Computational Design of Integrating Tetracycline Riboswitch into the 5’UTR of ERG11 Gene in Komagataella phaffii using CRISPR-Cas9 System

Subject Areas : Mycology

Sajjad Yazdanpanah ¹ , حسن محبت کار ^{2 *} , Mohammad Barshan-tashnizi ³ , Sareh Arjmand ⁴

1 -
2 - استاد-دانشگاه اصفهان
3 - Department of Life Science Engineering (LSE), Faculty of New Sciences & Technologies (FNST), University of Tehran, Tehran, Iran.
4 - Protein Research Center, Shahid Beheshti University, Tehran, Iran

Received: 2025-07-03 Accepted : 2025-08-26 Published : 2025-09-11

Keywords: Recombinant Proteins, Komagataella phaffi, Ergosterol, Riboswitch, CRISPR-Cas9, Membrane Fluidity. ,

Abstract :

Background and Objectives: Komagataella phaffii (K. phaffii) is widely recognized as a top choice for producing recombinant proteins. However, its efficiency in secreting these proteins remains less than ideal. One limiting factor is the rigidity of the yeast cell membrane, which is largely influenced by ergosterol levels. The ERG11 gene is responsible for coding a crucial enzyme in the ergosterol biosynthesis pathway. Reducing the expression of  ERG11 could increase membrane fluidity, potentially improving protein secretion. In this study, we aim to design a method to dynamically control the expression of ERG11 by integrating a tetracycline riboswitch (TcRs) into its 5′ untranslated region (5′UTR).

Materials and Methods: Computational techniques, including RNA secondary structure prediction and molecular docking, were utilized to identify optimal sites for riboswitch insertion. Additionally, a CRISPR/Cas9-based knock-in strategy was designed to enable precise integration of the TcRs upstream of  ERG11 through homologous recombination.

Results: Based on computational analyses, four suitable insertion sites for TcRs were identified, and a DNA donor construct comprising TcRs with two homologous arms was designed for precise integration. Additionally, three single-guide RNAs (sgRNAs) were strategically designed to target sequences adjacent to the riboswitch insertion sites, thereby enhancing the efficiency and accuracy of knock-in via homologous recombination.

 Conclusion: This work highlights effective insertion sites and provides a validated approach for introducing a regulatory riboswitch into the ERG11 locus. This method offers a potential means to modulate membrane fluidity dynamically, enhancing the secretion of recombinant proteins in K. phaffii.

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