Burden Imposed by Heterologous Protein Production in Two Major Industrial Yeast Cell Factories: Identifying Sources and Mitigation Strategies

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.

Keywords: biotechnology; burden; heterologous protein production; metabolism; strain engineering; yeast.

Figure 1

Burden-triggering bottlenecks in yeast cell factories during heterologous protein synthesis and secretion. Schematic representation of yeast cell factory engineered to synthesize and secrete a heterologous protein of interest (POI), encoded by a gene of interest (GOI). The GOI is transcribed into messenger RNA (mRNA), later translated by ribosomes into a peptide that is translocated into endoplasmic reticulum (ER), where it is folded and modified. Via anterograde transport, the POI reaches the golgi complex for further modifications and secretion signal cleavage. Via vesicle exocytosis, the POI is secreted to the extracellular medium. Meanwhile, heterologous protein production is fueled by metabolic pathways such as glycolysis, pentose phosphate pathway (PPP), the tricarboxylic acid cycle (TCA), and the electron transport chain (ETC) which deliver energy (ATP), redox cofactors (NADPH, NADH), nucleotides, and amino acids required for heterologous protein production. Different engineering targets (ETs) that have been highlighted in literature to mitigate the burden that heterologous protein production imposes on the host cell are marked with a target symbol. ET1: Gene dosage, promoter strength, plasmid vs. genome integration. ET2: Codon optimization, co- or post-translational translocation. ET3: Tuning genes involved in unfolded protein response, oxidative stress response, ER-associated protein degradation pathways. ET4: Direct POI for secretion or express intracellularly. ET5: Process engineering by adding certain amino acids to the medium, changing the medium or carbon source. ET6: Redirecting metabolic fluxes by tuning relevant target genes. Synthetic circuit engineering for dynamic regulation may be simultaneously aimed at any combination of ETs 1–4 and/or 6.