Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

Abstract

In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 µg/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.

Designing Modular Cell-free Systems for Tunable Biotransformation of l-phenylalanine to Aromatic Compounds

“…For example, the conversion of L-Phe to 2-PE was not complete within 24 h, which is likely due to the low expression of soluble StyC and the resulting low catalytic efficiency. Therefore, given the flexibility of cell-free systems, one might switch from the E. coli CFPS system to many other CFPS systems ( Kelwick et al, 2016 ; Li et al, 2017 ; Des Soye et al, 2018 ; Wang et al, 2018 ; Xu et al, 2020 ; Zhang et al, 2020 ) to better mimic the physicochemical environment of native hosts for the expression of related enzymes with high (soluble) yields and high activities. In other words, various CFPS systems can be used to express highly active enzymes in different cell-free modules and then mix them for efficient biotranformations, thus giving rise to high productivity.…”

Methods for Expression of Recombinant Proteins Using a Pichia pastoris Cell‐Free System

“…The reaction mix outlined below has been improved using Design of Experiments (DOE) to result in increased protein synthesis (Spice et al, 2020a). Alternatively, a standard reaction mix more closely aligned to that used for CFPS with S. cerevisiae and P. pastoris in other published papers (Aw & Polizzi, 2019;Hodgman & Jewett, 2013;Zhang, Liu, & Li, 2020) can be used. This protocol requires precise pipetting, and the order in which components are added is essential for the function of the CFPS reaction (Fig.…”

“…For assessing the P. pastoris CFPS system, a convenient model protein such as luciferase is used. It is not possible to use a GFP reporter, unless it is allowed to fold correctly into a refolding buffer before reading for at least 2 hr (Zhang et al., 2020). As this is more time consuming than a luciferase assay in our hands, luciferase remains our preferred reporter.…”

“…It is worth noting that a P. pastoris cell‐free system was very recently developed by another group using the protease‐deficient strain SMD1163. Although the extract preparation methodologies and reaction mix composition they describe have similarities to the protocol described here, the yields reported for superfolder green fluorescent protein (sfGFP) in their system are considerably lower, potentially due to their use of the more readily accessible sonication method for lysis (Zhang et al., 2020).…”

Design, Development and Optimization of a Functional Mammalian Cell-Free Protein Synthesis Platform

“…Among CFPS platforms, eukaryotic systems are of increasing interest due to their ability to produce post-translationally modified proteins (Mikami et al, 2006a ; Brödel et al, 2013 ; Zemella et al, 2018 ). Systems have been developed based on yeast (Hodgman and Jewett, 2013 ; Gan and Jewett, 2014 ; Aw and Polizzi, 2019 ; Zhang et al, 2020 ), plants (Madin et al, 2000 ; Murota et al, 2011 ; Buntru et al, 2014 , 2015 ), insect (Ezure et al, 2006 , 2010 ; Madono et al, 2011 ; Richter et al, 2014 ; Stech et al, 2014 ), and mammalian cells (Stavnezer and Huang, 1971 ; Shields and Blobel, 1978 ; Jackson and Hunt, 1983 ; Starr and Hanover, 1990 ; Katzen and Kudlicki, 2006 ; Mikami et al, 2006a , b ; Mikami et al, 2008 ; Brödel et al, 2014 ; Yadavalli and Sam-Yellowe, 2015 ; Thoring et al, 2017 ; Burgenson et al, 2018 ; Thoring and Kubick, 2018 ). Mammalian systems are of particular interest due to their ability to produce glycoproteins with human-like N-linked glycosylation.…”