OAZ1/CASP8AP2 double knockout enhances recombinant protein production in HEK293 cells through metabolic reprogramming and antiapoptotic effects

Abstract

Human embryonic kidney (HEK) 293 cells are widely used for recombinant protein production because of their efficient posttranslational modification capabilities. However, their large-scale culture is often limited by metabolic stress and early apoptosis, leading to insufficient protein yields. In this study, we aim to increase protein expression through the coordinated modulation of metabolic and apoptotic pathways. Using CRISPR/Cas9 technology, we target and knockout the genes of ornithine decarboxylase antizyme 1 (OAZ1), which regulates polyamine metabolism, and caspase 8-associated protein 2 (CASP8AP2), an apoptosis-related protein. We successfully construct an OAZ1/CASP8AP2 double-knockout HEK293 cell line. Following transfection with the knockout vector and screening of single-cell clones, multiple levels of validation confirm the successful gene knockout. The results show that the double-knockout cells exhibit significantly reduced apoptosis rates. Furthermore, the production of recombinant secreted alkaline phosphatase (SEAP) and vitronectin (VN) increases by 2.1 folds and 2.9 folds, respectively, compared with those in wild-type cells. Metabolic profiling reveals that the cell cycle is arrested in the G1/G0 phase, accompanied by increased specific consumption and production rates of key metabolites. This study demonstrates that concurrent inhibition of apoptosis and optimization of metabolism effectively enhances recombinant protein production in HEK293 cells, suggesting a novel strategy for improving HEK293 cell-based expression.

1. Introduction

Recombinant proteins serve as core therapeutic molecules in the biopharmaceutical field, playing an irreplaceable role in antibody drug development, gene therapy vector production, and vaccine research [1]. Mammalian cell expression systems, owing to their precise posttranslational modification capabilities, have become the preferred platform for the industrial production of complex structural proteins, such as glycosylated antibodies and transmembrane receptors. Among these, the HEK293 cell line has significant advantages in recombinant protein expression owing to its highly humanized glycosylation profile, adaptability to suspension culture, and rapid proliferation [2]. However, three major bottlenecks hinder its performance in large-scale cultures: limited tolerance to metabolic stress, leading to inefficient nutrient utilization; premature activation of extrinsic apoptotic pathways; and constrained recombinant protein expression levels [3–5]. These challenges significantly restrict its application efficiency in large-scale biopharmaceutical manufacturing, necessitating systematic optimization of cellular performance via genetic engineering strategies.

The advent of CRISPR/Cas9 technology has provided efficient and precise gene editing tools for cell engineering. By inducing targeted DNA double-strand breaks and leveraging nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms [6,7], this technology enables gene knockout, insertion, and site-specific modification [8]. Compared with traditional gene editing methods, CRISPR/Cas9 offers advantages such as multiplexed gene editing, ease of use, and high-throughput screening and has been widely applied in cell line engineering [9]. However, current studies often focus on single-gene regulation, which is insufficient to coordinate the complex interplay among cellular metabolism, apoptosis, and protein secretion pathways. Therefore, developing multitarget synergistic editing strategies is a critical research direction for enhancing recombinant protein production [10–12].

In industrial-scale recombinant protein production, controlling apoptosis is a central challenge. High-density bioreactor cultures are susceptible to apoptosis triggered by factors such as nutrient gradients, the accumulation of metabolic byproducts, and osmotic fluctuations, resulting in decreased cell viability and reduced protein yields [13–15]. Studies have shown that inhibiting key apoptotic signaling nodes can significantly extend the culture duration and increase product accumulation [16]. Concurrently, the metabolic characteristics of mammalian cells directly impact their protein synthesis capacity. For example, HEK293 cells exhibit high levels of glycolysis, which support rapid cell proliferation but lead to inefficient glucose utilization and excessive lactate accumulation, forming a vicious lactate-glucose cometabolism cycle [17–20].