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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., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

More than 200 million tons of plant oils and animal fats are produced annually worldwide from oil, crops, and the rendered animal fat industry. Triacylglycerol, an abundant energy-dense compound, is the major form of lipid in oils and fats. While oils or fats are very important raw materials and functional ingredients for food or related products, a significant portion is currently diverted to or recovered as waste. To significantly increase the value of waste oils or fats and expand their applications with a minimal environmental footprint, microbial biomanufacturing is presented as an effective strategy for adding value. Though both bacteria and yeast can be engineered to use oils or fats as the biomanufacturing feedstocks, the yeast Yarrowia lipolytica is presented as one of the most attractive platforms. Y. lipolytica is oleaginous, generally regarded as safe, demonstrated as a promising industrial producer, and has unique capabilities for efficient catabolism and bioconversion of lipid substrates. This review summarizes the major challenges and opportunities for Y. lipolytica as a new biomanufacturing platform for the production of value-added products from oils and fats. This review also discusses relevant cellular and metabolic engineering strategies such as fatty acid transport, fatty acid catabolism and bioconversion, redox balances and energy yield, cell morphology and stress response, and bioreaction engineering. Finally, this review highlights specific product classes including long-chain diacids, wax esters, terpenes, and carotenoids with unique synthesis opportunities from oils and fats in Y. lipolytica., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Microdroplet screening of microorganisms can improve the rate of strain selection and characterization within the canonical design-build-test paradigm. However, a full analysis of the microdroplet environment and how well these conditions translate to culturing conditions and techniques is lacking in the field. Quantification of three different biosensor/analyte combinations at 12 hour timepoints reveals the potential for extended dose-response ranges as compared to traditional in vitro conditions. Using these dynamics, we present an application and analysis of microfluidic droplet screening utilizing whole-cell biosensors, ultimately identifying an altered productivity profile of itaconic acid in a Yarrowia lipolytica -based piggyBac transposon library. Specifically, we demonstrate that the timepoint for microdroplet selection can influence the outcome of the selection and thus shift the identified strain productivity and final titer. In this case, strains selected at earlier timepoints showed increased early productivity in flask scale, with the converse true as well. Differences in response indicate microdroplet assays require tailored development to more accurately sort for phenotypes that are scalable to larger incubation volumes. Likewise, these results further highlight that screening conditions are critical parameters for success in high-throughput applications.

Hydrothermal liquefaction (HTL) is an emerging method for thermochemical conversion of wet organic waste and biomass into renewable biocrude. HTL also produces an aqueous phase (HTL-AP) side stream containing 2-4% light organic compounds that require treatment. Although anaerobic digestion (AD) of HTL-AP has shown promise, lengthy time periods were required for AD microbial communities to adapt to metabolic inhibitors in HTL-AP. An alternative for HTL-AP valorization was recently demonstrated using two engineered strains of Yarrowia lipolytica, E26 and Diploid TAL, for the overproduction of lipids and the polyketide triacetic acid lactone (TAL) respectively. These strains tolerated up to 10% HTL-AP (v/v) in defined media and up to 25% (v/v) HTL-AP in rich media. In this work, adaptive laboratory evolution (ALE) of these strains increased the bulk population tolerance for HTL-AP to up to 30% (v/v) in defined media and up to 35% (v/v) for individual isolates in rich media. The predominate organic acids within HTL-AP (acetic, butyric, and propionic) were rapidly consumed by the evolved Y. lipolytica strains. A TAL-producing isolate (strain 144-3) achieved a nearly 3-fold increase in TAL titer over the parent strain while simultaneously reducing the chemical oxygen demand (COD) of HTL-AP containing media. Fermentation with HTL-AP as the sole nutrient source demonstrated direct conversion of waste into TAL at 10% theoretical yield. Potential genetic mutations of evolved TAL production strains that could be imparting tolerance were explored. This work advances the potential of Y. lipolytica to biologically treat and simultaneously extract value from HTL wastewater. KEY POINTS: • Adaptive evolution of two Y. lipolytica strains enhanced their tolerance to waste. • Y. lipolytica reduces chemical oxygen demand in media containing waste. • Y. lipolytica can produce triacetic acid lactone directly from wastewater., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microbial fermentation offers a sustainable source of fuels, commodity chemicals, and pharmaceuticals, yet strain performance is influenced greatly by the growth media selected. Specifically, trace metals (e.g., iron, copper, manganese, zinc, and others) are critical for proper growth and enzymatic function within microorganisms yet are non-standardized across media formulation. In this work, the effect of trace metal supplementation on the lipid production profile of Yarrowia lipolytica was explored using tube scale fermentation followed by biomass and lipid characterization. Addition of iron (II) to the chemically defined Yeast Synthetic Complete (YSC) medium increased final optical density nearly twofold and lipid production threefold, while addition of copper (II) had no impact. Additionally, dose-responsive changes in lipid distribution were observed, with the percent of oleic acid increasing and stearic acid decreasing as initial iron concentration increased. These changes were reversible with subsequent iron-selective chelation. Use of rich Yeast Peptone Dextrose (YPD) medium enabled further increases in the production of two specialty oleochemicals ultimately reaching 63 and 47% of the lipid pool as α-linolenic acid and cyclopropane fatty acid, respectively, compared to YSC medium. Selective removal of iron (II) natively present in YPD medium decreased this oleochemical production, ultimately aligning the lipid profile with that of non-supplemented YSC medium. These results provide further insight into the proposed mechanisms for iron regulation in yeasts especially as these productions strains contain a mutant allele of the iron regulator, mga2. The work presented here also suggests a non-genetic method for control of the lipid profile in Y. lipolytica for use in diverse applications. KEY POINTS: • Iron supplementation increases cell density and lipid titer in Yarrowia lipolytica. • Iron addition reversibly alters lipid portfolio increasing linolenic acid. • Removal of iron from YPD media provides a link to enhanced oleochemical production., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A mutant excision + /integration - piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.

