'Small volume-big problem': culturing Yarrowia lipolytica in high-throughput micro-formats.

With the current progress in the 'design' and 'build' stages of the 'design-build-test-learn' cycle, many synthetic biology projects become 'test-limited'. Advances in the parallelization of microbes cultivations are of great aid, however, for many species down-scaling leaves a metabolic footprint. Yarrowia lipolytica is one such demanding yeast species, for which scaling-down inevitably leads to perturbations in phenotype development. Strictly aerobic metabolism, propensity for filamentation and adhesion to hydrophobic surfaces, spontaneous flocculation, and high acidification of media are just several characteristics that make the transfer of the micro-scale protocols developed for the other microbial species very challenging in this case. It is well recognized that without additional 'personalized' optimization, either MTP-based or single-cell-based protocols are useless for accurate studies of Y. lipolytica phenotypes. This review summarizes the progress in the scaling-down and parallelization of Y. lipolytica cultures, highlighting the challenges that occur most frequently and strategies for their overcoming. The problem of Y. lipolytica cultures down-scaling is illustrated by calculating the costs of micro-cultivations, and determining the unintentionally introduced, thus uncontrolled, variables. The key research into culturing Y. lipolytica in various MTP formats and micro- and pico-bioreactors is discussed. Own recently developed and carefully pre-optimized high-throughput cultivation protocol is presented, alongside the details from the optimization stage. We hope that this work will serve as a practical guide for those working with Y. lipolytica high-throughput screens.

'Mother(Nature) knows best' - hijacking nature-designed transcriptional programs for enhancing stress resistance and protein production in Yarrowia lipolytica; presentation of YaliFunTome database.

BACKGROUND: In the era of rationally designed synthetic biology, heterologous metabolites production, and other counter-nature engineering of cellular metabolism, we took a step back and recalled that 'Mother(-Nature) knows best'. While still aiming at synthetic, non-natural outcomes of generating an 'over-production phenotype' we dug into the pre-designed transcriptional programs evolved in our host organism-Yarrowia lipolytica, hoping that some of these fine-tuned orchestrated programs could be hijacked and used. Having an interest in the practical outcomes of the research, we targeted industrially-relevant functionalities-stress resistance and enhanced synthesis of proteins, and gauged them over extensive experimental design's completion. RESULTS: Technically, the problem was addressed by screening a broad library of over 120 Y. lipolytica strains under 72 combinations of variables through a carefully pre-optimized high-throughput cultivation protocol, which enabled actual phenotype development. The abundance of the transcription program elicitors-transcription factors (TFs), was secured by their overexpression, while challenging the strains with the multitude of conditions was inflicted to impact their activation stratus. The data were subjected to mathematical modeling to increase their informativeness. The amount of the gathered data prompted us to present them in the form of a searchable catalog - the YaliFunTome database ( https://sparrow.up.poznan.pl/tsdatabase/ )-to facilitate the withdrawal of biological sense from numerical data. We succeeded in the identification of TFs that act as omni-boosters of protein synthesis, enhance resistance to limited oxygen availability, and improve protein synthesis capacity under inorganic nitrogen provision. CONCLUSIONS: All potential users are invited to browse YaliFunTome in the search for homologous TFs and the TF-driven phenotypes of interest.

Transcription factors enhancing synthesis of recombinant proteins and resistance to stress in Yarrowia lipolytica.

Resistance to environmental stress and synthesis of recombinant proteins (r-Prots) are both complex, strongly interconnected biological traits relying on orchestrated contribution of multiple genes. This, in turn, makes their engineering a challenging task. One of the possible strategies is to modify the operation of transcription factors (TFs) associated with these complex traits. The aim of this study was to examine the potential implications of selected five TFs (HSF1-YALI0E13948g, GZF1-YALI0D20482g, CRF1-YALI0B08206g, SKN7-YALI0D14520g, and YAP-like-YALI0D07744g) in stress resistance and/or r-Prot synthesis in Yarrowia lipolytica. The selected TFs were over-expressed or deleted (OE/KO) in a host strain synthesizing a reporter r-Prot. The strains were subjected to phenotype screening under different environmental conditions (pH, oxygen availability, temperature, and osmolality), and the obtained data processing was assisted by mathematical modeling. The results demonstrated that growth and the r-Prot yields under specific conditions can be significantly increased or decreased due to the TFs' engineering. Environmental factors "awakening" individual TFs were indicated, and their contribution was mathematically described. For example, OE of Yap-like TF was proven to alleviate growth retardation under high pH, while Gzf1 and Hsf1 were shown to serve as universal enhancers of r-Prot production in Y. lipolytica. On the other hand, KO of SKN7 and HSF1 disabled growth under hyperosmotic stress. This research demonstrates the usefulness of the TFs engineering approach in the manipulation of complex traits and evidences newly identified functions of the studied TFs. KEY POINTS: • Function and implication in complex traits of 5 TFs in Y. lipolytica were studied. • Gzf1 and Hsf1 are the universal r-Prots synthesis enhancers in Y. lipolytica. • Yap-like TF's activity is pH-dependent; Skn7 and Hsf1 act in osmostress response.

Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load.

While overproduction of recombinant secretory proteins (rs-Prots) triggers multiple changes in the physiology of the producer cell, exposure to suboptimal growth conditions may further increase that biological response. The environmental conditions may modulate the efficiency of both the rs-Prot gene transcription and translation but also the polypeptide folding. Insights into responses elicited by different environmental stresses on the rs-Prots synthesis and host yeast physiology might contribute to a better understanding of fundamental biology processes, thus providing some clues to further optimise bioprocesses. Herein, a series of batch cultivations of Yarrowia lipolytica strains differentially metabolically burdened by the rs-Prots overproduction have been conducted. Combinations of different stress factors, namely pH (3/7) and oxygen availability (kLa 28/110 h-1), have been considered for their impact on cell growth and morphology, substrate consumption, metabolic activity, genes expression, and secretion of the rs-Prots. Amongst others, our data demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth. Moreover, the observed decrease in rs-Prot production under adverse environmental conditions rather results from the emergence of a less-producing cell subpopulation than from the decrease of the synthetic capacity of the whole cell population.

Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling.

Nowadays considerable effort is being pursued towards development of consolidated microbial biocatalysts that will be able to utilize complex, non-pretreated substrates and produce valuable compounds. In such engineered microbes, synthesis of extracellular hydrolases may be fine-tuned by different approaches, like strength of promoter, type of secretory tag, and gene copy number. In this study, we investigated if organization of a multi-element expression cassette impacts the resultant Yarrowia lipolytica transformants' phenotype, presuming that different variants of the cassette are composed of the same regulatory elements and encode the same mature proteins. To this end, Y. lipolytica cells were transformed with expression cassettes bearing a pair of genes encoding exactly the same mature amylases, but fused to four different signal peptides (SP), and located interchangeably in either first or second position of a synthetic DNA construction. The resultant strains were tested for growth on raw and pretreated complex substrates of different plant origin for comprehensive examination of the strains' acquired characteristics. Optimized strain was tested in batch bioreactor cultivations for growth and lipids accumulation. Based on the conducted research, we concluded that the positional order of transcription units (TU) and the type of exploited SP affect final characteristics of the resultant consolidated biocatalyst strains, and thus could be considered as additional factors to be evaluated upon consolidated biocatalysts optimization. KEY POINTS: • Y. lipolytica growing on raw starch was constructed and tested on different substrates. • Impact of expression cassette design and SP on biocatalysts' phenotype was evidenced. • Consolidated biocatalyst process for lipids production from starch was conducted.

Impact of oxygen availability on heterologous geneexpression and polypeptide secretion dynamics in Yarrowia lipolytica-based protein production platforms.

Industrially relevant traits of Yarrowia lipolytica, like high growth rate, capacity to grow at high cell density or to synthesize biomolecules with high productivities, strongly rely on sufficient oxygen provision. Although the impact of oxygen availability (OA) on the physiology of Y. lipolytica has been already studied, its influence on recombinant protein (rProt) synthesis and secretion has been largely neglected to date. With the aim to fill this gap, a fluorescent reporter protein (yellow fluorescent protein [YFP]) was used herein as a proxy to follow simultaneously rProt synthesis and secretion in Y. lipolytica under different OAs. This study covers the analysis of the reporter gene expression through reverse transcription quantitative polymerase chain reaction, polypeptide synthesis and its retention-to-secretion ratio using flow cytometry and fluorymetry during shake flasks and bioreactor cultivations under different OA. The results gathered demonstrate that OA has a dramatic impact on the kinetics of intracellular and extracellular YFP accumulation. Higher rProt production and secretion were favoured under high OA, and were largely related to OA and not to cell growth. Our observations also suggest the existence of some upper limit of secretory protein accumulation inside the cells above which massive secretion is initiated. Moreover, at low OA, the first bottleneck in rProt synthesis occurs as early as at transcription level, which could results from a lower availability of transcriptional machinery elements. Finally, using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis.