Firstly, three promising lipases—CALB (from Candida antarctica), GGL (from Galactomyces geotrichum Y05), and LipZ01 (from oil-contaminated soil)—were selected for their distinct advantages in activity and stability (Table 1).

Table 1 Three lipase used in this project.

After the company synthesized three lipases that had been optimized for codons, we inserted them into a P. pastoris expression vector pPICZαA, which includes the methanol-inducible AOX1 promoter, the α-factor secretion signal, and a C-terminal His-tag, followed by the AOX1 terminator. The Zeocin resistance gene enables selection in transformants (Figure 2).

Figure 2 Plasmid Design diagram in cycle 1.

We firstly amplified the pPICZαA and three lipase gene, followed by agarose gel electrophoresis. The results showed the successful amplification of pPICZαA vector (3476 bp), CALB (954 bp), GGL (1692 bp), and LipZ01 (1428 bp)—with bands corresponding to their expected sizes under UV illumination (Figure 3).

Figure 3 The amplification results of the DNA fragments.

Following the amplification of the lipase genes and plasmid vector, the transformation plates confirmed efficient plasmid introduction into E. coli DH5α competent cell (Figure 4A). Colony PCR analysis further validated the presence of the correct insert sizes, with distinct bands corresponding to CALB (1443 bp), GGL (2179 bp), and LipZ01 (1912 bp) observed across multiple clones (Figure 4B). Sequencing results confirmed the precise integration of each expression cassette (Figure 4C). These results collectively demonstrate the accurate assembly of all three plasmids.

Figure 4 Construction result of three plasmids.

(A) Transformation plates. (B) Colony PCR result. (C)Sequencing result.

The recombinant plasmids pPICZαA-CALB, pPICZαA-GGL, and pPICZαA-LipZ01 were successfully extracted and linearized by SacI enzyme prior to transformation, as confirmed by agarose gel electrophoresis (Figure 5A). Linearizing the plasmid is a critical step because it exposes homologous ends that facilitate targeted integration into the P. pastoris genome via homologous recombination, significantly increasing transformation efficiency compared to circular plasmids.

Next, we used the lithium chloride transformation method to transfer the linearized plasmid into P. pastoris. This method can generate active yeast cells with permeable cell membranes, enabling the linearized plasmid to smoothly enter the cells. For the pPICZαA vector, the DNA that has been linearized will specifically integrate into the yeast genome through homologous recombination at the AOX1 locus in the yeast genome. This process ensures that the target gene can be stably expressed under the regulation of the inducible AOX1 promoter.

As shown in Figure 5B, the linearized plasmids were individually transformed into P. pastoris strains, with successful transformants growing on selective YPD plate. The yeast colony PCR results further verified the correct genomic integration of each lipase gene, showing specific amplification products of 954 bp (CALB), 1692 bp (GGL), and 1428 bp (LipZ01) (Figure 5C). These results collectively demonstrate the successful construction of three recombinant P. pastoris strains capable of expressing the target lipases, providing a solid foundation for subsequent protein expression and functional studies.

Figure 5 Construction result of P. pastoris containing lipase.

(A) Plasmid Linearization result. (B) Transformation plates. (C) Yeast colony PCR result.

• Lipase secretion and purification

Subsequently, we inoculated the yeast transformants containing the CALB gene and activated them in the BMGY medium. Then, using the BMMY medium, we began to induce the secretion of CALB. Figure 6 presented the SDS-PAGE analysis of recombinant lipase secretion in P. pastoris after 72-hour fermentation. A distinct protein band at approximately 44 kDa, corresponding to the expected molecular weight of CALB (43.8 kDa), was clearly observed in the culture supernatant of all three tested strains at 48 h and 72 h, confirming successful extracellular secretion (Figure 6A). In contrast, no visible bands were detected at the predicted molecular weights for GGL (63.1 kDa) or LipZ01 (51.1 kDa) in any strain or time point (Figure 6B-C).

The successful secretion of CALB can be attributed to its compatibility with the α-factor secretion signal and efficient folding in the secretory pathway. The absence of GGL and LipZ01 bands suggests potential issues such as inefficient signal peptide recognition, protein misfolding leading to endoplasmic reticulum retention, or intracellular degradation. These results indicate that while the expression system supports CALB secretion, further optimization of the secretion signal or co-expression of chaperones may be necessary for efficient production of GGL and LipZ01.

Figure 6 SDS-PAGE results of (A) CALB, (B)GGL, and (C) LipZ01 secretion.

