# Analysis of the aspartic protease gene family in Nicotiana benthamiana and its application in recombinant protein expression

## TL;DR

This study identifies and characterizes aspartic protease genes in Nicotiana benthamiana, showing how silencing specific genes can improve recombinant protein production in plants.

## Contribution

The first comprehensive genomic analysis of aspartic protease genes in Nicotiana benthamiana and experimental validation of their impact on recombinant protein accumulation.

## Key findings

- 89 aspartic protease (NbAP) genes were identified in Nicotiana benthamiana.
- Silencing specific NbAP genes (e.g., NbAP46, NbAP47, NbAP79) significantly increased recombinant protein accumulation.
- Segmental duplication is the main mechanism for NbAP gene family expansion.

## Abstract

Aspartic proteases (APs) play crucial roles in plant growth, stress responses, and protein metabolism. However, endogenous APs in Nicotiana benthamiana, a key plant molecular farming platform, pose a significant challenge by degrading valuable recombinant proteins, thereby limiting production yields.

A comprehensive understanding of the AP gene family in N. benthamiana is essential for developing strategies to mitigate this degradation. In this study, multiple bioinformatics approaches were employed to identify the NbAPs gene family in the Nicotiana benthamiana genome. The analysis encompassed the protein characteristics, phylogenetic relationships, gene structures, conserved motifs, gene duplication events, and chromosomal distribution of family members. Additionally, the role of NbAPs members in recombinant protein expression was investigated.

We performed a genome-wide analysis and identified 89 Aspartic Protease (NbAP) genes in N. benthamiana. Phylogenetic analysis classified these genes into three subfamilies-typical, nucellin-like, and atypical-revealing both evolutionary conservation and diversification. The NbAP members exhibited diverse gene structures, conserved motifs, and subcellular localizations, with significant enrichment predicted for the vacuole and chloroplasts. Segmental duplication was identified as the primary mechanism of NbAP family expansion. Promoter analysis revealed the presence of cis-elements associated with stress responses, hormone regulation, and developmental processes. Transcriptome and RT-qPCR analyses showed dynamic expression patterns of NbAPs following Agrobacterium infiltration, with genes such as NbAP46, NbAP47, or NbAP79 showing sustained upregulation. Functional validation using Virus-Induced Gene Silencing (VIGS) demonstrated that silencing NbAP46, NbAP47, or NbAP79 significantly enhanced the accumulation of transiently expressed GFP protein.

This study provides the first comprehensive genomic identification and characterization of the AP family in N. benthamiana. We highlight the diversity of NbAPs and their potential biological roles, and experimentally validate that specific members negatively influence recombinant protein stability. Silencing these proteases leads to enhanced accumulation of foreign proteins. Overall, our findings establish a foundational resource and identify potential targets for genetic engineering to further optimize N. benthamiana as a superior bioreactor for plant molecular farming.