Contribution

Overview

In this project, we developed and validated multiple genetic parts for expression in Pichia pastoris. These parts include basic elements (promoters, terminators, tags, coding sequences, and plasmid backbones) as well as composite constructs that integrate these elements for functional expression of β-T-galactosidase (β-T-Gal) and glucose oxidase (GOD). The system aims to produce lactose-free and sugar-reduced dairy products. We validated each component experimentally, including PCR amplification, plasmid construction, yeast transformation, protein expression, purification, and functional verification.

Table 1. Part Contributions

Part Number	Part Name	Part Type	Part Function
BBa_K945000	AOX1 promoter	basic part	Regulatory
BBa_K863203	AOX1 terminator	basic part	Terminator
BBa_K3141000	pPICZαA	basic part	Plasmid Backbone
BBa_K3584001	His tag-Lac operate-T7 tag	basic part	Tag
BBa_252LAY4D	Beta-T-Galactosidase	basic part	Coding
BBa_258NI5BN	Glucose oxidase	basic part	Coding
BBa_25RILBZU	pPLCZαA-Beta-T-Galactosidase	composite part	Plasmid
BBa_25DDF4OS	pPLCZɑA-Glucose oxidase	composite part	Plasmid

1. Basic Parts

1.1 AOX1 Promoter (BBa_K945000)

Base Pairs: 939 bp

Origin: Native promoter from Pichia pastoris.

Usage and Biology: The AOX1 promoter is a natural component of Pichia pastoris and regulates the expression of the AOX1 gene, which encodes alcohol oxidase. Widely utilized in the production of recombinant proteins (e.g., vaccines, antibodies, and industrial enzymes), this promoter was employed in our experiments to drive the expression of β-T-Gal and GOD, two key enzymes for lactose-free dairy applications.

Fig 1. Gene map of the AOX1 promoter

Properties and functions: The AOX1 promoter exhibits high-efficiency protein expression capability, typically yielding recombinant protein at 1–5 g/L under optimized conditions. For instance, in high-density fermentation, β-T-galactosidase (approximately 80 kDa) yields 2.3 g/L, while glucose oxidase (approximately 48 kDa) reaches 1.8 g/L. This promoter supports eukaryotic-specific post-translational modifications such as glycosylation, which helps maintain protein structural and functional integrity. Its expression regulation is stringent, being completely repressed in glucose or glycerol media and efficiently induced only by methanol. To further enhance system applicability, future research may explore the following directions: testing glucose-sensitive promoters (e.g., GAP) to construct methanol-free autoinductive systems; investigating mixed carbon sources for staged expression regulation; and developing thermotolerant promoter variants to support high-temperature fermentation, thereby improving production safety and process efficiency.

1.2 AOX1 Terminator (BBa_K863203)

Base Pairs: 247 bp

Origin: Native promoter from Pichia pastoris.

Usage and Biology: It is mainly used to ensure efficient and stable gene expression in Pichia pastoris. In our experiment, we used AOX1 Terminator and AOX1 Promoter together to construct a complete β-T-Gal and GOD expression cassette. It not only ensures the correct expression of genes and makes the system more standardized, but also improves protein production.

Fig 2. Gene map of AOX1 terminator

Properties and functions: Regarding transcription termination, the AOX1 terminator located downstream of target genes (such as β-T-Gal or GOD) precisely terminates RNA polymerase-mediated transcription. This effectively prevents “read-through transcription,” avoiding interference with downstream genes or vector backbone stability. This terminator contains specific sequence signals (e.g., polyadenylation signals) that promote 3'-end processing of mRNA, prolonging its half-life and thereby enhancing target protein expression levels. The stable mRNA structure also facilitates ribosome binding and translation processes, ensuring efficient protein synthesis. Future studies may test and compare the effects of different terminators on gene expression efficiency and mRNA stability to further optimize target protein yield.

1.3 pPICZαA (BBa_K3141000)

Base Pairs: 3600 bp

Type: Plasmid Backbone

Usage and Biology: We provide complete plasmid designs for pPICZαA-β-T-Gal and pPICZαA-GOD, which can be directly used by future research teams to develop engineered lactose-degrading bacteria and optimize the expression of other enzymes inPichia pastoris. Additionally, we have verified the entire workflow, from plasmid construction and yeast transformation to protein induction.