We found that the purity of the lipase secreted into the supernatant was not high enough. To avoid affecting the subsequent quantitative analysis of the lipase and the enzyme activity test, we carried out nickel column purification on it. Since the crude supernatant contained impurities that could interfere with accurate quantification and activity assays, we concentrated it using an ultrafiltration device and exchanged the buffer to PBS (pH 7.4) to optimize binding efficiency. Following washing and elution steps, a distinct band at approximately 44 kDa, corresponding to CALB, was clearly observed in the elution fractions (Figure 7, E1 and E2), confirming successful purification of the target protein to high purity. In contrast, no specific bands were detected at the expected molecular weights for GGL (63.1 kDa) or LipZ01 (51.1 kDa) in their respective elution lanes, despite applying the same concentration and purification procedure. This result is consistent with the earlier secretion data and further confirms that these two lipases were not successfully secreted in a soluble, His-tagged form, or were present at levels below the detection limit.

Figure 7 Purification results of three lipase.

• Quantitative analysis of lipase

Next, we use the BCA method to determine the concentration of the lipase. The BCA method relies on the reduction of Cu²⁺ to Cu⁺ by peptide bonds in an alkaline medium, with the resulting Cu⁺ ions forming a purple complex with BCA that can be measured at 562 nm. A series of bovine serum albumin (BSA) standards (25-500 μg/mL) were used to establish a linear calibration curve, which exhibited excellent linearity with the equation Y = 0.0009982*X + 0.1326 and a coefficient of determination (R²) of 0.9934 (Figure 8). The high R² value indicateed a strong correlation between absorbance and protein concentration, validating the reliability of this standard curve for subsequent quantification of lipase samples.

Figure 8 The BSA standard curve results of the BCA method

This standardized protocol was applied to determine the concentration of purified lipases in the culture supernatant, enabling accurate comparison of expression levels among CALB, GGL, and LipZ01 under different experimental conditions. The BCA assay results quantitatively confirmed the successful secretion of recombinant CALB, which achieved a concentration of 68.21 μg/mL, significantly higher than the negative control (0.96 μg/mL). In contrast, GGL and LipZ01 showed only minimal concentrations (4.89 and 1.61 μg/mL, respectively), consistent with earlier SDS-PAGE and purification data indicating their secretion failure (Figure 9). These results validated the effectiveness of the expression and purification system for CALB, while underscoring the need for further optimization to enable detectable secretion of GGL and LipZ01.

Figure 9 The quantitative results of the concentration of purified lipase.

• Enzyme activity test of lipase

We determined the activity of the lipase using the p-nitrophenol method. This assay is based on the hydrolysis of p-Nitrophenyl esters by lipase, releasing yellow-colored 4-Nitrophenol (4-NP) that absorbs at 450 nm. A series of 4-NP standards (0–0.1 mmol/L) were prepared, and their absorbance values were measured to establish a linear calibration curve. The fitted equation Y = 5.924x + 0.01800 exhibited excellent linearity (R² = 0.9857), confirming a strong correlation between absorbance and 4-NP concentration (Figure 10). This reliable standard curve enables accurate quantification of lipase activity in subsequent enzymatic assays.

Figure 10 Standard curve of 4-Nitrophenol

Finally, the lipase activity of the three recombinant strains was measured. The CALB-expressing strain demonstrated the highest enzymatic activity at 28.59 ± 0.37 U/mL, which was approximately 3.5-fold higher than the activities of GGL (8.15 ± 0.14 U/mL) and LipZ01 (8.41 ± 0.09 U/mL). Statistical analysis (​​​​p < 0.0001) confirmed that CALB activity was significantly higher than both GGL and LipZ01, while no significant difference (ns) was observed between GGL and LipZ01. The CALB-expressing strain showed significantly higher activity than the negative control (pPICZαA, 5.99 ± 0.70 U/mL), validating the successful expression of it (Figure 11). These results indicate that CALB exhibits superior catalytic efficiency under the tested conditions.

Figure 11 Lipase activity test result.

During this round of DBTL cycle, we found that CLAB was able to successfully be secreted from the cells of P. pastoris into the supernatant of the culture medium. The enzymatic activity revealed that only CALB was successfully secreted extracellularly, demonstrating measurable enzymatic activity (reaching 28.59 ± 0.37 U/mL). Therefore, in the subsequent optimization phase, we will focus on CALB, specifically targeting the enhancement of its expression and secretion efficiency through promoter and signal peptide optimization.

