asp329) demonstrated biocompatibility and cell adhesion properties (Yan et al. 2024). Wang et al. (2025) used 16 repeats of the type III collagen G483-R509 fragment, which demonstrated good cell adhesion and migration functions and can promote the regeneration of skin collagen. SynthCol1 outperformed bovine type I collagen in cell adhesion, showing higher adherent cell density (Fig. 6). This functional superiority may stem from SynthCol1’s enriched integrin recognition sites—a consequence of its optimized amino acid sequence design. On the other hand, the formation of polymers may play an important role during the process of cell adhesion. The self-assembling property is also found in other human-like collagens (Xing et al. 2012), which indicates that this might be a common characteristic of recombinant collagens. Moreover, SynthCol1 exhibited distinct temperature-dependent rheological properties. At high concentrations (~ 5.0 mg/mL), the solution formed a gel-like state at low temperature (4 °C), demonstrating reversible thermoresponsive behavior (Fig. S3). This reversible gelation behavior indicates its potential for use in temperature-modulated biomaterial applications.

Functional characterization revealed that SynthCol1 promoted cellular bioactivity and demonstrated significant skin-repair efficacy in a reconstructed human skin model, confirming its potential as a biomedical material for wound healing. Collagen constitutes the predominant component of dermal proteins, forming a reticular fiber network that provides structural support, elasticity, and hydration capacity to the skin (Zhao et al. 2025). With advancing age, the rate of collagen synthesis declines relative to its degradation, leading to a net loss of dermal collagen. Concurrently, UVA radiation penetrates the dermis, induces the activation of matrix metalloproteinases (MMPs)—notably MMP-1 and MMP-3—and accelerates collagen breakdown (Feng et al. 2024; Nan et al. 2025). This progressive loss of structural integrity compromises skin firmness and contributes to wrinkle formation, thereby accelerating the visible aging process (Fig. 9) (Bellavite and Imbriano 2025; Salamito et al. 2025). Owing to its exceptional biocompatibility and bioactivity, recombinant collagen is widely used in skincare biomaterials (Chen et al. 2024). Research on recombinant collagen’s reparative effects typically utilizes photoaged skin models, where types I and III demonstrate efficacy in restoring photodamaged tissue (Liu et al. 2024; Wang et al. 2022). Here, using a UVA-injured reconstructed 3D skin model, we established that SynthCol1 orchestrates repair through three synergistic mechanisms (Fig. 9): (1) Structural reconstitution via upregulation of basement membrane components (collagens IV, VII, XV; loricrin) and hydration regulator AQP3; (2) Barrier regeneration evidenced by enhanced ceramide NP synthesis and normalized stratum corneum ultrastructure (Fig. 7I and Fig. 8); and (3) Microenvironment modulation through suppression of pro-inflammatory IL-1α and oxidative stress marker ROS (Fig. 7G–H). Notably, SynthCol1 demonstrated a 25% increase in cell adhesion compared to natural type I collagen (Fig. 6), coupled with superior basement membrane restoration—demonstrating the biofunctional advantage conferred by its engineered 9-mer integrin-binding domains.

Fig. 9.

Fig. 9

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Schematic illustration of the multi-target mechanisms by which SynthCol1 protects skin cells against UVA-induced damage. SynthCol1 acts through three synergistic pathways: (1) structural reconstitution of the basement membrane via upregulation of Col IV, VII, XVII, Loricrin, and AQP3; (2) barrier regeneration through increased ceramide NP synthesis and normalization of stratum corneum architecture; and (3) modulation of the inflammatory microenvironment via suppression of IL-1α and ROS. MMPs, matrix metalloproteinases

Conclusions

This study established a high-yield (15.3 g/L in a 500 L bioreactor) production of the recombinant humanized collagen SynthCol1 in P. pastoris GS115. Its engineered 9-mer repeat domain, which incorporates specific integrin-binding motifs (GFPGER/GMPGER), was demonstrated to enhance cellular adhesion significantly over natural collagen. Furthermore, in full-thickness skin models, SynthCol1 demonstrated robust photoprotection and reparative efficacy, effectively restoring UVA-induced damage. However, due to limitations in current production technologies, the recombinant collagens lack a complete or correct triple-helix structure. This structural discrepancy may compromise their bioactivity, resulting in diminished efficacy. Overall, with scalable microbial production and functional fidelity, SynthCol1 has the potential to serve as a versatile platform biomaterial for medical-grade devices, advanced cosmeceuticals, and targeted drug delivery systems.

Supplementary Information

Below is the link to the electronic supplementary material.

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(DOCX 315 KB)

ESM 2 (793.3KB, pdf)

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Author contribution

Z. Zhang and H. Yu performed project conception and experiment design; Z. Chen, H. Du, J. Li, W. Xia and Y. Wang conducted the experiments and analyzed the data; J. Li and Z. Zhang wrote the original manuscript; H. Yu thoroughly revised and finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was sponsored by Zhejiang Chumsun Biological Products Co., Ltd.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Informed consent

Not applicable.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher’s Note

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Supplementary Materials

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Data Availability Statement

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