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Industrial waste streams can be of diverse nature and many industrial waste streams have a biodegradable fraction that can be considered a sustainable source of biomass. Waste valorization via production of energy or commodity chemicals would result in decreasing the volume of waste that needs to be managed, in minimizing the environmental impact and in generating added value through the synthesis of a wide range of products. In this work we present two examples of industrial waste valorization based on the use of yeast cell factories. The first case demonstrates the possibility to valorize cotton fabric waste from an apparel textile manufacturing company. In particular, the use of the glucose-rich stream obtained upon cotton pretreatment has been used to produce lactic acid by the recombinant Saccharomyces cerevisiae CEN.PK m850 (1) with a maximum yield of 82.7% on cotton-derived glucose. The second case we present is based on the use of the glycerol-rich waste stream deriving from biodiesel production. The crude glycerol present at very high concentration has been used as substrate to produce citric acid using the yeast Yarrowia lipolytica. The produced citric acid has been used for the synthesis of Carbon Dots (CDs), that is a new type of carbon-based nanomaterials that have attracted broad research interest due to their excellent and tunable photoluminescence, high quantum yield, low toxicity, small size, appreciable biocompatibility (2). We demonstrated that CDs produced with glycerol from biodiesel waste have physico-chemical properties equal to the ones of CDs produced with pure citric acid; thus proving that glycerol-reach waste streams can be valorised via microbial cell factories for production of materials that find their application in diverse fields, such as biomedical and optoelectronics.

Background Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. Results In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. Conclusions A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Interest in replacing fish oil and fish meal as fish feed ingredients with more socially, environmen-tally, and economically sustainable alternatives has increased in recent years. One such alternative could be the use of yeast biomass. In this report, the growth characteristics of the yeast Yarrowia lipolytica on a hydrolysate originating from food waste have been analyzed. The yeast was grown on a filtrated hydrolysate, which was produced in several filtering steps using paper and glass fiber filters, where two degrees of purity were used for the cultivations, e.g., minimally, and sterile filtered hydrolysate. The yeast was cultivated in small bioreactors where the growth characteristics on the different substrates were analyzed. Another parameter that was investigated was the pH (6.2 and 5.5) effect on the yeast growth. Also, the impact of pH at harvest was analyzed by harvesting the biomass at low and high pH levels. During all cultivations, samples were taken for the analysis of volatile fatty acids and lactate by High-Performance Liquid Chromatography (HPLC). The main findings of this project were firstly, that Y. lipolytica could grow on both sterile and minimally fil-tered hydrolysate. Secondly, the yeast seemed to prefer acetate and propionate over lactate. Thirdly, that the method used for filtration worked well for the preparation of yeast cultivation medium., Användandet av fiskolja och fiskmjöl står under senare tid inför ett skifte för att utvecklas till mer socialt, miljömässigt och ekonomiskt mer hållbara alternativ. Ett av dessa alternativ kan vara att ersätta tidigare råvaror genom biomassan från jäst. I denna rapport har Yarrowia lipolyticas tillväxt-faktorer på ett hydrolysat från matavfall analyserats. För att jästen ska ha kunnat växa på hydroly-satet har det först filtrerats i olika steg med hjälp av papper- och glasfiberfilter där två alternativ av renhet valts ut för kultivering (minimalt filtrerat och sterilfiltrerat hydrolysat). Jästen har vuxit i små bioreaktorer där tillväxtfaktorer har analyserats. Andra parametrar som även undersökts är vad olika pH (6.2 och 5.5) har för påverkan på tillväxten. pH-värdens påverkan vid skörd av biomassan har också undersökts. Under alla kultiveringar har prover tagits som analyserats för flyktiga fettsyror och laktat genom High-Performance Liquid Chromatography (HPLC). Resultaten av dessa experi-ment har för det första varit att det är möjligt för Y. lipolytica att växa på de båda substraten. För det andra, verkar jästen föredra de två fettsyrorna acetat och propionat framför laktat. För det tredje, att filtreringsprocessen som togs fram visade sig vara en bra metod för att preparera odlingsmedium.

A novel fermentation process was developed in which renewable electricity is indirectly used as an energy source in fermentation, synergistically decreasing both the consumption of sugar as a first generation carbon source and emission of the greenhouse gas CO2. As an illustration, a glucose-based process is co-fed with formic acid, which can be generated by capturing CO2 from fermentation offgas followed by electrochemical reduction with renewable electricity. This “closed carbon loop” concept is demonstrated by a case study in which cofeeding formic acid is shown to significantly increase the yield of biomass on glucose of the industrially relevant yeast species Yarrowia lipolytica. First, the optimal feed ratio of formic acid to glucose is established using chemostat cultivations. Subsequently, guided by a dynamic fermentation process model, a fed-batch protocol is developed and demonstrated on laboratory scale. Finally, the developed fed-batch process is tested and proven to be scalable at pilot scale. Extensions of the concept are discussed to apply the concept to anaerobic fermentations, and to recycle the O2 that is co-generated with the formic acid to aerobic fermentation processes for intensification purposes., BT/Industrial Microbiology, BT/Bioprocess Engineering

The use of low-value organic residues as a component of microbial growth media has potential to lower input costs for industrial fermentation. Wastewaters generated from meat rendering plants are enriched with high levels of organic matter and nutrients. The thesis focused on understanding the significance of using red meat processing wastewater as a microbial growth substrate for nutrient recovery, via the production of value-added products such as fatty acids, amino acids, and lipases. The study included detailed phenotypic and genotypic characterisation of five uncharacterised Yarrowia lipolytica, two Yarrowia deformans and one Starmerella meliponinorum regarding the production of industrially relevant compounds.

