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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.

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.

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.

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.

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.

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.

Background The oleaginous yeast Yarrowia lipolytica is increasingly used as alternative cell factory for the production of recombinant proteins. At present, several promoters with different strengths have been developed based either on the constitutive pTEF promoter or on oleic acid inducible promoters such as pPOX2 and pLIP2. Although these promoters are highly efficient, there is still a lack of versatile inducible promoters for gene expression in Y. lipolytica. Results We have isolated and characterized the promoter of the EYK1 gene coding for an erythrulose kinase. pEYK1 induction was found to be impaired in media supplemented with glucose and glycerol, while the presence of erythritol and erythrulose strongly increased the promoter induction level. Promoter characterization and mutagenesis allowed the identification of the upstream activating sequence UAS1 EYK1 . New hybrid promoters containing tandem repeats of either UAS1 XPR2 or UAS1 EYK1 were developed showing higher expression levels than the native pEYK1 promoter. Furthermore, promoter strength was improved in a strain carrying a deletion in the EYK1 gene, allowing thus the utilization of erythritol and erythrulose as free inducer. Conclusions Novel tunable and regulated promoters with applications in the field of heterologous protein production, metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in the yeast Y. lipolytica .

Oleaginous microorganisms, such as Yarrowia lipolytica, accumulate lipids that can have interesting applications in food biotechnology or the synthesis of biodiesel. Y. lipolytica yeast can have many advantages such as wide substrate range usage and robustness to extreme conditions, while under several culture conditions it can produce high lipid productivity. Based on this assumption, in this study, 12 different Yarrowia lipolytica strains were used to investigate microbial lipid production using a glucose-based medium under nitrogen-limited conditions in shake-flask cultivations. Twelve wild-type or mutant strains of Yarrowia lipolytica which were newly isolated or belonged to official culture collections were tested, and moderate lipid quantities (up to 1.30 g/L) were produced; in many instances, nitrogen limitation led to citric acid production in the medium. Lipids were mainly composed of C16 and C18 fatty acids. Most of the fatty acids of the microbial lipid were unsaturated and corresponded mainly to oleic, palmitic and linoleic acids. Linolenic acid (C18:3) was produced in significant quantities (between 10% and 20%, wt/wt of dry cell weight (DCW)) by strains H917 and Po1dL.

Background Phytopathogenic fungi affecting crop and post-harvested vegetables are a major threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management. Results The genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42. Conclusion The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents.

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.