Whole synthetic pathway engineering of recombinant protein production

Abstract

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass.In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.

Phaeodactylum tricornutum: A Diatom Cell Factory

“…Microbial cell factory hosts have predominantly been proposed for high-value products, such as the production of recombinant vaccines and therapeutics for the prevention and treatment of diseases. Escherichia coli has often remained the choice for development as a microbial cell chassis, but eukaryotic platforms, such as Saccharomyces cerevisiae [2], Chinese hamster ovary (CHO) cell lines [3], and plants [4] are also well developed. However, with the ever-increasing need to provide environmentally sustainable solutions to manufacturing that are also economically viable for a multitude of products including low-value high-volume products, alternative platforms with diverse capabilities are required.…”

Key Challenges in Designing CHO Chassis Platforms

“…Developing strong modular mammalian expression platforms is an important R&D goal of the biopharmaceutical industry. As the development of stable CHO production cell lines is still a challenging and slow process, the establishment of pre-optimised chassis cell lines with plug-and-play modules that can be combined at will and as needed is a promising approach [215], even if it is more challenging than in simpler microbial systems.…”

“…Finally, simply speeding up cell line development by new and more efficient selection mechanisms or by the above mentioned use of targeted integration and of pre-optimised cell lines, will reduce the number of divisions that a cell line will undergo until ready for manufacturing, thus also reducing the accumulation of variants or mutations. Finally, in all these approaches the impact of the product gene and its specific requirements for translation, processing and secretion will have to be taken into account [215,234]. Steps towards creating a Chinese hamster ovary (CHO) chassis cell line.…”

Protein-specific signal peptides for mammalian vector engineering

“…Advances in the synthetic biology field, particularly in DNA sequence engineering, have significantly expanded opportunities to improve CHO cell expression vector design [10]. While some vector components have been widely studied, with associated availability of DNA part libraries [11], mammalian signal peptides remain relatively unexplored.…”