Microbial biochemical production as a renewable alternative to traditional methods is a rapidly growing sector of industrial biotechnology. Non-conventional microbes are attractive targets for metabolic engineering to produce biochemicals as they can present a range of desirable traits that may help avoid complex and intensive engineering of less suitable model hosts. Yarrowia lipolytica is one such non-conventional yeast with an abundant acetyl-CoA pool and native capacity to produce and accumulate lipids to high levels. While there have been significant advances in the metabolic engineering of this yeast for the biosynthesis of oleochemicals and other value-added products, there is also a dearth of synthetic biology tools for genome engineering, functional genomic screening and rapid strain development. We have sought to overcome these limitations by developing CRISPR-Cas9 and Cas12a systems for multiplexed gene knockout, integration, regulation, and genome-wide screening. However, prediction of highly active guide RNA (gRNA) which are crucial in effective genome editing and improving confidence in hit calling, remains a challenge. To address this, we constructed two genome-wide libraries, one using SpCas9 and the other using LbCas12a, to target all protein coding sequences. A negative selection screen in the absence of DNA repair, was used to generate gRNA activity scores for both endonucleases. This genome-wide data served as input to a deep learning algorithm, DeepGuide, that could accurately predict high activity gRNA for both Cas9 and Cas12a. Another critical challenge in accurately assessing screening outcomes is accounting for the variability in gRNA activity. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Thus, we also developed acCRISPR, an end-to-end pipeline that used gRNA activity scores to provide an activity correction to the screening outcomes, thus accurately

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1, BcABA2, BcABA3, BcABA4, and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of mevalonate pathway and ABA-biosynthesis encoding genes, and we expressed plant ABA-transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g DCW ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA, however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms that Y. lipolytica as a potential alternative host for the abscisic acid production.

Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree-negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg/L of ergothioneine in small-scale cultivation, with an additional copy of each gene improving the titer to 205 mg/L. The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate-limited fed-batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g/L ergothioneine in 220 hours, corresponding to an overall volumetric productivity of 7.41 mg L-1 h-1 , showing that Yarrowia lipolytica is a promising host for ergothioneine production.

[Abstract] Volatile fatty acids (VFAs) can be considered as low-cost carbon substrates for lipid accumulation by oleaginous yeasts. This study demonstrates that a common mixture of VFAs, typically obtained from the anaerobic fermentation of C1-gases by some acetogenic bacteria, can be used in a second aerobic fermentation with the yeast Yarrowia lipolytica to obtain lipids as precursors of biodiesel. In the batch experiments, the preference of Yarrowia lipolytica W29 for acetic acid over butyric and caproic acids was demonstrated, with the highest consumption rate reaching 0.664 g/L·h. In the bioreactor experiments, the amount initial biomass inoculated, as well as the initial acid concentration, were found to have a significant influence on the process. Though the lipid content was relatively low, it can be optimized and further improved. Oleic, linoleic and palmitic acids accounted for about 80 % of the fatty acids in the lipids, which makes them suitable for biodiesel.

Several plant triterpenoids have valuable pharmaceutical properties, but their production and usage is limited since extraction from plants can burden natural resources, and result in low yields and purity. Here, we engineered oleaginous yeast Yarrowia lipolytica to produce three valuable plant triterpenoids (asiatic, madecassic, and arjunolic acids) by fermentation. First, we established the recombinant production of precursors, ursolic and oleanolic acids, by expressing plant enzymes in free or fused versions in a Y. lipolytica strain previously optimized for squalene production. Engineered strains produced up to 11.6 mg/g DCW ursolic acid or 10.2 mg/g DCW oleanolic acid. The biosynthetic pathway from ursolic acid was extended by expressing the Centella asiatica cytochrome P450 monoxygenases CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p, resulting in the production of trace amounts of asiatic acid and 0.12 mg/g DCW madecassic acid. Expressing the same C. asiatica cytochromes P450 in oleanolic acid-producing strain resulted in the production of oleanane triterpenoids. Expression of CaCYP716C11p in the oleanolic acid-producing strain yielded 8.9 mg/g DCW maslinic acid. Further expression of a codon-optimized CaCYP714E19p resulted in 4.4 mg/g DCW arjunolic acid. Lastly, arjunolic acid production was increased to 9.1 mg/g DCW by swapping the N-terminal domain of CaCYP714E19p with the N-terminal domain from a Kalopanax septemlobus cytochrome P450. In summary, we have demonstrated the production of asiatic, madecassic, and arjunolic acids in a microbial cell factory. The strains and fermentation processes need to be further improved before the production of these molecules by fermentation can be industrialized.