Fig 3. Plamid map of pPICZαA

Properties and functions: This expression vector is specifically designed for Pichia pastoris but can also be cloned and amplified in Escherichia coli. The Zeocin resistance gene carried by the vector enables dual selection in both yeast and bacteria. During recombinant plasmid construction, the target gene (e.g., β-T-Gal/GOD) was inserted into the multiple cloning site of the pPICZαA vector via restriction enzyme digestion or homologous recombination. The correct recombinant plasmid pPICZαA-β-T-Gal/GOD was validated. Subsequently, the SacI-linearized vector was integrated into the Pichia pastoris GS115 genome via homologous recombination. Positive clones were selected using Zeocin resistance, ensuring stable inheritance of the exogenous gene. Finally, methanol induction of the AOX1 promoter drives efficient expression of the target protein. To optimize the system, testing alternative inducible promoters may reduce methanol dependency in future studies.

1.4 His tag-Lac operator-T7 tag (BBa_K3584001)

Base Pairs: 18 bp

Usage and Biology: In this project, the tag was integrated into the pPICZαA vector to facilitate the purification of β-T-Gal and GOD proteins, thereby realizing an integrated purification, detection, and regulation system.

Properties and functions: This tag can specifically bind to Ni²⁺ on Ni-NTA resin, enabling efficient capture of the target protein through one-step affinity chromatography while effectively removing untagged contaminating proteins. A short peptide spacer has been introduced between the tag and the target protein to minimize potential steric hindrance effects on binding efficiency. In the future, protease cleavage sites can be introduced within this spacer region to remove the tag post-purification, yielding target proteins with structure and function closer to their native state.

1.5 Beta-β-T-Galactosidase (BBa_252LAY4D)

Base Pairs: 2209 bp

Origin: Kibdelosporangium sp. MJ126-NF4

Usage and Biology: The dual catalytic activities of glycosyltransferase-type β-T-galactosidase are defined as follows: (1) Hydrolytic activity: Using water molecules as acceptors, it cleaves the β-1,4-glycosidic bond in lactose, yielding glucose and galactose (lactose + H₂O → glucose + galactose). (2) Glycosyltransferase activity: Using other sugar molecules as substrates, the enzyme first forms a covalent intermediate with lactose, then transfers the galactose moiety to the recipient sugar, synthesizing a new glycosidic bond and yielding galactosyl oligosaccharides (GOS). Glycosyltransferase activity is key to achieving high-value conversion with this enzyme.

The value of glycosyltransferase-type β-T-galactosidase lies in its controllable catalytic directionality. By precisely regulating reaction conditions (e.g., substrate concentration), its catalytic pathway can be guided: at low substrate concentrations, it favors lactose hydrolysis; at high substrate concentrations, its glycosyltransferase activity is significantly enhanced, enabling efficient synthesis of prebiotic oligosaccharides (GOS).

Design

For the genetic design of β-T-Gal, we adopted a synthetic biology strategy. Its sequence (2199 bp, encoding 783 amino acids) was newly optimized rather than directly derived from traditional microbial sources of β-T-galactosidase. His tag (BBa_K3584001) was incorporated at the C-terminus to facilitate purification. The sequence was optimized for enhanced expression in the Pichia pastoris expression system. Subsequent thin-layer chromatography experiments confirmed that this enzyme exhibits over 90% lactose degradation efficiency at 40°C, demonstrating its structural and functional innovation.

Fig 4. Gene map of β-T-Gal

1.6 Glucose Oxidase (BBa_258NI5BN)

Base Pairs: 1294 bp

Origin: Aspergillus niger

Usage and Biology: Glucose oxidase is a highly specific aerobic dehydrogenase that efficiently catalyzes the oxidation of β-D-glucose. Its core catalytic mechanism is: β-D-glucose + O₂ + H₂O → D-gluconic acid δ-lactone + H₂O₂. The resulting D-gluconic acid δ-lactone spontaneously hydrolyzes to gluconic acid. This reaction simultaneously consumes oxygen and produces hydrogen peroxide. Therefore, in the GOD-POD coupled assay system, the generated H₂O₂ can be further utilized by peroxidase to oxidize colorless substrates (such as o-anisidine dihydrochloride) into colored products, thereby enabling quantitative analysis of GOD activity. This enzyme exhibits strong substrate specificity toward β-D-glucose and shows negligible catalytic activity toward other sugars. This characteristic provides a crucial foundation for its application in biosensing and food analysis.

