Revision as of 03:20, 10 September 2024

P_GTH1-Xylanase-Pir1

CONSTRUCTION

Pir1 is an anchor protein of Saccharomyces cerevisiae, widely utilized in yeast display systems. To explore its potential compared to GCW61, we aimed to construct a Xylanase-Pir1 fusion protein. Using the same gene expression strategy as with GCW61, the Pir1 sequence from Saccharomyces cerevisiae was synthesized by Integrated DNA Technologies (IDT) with a GS linker at the N-terminus, flanked by EcoRI-XbaI-NgoMIV as a prefix and AgeI-SpeI-PstI as a suffix. This basic part, GS-Pir1, was assembled into the pSB1C3 vector ([[BBa_K5049002]]), and the composite part with Xylanase gene, Xylanase-Pir1 (BBa_K5049005), was generated. The final functional composite part of PGTH1-Xylanase-Pir1/pSB1C3 was then created (BBa_K5049007). To evaluate its performance in yeast and compare it with the GCW61 anchor, the PGTH1-Xylanase-Pir1 construct was cloned into the pZAHR vector. The construct was verified by colony PCR (Figure 3) and confirmed by DNA sequencing.

Figure 1 | Verification of PGTH1-Xylanase-Pir1/pZAHR construct. Colony PCR using a GTH1-specific forward primer (5’- CCCCAAACATTTGCTCCCCCTAG-3’) and a Pir1-specific reverse primer (5’-AGAAGTTAAAGTTGTGGCTTG-3’) yielded an expected 2346-bp DNA fragment. The numbers indicate selected colonies, with lane 1 showing a 1kb DNA marker on 1% agarose gel in 0.5x TAE buffer (FluoroBand™ 1 KB (0.25-10 kb) Fluorescent DNA Ladder, SMOBIO Technology, Inc.).

FUNCTIONAL ASSAY

In order to verify the activity of xylanase displayed on the yeast surface, we conducted a DNS assay to compare the activities of Xylanase-GCW61 and Xylanase-Pir1. Following the same procedure as in previous experiments, glucose-induced whole cells of Pichia pastoris harboring the respective plasmid DNAs were collected at 24-hour intervals over a 96-hour period. The cells were mixed with 5% xylan and incubated at 37°C for 15 minutes. Subsequently, DNS solution was added, and the mixture was heated at 100°C for 15 minutes to halt the enzyme activities and facilitate color development. After cooling the samples on ice for 5 minutes, the extent of color change was measured at OD540. The results were converted to enzyme activity, expressed as Units/mL/min, consistent with our prior measurements.

As shown in Figure 5, the enzyme activities of Xylanase-GCW61 and Xylanase-Pir1 displayed on the surface of Pichia pastoris exhibited distinct trends. Xylanase-GCW61 activity peaked at approximately 109 Units/mL/min after 48 hours but rapidly declined over the subsequent two days. In contrast, Xylanase-Pir1 activity showed a gradual increase, reaching around 91 Units/mL/min at 72 hours before slightly decreasing. And the activities of wild-type xylanase (Xylanase-WT) without an anchor protein showed a background level of around 22-24 Units/mL/min, demonstrating the effectiveness of using either GCW61 or Pir1 as anchor proteins in the yeast surface display system. Given our objective of using yeast carriers as animal feed additives, where rapid protein induction and enzymatic activity are critical for cost efficiency, we selected Xylanase-GCW61 for further experimentation. This upcoming study is designed to serve as a proof-of-concept, demonstrating the practical application and effectiveness of this approach in real-world feed additive scenarios.

Figure 5 | Comparison of Xylanase Activities in Yeast Surface Display System. This figure illustrates the xylanase activities of wild-type xylanase (Xylanase-WT) without an anchor protein (yellow line), Xylanase-GCW61 (blue line), and Xylanase-Pir1 (red line) expressed in Pichia pastoris. The yeast strains carrying these genes were cultured in BMY media and induced with 0.005% glucose (0.05g/L) for 96 hours, with samples collected at 24-hour intervals. Xylanase activity was determined using DNS assays, with methods and calculations as previously described. Error bars represent the standard error from three independent experiments, demonstrating the reproducibility of the findings.

Sequence and Features