Hydrothermal liquefaction is a promising technology to upgrade wet organic waste into a biocrude oil for diesel or jet fuel; however, this process generates an acid-rich aqueous phase which poses disposal issues. This hydrothermal liquefaction aqueous phase (HTL-AP) contains organic acids, phenol, and other toxins. This work demonstrates that Y. lipolytica as a unique host to valorize HTL-AP into a variety of co-products. Specifically, strains of Y. lipolytica can tolerate HTL-AP at 10% in defined media and 25% in rich media. The addition of HTL-AP enhances production of the polymer precursor itaconic acid by 3-fold and the polyketide triacetic acid lactone at least 2-fold. Additional co-products (lipids and citric acid) were produced in these fermentations. Finally, bioreactor cultivation enabled 21.6 g/L triacetic acid lactone from 20% HTL-AP in mixed sugar hydrolysate. These results demonstrate the first use of Y. lipolytica in HTL-AP valorization toward production of a portfolio of value-added compounds., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α-linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose-utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52 g L -1 ALA, 96.6 mg L -1 riboflavin, and 2.9 g L -1 TAL, are obtained from xylose in flask cultures and 1.42 g L -1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polyketides are a diverse class of molecules sought after for their valuable properties, including as potential pharmaceuticals. Previously, we demonstrated that the oleaginous yeast Yarrowia lipolytica is an optimal host for production of the simple polyketide, triacetic acid lactone (TAL). We here expand the capacities of this host by overcoming previous media challenges and enabling production of more complex polyketides. Specifically, we employ a β-oxidation related strategy to improve polyketide production directly from defined media. Beyond TAL production, we establish biosynthesis of the 4-coumaroyl-CoA derived polyketides: naringenin, resveratrol, and bisdemethoxycurcumin, as well as the diketide intermediate, (E)-5-(4-hydroxyphenyl)-3-oxopent-4-enoic acid. In this background, we enable high-level de novo production of naringenin through import of both a heterologous pathway and a mutant Y. lipolytica allele. In doing so, we generated an averaged maximum titer of 898 mg/L naringenin, the highest titer reported to date in any host. These results demonstrate that Y. lipolytica is an ideal polyketide production host for more complex 4-coumaroyl-CoA derived products., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Microbial production of fuels and chemicals offers a means by which sustainable product manufacture can be achieved. In this regard, Yarrowia lipolytica is a unique microorganism suitable for a diverse array of biotechnological applications. As a robust oleaginous yeast, it has been well studied for production of fuels and chemicals derived from fatty acids. However, thanks in part to newfound genetic tools and metabolic understanding, Y. lipolytica has been explored for high-level production of a variety of non-lipid products. This mini-review will discuss some of the recent research surrounding the ability of Y. lipolytica to support bio-based chemical production outside the realm of fatty acid metabolism including polyketides, terpenes, carotenoids, pentose phosphate-derived products, polymers, and nanoparticles.

Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C). We subsequently enhance tolerance in S. cerevisiae through directed evolution of YlILT1 using growth-based selection, leading to identification of mutants that grow in up to 3.5% v/v ionic liquid. Lastly, we demonstrate that strains expressing YlILT1 variants demonstrate improved growth rate and ethanol production in the presence of residual IIL. This shows that dominant overexpression of a heterologous protein (wild type or evolved) from an IIL-tolerant yeast can increase tolerance in S. cerevisiae at concentrations relevant to bioethanol production from IIL-treated biomass.

