'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.

Molecular background of HAC1-driven improvement in the secretion of recombinant protein in Yarrowia lipolytica based on comparative transcriptomics.

While the unfolded protein response (UPR) and its major regulator - transcription factor Hac1 are well-conserved across Eukarya, species-specific variations are repeatedly reported. Here we investigated molecular mechanisms by which co-over-expression of HAC1 improves secretion of a recombinant protein (r-Prot) in Yarrowia lipolytica, using comparative transcriptomics. Co-over-expression of HAC1 caused an >2-fold increase in secreted r-Prot, but its intracellular levels were decreased. The unconventional splicing rate of the HAC1 mRNA was counted through transcript sequencing. Multiple biological processes were affected in the HAC1-and-r-Prot co-over-expressing strain, including ribosome biogenesis, nuclear and mitochondrial events, cell cycle arrest, attenuation of gene expression by RNA polymerase III and II, as well as modulation of proteolysis and RNA metabolism; but whether the HAC1 co-over-expression/induction was the actual causative agent for these changes, was not always clear. We settled that the expression of the "conventional" HAC1 targets (KAR2 and PDI1) is not affected by its over-expression.

Multiple region high resolution melting-based method for accurate differentiation of food-derived yeasts at species level resolution.

Yeasts comprise a divergent group of microorganisms playing an important role in foods production. To monitor the production processes, validate authenticity and safety of foods, reliable methods of yeast identification and differentiation are needed. Nowadays, traditional PCR/sequencing-based methods are replaced by rapid techniques not requiring post-PCR processing. In the present study, we developed a three region-based qPCR-HRM protocol, for rapid differentiation of yeast species isolated from food products. The three targeted fragments (26S rDNA, 18S rDNA, ITS) were carefully analyzed to dock the primers at inter-species conservative regions, flanking polymorphic regions of ∼200 bp. Thirty eight yeast strains were used as a training material. The collection of yeast spanned Pichia, Clavispora, Candida, Yarrowia, Kluyveromyces, Saccharomyces, and Wickerhamomyces genera. MALDI-TOF mass spectrometry and conventional rDNA sequencing were used for validation. Conducted studies demonstrated that when used individually, each of the three regions analyzed by qPCR-HRM possessed its own yeast species-specific limitations, sometimes leading to inaccurate taxonomic classification. On the other hand, simultaneous analysis of the three proposed regions resulted in rapid and adequate differentiation of all the yeast strains at species-level resolution.

A unique, newly discovered four-member protein family involved in extracellular fatty acid binding in Yarrowia lipolytica.

BACKGROUND: Yarrowia lipolytica, a nonconventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacities. Y. lipolytica is used as a chassis for the production of usual and unusual lipids and lipid derivatives. While the genes involved in the intracellular transport and activation of fatty acids in different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified thus far. In this study, we identified secreted proteins involved in extracellular fatty acid binding. RESULTS: Recent analysis of the Y. lipolytica secretome led to the identification of a multigene family that encodes four secreted proteins, preliminarily named UP1 to UP4. These proteins were efficiently overexpressed individually in wild-type and multideletant strain (Q4: Δup1Δup2Δup3Δup4) backgrounds. Phenotypic analysis demonstrated the involvement of these proteins in the binding of extracellular fatty acids. Additionally, gene deletion and overexpression prevented and promoted sensitivity to octanoic acid (C8) toxicity, respectively. The results suggested binding is dependent on aliphatic chain length and fatty acid concentration. 3D structure modeling supports the proteins' role in fatty acid assimilation at the molecular level. CONCLUSIONS: We discovered a family of extracellular-fatty-acid-binding proteins in Y. lipolytica and have proposed to name its members eFbp1 to eFbp4. The exact mode of eFbps action remains to be deciphered individually and synergistically; nevertheless, it is expected that the proteins will have applications in lipid biotechnology, such as improving fatty acid production and/or bioconversion.

"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.

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.

Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica-molecular background revealed by quantitative proteomics.

In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg-1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo-keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided. KEY POINTS: • Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica. • Enhanced abundance of HSPs and osmolytes is overweighted by limited translation. • Global proteome under Osm, Temp and Osm Temp treatments was studied.

Epigenetic Response of Yarrowia lipolytica to Stress: Tracking Methylation Level and Search for Methylation Patterns via Whole-Genome Sequencing.