Design

High-efficiency secretion expression of glucose oxidase was achieved for the first time in Pichia pastoris. Through codon optimization, its expression level was enhanced compared to the wild-type gene. GOD catalyzes the highly specific oxidation of β-D-glucose to gluconic acid and H₂O₂, requiring FAD as a cofactor. Building upon this, we engineered a sequential dual-enzyme control system: β-T-Gal first degrades lactose into glucose, followed by GOD efficiently removing glucose, thereby synergistically achieving dual functions of “lactose degradation and glucose clearance.”

Fig 5. Gene Map of GOD

2. Composite Parts

2.1 pPICZαA-β-T-Galactosidase (BBa_25RILBZU)

The recombinant expression plasmid pPICZαA-β-T-Galactosidase was successfully constructed for heterologous expression in Pichia pastoris. The gene encoding the synthetic β-T-Galactosidase was amplified via PCR, generating a distinct fragment of approximately 2209 bp. As shown in Fig. 6, agarose gel electrophoresis analysis of the PCR product revealed a single, clear band that aligned with the expected molecular size, confirming the specificity and success of the amplification. The correct fragment was subsequently ligated into the pPICZαA vector for downstream applications.

Fig 6. Electrophoresis result of β-T-Gal

Following transformation into E. coli DH5α, positive clones were selected from the zeocin-resistant colonies (Fig. 7A) and subjected to colony PCR for initial verification. The analysis revealed a specific amplification product of approximately 2209 bp, which corresponds to the expected size of the β-T-Gal gene (Fig. 7B). Subsequent DNA sequencing of the plasmid from a positive clone confirmed the precise insertion of the target sequence into the pPICZαA vector without mutations, thereby validating the successful construction of the pPICZαA-β-T-Gal plasmid (Fig. 7C).

Fig 7. pPICZαA-β-T-Gal Recombinant Plasmid Identification Map

Before yeast transformation, the recombinant plasmids were linearized via restriction digestion. Fig. 8 presents the electrophoresis result of the linearized DNA, with bands matching the expected size (~5693 bp for pPICZαA-β-T-Gal).

Fig 8. Electrophoresis Result of Linearized pPICZαA-β-T-Gal

The linearized plasmid was transformed into Pichia pastoris cells pre-cultured in YPD medium. Positive transformants were selected on YPD plates containing zeocin, as shown in Fig. 9A, indicating successful integration of the expression cassette into the yeast genome. To further verify this, genomic DNA was extracted from the transformants and analyzed by PCR. As demonstrated in Fig. 9B, a DNA fragment of approximately 2209 bp, consistent with the expected size of the β-T-Gal gene, was amplified, confirming the stable incorporation of the target expression cassette into the yeast chromosome.

Fig 9. Validation of yeast transformation results

The expression and purification of β-T-Galactosidase were assessed by SDS-PAGE. As depicted in Fig. 10, the purified protein preparation exhibited a single, distinct band migrating at a position consistent with the theoretical molecular weight of ~80 kDa when compared to the protein ladder, confirming the integrity and homogeneity of the recombinant product.

Fig 10. β-T-Gal Protein Expression Map

To evaluate the catalytic activity of the purified β-T-galactosidase (β-T-Gal), this study utilized its specific hydrolysis of o-nitrophenyl-β-D-galactoside (ONPG) to produce the yellow product o-nitrophenol (ONP). Enzyme activity was quantified by measuring the characteristic absorbance of ONP at 420 nm. The experimental method is briefly described as follows: Enzyme solutions of β-T-Gal at different concentrations were reacted with ONPG substrate at 40°C and pH 7.0 for a specific duration. The reaction was terminated by adding sodium carbonate solution, followed by immediate absorbance measurement using a microplate reader. As shown in Table 1, three parallel experiments were conducted at each concentration to ensure data reliability.