However, GGL and LipZ01 did not show secretion. Through literature research, we summarized the possible reasons and solutions, and can optimize this expression system in future studies (Table 2).

Table 2 Reasons and solutions for the failure of GGL and LipZ01 secretion.

Achieving efficient expression of lipases in P. pastoris requires careful selection and optimization of promoters, as they directly govern the efficiency and intensity of target gene transcription initiation [9]. The pPIC9K vector is a commonly used tool in the P. pastoris expression system, which incorporates the potent methanol-inducible AOX1 promoter, and utilizes the α-factor signal peptide to direct the secretory expression of recombinant proteins. This vector is marked for prokaryotic screening in E. coli with ampicillin (Amp) and kanamycin (Kan) resistance, while screening in P. pastoris utilizes the HIS4 marker. (Figure 12).

Figure 12 Plasmid Design diagram in cycle 2a.

To systematically evaluate the impact of different promoters on lipase expression levels, we replaced the original AOX1 promoter with three candidate promoters—AOX713, FDH1, and FLD1 (Table 3). Through this strategy, we aimed to identify the optimal promoter capable of maximizing lipase transcription levels and final yield, laying the foundation for constructing an efficient P. pastoris. cell factory.

Table 3 Promoters and signal peptides used in this project.

Firstly, we performed PCR amplification on each promoter, the CALB gene, and the plasmid backbone. The results of agarose gel electrophoresis showed that all target fragments were successfully amplified. The promoter fragments (AOX713, FDH1, FLD1) and the Kan resistance gene fragment all presented clear bands, with sizes consistent with expectations (approximately 4366 bp, 4244 bp, and 4699 bp), and the CALB gene fragment (996 bp) was also successfully amplified (Figure 13). After gel excision, these fragments were used for the subsequent construction of the recombinant vector.

Figure 13 The amplification results of the DNA fragments.

Following the amplification of the different promoters, CALB gene, and plasmid vector, we combined them through homologous recombination. the transformation plates confirmed efficient plasmid introduction into E. coli DH5α competent cell. Colony PCR analysis demonstrated distinct bands of expected sizes: 2188 bp for AOX713-CALB, 2154 bp for FDH1-CALB, and 2169 bp for FLD1-CALB. Sequencing results further verified the precise integration of each expression cassette with correct promoter-signal peptide-CALB organization. These results collectively confirmed the accurate assembly of three plasmid constructs, providing a reliable foundation for subsequent yeast transformation and protein expression studies (Figure 14).

Figure 14 The colony PCR and sequencing results of plasmids.

The recombinant plasmids were then successfully extracted and linearized by SalI enzyme before transformation, as confirmed by agarose gel electrophoresis. Each plasmid displayed a single, clear band after single enzyme digestion, corresponding to the expected size of the linearized vector (Figure 15A). After enzyme digestion, the linearized plasmids were transformed into P. pastoris competent cell, respectively. Figure 15B showed that transformants of P. pastoris growing on selective plates for six different constructs. Further colony PCR analysis confirmed genomic integration, with distinct bands observed at the expected size of 954 bp for all constructs when compared to the DNA ladder (Figure 15C). These results demonstrated the successful integration of the CALB gene expression cassettes into the yeast genome, validating the effectiveness of the transformation procedure and providing a reliable foundation for subsequent protein expression studies.

Figure 15 Construction result of P. pastoris containing lipase.

(A) The linearization results of the plasmids. (B) Transformation plates. (C) Yeast colony PCR results.

• Lipase secretion and quantitative analysis

Next, we conducted fermentation on each of these three strains. The SDS-PAGE analysis revealed distinct protein bands at approximately 43.8 kDa in the 72-hour samples, corresponding to the expected molecular weight of CALB, while no such bands were observed at 0 hours or in the negative control (pPIC9K) (Figure 16A). Quantitative analysis using the BCA method demonstrated significant differences in CALB secretion levels among the promoters. The FDH1 promoter yielded the highest concentration (93.82 ± 4.16 μg/mL), followed by AOX713 (86.04 ± 8.53 μg/mL), AOX1 (68.21 ± 4.94 μg/mL), and FLD1 (61.25 ± 3.32 μg/mL). Statistical analysis indicated that FDH1-driven expression was significantly higher than both AOX1 and FLD1 (​​P ≤ 0.01) (Figure 16B).

Figure 16 (A)SDS-PAGE verification and (B) quantification of CALB under different promoter regulation.

