Yeast Yarrowia Lipolytica Research Articles

A 37-amino acid loop in the Yarrowia lipolytica hexokinase impacts its activity and affinity and modulates gene expression

The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products. Nonetheless, there are still gaps in our understanding of its central carbon metabolism. Here we present an in-depth study of Y. lipolytica hexokinase (YlHxk1), a structurally unique protein. The greatest peculiarity of YlHxk1 is a 37-amino acid loop region, a structure not found in any other known hexokinases. By combining bioinformatic and experimental methods we showed that the loop in YlHxk1 is essential for activity of this protein and through that on growth of Y. lipolytica on glucose and fructose. We further proved that the loop in YlHxk1 hinders binding with trehalose 6-phosphate (T6P), a glycolysis inhibitor, as hexokinase with partial deletion of this region is 4.7-fold less sensitive to this molecule. We also found that YlHxk1 devoid of the loop causes strong repressive effect on lipase-encoding genes LIP2 and LIP8 and that the hexokinase overexpression in Y. lipolytica changes glycerol over glucose preference when cultivated in media containing both substrates.

Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica.

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 ± 1.6mg 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 ± 11.7mg L-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1±13.4mg 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 showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.

Statistical optimization of recombinant glucose oxidase production by yeast Yarrowia lipolytica using response surface methodology

مقدمه: گلوکز اکسیداز به عنوان یک آنزیم تجاری در صنایع دارویی، دارویی و غذایی مورد توجه است. بهینه سازی محیط و شرایط تخمیر برای افزایش تولید گلوکز اکسیداز هترولوگ مهم است. هدف از مطالعه حاضر بهینه سازی تولید گلوکز اکسیداز در یک سویه نوترکیب مخمر یارروویا لیپولیتیکا از طریق روش‌های آماری بود.مواد و روش ها: روش سطح پاسخ به عنوان یک روش آماری برای طراحی آزمایش برای بهینه سازی تولید گلوکز اکسیداز نوترکیب توسط مخمر یارروویا لیپولیتیکا استفاده شد. سپس، از شرایط بهینه شده برای تولید گلوکز اکسیداز در کشت بیوراکتور استفاده شد.یافته ها: تولید گلوکز اکسیداز پس از بهینه سازی محیط در کشت ارلن تا 570 واحد در لیتر افزایش یافت که 2 برابر بیشتر از محیط غیربهینه شده است. در کشت بیوراکتور با استفاده از محیط بهینه شده حاوی گلوکز و ساکارز به عنوان منابع کربن، تولید گلوکز اکسیداز به ترتیب پس از دو و شش روز به 720 و 730 واحد در لیتر رسید.بحث و نتیجه گیری: نتایج این مطالعه اطلاعات ارزشمندی در مورد بهینه سازی آماری توسعه فرآیند زیستی برای تولید پروتئین هترولوگ در مخمر یارروویا لیپولیتیکا ارائه می‌دهد.

Simultaneous Improvement of Limonene Production and Tolerance in Yarrowia lipolytica through Tolerance Engineering and Evolutionary Engineering.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y.lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y.lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y.lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

Probiotic and Immunomodulatory Activity of Marine Yeast Yarrowia lipolytica Strains and Response Against Vibrio parahaemolyticus in Fish.

Yarrowia lipolytica has been widely used in food industry but scarcely explored as probiotics. Thus, the aims of this study were to characterize in vitro the probiotic potential, antioxidant capacity, and antimicrobial activity of the marine yeast Y. lipolytica D-1 and N-6 strains. Dietary administration effect was evaluated in vivo on immunological parameters in serum, skin-mucus, intestine, and fish leukocytes upon challenge with Vibrio parahaemolyticus. The results showed that Y. lipolytica D-1 and N-6 strains grew with NaCl or bile salts but were sensitive to low pH. Each of the Y. lipolytica strains had a distinctive antioxidant capacity and fatty acid profile, but their antimicrobial activity was similar against fish bacterial pathogens. Fish (Lutjanus peru) supplemented with Y. lipolytica strains showed normal intestinal morphology, high IgM levels, and antioxidant enzyme activities. Immune-related genes were modulated in fish fed Y. lipolytica in a strain-dependent fashion. In addition, leucocytes from fish fed Y. lipolytica challenged with V. parahaemolyticus increased innate immune and antioxidant parameters compared with the control groups. In conclusion, the marine yeast Y. lipolytica D-1 and N-6 strains may be potential probiotics for fish by exerting free-radical scavenging, antimicrobial activity, and improved immune-protective responses against V. parahaemolyticus infection.

Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

Methyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

Phylogenomic and biochemical analysis reassesses temperate marine yeast Yarrowia lipolytica NCIM 3590 to be Yarrowia bubula

Yarrowia clade contains yeast species morphologically, ecologically, physiologically and genetically diverse in nature. Yarrowia lipolytica NCIM 3590 (NCIM 3590), a biotechnologically important strain, isolated from Scottish sea waters was reinvestigated for its phenotypic, biochemical, molecular and genomic properties as it exhibited characteristics unlike Y. lipolytica, namely, absence of extracellular lipolytic activity, growth at lower temperatures (less than 20 °C) and in high salt concentrations (10% NaCl). Molecular identification using ITS and D1/D2 sequences suggested NCIM 3590 to be 100% identical with reference strain Yarrowia bubula CBS 12934 rather than Y. lipolytica CBS 6124 (87% identity) while phylogenetic analysis revealed that it clustered with Y. bubula under a separate clade. Further, whole genome sequencing of NCIM 3590 was performed using Illumina NextSeq technology and the draft reported here. The overall genome relatedness values obtained by dDDH (94.1%), ANIb/ANIm (99.41/99.42%) and OrthoANI (99.47%) indicated proximity between NCIM 3590 and CBS 12934 as compared to the reference strain Y. lipolytica. No extracellular lipase activity could be detected in NCIM 3590 while LIP2 gene TBLASTN analysis suggests a low 42% identity with e value 2 e−77 and 62% coverage. Hence molecular, phylogenetic, genomics, biochemical and microbial analyses suggests it belongs to Yarrowia bubula.

High-efficiency production of bisabolene from waste cooking oil by metabolically engineered Yarrowia lipolytica.

SummaryThe natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non‐economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof‐of‐principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α‐bisabolene, β‐bisabolene and γ‐bisabolene through heterologous expression of the α‐bisabolene synthase from Abies grandis, the β‐bisabolene synthase gene from Zingiber officinale and the γ‐bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher‐level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.

Studies on Upgradation of Waste Fish Oil to Lipid-Rich Yeast Biomass in Yarrowia lipolytica Batch Cultures

The aim of the study was to evaluate the possibility to utilize a fish waste oil issued from the industrial smoking process in nitrogen-limited Yarrowia lipolytica yeast batch cultures. The waste carbon source was utilized by the yeast and stimulated the single cell oil production via an ex novo pathway. The yeast biomass contained lipids up to 0.227 g/g d.m.. Independently from culture conditions, high contents of very long chain fatty acids were quantified in yeast biomass including docosahexaenoic (DHA), eicosapentaenoic acid (EPA), eicosenic and erucic acids. The pH regulation did not influence the cellular lipids yield (0.234 g/g d.m.). Meanwhile, the intensification of the oxygenation of medium by changing the mixing speed (maximum concentration of lipids produced 4.64 g/dm3) and decreasing the amount of inoculum had a positive effect on the culture parameters in waste fish oil medium. Further work on upgradation of the original waste is advisable, especially because the oil indicated high content of polyphenols and lower susceptibility to oxidation than microbial oil derived from control olive oil medium.

Biomanufacturing of value‐added products from oils or fats: A case study on cellular and fermentation engineering of Yarrowia lipolytica

The United States produces more than 10 million tons of waste oils and fats each year. This paper aims to establish a new biomanufacturing platform that converts waste oils or fats into a series of value-added products. Our research employs the oleaginous yeast Yarrowia lipolytica as a case study for citric acid (CA) production from waste oils. First, we conducted the computational fluid dynamics (CFD) simulation of the bioreactor system and identified that the extracellular mixing and mass transfer is the first limiting factor of an oil fermentation process due to the insolubility of oil in water. Based on the CFD simulation results, the bioreactor design and operating conditions were optimized and successfully enhanced oil uptake and bioconversion in fed-batch fermentation experiments. After that, we investigated the impacts of cell morphology on oil uptake, intracellular lipid accumulation, and CA formation by overexpressing and deleting the MHY1 gene in the wild type Y. lipolytica ATCC20362. Fairly good linear correlations (R2 > 0.82) were achieved between cell morphology and productivities of biomass, lipid, and CA. Finally, fermentation kinetics with both glucose and oil substrates were compared and the oil fermentation process was carefully evaluated. Our study suggests that waste oils or fats can be economical feedstocks for biomanufacturing of many high-value products.