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments show

β-Carotene is a yellow-orange-red pigment used in food, cosmetics, and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB, and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained on mineral medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts., BT/Industrial Microbiology

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway. We then built a GA4-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA4. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA7 (trace amounts) and GA3 (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA3-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes. Classification: Biological Sciences, Applied Biological Sciences.

The production of β-carotene has become increasingly interesting within the biotechnological industry due to a rising demand for safer and more natural colorants, nutritional supplements, and antioxidants. A recent study has described the potential of Yarrowia lipolytica as a β-carotene-producing cell factory, reporting the highest titer of recombinant β-carotene produced to date. Finding the best conditions to maximize production and scaling up the process to full scale, a costly and time-consuming process, it is often a bottleneck in biotechnology. In this work, we explored the benefits of using micro-fermentation equipment to significantly reduce the time spent on design and optimization of bioreaction conditions, especially in the early stages of process development. In this proof-of-concept study, a β-carotene producing Y. lipolytica strain was tested in micro-fermentations partly to assess the robustness of the cell factory design and partly to perform media optimization. The medium optimization led us to an improvement of up to 50% in the yield of β-carotene production in the best of the conditions. Overall, the micro-fermentation system had a high degree of reliability in all tests.

Erythritol is a polyol produced by Yarrowia lipolytica under hyperosmotic stress. In this study, the osmo-sensitive strain Y. lipolytica yl-hog1Δ was subjected to stress, triggered by a high concentration of carbon sources. The strain thrived on 0.75 M erythritol medium, while the same concentrations of glucose and glycerol proved to be lethal. The addition of 0.1 M erythritol to the medium containing 0.75 M glucose or glycerol allowed the growth of yl-hog1Δ. Supplementation with other potential osmolytes such as mannitol or L-proline did not have a similar effect. To examine whether the osmoprotective effect might be related to erythritol accumulation, we deleted two genes involved in erythritol utilization, the transcription factor Euf1 and the enzyme erythritol dehydrogenase Eyd1. The strain eyd1Δ yl-hog1Δ, which lacked the erythritol utilization enzyme, reacted to the erythritol supplementation significantly better than yl-hog1Δ. On the other hand, the strain euf1Δ yl-hog1Δ became insensitive to supplementation, and the addition of erythritol could no longer improve the growth of this strain in hyperosmotic conditions. This indicates that Euf1 regulates additional, still unknown genes involved in erythritol metabolism.

Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via β-oxidation to preferentially twelve or ten carbons. The strains could produce γ-dodecalactone from oleic acid and δ-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L γ-dodecalactone in a fed-batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.

Research in cell adhesion has important implications in various areas, such as food processing, medicine, environmental engineering, biotechnological processes. Cell surface characterization and immobilization of microorganisms on solid surfaces can be performed by promoting cell adhesion, in a relatively simple, inexpensive, and quick manner. The adhesion of Yarrowia lipolytica IMUFRJ 50682 to different surfaces, especially potential residual plastics (polystyrene, poly(ethylene terephthalate), and poly(tetrafluoroethylene)), and its use as an immobilized biocatalyst were tested. Y. lipolytica IMUFRJ 50682 presented high adhesion to different surfaces such as poly(tetrafluoroethylene) (Teflon), polystyrene, and glass, independent of pH, and low adhesion to poly(ethylene terephthalate) (PET). The adhesion of the cells to polystyrene was probably due to hydrophobic interactions involving proteins or protein complexes. The adhesion of the cells to Teflon might be the result not only of hydrophobic interactions but also of acid-basic forces. Additionally, the present work shows that Y. lipolytica cell extracts previously treated by ultrasound waves (cell debris) maintained their enzymatic activity (lipase) and could be attached to polystyrene and PET and used successfully as immobilized biocatalysts in hydrolysis reactions.

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations. Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits. Here we demonstrate a biotechnological method for the production of (Z)-hexadec-11-en-1-ol and (Z)-tetradec-9-en-1-ol, using engineered yeast cell factories. These unsaturated fatty alcohols are pheromone components or the immediate precursors of pheromone components of several economically important moth pests. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting yeast-derived pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a conventionally produced synthetic pheromone mixture. We further demonstrated the production of (Z)-tetradec-9-en-1-yl acetate, the main pheromone component of the fall armyworm Spodoptera frugiperda. Taken together our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control via yeast fermentation.