• Enzyme activity test of lipase

Finally, we determined the lipase activity of the recombinant P. pastoris strains expressing CALB under different promoter controls using the p-nitrophenol method. The enzyme activity produced by the FDH1 promoter was the highest, at 31.68 ± 0.24 U/mL, significantly higher than that obtained from the AOX1 (28.59 ± 0.37 U/mL) and FLD1 (25.88 ± 1.12 U/mL) promoters (P ≤ 0.01). It is noteworthy that the activity levels driven by the AOX713 (25.37 ± 0.85 U/mL) and FLD1 promoters were even lower than that of the commonly used AOX1 promoter (25.37 ± 0.85 U/mL). The activity of all the recombinant strains was significantly higher than that of the negative control (pPIC9K empty vector, 0.96 ± 0.67 U/mL). These results clearly indicated that the choice of promoter has a crucial impact on the production of lipase. Under the tested conditions, the FDH1 promoter was proven to be the most effective choice for achieving high CALB activity (Figure 17).

Figure 17 Lipase activity test result.

In this cycle, we systematically evaluated the performance of three promoters (AOX713, FDH1, FLD1) for the expression of CALB in P. pastoris and enzyme activity. Therefore, this promoter will be included in the final round of plasmid construction to maximize the yield of the recombinant CALB.

In this round of experiments, we realized that at the transcriptional level, the strength of the promoter is the main determinant of the production of the recombinant protein, as it directly controls the amount of the synthesized mRNA [14]. Compared with the traditional AOX1 promoter and other tested candidate promoters, under the fermentation conditions adopted, the FDH1 promoter is likely to exhibit stronger or more stable transcriptional activity, thereby promoting the accumulation of higher levels of CALB mRNA and ultimately leading to an increase in protein synthesis and secretion.

In the P. pastoris expression system, the signal peptide is crucial for guiding the secretion of heterologous lipases to the extracellular space. It not only simplifies the downstream purification process but also significantly increases the yield of the target protein [15]. In P. pastoris, the secretion of proteins by signal peptides begins when the proteins are recognized by the signal recognition particles and the ribosome complexes are directed to the endoplasmic reticulum. The newly synthesized peptide chains enter the endoplasmic reticulum lumen through the translocation site, the signal peptide is removed, and the protein undergoes folding and processing in the Golgi apparatus before being released into the extracellular space through exocytosis [16]. However, the compatibility of the signal peptides with different exogenous proteins varies, and its cutting efficiency may differ depending on the N-terminal amino acid sequence characteristics of the target protein, resulting in suboptimal secretion efficiency for some proteins or the presence of additional amino acids at the N-terminus.

In this cycle, we obtained three hybrid signal peptides through literature research. These signal peptides were all modified from the α-factor of S. cerevisiae, which is the most commonly used signal sequence in P. pastoris for recombinant protein secretion. These signal precursor regions consist of two parts: the pro-peptide located at the N-terminal of the precursor region, which functions as a signal peptide; and the precursor peptide (pre-peptide) located at the C-terminal, which plays a role in promoting protein secretion (Table 4).

Table 4 Signal peptides used in this project.

To optimize the secretion efficiency of lipase in P. pastoris, this study replaced the original α-factor signal peptide of the pPIC9K vector with engineered ones. We introduced three highly efficient secretion signal peptides that have been verified in the literature: 0030α-factor, SWP1-α-factor, and KRE1-α-factor, which retained the AOX1 promoter on the pPIC9K vector (Figure 18).

Figure 18 Plasmid Design diagram in cycle 2b.

Firstly, we performed PCR amplification on each signal peptide, the CALB gene, and the plasmid backbone. The agarose gel electrophoresis results demonstrated successful amplification of all target DNA fragments with the expected length: the plasmid backbone fragments (Amp-0030α, Amp-SWP1α, Amp-KRE1α) are approximately 4.2 kb, the signal peptide-CALB fusion fragments (0030α-CALB, SWP1α-CALB, KRE1α-CALB) are approximately 1.0 kb, and the Kanamycin resistance gene (Kan) is approximately 4.7 kb (Figure 19).

Figure 19 The amplification results of the DNA fragments.

Following the amplification of the different signal peptides, CALB gene, and plasmid vector, we combined them through homologous recombination. the transformation plates confirmed efficient plasmid introduction into E. coli DH5α.