DNA methylation is a common, but not universal, epigenetic modification that plays an important role in multiple cellular processes. While definitely settled for numerous plant, mammalian, and bacterial species, the genome methylation in different fungal species, including widely studied and industrially-relevant yeast species, Yarrowia lipolytica, is still a matter of debate. In this paper, we report a differential DNA methylation level in the genome of Y. lipolytica subjected to sequential subculturing and to heat stress conditions. To this end, we adopted repeated batch bioreactor cultivations of Y. lipolytica subjected to thermal stress in specific time intervals. To analyze the variation in DNA methylation between stressed and control cultures, we (a) quantified the global DNA methylation status using an immuno-assay, and (b) studied DNA methylation patterns through whole-genome sequencing. Primarily, we demonstrated that 5 mC modification can be detected using a commercial immuno-assay, and that the modifications are present in Y. lipolytica's genome at ~0.5% 5 mC frequency. On the other hand, we did not observe any changes in the epigenetic response of Y. lipolytica to heat shock (HS) treatment. Interestingly, we identified a general phenomenon of decreased 5 mC level in Y. lipolytica's genome in the stationary phase of growth, when compared to a late-exponential epigenome. While this study provides an insight into the subculturing stress response and adaptation to the stress at epigenetic level by Y. lipolytica, it also leaves an open question of inability to detect any genomic DNA methylation level (either in CpG context or context-less) through whole-genome sequencing. The results of ONT sequencing, suggesting that 5 mC modification is either rare or non-existent in Y. lipolytica genome, are contradicted with the results of the immunoassay.

Secretory helpers for enhanced production of heterologous proteins in Yarrowia lipolytica.

Depending on the suboptimal factor, the target protein secretion can be over 1000-fold below the theoretical maximum. The bottlenecks may be alleviated by co-overexpression of "secretory helpers" (SHs). Here we proposed twelve SHs, functionally spanning the whole transcription-translation-translocation-folding-maturation-excretion pipeline. The genes were co-transformed with an easy-to-track reporter, and tested less than two temperatures. Our results indicated a clear distinction in the effects triggered by SHs involved in either synthesis or trafficking of the heterologous polypeptides. For superior operation of synthesis-related SHs, namely RPL3, SSA5 and SSA8, the secretory pathway's capacity must be released by applying decreased temperature (25 °C). The other SHs considered (e.g. SSO1, CWP11) did not give such spectacular results in the amounts of the target heterologous polypeptide, but allowed to maintain secretory capacity under unfavorable thermal conditions. This study provides generalizable guidelines for cloning/culturing strategies aiming at enhancement of heterologous protein secretion in Y. lipolytica.

Thermal treatment improves a process of crude glycerol valorization for the production of a heterologous enzyme by Yarrowia lipolytica.

Valorization of crude glycerol requires a potent bifunctional biocatalyst, such as Yarrowia lipolytica, capable of high-density growth on this substrate, and having i.a. high propensity for heterologous protein synthesis. Increasing evidence suggests that controlled administration of stress, i.a. thermal treatment, has a positive impact on bioprocess performance. In this study, we systematically adjusted thermal treatment conditions (20 to 42 °C) in order to maximize heterologous protein production by Y. lipolytica growing in crude glycerol-based medium. Our results showed nearly 30% enhancement in the enzyme production triggered by temporary exposure to decreased temperature. Here developed mathematical model indicated optimal treatment conditions (20 °C, 153') that were later applied to a process with biodiesel-derived glycerol and technical substrates. Techno-economic analysis of a pilot-scale-waste-free process was conducted. Quantitative description of the associated costs and economic gain due to exploitation of industrial substrates, as well as indication of current bottlenecks of the process, are also provided.

Global transcriptome profiling reveals genes responding to overproduction of a small secretory, a high cysteine- and a high glycosylation-bearing protein in Yarrowia lipolytica.

Investigation of the yeast cell's response to recombinant secretory protein (rs-Prot) overproduction is relevant for both basic and applied research. Imbalance, overloading or stress within this process impacts the whole cell. In the present study, by using steady-state cultures and transcriptomics, we investigated the cellular response of Yarrowia lipolytica challenged with high-level expression of genes encoding proteins with significantly different biochemical characteristics: a small protein retained within the cell i) or secreted ii), a medium size secretory protein with a high number of disulfide bonds iii), or glycosylation sites iv). Extensive analysis of omics data, supported by careful manual curation, led to some anticipated observations on oxidative and unfolded protein stress (CTT1, PXMP2/4, HAC1), glycosylation (ALGs, KTRs, MNTs, MNNs), folding and translocation (SSAs, SSEs) but also generated new exciting knowledge on non-conventional protein secretion (NCE102), transcriptional regulators (FLO11, MHY1, D01353 g, RSFA, E23925g or MAF1), vacuolar proteolysis targets in Y. lipolytica (ATGs, VPSs, HSE1, PRB1, PRC1, PEP4) or growth arrest (CLN1) upon rs-Prots overproduction.