Table 1. Absorbance measurement at 420 nm

mg/ml	A420
0	0.032	0.034	0.031
1	0.071	0.078	0.077
2	0.084	0.089	0.091
5	0.126	0.133	0.132
10	0.232	0.239	0.239
15	0.305	0.289	0.295
20	0.296	0.299	0.298

Results demonstrated that the purified β-T-Gal exhibited significant catalytic activity. As shown in Fig. 11, the absorbance value of the reaction system increased significantly with rising enzyme concentration, exhibiting a good linear relationship between the two. This linear trend clearly confirmed that ONP production is proportional to enzyme concentration, indicating the successful purification of β-T-Gal with highly efficient and concentration-dependent catalytic activity.

Fig 11. Results of β-T-Gal Hydrolysis Activity Test

Thin-layer chromatography (TLC) was used to assess β-T-Gal activity under two experimental conditions: (1) fixed β-T-Gal concentration with gradient lactose concentrations; (2) fixed lactose concentration with gradient time. Figure 12 shows the results of the lactose-gradient experiment: the migration distance of samples increased with increasing lactose concentration. This indicates that when the substrate is insufficient and β-T-Gal is not saturated, the lactose concentration acts as the limiting factor for the hydrolysis reaction, and the amount of product increases with the increase in substrate. As the reaction time extends from 0 min to 60 min, there is little difference in the intensity of the "lactose substrate spot" on the TLC plate, and no obvious distinction in the resolution of spots between adjacent time points. This may be because β-T-Gal has completed the hydrolysis of most lactose within 15 min; the change in the amount of substrate is minimal in the subsequent time, resulting in insignificant differences between spots at adjacent time points and making it impossible to clearly distinguish the degree of hydrolysis. The time gradient needs to be adjusted in subsequent experiments to enable such a distinction.

Fig 12. TLC Analysis of β-T-Gal Activity

At a fixed enzyme concentration, increasing lactose concentration not only significantly elevated the yield of the primary hydrolysis product glucose. Notably, in the high-concentration lactose system (5%), galacto-oligosaccharides (GOS) were also observed to form alongside glucose. Moreover, GOS production increased with rising lactose concentration, indicating that high substrate concentration promotes β-T-Gal-catalyzed glycosylation reactions, thereby synthesizing GOS.

To investigate the efficiency of lactose hydrolysis under the synergistic action of β-T-Gal and GOD, we comparatively analyzed the hydrolysis effects in both pure lactose solution and fresh milk. Thin Layer Chromatography (TLC) results demonstrated that, under the synergistic action of the two enzymes, lactose was hydrolyzed into glucose and galactooligosaccharides (GOS). Moreover, the hydrolysis effect in fresh milk was identical to that in the pure lactose solution (Fig. 13). This confirms that the synergistic effect of β-T-Gal and GOD is feasible in fresh milk.

Fig 13. TLC Results of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity

To quantitatively compare the decomposition efficiency of lactose in milk under the synergistic action of dual enzymes (β-T-Gal and GOD), we employed High-Performance Liquid Chromatography (HPLC) to dynamically monitor the decrease in lactose concentration and the increase in glucose concentration in milk during the reaction. The experimental setup was as follows: β-T-Gal and GOD were mixed at a 1:1 ratio and added to the milk system; the reaction was initiated under three temperature conditions (25°C, 37°C, and 42°C), and samples were taken at 10-minute, 20-minute, and 60-minute intervals after the start of the reaction. The concentrations of lactose and glucose at each time point were determined by HPLC (Table 2 and Table 3), and the relevant results are shown in Fig. 14.