Non-conventional yeasts are an excellent option for a number of different industrial bioprocesses. They possess beneficial natural phenotypes, which translates to several fermentation advantages when compared to traditional hosts, like Saccharomyces cerevisiae. The non-conventional yeasts Yarrowia lipolytica, Trichosporon oleaginosus, Kluyveromyces marxianus, Dekkera bruxellensis, Pichia kudriavzevii, Debaryomyces hansenii and Hansenula polymorpha, are considered desirable industrial hosts due to their natural characteristics, including tolerance to several by-products and inhibitors, thermotolerance, salt resistance or osmo- and xerotolerance. Therefore, they are a great alternative for the industrial production of bioethanol, fine chemicals, lipids and recombinant proteins, among others. In this review, we summarize the best natural characteristics of those seven non-conventional yeasts and their use in industrial biotechnology, as well as the molecular/synthetic biology tools available for their genetic modification. Moreover, possible limitations regarding their performance in industrial fermentations and a list of challenges to overcome in the future are also discussed.

Despite all the progresses made by metabolic engineering, still only a few biotechnological processes are running at an industrial level. In order to boost the biotechnological sector, integration strategies as well as long-term views are needed. The aim of the present review is to identify the main drawbacks in biotechnological processes, and to propose possible solutions to overcome the issues in question. Novel cell factories and bioreactor design are discussed as possible solutions. In particular, the following microorganisms: Yarrowia lipolytica, Trichosporon oleaginosus, Ustilago cynodontis, Debaryomyces hansenii along with sequential bioreactor configurations are presented as possible cell factories and bioreactor design solutions, respectively.

Current energy security and climate change policies encourage the development and utilization of bioenergy. Oleaginous yeasts provide a particularly attractive platform for the sustainable production of biofuels and industrial chemicals due to their ability to accumulate high amounts of lipids. In particular, microbial lipids in the form of triacylglycerides (TAGs) produced from renewable feedstocks have attracted considerable attention because they can be directly used in the production of biodiesel and oleochemicals analogous to petrochemicals. As an oleaginous yeast that is generally regarded as safe, Yarrowia lipolytica has been extensively studied, with large amounts of data on its lipid metabolism, genetic tools, and genome sequencing and annotation. In this review, we highlight the newest strategies for increasing lipid accumulation using metabolic engineering and summarize the research advances on the overaccumulation of lipids in Y. lipolytica. Finally, perspectives for future engineering approaches are proposed.

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

Various solutions that have been proposed to address the contamination problem of leather tanning wastewater at El Cerrito (Valle del Cauca, Colombia), have generated conflicts between stakeholders. Therefore, with the purpose of selecting a sustainable alternative for the reduction of chromium present in liquid effluents of the local tanning sector, the Social Multi-Criteria Evaluation (SMCE) method was used to analyze three proposed treatment options. These consisted of a physicochemical treatment based on MgO precipitation and acid re-dissolution, a biotechnological treatment with native yeasts coupled to Cleaner Production (CP) and a non-treatment option. During the evaluation, the social, economic, environmental and technical criteria considered, allowed for the selection of the biotechnology alternative, as the treatment option with the highest sustainability. The physicochemical alternative demonstrated a low acceptance while the non-treatment option proved unfeasible, by affecting the natural environment and social component and was favored only by the economic criterion, Varias soluciones que se han propuesto para abordar el problema de contaminación de las aguas residuales del curtido de pieles en El Cerrito (Valle del Cauca, Colombia) han generado conflictos de intereses entre las partes involucradas. Por lo tanto, con el objetivo de seleccionar una alternativa sostenible para la reducción de cromo presente en los efluentes líquidos del sector curtidor, se utilizó el método de Evaluación Multicriterio Social (EMCS) para analizar tres opciones de tratamiento propuestas. Estas consistieron en un tratamiento fisicoquímico basado en la precipitación con MgO y redisolución ácida, un tratamiento biotecnologico con levaduras nativas acopladas a Producción Más Limpia (CP) y una opción sin tratamiento. Durante la evaluación, los criterios sociales, económicos, ambientales y técnicos considerados, permitieron seleccionar la alternativa biotecnológica, como la opción con mayor sostenibilidad. La alternativa fisicoquímica demostró una baja aceptación, mientras que la opción sin-tratamiento resultó inviable, al afectar el entorno natural y el componente social, favorecida solo por el criterio económico.

CRISPR is a revolutionary method to effectively and efficiently alter the genomic make-up of an organism. Unlike any other genetic engineering tool or technique, CRISPR is remarkably cheaper, simpler and faster to perform. In biotechnology, the best eukaryotic organism for research is yeast, due to their fast growth rate and ease of manipulation compared to multicellular organisms. Hence the aim of the study was the development of a wide range CRISPR-Cas9 system for a wide variety of different yeasts for easy and fast gene editing. The system were validated in Saccharomyces cerevisiae and six other non-conventional yeast. System construction began with the incorporation (separately) of three different optimized CAS9 (optimized for expression in Pichia pastoris, Candida albicans and Homo sapiens) genes into the wide range pKM180 vector. The three different CAS9 construct were then tested for correct expression of the Cas9 protein and the effects thereof in all the yeasts. Through western blot analysis it was observed that all three of the different Cas9 proteins were expressed successfully in the different yeasts. However, all of the Cas9 proteins had a negative effect on the growth of the yeast. For the completion of the CRISPR-Cas9 system, a Ribozyme‐gRNA‐Ribozyme cassette was incorporated into the wide range CAS9 vector, containing the C. albicans optimized CAS9. The system was then validated with successful disruption of the ADE2 gene in all of the yeasts. This proved that the wide range CRISPR-Cas9 system was applicable in a wide variety of different yeasts, thus allowing for rapid, cost-effective genetic manipulation of biotechnologically relevant yeast strains., National Research Foundation (NRF)