Colony PCR analysis demonstrated distinct bands of expected sizes: 0030-α-CALB (2426 bp), SWP1-α-CALB (2411 bp), and KRE1-α-CALB (2411 bp). Sequencing results further verified the precise integration of each expression cassette with correct promoter-signal peptide-CALB organization. These results collectively confirmed the accurate assembly of three plasmid constructs, providing a reliable foundation for subsequent yeast transformation and protein expression studies (Figure 20).

Figure 20 The colony PCR and sequencing results of plasmids.

The recombinant plasmids were then successfully extracted and linearized by SalI enzyme before transformation, as confirmed by agarose gel electrophoresis. Each plasmid displayed a single, clear band after single enzyme digestion, corresponding to the expected size of the linearized vector. These linearized plasmids were then transferred into the competent cells of P. pastoris. After 48 hours of cultivation, transformants of the yeast grew on the plates. We conducted PCR on yeast colonies, the results showed the genomic integration, displaying specific bands of the expected size (~954 bp) for all three signal peptide variants (0030α, SWP1α, KRE1α). These results collectively verified the accurate construction of the engineered yeast strains (Figure 21).

Figure 21 Construction result of P. pastoris containing lipase.

(A) The linearization results of the plasmids. (B) Transformation plates. (C) Yeast colony PCR results.

• Lipase secretion and quantitative analysis

Next, we conducted fermentation on each of these three strains. The SDS-PAGE results showed distinct protein bands at approximately 43.8 kDa in the 72-hour samples for all engineered strains, corresponding to secreted CALB, while absent in the negative control (pPIC9K) and pre-induction samples. Quantitative analysis revealed that the 0030-α signal peptide yielded the highest CALB concentration (90.76 ± 3.65 μg/mL), significantly outperforming the standard α-factor signal peptide (68.21 ± 4.94 μg/mL, ​​P ≤ 0.001). The SWP1-α variant also showed improved secretion (69.62 ± 5.13 μg/mL), whereas KRE1-α resulted in lower yield (56.28 ± 1.79 μg/mL). These results demonstrated that signal peptide engineering significantly enhances CALB secretion efficiency in P. pastoris.

Figure 22 (A)SDS-PAGE verification and (B) quantification of CALB under different promoter regulation.

• Enzyme activity test of lipase

The enzyme activity test results showed that the enzyme activity of 0030-α-CALB was the highest (31.25 U/mL), which was significantly higher than that of the standard α-factor control group (28.59 U/mL), with a statistically significant difference (*P ≤ 0.05). The activities of SWP1-α-CALB (27.32 U/mL) and KRE1-α-CALB (27.08 U/mL) variants were comparable to that of the α-factor control group, and no significant differences were observed between them (ns). These results indicate that the 0030-α signal peptide enhances the catalytic efficiency of CALB (Figure 23).

Figure 23 Lipase activity test result.

This study systematically evaluated the efficiency of three engineered signal peptides (0030α-factor, SWP1-α-factor, and KRE1-α-factor) in directing CALB secretion in P. pastoris. Recombinant plasmids containing different signal peptides were successfully constructed and transformed into P. pastoris. SDS-PAGE and quantitative analysis revealed that the 0030α-signaling peptide induced the highest CALB secretion concentration (90.76 ± 3.65 μg/mL), significantly outperforming the traditional α-factor signaling peptide. Enzyme activity assays further confirmed that 0030α-CALB exhibited markedly higher activity (31.25 U/mL) compared to the control group. Therefore, 0030α-signal peptide was identified as the optimal signal peptide and will be used for final recombinant strain construction.

The best-performing combinations were finally integrated into expression cassettes for recombinant strain construction. As shown in Figure 24, the expression vector pPIC9K was engineered to contain the optimal FDH1 promoter and 0030α-factor signal peptide fused with the CALB gene.

Figure 24 Plasmid Design diagram in cycle 3.

The agarose gel electrophoresis results (Figure 25A) confirmed successful amplification of both the vector backbone (Amp-FDH1, ~4402 bp) and the insert fragment (0030α-CALB-Kan, ~5954 bp), with bands matching expected sizes. Colony PCR verification demonstrated successful transformation of E. coli DH5α competent cell, showing specific bands of approximately 2495 bp corresponding to the target expression cassette in multiple transformants (Figure 25B-C). The sequencing results validated the accurate assembly of the optimized expression system combining the FDH1 promoter and 0030α-factor signal peptide (Figure 25D).

Figure 25 The amplification results of the DNA fragments.

(A) Agarose gel electrophoresis result. (B-D) Colony PCR and sequencing results.

