Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

Abstract

Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three‐fold increase in recombinant protein yield compared with those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml −1. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System

“…Pichia pastoris cells were grown and extracts were prepared as previously described (Aw and Polizzi, 2019). Briefly, overnight cultures of P. pastoris strain FHL1 were grown in 5 ml of YPD medium and then used to inoculate 200 ml of YPD medium to an OD 600 of 0.1.…”

“…Cell growth was allowed to proceed to an OD 600 of 18-20 at 30 • C, 250 rpm. Cells were harvested, washed, homogenized and prepared using the previously described methodology (Aw and Polizzi, 2019). Cellfree protein synthesis reactions were performed by a coupled in vitro transcription/translation system at a volume of 50 µL at room temperature for 3 h with no shaking as previously detailed (Aw and Polizzi, 2019).…”

“…Developing new extracts is still of great interest, unlocking access to advantageous properties of the organisms from which they are derived. The available repertoire of developed CFPS extracts is still expanding and now includes HEK293 (Bradrick et al, 2013) Saccharomyces cerevisiae (Gan and Jewett, 2014), BY-2 tobacco cells (Buntru et al, 2014), Streptomyces venezuelae (Moore et al, 2017), Bacillus megaterium (Moore et al, 2018), Vibrio natriegens (Gan and Jewett, 2014) and most recently Pichia pastoris (Aw and Polizzi, 2019).…”

Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

“…In addition, cell‐free biosensors can use CFPS systems other than E. coli , such as yeast and mammalian cells, to develop more sophisticated biosensors. [ 114,115 ] By improving the sensor recognition mechanisms and combining cell‐free systems with other materials (e.g., silicon), more types of functional cell‐free biosensors can be developed. It might expand the detection range of cell‐free biosensors to sense the odor, air, temperature, light, and osmotic pressure.…”

Biological Materials: The Next Frontier for Cell-Free Synthetic Biology

“…(iv) Cell extract-based cell-free protein synthesis reactions utilize the transcription and translation machinery within cell lysates, along with exogenously added energy mix components (e.g., amino acids) and DNA templates for in vitro protein production. different host cells have been used to develop these reactions, including bacteria such as Bacillus subtilis (Kelwick et al, 2016), Streptomyces venezuelae (Moore et al, 2017a;Li et al, 2018) and E. coli (Sun et al, 2013) as well as insect (Ezure et al, 2006), wheat germ (Harbers, 2014), yeast (Hodgman and Jewett, 2013;Aw and Polizzi, 2019), protozoans such as Leishmania tarentolae (Mureev et al, 2009;Kovtun et al, 2010Kovtun et al, , 2011 and mammalian cells (Weber et al, 1975;Martin et al, 2017).…”