Enhanced Production of Erythritol from Glucose by the Newly Obtained UV Mutant Yarrowia lipolytica K1UV15.

Erythritol is a polyol with a sweet taste but low energy value. Thanks to its valuable properties, as well as growing social awareness and nutritional trends, its popularity is growing rapidly. The aim of this study was to increase the effectiveness of erythritol production from glucose using new UV mutants of the yeast Yarrowia lipolytica obtained in the Wratislavia K1 strain. The ability of the new strains to biosynthesize erythritol and utilize this polyol was examined in shake-flask cultures and fed-batch processes conducted in a stirred tank reactor with a total glucose concentration of 300 and 400 g/L. The Wratislavia K1 strain produced erythritol most efficiently (97.5 g/L; 192 h) at an initial glucose concentration of 250 g/L (total: 300 g/L). New strains were assessed under such conditions, and it was noted that the highest erythritol concentration (145 g/L; 183 h) was produced by the K1UV15 strain. A significant improvement in the erythritol biosynthesis efficiency (148 g/L; 150 h) was achieved upon the increase in (NH4)2SO4 to 3.6 g/L. Further, in the culture with such a concentration of the nitrogen source and increased total glucose level (400 g/L), the K1UV15 strain produced 226 g/L of erythritol within 281 h.

The isoprenyl chain length of coenzyme Q mediates the nutritional resistance of fungi to amoeba predation.

Amoebae are environmental predators feeding on bacteria, fungi, and other eukaryotic microbes. Predatory interactions alter microbial communities and impose selective pressure toward phagocytic resistance or escape which may, in turn, foster virulence attributes. The ubiquitous fungivorous amoeba Protostelium aurantium has a wide prey spectrum in the fungal kingdom but discriminates against members of the Saccharomyces clade, such as Saccharomyces cerevisiae and Candida glabrata. Here, we show that this prey discrimination among fungi is solely based on the presence of ubiquinone as an essential cofactor for the predator. While the amoeba readily fed on fungi with CoQ presenting longer isoprenyl side chain variants CoQ8-10, such as those from the Candida clade, it failed to proliferate on those with shorter CoQ variants, specifically from the Saccharomyces clade (CoQ6). Supplementing non-edible yeast with CoQ9 or CoQ10 rescued the growth of P. aurantium, highlighting the importance of a long isoprenyl side chain. Heterologous biosynthesis of CoQ9 in S. cerevisiae by introducing genes responsible for CoQ9 production from the evolutionary more basic Yarrowia lipolytica complemented the function of the native CoQ6. The results suggest that the use of CoQ6 among members of the Saccharomyces clade might have originated as a predatory escape strategy in fungal lineages and could be retained in organisms that were able to thrive by fermentation. IMPORTANCE: Ubiquinones (CoQ) are universal electron carriers in the respiratory chain of all aerobic bacteria and eukaryotes. Usually 8-10 isoprenyl units ensure their localization within the lipid bilayer. Members of the Saccharomyces clade among fungi are unique in using only 6. The reason for this is unclear. Here we provide evidence that the use of CoQ6 efficiently protects these fungi from predation by the ubiquitous fungivorous amoeba Protostelium aurantium which lacks its own biosynthetic pathway for this vitamin. The amoebae were starving on a diet of CoQ6 yeasts which could be complemented by either the addition of longer CoQs or the genetic engineering of a CoQ9 biosynthetic pathway.

Mining novel gene targets for improving tolerance to furfural and acetic acid in Yarrowia lipolytica using whole-genome CRISPRi library.

Abundant renewable resource lignocellulosic biomass possesses tremendous potential for green biomanufacturing, while its efficient utilization by Yarrowia lipolytica, an attractive biochemical production host, is restricted since the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate. Given deficient understanding of inherent interactions between inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using clustered regularly interspaced short palindromic repeats interference library in Y. lipolytica, achieving tolerance to 0.35 % (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15 % (v/v) acetic acid. The tolerance mechanism might involve improvement of cell division and decrease of reactive oxygen species level. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for improving microbial tolerance to lignocellulose-derived inhibitors and revealing underlying mechanism.

The mechanisms of yeast extracellular metabolites in stimulating microbial degradation of trichloroethylene: Physiological characteristics and omics analysis.

