Abstract
Efficient production hosts are a key requirement for bringing biopharmaceutical and biotechnological innovations to the market. In this work, a truly universal high-throughput platform for optimization of microbial protein production is described. Using droplet microfluidics, large genetic libraries of strains are encapsulated into biocompatible gel beads that are engineered to selectively retain any protein of interest. Bead-retained products are then fluorescently labeled and strains with superior production titers are isolated using flow cytometry. The broad applicability of the platform is demonstrated by successfully culturing several industrially relevant bacterial and yeast strains and detecting peptides or proteins of interest that are secreted or released from the cell via autolysis. Lastly, the platform is applied to optimize cutinase secretion in Komagataella phaffii (Pichia pastoris) and a strain with 5.7-fold improvement is isolated. The platform permits the analysis of >106 genotypes per day and is readily applicable to any protein that can be equipped with a His6 -tag. It is envisioned that the platform will be useful for large screening campaigns that aim to identify improved hosts for large-scale production of biotechnologically relevant proteins, thereby accelerating the costly and time-consuming process of strain engineering.
Highlights
This page was generated automatically upon download from the ETH Zurich Research Collection
The platform relies on the cultivation of a genetically diversified population of a microbial strain expressing and releasing a His6-tagged protein of interest within picoliter-volume agarose gel beads, which we refer to as picoliter reactors
The protein of interest is affinitycaptured within the picoliter reactors (pLRs) by nickel-nitriloacetic acid (Ni-NTA)functionalized capturing microparticles and subsequently labeled by a His6-tag-specific fluorescent probe
Summary
This page was generated automatically upon download from the ETH Zurich Research Collection. Microbes feature high The yields are connected to the cellular machinery genetic tractability, which enables facile engineering for directly involved in the synthesis or translocation of the enhanced yields, as well as relatively inexpensive and fast fer- product, which is in itself complex, and intimately intermentation processes. These multi-faceted benefits have made twined with the broader physiology of the host organism,[21]
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