Hydrolytic secretome engineering in Yarrowia lipolytica for consolidated bioprocessing on polysaccharide resources: review on starch, cellulose, xylan, and inulin.

Consolidated bioprocessing (CBP) featuring concomitant hydrolysis of renewable substrates and microbial conversion into value-added biomolecules is considered to bring substantial benefits to the overall process efficiency. The biggest challenge in developing an economically feasible CBP process is identification of bifunctional biocatalyst merging the ability to utilize the substrate and convert it to value-added product with high efficiency. Yarrowia lipolytica is known for its exceptional performance in hydrophobic substrates assimilation and storage. On the other hand, its capacity to grow on plant-derived biomass is strongly limited. Still, its high potential to simultaneously overproduce several secretory proteins makes Y. lipolytica a platform of choice for expanding its substrate range to complex polysaccharides by engineering its hydrolytic secretome. This review provides an overview of different genetic engineering strategies advancing development of Y. lipolytica strains able to grow on the following four complex polysaccharides: starch, cellulose, xylan, and inulin. Much attention has been paid to genome mining studies uncovering native potential of this species to assimilate untypical sugars, as in many cases it turns out that dormant pathways are present in Y. lipolytica's genome. In addition, the magnitude of the economic gain by CBP processing is here discussed and supported with adequate calculations based on simulated process models. KEY POINTS: • The mini-review updates the knowledge on polysaccharide-utilizing Yarrowia lipolytica. • Insight into molecular bases founding new biochemical qualities is provided. • Model industrial processes were simulated and the associated costs were calculated.

A new set of reference genes for comparative gene expression analyses in Yarrowia lipolytica.

Accurate quantitation of gene expression levels require sensitive, precise and reproducible measurements of specific transcripts. Normalization to a reference gene is the most common practice to minimize the impact of the uncontrolled variation. The fundamental prerequisite for an accurate reference gene is to be stably expressed amongst all the samples included in the analysis. In the present study we aimed to assess the expression level and stability of a panel of 21 genes in Yarrowia lipolytica throughout varying conditions, covering composition of the culturing medium, growth phase and strain-wild type and recombinant burdened with heterologous protein overexpression. The panel of the selected candidate genes covered those essential for growth and maintenance of metabolism and homologs of commonly used internal references in RT-qPCR. The candidate genes expression level and stability were assessed and the data were processed using dedicated computational tools (geNorm and NormFinder). The results obtained here indicated genes unaffected by the burden of overexpression (TEF1, TPI1, UBC2, SRPN2, ALG9-like, RYL1) or by the culture medium used (ACT1, TPI1, UBC2, SEC61, ODC, CLA4, FKS1, TPS1), as well as those the least (SSDH, ODC, GPD) and the most (SEC62, TPI1, IPP1) suitable for normalization of RT-qPCR data in Y. lipolytica.

Impact of overproduced heterologous protein characteristics on physiological response in Yarrowia lipolytica steady-state-maintained continuous cultures.

Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27-65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein's post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed.Key Points• Eight expression systems, producing different reporter proteins were analyzed.• The cells were maintained in steady-state by continuous chemostat culturing.• Protein- and promoter-specific effects were observed.

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.

Production of Raw Starch-Digesting Amylolytic Preparation in Yarrowia lipolytica and Its Application in Biotechnological Synthesis of Lactic Acid and Ethanol.

Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.

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.

Feeding strategy impacts heterologous protein production in Yarrowia lipolytica fed-batch cultures-Insight into the role of osmolarity.

Fed-batch cultivation is the preferred bioprocessing strategy applied in microbial production of proteins. Feeding strategy is crucial parameters to be optimized upon development of a fed-batch process. In this study, we investigated impact of different feeding strategies on production of recombinant enzymatic protein in Yarrowia lipolytica cultures. From amongst tested strategies, comprising intermittent and continuous feedings, also in cascade with respiratory factors, intermittent feeding executed after complete exhaustion of glycerol from the medium, with moderate amplitude of osmolarity, was the most beneficial in terms of the secretory enzyme amount, its volumetric productivity and specific activity. Because adopted feeding strategies strongly modulated osmolarity of the cultures, the effect of osmotic pressure on production of the target heterologous protein was investigated in a series of batch cultivations with addition of osmoactive compounds (NaCl, sorbitol, sucrose, and glycerol) at different concentrations. Although obvious promoting effect of the osmoactive substances on the enzyme production was clear, no straightforward correlation between the medium osmolarity and the target enzyme's specific activity could be observed. These results suggest that not only the level of osmolarity but also chemical character of the osmoactive compound have both important impact on the production of secretory proteins in Y. lipolytica cultures.