The biodegradation of Trichloroethylene (TCE) is limited by low microbial metabolic capacity but can be enhanced through biostimulation strategies. This study explored the physiological effects and potential molecular mechanisms of the yeast Yarrowia lipolytica extracellular metabolites (YEMs) on the degradation of TCE by Acinetobacter LT1. Results indicated that YEMs stimulated the efficiency of strain LT1 by 50.28%. At the physiological level, YEMs exhibited protective effects on cell morphology, reduced oxidative stress, lessened membrane damage, and enhanced energy production and conversion. Analysis of omics results revealed that the regulation of various metabolic pathways by YEMs improved the degradation of TCE. Furthermore, RT-qPCR showed that the genes encoding YhhW protein in TCE stress and YEMs stimulation groups were 1.72 and 3.22 times the control group, respectively. Molecular docking results showed that the conformation of YhhW after binding to TCE changed into a more active form, which enhanced enzyme activity. Therefore, it is speculated that YhhW is the primary degradative enzyme involved in the process of YEMs stimulating strain LT1 to degrade TCE. These results reveal how YEMs induce strain LT1 to enhance TCE degradation.

Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering.

Gastrodin, 4-hydroxybenzyl alcohol-4-O-ß-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

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.

Remodeling the Homologous Recombination Mechanism of Yarrowia lipolytica for High-Level Biosynthesis of Squalene.

Squalene is a high-value antioxidant with many commercial applications. The use of microbial cell factories to produce squalene as an alternative to plant and animal extracts could meet increasing market demand. Yarrowia lipolytica is an excellent host for squalene production due to its high levels of acetyl-CoA and a hydrophobic environment. However, the need for precise and complicated gene editing has hindered the industrialization of this strain. Herein, the rapid construction of a strain with high squalene production was achieved by enhancing the homologous recombination efficiency in Y. lipolytica. First, remodeling of the homologous recombination efficiency resulted in a 10-fold increase in the homologous recombination rate. Next, the whole mevalonate pathway was integrated into the chromosome to enhance squalene production. Then, a higher level of squalene accumulation was achieved by increasing the level of acetyl coenzyme A and regulating the downstream steroid synthesis pathway. Finally, the squalene production reached 35 g/L after optimizing the fermentation conditions and performing a fed-batch culture in a 5 L jar fermenter. This is the highest squalene production ever reported to date by de novo biosynthesis without adding any inhibitors, paving a new path toward the industrial production of squalene and its downstream products.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

Strain improvement for enhanced erythritol production by Moniliella pollinis Mutant-58 using jaggery as a cost-effective substrate

Erythritol has been produced by various microorganisms including Yarrowia, Moniliella, Aureobasidium, and Candida strains. Due to its relatively high price, erythritol sweetener is used lesser than other polyols despite having many advantages. Therefore, in this study, Moniliella pollinis strain was improved for erythritol production by chemical mutagenesis and subsequently screening for cost-effective carbon sources for the enhanced erythritol yield. M. pollinis was subjected to N-methyl N-nitro N-nitroso guanidine (NTG), ethyl methyl sulfonate (EMS), and UV mutagenesis for improved erythritol production. The fmutant strains were evaluated for enhanced erythritol production medium optimization by using different carbon substrates at the shake flask level. To enhance the production of erythritol and statistical media, optimization was carried out using a central composite design (CCD). Among 198 isolated mutants, Mutant-58 strain generated by EMS mutagenesis was selected for further assessment. The Mutant-58 strain showed significant morphological changes as compared to the parent strain. Furthermore, statistically optimized media composition resulted in the higher production of erythritol (91.2 ± 3.4 g/L) with a yield of 40.7 ± 3.4 % in shake flask experiments. The optimized medium composition for erythritol production constitutes (g/L) 225 jaggery, 4.4 yeast extract (YE), 4.4 KH2PO4, 0.31 MgSO4, and pH 5.5. The present study demonstrated strain improvement, media, and process optimization resulting in a 30% increase in the erythritol production in the Mutant-58 as compared to the parent strain. This is also the first instance where jaggery has been used as a cost-effective carbon source alternative to glucose for industrial-scale erythritol production. (AU)

Construction and Regulation of the Abscisic Acid Biosynthesis Pathway in Yarrowia lipolytica.