Sustainability assessment using a life‐cycle approach is indispensable to contemporary bioprocess development. This assessment is particularly important for early‐stage bioprocess development. As early‐stage investigations of bioprocesses involve the evaluation of their ecological and socioeconomic effects, they can be adjusted more effectively and improved towards sustainability, thereby reducing environmental risk and production costs. Early‐stage sustainability assessment is an important precautionary practice and, despite limited data, a unique opportunity to determine the primary impacts of bioprocess development. To this end, a simple and robust method was applied based on the standardized life‐cycle sustainability assessment methodology and commercially available datasets. In our study, we elaborated on the yeast‐based citric acid production process with Yarrowia lipolytica assessing 11 different substrates in different process modes. The focus of our analysis comprised both cultivation and down‐stream processing. According to our results, the repeated batch raw glycerol based bioprocess alternative showed the best environmental performance. The second‐ and third‐best options were also glycerol‐based. The least sustainable processes were those using molasses, chemically produced ethanol, and soy bean oil. The aggregated results of environmental, economic, and social impacts display waste frying oil as the best‐ranked alternative. The bioprocess with sunflower oil in the batch mode ranked second. The least favorable alternatives were the chemically produced ethanol‐, soy oil‐, refined glycerol‐, and molasses‐based citric acid production processes. The scenario analysis demonstrated that the environmental impact of nutrients and wastewater treatment is negligible, but energy demand of cultivation and down‐stream processing dominated the production process. However, without energy demand the omission of neutralizers almost halves the total impact, and neglecting

Textile waste contains biodegradable fraction which can be used as an alternative feedstock for succinic acid production. The feasibility of bioconversion of biochar-treated textile waste hydrolysate into succinic acid was evaluated. A substrate loading of 9% was applied to hydrolyze mixed textile waste and the resultant glucose-rich hydrolysate with dyestuff was collected after enzymatic hydrolysis. Biochar from different pyrolysis temperatures (400–700 °C)and different dosages of the selected biochar (1–5%)were applied to remove colorants. The results showed that biochar had a good performance at a dosage of 2 w/w % on removal of colorant inhibitors. There was no negative effect observed during the subsequent fermentation. After optimization of fermentation media in shake flasks, the resultant succinic acid titer reached 19.6 g/L with a SA yield of 0.76 g/g, supplemented with 30 g/L tryptone. The in situ fibrous bed bioreactor (isFBB)can further improve SA titer, up to 28.8 g/L corresponding to a yield of 0.61 g/g without pH control. Furthermore, a shorter lag phase during succinic acid fermentation in isFBB was observed. It can be concluded that a novel textile waste-based biorefinery approach for succinic acid production aided by biochar sorption was successfully developed. © 2019 Elsevier B.V.

Glycerol is considered as a promising substrate for biotechnological applications and the non-conventional yeast Yarrowia lipolytica has been used extensively for the valorization of this compound. Contrary to S. cerevisiae, Y. lipolytica seems to prefer glycerol over glucose and it has been reported previously that the presence of glycerol can suppress the consumption of glucose in co-substrate fermentations. Based on these observations, we hypothesized glycerol repression-like effects in Y. lipolytica, which are converse to well described carbon repression mechanisms ensuring the prioritized use of glucose (e.g. in S. cerevisiae). We therefore aimed to investigate this effect on the level of transcription. Strains varying in the degree of glucose suppression were chosen and characterized in high-resolution growth screenings, resulting in the detection of different growth phenotypes under glycerol-glucose mixed conditions. Two strains, IBT and W29, were selected and cultivated in chemostats using glucose, glycerol and glucose/glycerol as carbon sources, followed by an RNA-Seq-based transcriptome analysis. We could show that several transporters were significantly higher expressed in W29, which is potentially related to the observed physiological differences. However, most of the expression variation between the strains were regardless of the carbon source applied, and cross-comparisons revealed that the strain-specific carbon source responses underwent in the opposite direction. A deeper analysis of the substrate specific carbon source response led to the identification of several differentially expressed genes with orthologous functions related to signal transduction and transcriptional regulation. This study provides an initial investigation on potentially novel carbon source regulation mechanisms in yeasts.

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal β-oxidation by chromosomal deletion of pot1 resulted in the biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C13-C23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8mg/L of total methyl ketones, representing more than a 6,000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones.

