"Fight-flight-or-freeze" - how Yarrowia lipolytica responds to stress at molecular level?

Yarrowia lipolytica is a popular yeast species employed in multiple biotechnological production processes. High resistance to extreme environmental conditions or metabolic burden triggered by synthetically forced over-synthesis of a target metabolite has its practical consequences. The proud status of an "industrial workhorse" that Y. lipolytica has gained is directly related to such a quality of this species. With the increasing amount of knowledge coming from detailed functional studies and comprehensive omics analyses, it is now possible to start painting the landscape of the molecular background behind stress response and adaptation in Y. lipolytica. This review summarizes the current state-of-art of a global effort in revealing how Y. lipolytica responds to both environmental threats and the intrinsic burden caused by the overproduction of recombinant secretory proteins at the molecular level. Detailed lists of genes, proteins, molecules, and biological processes deregulated upon exposure to external stress factors or affected by over-synthesis of heterologous proteins are provided. Specificities and universalities of Y. lipolytica cellular response to different extrinsic and intrinsic threats are highlighted. KEY POINTS: • Y. lipolytica as an industrial workhorse is subjected to multiple stress factors. • Cellular responses together with involved genes, proteins, and molecules are reviewed. • Native stress response mechanisms are studied and inspire engineering strategies.

Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein.

BACKGROUND: Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. RESULTS: We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture. CONCLUSIONS: We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.

Overexpressing target helper genes enhances secretion and glycosylation of recombinant proteins in Pichia pastoris under simulated microgravity.

In this study, the potential helper genes were identified through the data analysis of transcriptomic and proteomic profiling in recombinant Pichia pastoris cultured under simulated microgravity (SMG). Co-expressing of four genes PRX1, YAP1, AHA1, and YPT6, involved in the oxidative stress response and protein folding, exhibited promising helper factor effects on the recombinant protein yields in engineered P. pastoris, respectively. When two of the above genes were co-expressed simultaneously, ß-glucuronidase (PGUS) specific activity was further increased by 30.3-50.6 % comparing with that of single helper gene, particularly when the oxidative stress response and protein folding genes were both present in the combinations. In addition, co-expressing co-chaperone AHA1 and transcription factor YAP1 not only enhanced PGUS secretion, but also affected its glycosylation. Thus, through deep "omics" analysis of SMG effects, our results provided combined impact of new helper factors to improve the efficacy of recombinant protein secretion and glycosylation in engineered P. pastoris.

Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans.

Proteins secreted by Gram-positive bacteria are released into the culture medium with the obvious benefit that they usually retain their native conformation. This property makes these host cells potentially interesting for the production of recombinant proteins, as one can take full profit of established protocols for the purification of active proteins. Several state-of-the-art strategies to increase the yield of the secreted proteins will be discussed, using Streptomyces lividans as an example and compared with approaches used in some other host cells. It will be shown that approaches such as increasing expression and translation levels, choice of secretion pathway and modulation of proteins thereof, avoiding stress responses by changing expression levels of specific (stress) proteins, can be helpful to boost production yield. In addition, the potential of multi-omics approaches as a tool to understand the genetic background and metabolic fluxes in the host cell and to seek for new targets for strain and protein secretion improvement is discussed. It will be shown that S. lividans, along with other Gram-positive host cells, certainly plays a role as a production host for recombinant proteins in an economically viable way. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs.

Maternal secretion of recombinant proteins into chicken eggs may provide a viable approach for pharmaceutical production but remains limited by poor secretion efficiency through the membrane of oviduct cells, despite high expression levels. Here, we used site-specific integration of an EGFP fused to the OVAL gene by a rigid linker, (EAAAK)3, at the endogenous ovalbumin locus in chicken primordial germ cells to generate OVAL-E3-EGFP transgenic chickens, with transgenic chickens expressing CMV immediate enhancer/ß-actin-driven EGFP (CAG-EGFP) as a non-secreted control. In OVAL-E3-EGFP chickens, EGFP protein produced in maternal oviducts accumulates to high levels in eggs, but not in eggs of CAG-EGFP chickens. These results indicated that the secretion of foreign proteins can be substantially increased through fusion to the highly secreted endogenous ovalbumin. This study describes a basis for high yield recombinant protein expression in chicken eggs, enabling rapid and scalable production of numerous pharmaceutical proteins or metabolites.

Engineered Fusion Proteins for Efficient Protein Secretion and Purification of a Human Growth Factor from the Green Microalga Chlamydomonas reinhardtii.

Light-driven recombinant protein (RP) production in eukaryotic microalgae offers a sustainable alternative to other established cell-culture systems. RP production via secretion into the culture medium enables simple product separation from the cells adding a layer of process value in addition to the algal biomass, which can be separately harvested. For the model microalga Chlamydomonas reinhardtii, a broad range of molecular tools have been established to enable heterologous gene expression; however, low RP production levels and unreliable purification from secretion concepts have been reported. Domesticated C. reinhardtii strains used for genetic engineering are often cell-wall deficient. These strains nevertheless secrete cell-wall components such as insoluble (hydroxy)proline-rich glycoproteins into the culture media, which hinder downstream purification processes. Here, we attempted to overcome limitations in secretion titers and improve protein purification by combining fusion partners that enhance RP secretion and enable alternative aqueous two-phase (ATPS) RP extraction from the culture medium. Protein fusions were strategically designed to contain a stably folded peptide, which enhanced secretion capacities and gave insights into (some) regulatory mechanisms responsible for this process in the algal host. The elevated protein titers mediated by this fusion were then successfully applied in combination with a fungal hydrophobin tag, which enabled protein purification from the complex microalgal extracellular environment by ATPS, to yield functional recombinant human epidermal growth factor (hEGF) from the algal host.

Investigating the dynamics of recombinant protein secretion from a microalgal host.

Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.

In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

Protein production in yeasts is related to the specific growth rate µ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at µ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing µ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher µ. Mating and sporulation genes were most active at intermediate µ of 0.05 and 0.075 h(-1) . At very slow growth (µ = 0.015 h(-1) ) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high µ positively affects specific protein secretion rates by acting on multiple cellular processes.