Abscisic acid (ABA) is an important plant hormone with a variety of physiological functions such as regulating plant growth and helping plants to resist an adverse growth environment. However, at present, the ABA yield of heterologous biosynthesis by metabolic engineering is still low for industrial production. Therefore, five Botrytis cinerea genes (bcaba1, bcaba2, bcaba3, bcaba4, and bccpr1) related to ABA biosynthesis were expressed in Yarrowia lipolytica PO1h; its ABA production was 24.33 mg/L. By increasing the copy number of IDI and ERG12S, ERG20YMT, and bcaba3, bcaba1 genes, the yield of ABA was increased to 54.51 mg/L. By locating HMG-CoA reductase and HMG-CoA synthase in mitochondria, acetyl-CoA in mitochondria was converted into mevalonate; this increased the ABA yield to 102.12 mg/L. Finally, in the fed-batch fermentation process with the addition of dodecane, the ABA yield was up to 1212.57 mg/L, which is the highest yield of heterologous production of ABA by metabolic engineering.

Adsorption of azo dye by biomass and immobilized Yarrowia lipolytica; equilibrium, kinetic and thermodynamic studies.

One of the major environmental problems we have today is dye pollution, primarily caused by the textile industry. This pollution has detrimental effects on aquatic life, soil fertility, and human health. Many microbial biosorbents have been documented in the literature for the removal of a wide range of azo dyes commonly employed in the textile industry. However, Yarrowia lipolytica NBRC1658 is firstly used as both free and immobilized sorbents for the removal of Reactive yellow 18 (RY18), acid red 18 (AR18) and basic blue 41 (BB41) in this study. The effect of experimental conditions such as pH, biosorbent quantity, dye concentration, contact time, and temperature on dye removal capacity are examined. The research findings demonstrate that the adsorption capacity is higher in biomass compared to immobilized cells. The highest adsorption capacities are observed at pH 2 for RY18 and AR18, while pH 9 is optimal for BB41. Increasing the adsorbent dosage and initial concentration significantly improves the adsorption capacity. The Langmuir model best describes the adsorption process, indicating that the dye attaches to the biosorbent in a single layer, with a uniform biosorbent surface. The removal of the dye occurs through a chemical process on the biosorbent surface, as evidenced by the pseudo-second-order kinetic model. According to thermodynamic analysis, higher temperatures promote greater adsorption of dyes. Our study shows the effectiveness of Yarrowia lipolyica NBRC1658 as a biosorbent in the removal of a wide range of industrial dyes.

Transcriptome analysis reveals multiple targets of erythritol-related transcription factor EUF1 in unconventional yeast Yarrowia Lipolytica.

BACKGROUND: Erythritol is a four-carbon polyol with an unclear role in metabolism of some unconventional yeasts. Its production has been linked to the osmotic stress response, but the mechanism of stress protection remains unclear. Additionally, erythritol can be used as a carbon source. In the yeast Yarrowia lipolytica, its assimilation is activated by the transcription factor Euf1. The study investigates whether this factor can link erythritol to other processes in the cell. RESULTS: The research was performed on two closely related strains of Y. lipolytica: MK1 and K1, where strain K1 has no functional Euf1. Cultures were carried out in erythritol-containing and erythritol-free media. Transcriptome analysis revealed the effect of Euf1 on the regulation of more than 150 genes. Some of these could be easily connected with different aspects of erythritol assimilation, such as: utilization pathway, a new potential isoform of transketolase, or polyol transporters. However, many of the upregulated genes have never been linked to metabolism of erythritol. The most prominent examples are the degradation pathway of branched-chain amino acids and the glyoxylate cycle. The high transcription of genes affected by Euf1 is still dependent on the erythritol concentration in the medium. Moreover, almost all up-regulated genes have an ATGCA motif in the promoter sequence. CONCLUSIONS: These findings may be particularly relevant given the increasing use of erythritol-induced promoters in genetic engineering of Y. lipolytica. Moreover, use of this yeast in biotechnological processes often takes place under osmotic stress conditions. Erythritol might be produce as a by-product, thus better understanding of its influence on cell metabolism could facilitate processes optimization.

Adaptive responses of erythritol-producing Yarrowia lipolytica to thermal stress after evolution.

Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with 60Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains. KEY POINTS: • ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica • Genes encoding global regulators are mutated during thermal adaptive evolution • Ceramide and BCAA are critical molecules for cells to tolerate thermal stress.