A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Waste soybean frying oil for the production, extraction, and characterization of cell-wall-associated lipases from Yarrowia lipolytica.

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26W/cm2 for 1min for CBLF and 52W/cm2 for 2min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37°C; and > 70% residual activity for 60days at 4, - 4°C and for 15days at 30°C.

Біоконверсія жировмісної фази стоків рибопереробних підприємств у кормову добавку

The technology of utilization of fat-containing phase of fish processing effluents on the basis of combination of physical and chemical influence and biological oxidation is offered. It was found that the most promising biodestructor of fats is the yeast Yarrowia lipolytica, selected for the substrate. A basic technological scheme of bioconversion of the fat-containing phase of effluents of fish processing enterprises into yeast biomass for feed purposes has been developed.

Природные стильбеновые полифенолы как цитопротекторы клеток Yarrowia lipolytica при тепловом шоке

Using the extremophilic yeast of Yarrowia lipolytica, a new model has been proposed to study the protective properties of stilbene polyphenols, namely resveratrol and pinosylvin, under heat shock. It was shown that a short-term thermal exposure of yeast cells (55 C, 25 min) led to a 40% decrease in the colony-forming ability of the population, a fivefold decrease in the respiration rate, and a growth of cyanide resistance and catalase activity, which indicated the adaptive yeast response to heat stress. Under these conditions, natural biologically active stilbenes, resveratrol and pinosylvin, at a concentration of 10 μM each increased yeast survival by 28% and 13%, respectively. In heat shock, resveratrol additionally raised catalase activity, while pinosylvin increased the cell respiration rate and decreased cyanide resistance and catalase activity. The results obtained indicate that resveratrol acts as a mild pro-oxidant inducing antioxidant protection during the adaptive response of the yeast to heat shock. Unlike resveratrol, pinosylvin increases cell survival stabilizing mitochondrial function and preserving the ATP-generating component of respiration. Yarrowia lipolytica yeast, polyphenols, stilbenoids, resveratrol, pinosylvin, cellular respiratory activity, heat shock, superoxide dismutase, catalase

CRISPR Interference and Activation to Modulate Transcription in Yarrowia lipolytica.

Recent developments in RNA-guided nuclease technologies have advanced the engineering of a wide range of organisms, including the nonconventional yeast Yarrowia lipolytica. Y. lipolytica has been the focus of a range of synthetic biology and metabolic engineering studies due to its high capacity to synthesize and accumulate intracellular lipids. The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully adapted and used for genome editing in Y. lipolytica. However, as engineered strains are moved closer to industrialization, the need for finer control of transcription is still present. To overcome this challenge, we have developed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems to allow modulating the transcription of endogenous genes. We begin this protocol chapter by describing how to use the CRISPRi system to repress expression of any gene in Y. lipolytica. A second method describes how to use the CRISPRa system to increase expression of native Y. lipolytica genes. Finally, we describe how CRISPRi or CRISPRa vectors can be combined to enable multiplexed activation or repression of more than one gene. The implementation of CRISPRi and CRISPRa systems improves our ability to control gene expression in Y. lipolytica and promises to enable more advanced synthetic biology and metabolic engineering studies in this host.

Biotechnological valorization of biodiesel-derived glycerol: Trials with the non-conventional yeasts Yarrowia lipolytica and Rhodosporidium sp