Table 2. Lactose Concentration Detected by HPLC

Time (min)	25℃	37℃	42℃
0	3.5	2.7	2.4
10	2.6	2.1	2.5
20	2.5	1.7	1.4
60	2.1	2.1	1.2

Table 3. Glucose Concentration Detected by HPLC

Time (min)	25℃	37℃	42℃
0	0	0	0
10	0	2	3.5
20	0	3.4	6.1
60	3.6	5.6	6.7

In terms of the effect of temperature on reaction efficiency, both the lactose decomposition rate and glucose production rate at 42°C were significantly higher than those at 25°C and 37°C. This indicates that 42°C is the optimal reaction temperature for the dual-enzyme synergistic system to catalyze lactose decomposition in milk. A further analysis of the reaction kinetic characteristics at the optimal temperature (42°C) revealed that the first 20 minutes of the reaction were the stage with the fastest glucose production rate. This phenomenon suggests that during the initial stage of dual-enzyme synergy (the first 20 minutes), the binding efficiency between the enzymes and the substrate (lactose) is the highest, and the catalytic activity is at its peak, allowing lactose to be rapidly decomposed into glucose. However, as the reaction proceeds, the glucose production rate gradually slows down, which may be attributed to the decrease in substrate (lactose) concentration, the feedback inhibition caused by the accumulation of the product (glucose), or a slight decline in enzyme activity over the reaction time.

Fig 14. Quantitative Analysis of Dual-Enzyme Synergistic Products by HPLC

In summary, this study determined that the optimal reaction conditions for dual-enzyme synergistic lactose decomposition are 42°C, identifying the initial 20 minutes as the critical phase for high-efficiency conversion. During this period, enzyme-substrate binding is highly efficient, catalytic activity reaches its peak, and rapid lactose degradation is achievable. Therefore, controlling the reaction at 42°C for 20 minutes represents an optimized strategy balancing efficiency and energy consumption.

2.2 pPICZαA-Glucose oxidase (BBa_25DDF4OS)

The recombinant expression plasmid pPICZαA-Glucose Oxidase (pPICZαA-GOD) was constructed using homologous recombination. The gene encoding glucose oxidase (GOD) was amplified by polymerase chain reaction (PCR), resulting in a DNA fragment of approximately 1284 bp, which corresponds to the expected length of the full-length coding sequence. As shown in Fig. 15, agarose gel electrophoresis analysis of the PCR product displayed a single, clear band of the correct size, confirming the successful and specific amplification of the target gene prior to its insertion into the linearized pPICZαA vector.

Fig 15. Electrophoresis result of GOD

The recombinant plasmid was transformed into E. coli DH5α competent cells, and transformants were selected on zeocin-containing plates, as shown by the single colonies in Fig. 16A. Multiple colonies were initially screened by colony PCR to identify those carrying an insert of the expected size. A positive clone identified by PCR was subsequently subjected to DNA sequencing. The sequencing results confirmed that the glucose oxidase (GOD) gene was accurately inserted into the pPICZαA vector without mutations, thereby verifying the successful construction of the pPICZαA-Glucose Oxidase plasmid.

Fig 16. pPICZαA-GOD Recombinant Plasmid Identification Map

Before transforming into yeast, the recombinant plasmids were linearized by restriction digestion. Fig 17 shows the electrophoresis result of the linearized DNA, with bands of the expected size (~4778 bp for pPICZαA-GOD).

Fig 17. Electrophoresis result of pPICZαA-GOD after DNA linearization

The linearized pPICZαA-GOD plasmid was transformed into Pichia pastoris host cells pre-cultured in YPD medium. Successful transformants were selected on YPD plates supplemented with zeocin, with colony growth evident in Fig. 18A, indicating positive integration of the expression cassette into the yeast genome. To molecularly confirm the integration, genomic DNA was extracted from selected transformants and analyzed by PCR using gene-specific primers. As shown in Fig. 18B, an amplification product of approximately 1294 bp, corresponding to the expected size of the GOD gene fragment, was detected, verifying the precise and stable incorporation of the GOD expression cassette into the yeast chromosome.

Fig 18. Validation of yeast transformation results

The transformed yeast strains were cultured under inducing conditions to express the recombinant GOD protein. Subsequently, the secreted protein was purified from the culture supernatant using C-terminal His-tag technology. Analysis of the purification process by SDS-PAGE revealed a predominant band at approximately 48 kDa, which aligns with the expected molecular weight of the glycosylated GOD protein. The electrophoretic profile demonstrates successful purification, with the target protein band being the major component in the elution fraction, indicating a high purity level suitable for subsequent functional characterization.