High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit.

Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

[Advances in efficient biosynthesis of erythritol by metabolic engineering of Yarrowia lipolytica].

Erythritol is a novel 4-carbon sugar alcohol produced by microbes in the presence of hyper-osmotic stress. It has excellent potential to serve as an alternative sugar for people with diabetes and also a platform compound for synthesizing various C4 compounds, such as 1, 3-butadiene, 1, 4-butanediol, 2, 5-dihydrofuran and so on. Compared with other polyols, the fermentative production of erythritol is more challenging. Yarrowia lipolytica is the preferred chassis of erythritol biosynthesis for its high-titer and high-productivity. At present, there are still some bottlenecks in the production of erythritol by Y. lipolytica, such as weak metabolic activity, abundant by-products, and low industrial attributes. Progress has been made in tailoring high version strains according to industrial needs. For example, the highest titer of erythritol produced by the metabolically engineered Y. lipolytica reached 196 g/L and 150 g/L, respectively, by using glucose or glycerol as the carbon sources. However, further improving its production performance becomes challenging. This review summarizes the research progress in the synthesis of erythritol by Y. lipolytica from the perspectives of erythritol producing strains, metabolic pathways, modular modifications, and auxiliary strategies to enhance the industrial properties of the engineered strain. Key nodes in the metabolic pathway and their combination strategies are discussed to guide the research on promoting the production of erythritol by Y. lipolytica.

Inhibition of hyphal formation together with biochar addition promotes erythritol production by Yarrowia lipolytica.

This study aimed to investigate the effect of hyphal formation in Yarrowia lipolytica and biochar addition on erythritol production by submerged fermentation. Hyphal formation significantly inhibited erythritol production by Y. lipolytica. Transcriptome analysis suggested that the impaired erythritol synthesis of hyphal cells was associated with the differential expression of genes involved in amino acid metabolism, lipid metabolism, and cell wall stability. Deletion of RAS2 responsible for yeast-to-hypha transition and EYD1 included in erythritol degradation blocked hyphal formation and improved erythritol production. Biochar prepared from corncob, sugarcane bagasse (SB), corn straw, peanut shell, coconut shell, and walnut shell (WS) had a positive effect on erythritol production, of which WS pyrolyzed at 500°C (WSc) performed the best in flask fermentation. In a 3.7 L bioreactor, 220.20 ± 10 g/L erythritol with a productivity of 2.30 ± 0.10 g/L/h was obtained in the presence of 1.4% (w/v) WSc and 0.7% SBc (SB pyrolyzed at 500°C) within 96 h. These results suggest that inhibition of hyphal formation together with biochar addition is an efficient way to promote erythritol production.

Biosurfactants production by marine yeasts isolated from zoanthids and characterization of an emulsifier produced by Yarrowia lipolytica LMS 24B.

The present study investigates the potential for biosurfactant production of 19 marine yeast species obtained from zoanthids. Using the emulsification index test to screen the samples produced by the marine yeasts, we verified that five isolates exhibited an emulsification index ≥50%. Additional tests were performed on such isolates, including oil displacement, drop collapse, Parafilm M assay, and surface tension measurement. The tolerance of produced biosurfactants for environmental conditions was also analyzed, especially considering the media's temperature, pH, and salinity. Moreover, the surfactant's ability to emulsify different hydrocarbon sources and to metabolize kerosene as the sole carbon source was evaluated in vitro. Our results demonstrate that yeast biosurfactants can emulsify hydrocarbon sources under different physicochemical conditions and metabolize kerosene as a carbon source. Considering the Yarrowia lipolytica LMS 24B as the yeast model for biosurfactant production from the cell's wall biomass, emulsification indexes of 61.2% were obtained, even at a high temperature of 120 °C. Furthermore, the Fourier-transform middle infrared spectroscopy (FTIR) analysis of the biosurfactant's chemical composition revealed the presence of distinct functional groups assigned to a glycoprotein complex. Considering the status of developing new bioproducts and bioprocesses nowadays, our findings bring a new perspective to biosurfactant production by marine yeasts, especially Y. lipolytica LMS 24B. In particular, the presented results validate the relevance of marine environments as valuable sources of genetic resources, i.e., yeast strains capable of metabolizing and emulsifying petroleum derivatives.

Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.

Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.