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Recent advances in the synthesis of metal nanoparticles (MeNPs), and more specifically gold nanoparticles (AuNPs), have led to tremendous expansion of their potential applications in different fields, ranging from healthcare research to microelectronics and food packaging. The properties of functionalised MeNPs can be fine-tuned depending on their final application, and subsequently, these properties can strongly modulate their biological effects. In this review, we will firstly focus on the impact of MeNP characteristics (particularly of gold nanoparticles, AuNPs) such as shape, size, and aggregation on their biological activities. Moreover, we will detail different in vitro and in vivo assays to be performed when cytotoxicity and biocompatibility must be assessed. Due to the complex nature of nanomaterials, conflicting studies have led to different views on their safety, and it is clear that the definition of a standard biosafety label for AuNPs is difficult. In fact, AuNPs’ biocompatibility is strongly affected by the nanoparticles’ intrinsic characteristics, biological target, and methodology employed to evaluate their toxicity. In the last part of this review, the current legislation and requirements established by regulatory authorities, defining the main guidelines and standards to characterise new nanomaterials, will also be discussed, as this aspect has not been reviewed recently. It is clear that the lack of well-established safety regulations based on reliable, robust, and universal methodologies has hampered the development of MeNP applications in the healthcare field. Henceforth, the international community must make an effort to adopt specific and standard protocols for characterisation of these products.

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at a high level, and to secrete protein in large amounts. In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Though the construction of efficient cell factories for recombinant protein synthesis is important, the development of an efficient process for recombinant protein production in a bioreactor constitutes an equally vital aspect. Indeed, a sports car cannot drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering.

Background The methylotrophic yeast Komagataella phaffii is a premier host for recombinant protein (rProt) production, which traditionally relies on methanol induction of the alcohol oxidase 1 promoter (P AOX1 ). However, the flammability and associated industrial limitations of methanol have motivated the search for methanol-free induction systems. We recently demonstrated that disruption of formate dehydrogenase gene (FDH) in K. phaffii allows endogenous formate derived from tetrahydrofolate (THF)-mediated C1 metabolism to induce P AOX1 without the addition of external inducers. Therefore, we hypothesized that increasing intracellular formate production by enhancing serine biosynthesis could further improve promoter induction and rProt productivity. Results Overexpression of SER3 , encoding 3-phosphoglycerate dehydrogenase, the rate-limiting enzyme in serine synthesis, significantly increased P AOX1 -driven expression of an intracellular reporter protein (eGFP) and secreted glucose oxidase (Gox) from Aspergillus niger without compromising cell fitness. Enhanced formate accumulation and stronger P AOX1 induction were observed in both microbioreactor and bioreactor cultivations using sorbitol or glycerol-sorbitol mixtures. In GOX- m SER3 strain grown in bioreactor, SER3 overexpression led to a 30% increase in specific Gox activity compared with that of the parental FdhKO strain. Conclusions This study provides a cost-effective metabolic engineering strategy for methanol-free, self-inducible expression systems in K. phaffii based on P AOX1 , enabling safer and more sustainable industrial rProt production.

Background Functional sugar alcohols have been widely used in the food, medicine, and pharmaceutical industries for their unique properties. Among these, erythritol is a zero calories sweetener produced by the yeast Yarrowia lipolytica. However, in wild-type strains, erythritol is produced with low productivity and yield and only under high osmotic pressure together with other undesired polyols, such as mannitol or d-arabitol. The yeast is also able to catabolize erythritol in non-stressing conditions. Results Herein, Y. lipolytica has been metabolically engineered to increase erythritol production titer, yield, and productivity from glucose. This consisted of the disruption of anabolic pathways for mannitol and d-arabitol together with the erythritol catabolic pathway. Genes ZWF1 and GND encoding, respectively, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also constitutively expressed in regenerating the NADPH2 consumed during erythritol synthesis. Finally, the gene RSP5 gene from Saccharomyces cerevisiae encoding ubiquitin ligase was overexpressed to improve cell thermoresistance. The resulting strain HCY118 is impaired in mannitol or d-arabitol production and erythritol consumption. It can grow well up to 35 °C and retain an efficient erythritol production capacity at 33 °C. The yield, production, and productivity reached 0.63 g/g, 190 g/L, and 1.97 g/L·h in 2-L flasks, and increased to 0.65 g/g, 196 g/L, and 2.51 g/L·h in 30-m3 fermentor, respectively, which has economical practical importance. Conclusion The strategy developed herein yielded an engineered Y. lipolytica strain with enhanced thermoresistance and NADPH supply, resulting in a higher ability to produce erythritol, but not mannitol or d-arabitol from glucose. This is of interest for process development since it will reduce the cost of bioreactor cooling and erythritol purification.

In Komagataella phaffii (Pichia pastoris), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self‐induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF‐C1 metabolism, and it cannot be converted into carbon dioxide in a FdhKO strain. Under non‐repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF‐C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for a FdhKO strain on sorbitol and a non‐disrupted strain on sorbitol‐methanol. Considering a K. Phaffii FdhKO strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol‐free processes in K. phaffii. Formate from THF‐C1 metabolism induces pAOX1 in FDH formate dehydrogenase‐deficient P. pastoris under derepressed conditions.