Four non-conventional yeast strains belonging to the genera Yarrowia and Rhodosporidium were cultivated under nitrogen-limited conditions in shake flasks with biodiesel-derived glycerol employed as sole substrate. The strains R. toruloides DSM 4444 and Y. lipolytica ACA-DC 5033 displayed better performances and were further studied. At initial glycerol (Gly0) ≈ 50 g/L, the strain ACA-DC 5033 produced dry cell weight (DCW) ≈7.0 g/L, and also secondary extra-cellular metabolites as citric acid (CA) at ≈16 g/L and polyols (Pol) (mannitol, erythritol and arabitol) at ≈21 g/L. In double-media (besides nitrogen also Mg was limiting) for Gly0 ≈ 50 g/L, a shift towards CA production occurred (CAmax ≈ 33 g/L). The strain DSM 4444 at Gly0 ≈ 50 g/L produced DCW = 18.1 g/L containing lipids = 30.3% in DCW. In single nitrogen-limited media with Gly0 ≈ 90 g/L, the strain ACA-DC 5033 produced Pol ≈ 48 g/L and CA ≈ 20 g/L, while the strain DSM 4444 produced DCW = 27.3 g/L containing lipid = 54.5% w/w. At the late growth phases, metabolites were re-consumed. Balanced growth phase (trophophase) and phase of secondary metabolite synthesis (idiophase) were successfully simulated with the aid of a modified Velhlust-Aggelis model. Lipid extraction process was studied for these two strains by using two different extraction methods. Yeast lipids contained mostly oleic acid, constituting suitable precursors for the synthesis of 2nd generation biodiesel.

Western Blotting of Membrane-Bound Proteins in Yarrowia lipolytica.

With the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane. Once immobilized on these membranes, the protein of interest can be detected and visualized by exploiting antigen-antibody interactions. However, not all proteins are amenable to easy detection by Western blotting. Integral membrane proteins are a class of proteins that are attached to a biological membrane through a series of transmembrane segments that span the width of the membrane. Due to the inherent hydrophobicity of these proteins and their tendency to aggregate, the characterization and detection of these proteins can be challenging. In this methods chapter, we present a protocol for the easy detection and quantification of these proteins in the industrially important oleaginous yeast Yarrowia lipolytica. The first protocol describes a Western blotting procedure to quantify soluble cytosolic proteins of interest in Yarrowia lipolytica from its total cell lysate. The second protocol describes modifications to the first that are done to enhance detection and quantification of membrane-bound proteins in Yarrowia lipolytica from its total cell lysate, without the need for isolating the membrane-bound proteins, for use in Western blotting. The immunoblotting strategies described here should serve as an efficient and simple guide to quantify both cytosolic and the intractable membrane-bound proteins in Yarrowia lipolytica.

Genome Editing in Y. lipolytica Using TALENs.

TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied. Here we introduce how to use TALENs in the oleaginous yeast Yarrowia lipolytica by presenting a step-by-step method allowing to knock out or to introduce in vivo a point mutation in a gene of Yarrowia lipolytica. This chapter describes the material required, the transformation procedure, and the screening process.

Guide RNA Design for Genome-Wide CRISPR Screens in Yarrowia lipolytica.

Genome-wide functional genomic screens are essential to determining the genetic underpinning of a biological process. Novel and powerful tools for perturbing gene function, with the help of genetic and epigenetic information, have made it possible to systematically investigate the contribution of every gene to evolved and engineered phenotypes. Functional genomics and screening for enhanced phenotypes become ever more important when dealing with nonconventional hosts. Non-model organisms are valuable to metabolic engineering as they present a range of desirable phenotypes and can help in avoiding complex and intensive engineering of less suitable hosts that do not possess the desired phenotype(s). Domestication of such hosts however requires a suite of synthetic biology tools that allow for targeted genome engineering, regulation of gene expression, and genome-wide mutational screens. The widespread adoption of CRISPR-Cas9 and CRISPR-Cpf1 based systems has allowed for such screens in many organisms. Key considerations in any genome-wide CRISPR screen are the design of a set of unique guide RNAs targeting the required set of genes in the genome and the design of nontargeting guide RNAs that function as appropriate negative controls for the experiment. In this methods chapter, we present protocols for the design of guides for a CRISPR screen, targeting every gene in the genome of the industrially relevant oleaginous yeast Yarrowia lipolytica. The first set of protocols describes the algorithm for the design of genome targeting and nontargeting guides for a genome-wide CRISPR-Cpf1 screen. The second set of protocols describes modifications to the first for the design of guides for a CRISPR-Cas9 screen. The strategies described here should serve as an efficient guide to design a library of gRNAs for most genome-wide CRISPR screens.

Engineering Yarrowia lipolytica for sustainable production of the chamomile sesquiterpene (−)-α-bisabolol

The oleaginous yeast Yarrowia lipolytica was metabolically engineered to produce the chamomile sesquiterpene (−)-α-bisabolol in a green and sustainable way.