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Abstract
Production of heterologous proteins, especially biopharmaceuticals and industrial enzymes, in living cell factories consumes cellular resources. Such resources are reallocated from normal cellular processes toward production of the heterologous protein that is often of no benefit to the host cell. This competition for resources is a burden to host cells, has a negative impact on cell fitness, and may consequently trigger stress responses. Importantly, this often causes a reduction in final protein titers. Engineering strategies to generate more burden resilient production strains offer sustainable opportunities to increase production and profitability for this growing billion-dollar global industry. We review recently reported impacts of burden derived from resource competition in two commonly used protein-producing yeast cell factories: Saccharomyces cerevisiae and Komagataella phaffii (syn. Pichia pastoris). We dissect possible sources of burden in these organisms, from aspects related to genetic engineering to protein translation and export of soluble protein. We also summarize advances as well as challenges for cell factory design to mitigate burden and increase overall heterologous protein production from metabolic engineering, systems biology, and synthetic biology perspectives. Lastly, future profiling and engineering strategies are highlighted that may lead to constructing robust burden-resistant cell factories. This includes incorporation of systems-level data into mathematical models for rational design and engineering dynamical regulation circuits in production strains.
Highlights
Biomanufacturing of heterologous proteins from genetically engineered cell factories is a growing industry
If codon optimality is around the average tRNA adaptation index score [tAI score developed by dos Reis et al (2004)] of a gene in the specific host organism−0.37 in S. cerevisiae (Eguchi et al, 2018); 0.47 in K. phaffii (Xu et al, 2021)—the translation elongation rate is often not high enough for an overexpressed protein to reach the burden-limit triggering growth defects
While co-expressing single genes such as HRD1 or Homologous to Atf/Creb1 (HAC1) alleviates some of the cellular stress induced by protein folding burden, yeast can adapt to prolonged endoplasmic reticulum (ER) stress induced by folding burden through chromosomal duplication (Beaupere et al, 2018; Beaupere and Labunskyy, 2019)
Summary
Biomanufacturing of heterologous proteins from genetically engineered cell factories is a growing industry The first such biopharmaceutical product was approved by the FDA four decades ago (U.S Food Drug Administration, 1982; Nielsen, 2013), and over 300 different biopharmaceuticals are available (Walsh, 2018). Yeast cells have evolved to readily adapt to fluctuating environmental conditions, and may down-regulate protein production to match intracellular demands imposed by various external and internal conditions and stresses (Gasch, 2003). We will focus on burden conferred by heterologous soluble protein production in yeast cell factories from recombinant DNA replication through to the final secreted product. Many concepts discussed here will be relevant to burden in other production organisms, the unique challenges presented by non-yeast systems remain outside the scope of this review
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