Background Erythritol is a four-carbon sugar alcohol with sweetening properties that is used by the agro-food industry as a food additive. In the yeast Yarrowia lipolytica , the last step of erythritol synthesis involves the reduction of erythrose by specific erythrose reductase(s). In the earlier report, an erythrose reductase gene ( YALI0F18590g ) from erythritol-producing yeast Y. lipolytica MK1 was identified (Janek et al. in Microb Cell Fact 16:118, 2017 ). However, deletion of the gene in Y. lipolytica MK1 only resulted in some lower erythritol production but the erythritol synthesis process was still maintained, indicating that other erythrose reductase gene(s) might exist in the genome of Y. lipolytica . Results In this study, we have isolated genes g141.t1 ( YALI0D07634g ) and g3023.t1 ( YALI0C13508g ) encoding two novel erythrose reductases (ER). The biochemical characterization of the purified enzymes showed that they have a strong affinity for erythrose. Deletion of the two ER genes plus g801.t1 ( YALI0F18590g ) did not prevent erythritol synthesis, suggesting that other ER or ER-like enzymes remain to be discovered in this yeast. Overexpression of the newly isolated two genes (ER10 or ER25) led to an average 14.7% higher erythritol yield and 31.2% higher productivity compared to the wild-type strain. Finally, engineering NADPH cofactor metabolism by overexpression of genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, allowed a 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain. The best of our constructed strains produced an erythritol titer of 190 g/L in baffled flasks using glucose as main carbon source. Conclusions Our results highlight that in the Y. lipolytica genome several genes encode enzymes able to reduce erythrose into erythritol. The catalytic properties of these enzymes and their cofactor dependency are different from that of already known erythrose reductase of Y. lipolytica . Constitutive expression of the newly isolated genes and engineering of NADPH cofactor metabolism led to an increase in erythritol titer. Development of fermentation strategies will allow further improvement of this productivity in the future.

BackgroundMicrobial lipid production using renewable feedstock shows great promise for the biodiesel industry.ResultsIn this study, the ability of a lipid-engineered Yarrowia lipolytica strain JMY4086 to produce lipids using molasses and crude glycerol under different oxygenation conditions and at different inoculum densities was evaluated in fed-batch cultures. The greatest lipid content, 31% of CDW, was obtained using a low-density inoculum, a constant agitation rate of 800 rpm, and an oxygenation rate of 1.5 L/min. When the strain was cultured for 450 h in a chemostat containing a nitrogen-limited medium (dilution rate of 0.01 h−1; 250 g/L crude glycerol), volumetric lipid productivity was 0.43 g/L/h and biomass yield was 60 g CDW/L. The coefficient of lipid yield to glycerol consumption (YL/gly) and the coefficient of lipid yield to biomass yield (YL/X) were equal to 0.1 and 0.4, respectively.ConclusionsThese results indicate that lipids may be produced using renewable feedstock, thus providing a means of decreasing the cost of biodiesel production. Furthermore, using molasses for biomass production and recycling glycerol from the biodiesel industry should allow biolipids to be sustainably produced.

ABSTRACT The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the ‘conventional’ yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications. Non-conventional features render the yeast K. phaffii an attractive model organism and an efficient host for biotechnology applications.

In Komagataella phaffii, the use of formate as an AOX1 promoter (PAOX1) inducer in combination with sorbitol, a non‐repressive carbon source, has emerged as a promising alternative to methanol‐based expression systems. Recently, we demonstrated that formate derived from the tetrahydrofolate‐mediated one‐carbon (THF‐C1) metabolism accumulates in K. phaffii cells deficient in formate dehydrogenase (FdhKO) when grown in sorbitol‐based methanol‐free medium. Using the lipase CalB from Candida antarctica as a model protein, we observed that recombinant protein (rProt) productivity in an FdhKO strain grown on sorbitol was comparable to that of an Fdh‐proficient strain grown on methanol. However, sorbitol is inefficiently metabolised in K. phaffii, leading to a low growth rate and potentially limiting rProt productivity due to insufficient energy and carbon supply. Here, we increased the sorbitol uptake rate, and thus improved sorbitol metabolism, by overexpressing the gene encoding sorbitol dehydrogenase (SOR1) in an FdhKO strain. Our results demonstrate that while increased sorbitol metabolism promotes biomass formation, it reduces PAOX1 induction, as evidenced by lower formate accumulation and decreased rProt productivity, both for intracellular eGFP and secreted proteins namely CalB lipase and glucose oxidase (Gox) from Aspergillus niger in SOR1‐overexpressing strains. Additionally, oxygen availability for cells influences these dynamics, with lower oxygen transfer favouring higher PAOX1 induction due to increased formate accumulation in an FdhKO strain. Our data also suggest that at low oxygen transfer and low sorbitol uptake rate, the proportion of cells in an induced state increased significantly. This work provides valuable insights into the interplay between sorbitol metabolism and oxygen transfer conditions, contributing to the development of improved recombinant protein production strategies in K. phaffii. Sorbitol uptake and oxygen transfer modulate pAOX1 induction by formate in Komagataella phaffii lacking formate dehydrogenase under methanol‐free conditions.

Sugar alcohols and organic acids that derive from the metabolism of certain microorganisms have a panoply of applications in agro-food, chemical and pharmaceutical industries. The main challenge in their production is to reach a productivity threshold that allow the process to be profitable. This relies on the construction of efficient cell factories by metabolic engineering and on the development of low-cost production processes by using industrial wastes or cheap and widely available raw materials as feedstock. The non-conventional yeast Yarrowia lipolytica has emerged recently as a potential producer of such metabolites owing its low nutritive requirements, its ability to grow at high cell densities in a bioreactor and ease of genome edition. This review will focus on current knowledge on the synthesis of the most important sugar alcohols and organic acids in Y. lipolytica.