La production de biodiesel a attiré l’attention au cours des dernières décennies. Grâce à une réduction des émissions de gaz à effet de serre (GES), les biodiesels peuvent désormais compétitionner avec les sources de carburants à base de pétrole. Actuellement, de manière globale, les usines de production de biodiesel utilisent de l’huile végétale (comestible et non-comestible) et des graisses animales comme matière première. Par contre, ces usines cherchent des alternatives à plus faible coût pour remplacer ces matières. La production de lipides par des microorganismes oléagineux est une approche intéressante pour les chercheurs et les producteurs de biodiésel. Par contre, la production du biodiésel à partir de lipides demeure économiquement impraticable due au coût élevé de la matière première et des différentes étapes de production telles que la récolte des microorganismes, le séchage de la biomasse, la perturbation des parois cellulaires, l’utilisation de solvants organiques pour l’extraction lipidique, la transestérification et la purification. Dans cette étude, des matières destinées à l’enfouissement telles que de l’écume (usine d’épuration) et des boues primaires et secondaires ont été étudiées pour en extraire de l’huile. Due à la présence de seulement 30% m/m d’huile dans l’écume, les levures oléagineuses cultivées en présence de glycérol brut (Yarrowia lipolytica et Trichosporon aleaginous) ont été identifiées comme une meilleure alternative puisqu’elles croissent rapidement et qu’elles peuvent atteindre des concentrations de 80 g/L de biomasse avec 50 à 80% m/m de lipides. Par contre, la barrière technologique pour la production de biodiésel à partir de microorganismes oléagineux est le procédé de d’extraction et de purification. Donc, la récolte de biomasse s’est effectuée par la technologie de floculation avec l’utilisation de polymères extracellulaires (PEC) produit par fermentation des biosolides municipaux. Ensuite, une nouvelle approche de perturbat

Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2-pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen-limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut-and-paste transposon to mobilize and integrate multiple copies of the 2-pyrone synthase gene. Strain modifications and batch fermentation in nitrogen-limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.

The application of spent yeast for biogas production has been studied only in the context of breweries so far. This study is focused on the anaerobic digestion of concentrated yeast biomass (CYB), being a by‐product of citric acid biosynthesis. Two experimental set‐ups were used in order to test CYB as a mono‐substrate and co‐substrate for closing the loop in accordance with the ‘bioeconomy’ approach. The results show that CYB allows for obtaining a high biogas yield, with a maximum of 1.45 m3N/kgVS produced when CYB was used as a mono‐substrate. The average methane concentration was 66 ± 4%. However, anaerobic digestion of CYB alone was difficult to perform because of a tendency for over‐acidification, meaning that the maximum possible organic loading rate was 1 kg/(m3*d). Repeated clogging of tubes with coagulated biomass also disturbed continuous feeding. In contrast, the co‐digestion of CYB with waste frying fat at a ratio of 1:20 showed stable operation during a 70‐day fermentation period. The biogas yield using the substrate mixture was 1.42 m3/kgVS at an organic loading rate of 2 kg/(m3*d). The methane concentration reached 67 ± 4% and the acetate concentration did not exceed 30 mg/L during the entire fermentation.

The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, we present the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Background The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2. Results To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent. Conclusions Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powe

Astaxanthin is a red-colored carotenoid, used as food and feed additive. Astaxanthin is mainly produced by chemical synthesis, however, the process is expensive and synthetic astaxanthin is not approved for human consumption. In this study, we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation. First, we screened 12 different Y. lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis: bi-functional phytoene synthase/lycopene cyclase (crtYB) and phytoene desaturase (crtI) from the red yeast Xanthophyllomyces dendrorhous. The best strain produced 31.1 ± 0.5 mg/L β-carotene. Next, we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG1) and geranylgeranyl diphosphate synthase (GGS1/crtE) in the best producing strain and obtained 453.9 ± 20.2 mg/L β-carotene. Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1 ± 57.2 mg/L. Then we introduced β-carotene ketolase (crtW) from Paracoccus sp. N81106 and hydroxylase (crtZ) from Pantoea ananatis to convert β-carotene into astaxanthin. The constructed strain accumulated 10.4 ± 0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates, 5.7 ± 0.5 mg/L canthaxanthin, and 35.3 ± 1.8 mg/L echinenone. Finally, we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW (54.6 mg/L) of astaxanthin in a microtiter plate cultivation. Our study for the first time reports engineering of Y. lipolytica for the production of astaxanthin. The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y. lipolytica-based fermentation process for astaxanthin production.

Background: Yarrowia lipolytica is widely studied as a non-conventional model yeast owing to the high level of lipid accumulation. Therein, triacylglycerol (TAG) is a major component of liposome. In order to investigate the TAG biosynthesis mechanism at a systematic level, a novel genome-scale metabolic model of Y. lipolytica was reconstructed based on a previous model iYL619_PCP published by our lab and another model iYali4 published by Kerkhoven et al. Results: The novel model iYL_2.0 contains 645 genes, 1083 metabolites, and 1471 reactions, which was validated more effective on simulations of specific growth rate. The precision of 29 carbon sources utilities reached up to 96.6% when simulated by iYL_2.0. In minimal growth medium, 111 genes were identified as essential for cell growth, whereas 66 essential genes were identified in yeast extract medium, which were verified by database of essential genes, suggesting a better prediction ability of iYL_2.0 in comparison with other existing models. In addition, potential metabolic engineering targets of improving TAG production were predicted by three in silico methods developed in-house, and the effects of amino acids supplementation were investigated based on model iYL_2.0. Conclusions: The reconstructed model iYL_2.0 is a powerful platform for efficiently optimizing the metabolism of TAG and systematically understanding the physiological mechanism of Y. lipolytica. [Figure not available: see fulltext.]., QC 20181020