Fig 19. Result of the GOD Protein purification

The activity of purified glucose oxidase (GOD) was determined using a glucose oxidase (GOD)-peroxidase (POD) coupled reaction system. The principle is as follows: GOD catalyzes the oxidation of glucose to produce H₂O₂. Subsequently, POD catalyzes the decomposition of H₂O₂ in the presence of oxygen. The resulting reactive oxygen species oxidize colorless o-anisidine to form a brownish-yellow product. The absorbance of this product at a specific wavelength is positively correlated with GOD activity. A series of GOD dilutions was measured for reaction absorbance using a Multiskan™ FC microplate reader.

Table 4. Absorbance measurement at 500 nm

mg/ml	A500
0	0.076	0.074	0.075
0.5	0.178	0.179	0.178
1	0.184	0.183	0.183
2.5	0.469	0.467	0.465
5	0.725	0.723	0.721
10	1.251	1.249	1.247
20	1.508	1.503	1.5

Results demonstrated that although the absorbance curve was not strictly linear, its overall trend of significant increase with rising enzyme concentration clearly confirmed that the purified GOD retained potent catalytic activity.

Fig 20. Results of the GOD Hydrolysis Activity Test

To investigate the efficiency of lactose hydrolysis under the synergistic action of β-T-Gal and GOD, we comparatively analyzed the hydrolysis effects in both pure lactose solution and fresh milk. Thin Layer Chromatography (TLC) results demonstrated that, under the synergistic action of the two enzymes, lactose was hydrolyzed into glucose and galactooligosaccharides (GOS). Moreover, the hydrolysis effect in fresh milk was identical to that in the pure lactose solution. This confirms that the synergistic effect of β-T-Gal and GOD is feasible in fresh milk.

Fig 21. TLC Results of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity

Table 5. Lactose Concentration Detected by HPLC

Time (min)	25℃	37℃	42℃
0	3.5	2.7	2.4
10	2.6	2.1	2.5
20	2.5	1.7	1.4
60	2.1	2.1	1.2

Table 6. Glucose Concentration Detected by HPLC

Time (min)	25℃	37℃	42℃
0	0	0	0
10	0	2	3.5
20	0	3.4	6.1
60	3.6	5.6	6.7

In terms of the effect of temperature on reaction efficiency, both the lactose decomposition rate and glucose production rate at 42°C were significantly higher than those at 25°C and 37°C. This indicates that 42°C is the optimal reaction temperature for the dual-enzyme synergistic system to catalyze lactose decomposition in milk. A further analysis of the reaction kinetic characteristics at the optimal temperature (42°C) revealed that the first 20 minutes of the reaction were the stage with the fastest glucose production rate. This phenomenon suggests that during the initial stage of dual-enzyme synergy (the first 20 minutes), the binding efficiency between the enzymes and the substrate (lactose) is the highest, and the catalytic activity is at its peak, allowing lactose to be rapidly decomposed into glucose. However, as the reaction proceeds, the glucose production rate gradually slows down, which may be attributed to the decrease in substrate (lactose) concentration, the feedback inhibition caused by the accumulation of the product (glucose), or a slight decline in enzyme activity over the reaction time.

Fig 22. Quantitative Analysis of Dual-Enzyme Synergistic Products by HPLC

Other Contributions

We successfully obtained highly active microbial-derived recombinant glycosyltransferase-type β-T-galactosidase (β-T-Gal) and glucose oxidase (GOD) through a heterologous recombinant expression system. Building upon this foundation, we innovatively applied the synergistic catalysis of these two enzymes to develop a novel dairy processing technology. This process not only efficiently degrades lactose to alleviate lactose intolerance but also synthesizes galacto-oligosaccharides (GOS) through β-T-Gal's glycosyltransferase activity. Simultaneously, glucose is oxidatively removed using GOD, ultimately yielding functional dairy products with triple health benefits: low lactose, low glucose, and high GOS dietary fiber. This represents a technological leap from simple lactose hydrolysis to comprehensive nutritional fortification.