We then extracted and linearized the correct plasmids for yeast transformation. The agarose gel electrophoresis (Figure 26 A) confirmed successful linearization, showing a single clear band for the linearized plasmid (lane 2) compared to the negative control (lane 1). The transformation plates (Figure 26 B) exhibited robust colony growth, indicating efficient plasmid introduction into P. pastoris. Furthermore, yeast colony PCR analysis (Figure 26 C) validated the genomic integration, revealing a distinct band at approximately 954 bp corresponding to the FDH1-0030α-CALB expression cassette. These results collectively confirm the successful construction of the recombinant P. pastoris strain.

Figure 26 Construction result of P. pastoris containing lipase.

(A) Plasmid Linearization result. (B) Transformation plates. (C) Yeast colony PCR result.

• Lipase secretion and quantitative analysis

Next, we conducted fermentation on each of these strains (FDH1-0030α-CALB belongs to the experimental group, while FDH1-α-CALB and AOX1-0030α-CALB constitute the control group) and analyzed CALB secretion. The SDS-PAGE results (Figure 27A) clearly showed distinct protein bands at approximately 43.8 kDa in the experimental groups, corresponding to the expected molecular weight of CALB, while absent in the negative control. Quantitative analysis (Figure 27B) demonstrated that the combination of the FDH1 promoter and 0030-α-factor signal peptide yielded the highest secretion level (107.02 ± 3.95 μg/mL), significantly outperforming the other constructs (​​P ≤ 0.01). These results confirmed the synergistic effect of promoter and signal peptide optimization on CALB production.

Figure 27 SDS-PAGE verification of CALB secretion.

(A) SDS-PAGE result. (B) Quantitative results.

• Enzyme activity test of lipase

Similarly, we conducted an enzyme activity test. The results showed that compared with the negative control pPIC9K (NC), the lipase activity of all the recombinant strains was significantly increased. Among them, the combination of the FDH1 promoter and the 0030-α-factor signal peptide (FDH1-0030-α-factor-CALB) exhibited the highest enzyme activity (33.41 U/mL). Statistical analysis indicated that the enzyme activity of this combination was significantly higher than that of the strains that only optimized the promoter (FDH1-α-factor-CALB, 31.68 U/mL) or only optimized the signal peptide (AOX1-0030-α-factor-CALB, 31.25 U/mL) (P ≤ 0.05), confirming the positive effect of the synergistic optimization of the promoter and signal peptide on enhancing the catalytic efficiency of the lipase (Figure 28).

Figure 28 Lipase activity test results.

Finally, we investigated the temperature and pH values of the reaction to determine the optimal catalytic conditions of CALB. As shown in Figure 29A, the temperature optimization experiment indicated that CALB exhibited the highest enzymatic activity (approximately 45 U/mL) at 45°C, while the activity significantly decreased at low temperatures (25°C) or high temperatures (≥65°C). As shown in Figure 29B, the pH optimization results revealed that the enzyme maintained high activity within the range of 7.5–9.0, with the optimal pH being 8.0. It is noteworthy that CALB could still maintain over 70% relative activity within a wide pH range (6.0–10.0), indicating its good acid-base stability. These data clearly defined the optimal reaction conditions of CALB as 45°C and a neutral to weakly alkaline environment, providing key parameters for its industrial application.

Figure 29 Optimization of (A) reaction temperature and (B) pH for CALB.

In this project, we successfully established an efficient system for recombinant CALB production in P. pastoris. The optimized gene construct, which combines the FDH1 promoter and the 0030α factor signal peptide of the CALB gene, was successfully integrated into the yeast genome. The secretion level of CALB reached 107.02 ± 3.95 μg/mL, and the enzyme activity was 33.41 U/mL, which were 1.84 times and 1.17 times higher than those of the original strain (AOX1-α factor-CALB, secretion: 68.21 μg/mL, enzyme activity: 28.59 U/mL), respectively. In addition, we determined that the optimal catalytic conditions for CALB were 45°C and a pH value of 8.0. Under these conditions, the enzyme activity could reach up to 44.96 U/mL, and the enzyme showed high stability within a wide pH range (6.0-10.0). In conclusion, the collaborative optimization of the FDH1 promoter and the 0030α factor signal peptide is the key strategy for achieving high-yield active CALB in P. pastoris. This recombinant strain and the clearly defined optimal reaction parameters have laid a solid foundation for the industrial application of this biocatalyst.