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10 days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3 days incubation in control supernatant, while it was prevented for up to 10 days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant

P450s have significant industrial relevance in the field of biotechnology and sustainability. In cells, they allow for the utilization of various substrates for energy usage and storage. Their ability to oxidize various substrates can also be beneficial to create anything from biofuel precursors to oleochemicals. As Yarrowia lipolytica produces a wide variety of P450s that catalyze disparate reactions, it presents itself as an ideal target for enzyme production. Genetic engineering methods for Y. lipolytica have made enzyme overexpression in the yeast more obtainable. This project aims to utilize genetic engineering tools available to Y. lipolytica in an attempt to overproduce P450s. Specific genes relating to protein expression will be targeted. This research hopes to prove the viability and capacity of Y. lipolytica as a large scale producer of membrane associated proteins by proliferating ER creation through altering carbon flux in the organism. The experiments presented ascribe to quantify the potential of this microorganism with studies involving protein expression and cell morphology.

Microbial consortia have the potential to perform complex, industrially important tasks. The design of microbial consortia requires knowledge of the substrate preferences and metabolic outputs of each member, to allow understanding of potential interactions such as competition and beneficial metabolic exchange. Here, we used exometabolite profiling to follow the resource processing by a microbial co-culture of two biotechnologically relevant microbes, the bacterial cellulose degrader Cellulomonas fimi, and the oleaginous yeast Yarrowia lipolytica. We characterized the substrate preferences of the two strains on compounds typically found in lignocellulose hydrolysates. This allowed prediction that specific sugars resulting from hemicellulose polysaccharide degradation by C. fimi may serve as a cross-feeding metabolites to Y. lipolytica in co-culture. We also showed that products of ionic liquid-treated switchgrass lignocellulose degradation by C. fimi were channeled to Y. lipolytica in a co-culture. Additionally, we observed metabolites, such as shikimic acid accumulating in the co-culture supernatants, suggesting the potential for producing interesting co-products. Insights gained from characterizing the exometabolite profiles of individual and co-cultures of the two strains can help to refine this interaction, and guide strategies for making this an industrially viable co-culture to produce valuable products from lignocellulose material.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis in poultry and leads to economic losses in the poultry industry. Due to rising concerns of antibiotic resistance and antibiotic carry-over into food, bans have been implemented on antibiotic use in animal production. The process of discovering new antibiotics and having them registered and approved can take up to eight years, and their application will most likely be limited to human-use. Alternative therapies for the control of bacterial diseases in animals, including poultry, are therefore becoming increasingly important. Alternative treatment options could include the use of bacteriophages, bacteriophage enzymes, essential oils and vaccines. Bacterial vaccines are generally based on whole bacterial cells, and in the case of commensal bacteria such as E. coli, this can lead to poor gut health. Therefore, the need for highly specific bacterial vaccines are required, such as sub-unit vaccines containing only antigens found in pathogenic strains of E. coli. Various virulence genes have been found in APEC that contribute to the pathogenicity of the strains. Five of these genes have been found to be highly prevalent in most clinical cases of avian colibacillosis. In this study, two of these genes were selected as potential candidates for sub-unit vaccine development, namely increased serum survival (iss) and haemolysin F (hlyF). The increased serum survival gene, an outer-membrane protein of APEC, was successfully expressed in E. coli BL21 (DE3) using the pET28b(+) vector system, although the protein was mostly water-insoluble due to hydrophobic N -and C-terminals. The sequence of iss was then modified to create a truncated version of the gene encoding the hydrophilic region of the gene, also the potential epitope of the Iss antigen, to improve solubility during over-expression. Water-soluble truncated Iss was produced in conjunction with the full Iss protein, solubilised using a zwitteri, National Research Foundation (NRF)

A new, efficient method for the isolation of (2R,3S)-isocitric acid (ICA) from its fermentation solution was developed. It is noteworthy that this method is based on selective adsorption directly from the fermentation solution on activated carbon, followed by the release of both ICA and citric acid by means of elution with methanol and their final separation by known methods. Thereby, several disadvantages were overcome: Electrodialysis is no longer necessary to remove cations such as Na+ from the fermentation solution. Also, several hitherto accompanying dyestuffs were not observed with this method. Furthermore, removal of water by distillation is expendable. Eventually, the new crude product is of a quality that also avoids the use of a tedious slide vane rotary vacuum pump distillation of the trimethyl esters of both acids, which hitherto was the basis for the separation of ICA. In summary, the new method distinctly spares energy as well as time.

