Breakthrough in Komagataella phaffii cell-free protein synthesis: AOX1 promoter drives T7-independent expression efficiently

Abstract

This study develops a cell-free protein synthesis (CFPS) system based on the endogenous alcohol oxidase 1 promoter in Komagataella phaffii. The system avoids the dependence of the T7 promoter, thus eliminating the cost issues associated with the T7 RNA polymerase-dependent system in traditional CFPS systems. By integrating an alcohol oxidase 1 promoter-driven GFP expression cassette with optimized K. phaffii cell extract, key components are optimized via a one-factor-at-a-time experiment and a deterministic screening design. This study demonstrates that potassium glutamate and magnesium glutamate have a significant synergistic effect on this system. After optimization, the system achieves a GFP yield of 596.0 mg/L, providing a new record for GFP expression in K. phaffii CFPS systems. This work provides an important theoretical foundation for the further development of T7-independent K. phaffii CFPS systems and their potential applications in scalable bioproduction.

1. Introduction

Cell-free protein synthesis (CFPS) is an innovative synthetic biology technology that uses cell extracts and DNA or mRNA templates to generate proteins with the help of amino acids, enzymes, transcription factors, and nucleoside triphosphates [1]. The first CFPS system was established by Schweet et al. [2] in 1958 and used rabbit reticulocyte extract and 14C-labeled amino acids to synthesize small amounts of hemoglobin. Compared with cell-based systems, recent technological advances in CFPS enable rapid protein synthesis in hours rather than days, alongside high throughput screening capabilities and the modular assembly of synthetic biological components [3–7]. Owing to the low protein production efficiency and instability of the PURE CFPS, which is composed of purified transcription and translation machinery components [8,9], the development of cell extract-based CFPS systems has attracted the attention of researchers and industrial communities, such as rabbit reticulocytes [10], wheat germ [11], insect cells, E. coli, and yeast cell [12] extract-based CFPS systems.

Currently, E. coli CFPS dominates all the other CFPS systems because of its available components and well-established protocol of protein synthesis. E. coli cell extract-based CFPS also performs well in the biosynthesis of functional compounds containing chloro, alkene, and alkyne groups from unnatural amino acids [13], screening and identifying new enzymes from various species [14]. Additionally, E. coli cell extract-based CFPS was harnessed to investigate the interactions between proteins [15] and peptides with post-translation modification [16]. E. coli cell extract-based CFPS uses the T7 promoter and optimized T7 RNA polymerase [17] and works on a 10 mL scale successfully [18]. However, heterologous protein expression in E. coli often faces the issue of a lack of post-translational modifications, which can lead to misfolding, insolubility, or functional loss of heterologous proteins [19]. In recent years, other prokaryotic CFPS have also shown remarkable performance. Shin et al. [20] used the holoenzyme E(70) bacteriophage T7 to express 60 genes in a 40 kbp DNA fragment. Moore et al. [21] developed Streptomyces venezuelae CFPS via the Streptomyces kasOp* promoter and further demonstrated protein synthesis from high-GC-content genes [22]. Xu et al. [23] also reported Streptomyces cell extract-based CFPS after screening native promoters and ribosome binding sites, with a final yield of 515.7 ± 25.3 mg/L green fluorescent protein (GFP). However, prokaryotic CFPs generally lack eukaryotic post-translational modifications (PTMs), limiting their utility for complex proteins.

The common issue of low reporter expression when the native promoter is used is the low level of RNA polymerase, which is crucial for CFPS systems [24]. Therefore, CFPS with high RNA polymerase activity and efficient promoters from eukaryotic organisms has become a promising option for heterologous protein expression. Gupta et al. [25] used the tobacco Nicotiana tabacum BY‐2 cell extract CFPS, which was commercialized as ALiCE®, to express diverse, functional proteins at high yields of 1.5 mg/mL in 48 h. The methylotrophic yeast Komagataella phaffii, formerly known as Pichia pastoris, has been a famous cell factory for heterologous protein production for years [26]. It has also been used as a source of cell extract for K. phaffii CFPS by several researchers [27]. On the basis of the well-established protocol of E. coli-based CFPS, the K. phaffii CFPS system relies on optimized extract preparation and compatible T7 promoter-driven templates in E. coli CFPS [28,29]. Zhang et al. [30] simplified CFPS cell extract preparation and reaction conditions, achieving 50.16 ± 7.49 mg/L GFP in a 5 h batch reaction. Spice et al. [31] also developed a K. phaffii-based CFPS after reaction composition optimization through minimized experimental design and demonstrated the capacity of human serum albumin production.