L’érythritol est un polyol aux propriétés édulcorantes utilisé comme substitut de sucre par l’industrie agroalimentaire. Le but principal du projet est l’amélioration du procédé de production d’érythritol par génie métabolique. L’idée est de construire des souches surexprimant les gènes liés à la voie de synthèse de l’érythritol. L’objectif principal est donc d’identifier les gènes clés permettant d’augmenter la synthèse d’érythritol et d’évaluer cette dernière en bioréacteur.Parallèlement à cela, un autre objectif est d’identifier les gènes liés au catabolisme de l’érythritol. En effet, Y. lipolytica est capable de produire de l’érythritol, mais aussi de le reconsommer en cas d’absence d’autre source de carbone. L’objectif est donc d’identifier les gènes liés au catabolisme de l’érythritol afin de les déléter, et ainsi obtenir une souche capable de produire de grandes quantités d’érythritol sans le reconsommer en fin de culture.Les résultats obtenus ont permis d’identifier les étapes clés de la voie de synthèse de l’érythritol et d’obtenir des souches à haut rendement et productivité par génie génétique. Par ailleurs, deux gènes de la voie de dégradation de l’érythritol ont pu être identifiés pour la première fois chez une levure. En combinant la surexpression de gènes liés à la synthèse de l’érythritol et la délétion de gènes liés à sa dégradation, une souche présentant une productivité 74% plus importante que la souche sauvage a pu être créée. Par ailleurs, une souche capable de convertir l’érythritol en érythrulose, un autre composé d’intérêt, a également pu être construite., Doctorat en Sciences agronomiques et ingénierie biologique, info:eu-repo/semantics/nonPublished

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 UAS1EYK1. New hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 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., SCOPUS: ar.j, info:eu-repo/semantics/published

The original article was updated because several pieces of information were omitted. The following paragraph has been added in section Material and methods: Sequences analysis Genome sequences of Yarrowia species were assembled and annotated by Cécile Neuvéglise, Hugo Devillers and coworkers (to be published). Homologs of EYK1 in Yarrowia species were identified by Blast on the private site of GRYC (Genome Resources for Yeast Chromosomes; http://gryc.inra.fr) using EYK1 gene as template and retrieved using the download functionality developed by H. Devillers. The legend of Fig. S1 should be read as follows: Multiple alignments of YALY0F01606g genes with the DAK1, DAK2 and DAK3 in the lipolytica clade. Homologs of EYK1 in Yarrowia species were identified by Blast on the private site of GRYC using EYK1 gene as template and retrieved using the download functionality. Color code: red, similar in all gene; green, similar in two of the three genes; yellow highlighted, different in DAK3 group compared to DAK1 and DAK2 gene. Sequences are from strains YALI: Yarrowia lipolytica CLIB122; YAGA: Yarrowia galli CBS 9722 (96.77%); YAYA: Yarrowia yakushimensis CBS 10253 (91.62%); YAAL: Yarrowia alimentaria CBS 10151 (87.22%) and YAPH: Yarrowia phangngensis CBS 10407 (85.01%). Maximal identities with Yarrowia lipolytica EYK1 (YALI0F01606g) are indicated in bracket. The following information was missing in the Acknowledgements section: The genome sequence of Y. galli was funded by the project CALIN (Carburants Alternatifs et Systèmes d’Injection, grant N° 25331). The genomes of Y. yakushimensis, Y. alimentaria, and Y. phangngensis were funded by INRA in the frame of the AIP-Bioressources 2011 program (YALIP project granted to C. Neuvéglise)., SCOPUS: er.j, info:eu-repo/semantics/published

Cocoa butter (CB) extracted from cocoa beans is the main raw material for chocolate production. However, growing chocolate demands and limited CB production has resulted in a shortage of CB supply. CB is mainly composed of three different kinds of triacylglycerols (TAGs), POP (C16:0–C18:1–C16:0), POS (C16:0–C18:1–C18:0), and SOS (C18:0–C18:1–C18:0). The storage lipids of yeasts, mainly TAGs, also contain relative high-level of C16 and C18 fatty acids and might be used as CB-like lipids (CBL). In this study, we cultivated six different yeasts, including one non-oleaginous yeast strain, Saccharomyces cerevisiae CEN.PK113-7D, and five oleaginous yeast strains, Trichosporon oleaginosus DSM11815, Rhodotorula graminis DSM 27356, Lipomyces starkeyi DSM 70296, Rhodosporidium toruloides DSM 70398, and Yarrowia lipolytica CBS 6124, in nitrogen-limited medium and compared their CBL production ability. Under the same growth conditions, we found that TAGs were the main lipids in all six yeasts and that T. oleaginosus can produce more TAGs than the other five yeasts. Less than 3% of the total TAGs were identified as potential SOS in the six yeasts. However, T. oleaginosus produced 27.8% potential POP and POS at levels of 378 mg TAGs/g dry cell weight, hinting that this yeast may have potential as a CBL production host after further metabolic engineering in future.

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.

Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest., info:eu-repo/semantics/published