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation., (Copyright © 2019 International Metabolic Engineering Society. All rights reserved.)

Yarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6 × 104 transformants per μg DNA and 2.8 × 104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.

The nonconventional, oleaginous yeast, Yarrowia lipolytica is rapidly emerging as a valuable host for the production of a variety of both lipid and nonlipid chemical products. While the unique genetics of this organism pose some challenges, many new metabolic engineering tools have emerged to facilitate improved genetic manipulation in this host. This review establishes a case for Y. lipolytica as a premier metabolic engineering host based on innate metabolic capacity, emerging synthetic tools, and engineering examples. The metabolism underlying the lipid accumulation phenotype of this yeast as well as high flux through acyl-CoA precursors and the TCA cycle provide a favorable metabolic environment for expression of relevant heterologous pathways. These properties allow Y. lipolytica to be successfully engineered for the production of both native and nonnative lipid, organic acid, sugar and acetyl-CoA derived products. Finally, this host has unique metabolic pathways enabling growth on a wide range of carbon sources, including waste products. The expansion of carbon sources, together with the improvement of tools as highlighted here, have allowed this nonconventional organism to act as a cellular factory for valuable chemicals and fuels., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12-15 desaturase (Rk Δ12-15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.

Traditional synthesis of biodiesel competes with food sources and has limitations with storage, particularly due to limited oxidative stability. Microbial synthesis of lipids provides a platform to produce renewable fuel with improved properties from various renewable carbon sources. Specifically, biodiesel properties can be improved through the introduction of a cyclopropane ring in place of a double bond. In this study, we demonstrate the production of C19 cyclopropanated fatty acids in the oleaginous yeast Yarrowia lipolytica through the heterologous expression of the Escherichia coli cyclopropane fatty acid synthase. Ultimately, we establish a strain capable of 3.03 ± 0.26 g/L C19 cyclopropanated fatty acid production in bioreactor fermentation where this functionalized lipid comprises over 32% of the total lipid pool. This study provides a demonstration of the flexibility of lipid metabolism in Y. lipolytica to produce specialized fatty acids.

The oleaginous yeast Yarrowia lipolytica is quickly emerging as the most popular non-conventional (i.e., non-model organism) yeast in the bioproduction field. With a high propensity for flux through tricarboxylic acid (TCA) cycle intermediates and biological precursors such as acetyl-CoA and malonyl-CoA, this host is especially well suited to meet our industrial chemical production needs. Recent progress in synthetic biology tool development has greatly enhanced our ability to rewire this organism, with advances in genetic component design, CRISPR technologies, and modular cloning strategies. In this review we investigate recent developments in metabolic engineering and describe how the new tools being developed help to realize the full industrial potential of this host. Finally, we conclude with our vision of the developments that will be necessary to enhance future engineering efforts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Yarrowia lipolytica is a non-conventional yeast of interest to the biotechnology industry. However, the physiology, metabolism, and genetic regulation of Y. lipolytica diverge significantly from more well-studied and characterized yeasts such as Saccharomyces cerevisiae. To develop additional genetic tools for this industrially relevant host, the piggyBac transposon system to enable efficient generation of genome-wide insertional mutagenesis libraries and introduction of scarless, footprint-free genomic modifications in Y. lipolytica. Specifically, we demonstrate piggyBac transposition in Y. lipolytica, and then use the approach to screen transposon insertion libraries for rapid isolation of mutations that confer altered canavanine resistance, pigment formation, and neutral lipid accumulation. We also develop a variety of piggyBac compatible selection markers for footprint-free genome engineering, including a novel dominant marker cassette (Escherichia coli guaB) for effective Y. lipolytica selection using mycophenolic acid. We utilize these marker cassettes to construct a piggyBac vector set that allows for auxotrophic selection (uracil or tryptophan biosynthesis) or dominant selection (hygromycin, nourseothricin, chlorimuron ethyl, or mycophenolic acid resistance) and subsequent marker excision. These new genetic tools and techniques will help to facilitate and accelerate the engineering of Y. lipolytica strains for efficient and sustainable production of a wide variety of small molecules and proteins., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica ) can overcome such production limitations owing to a natural propensity for high flux through acetyl-CoA. By exploring three distinct metabolic engineering strategies for acetyl-CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression ( PEX10 ) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials., Competing Interests: The authors declare no conflict of interest.

The conversion of lignocellulosic sugars, in particular xylose, is important for sustainable fuels and chemicals production. While the oleaginous yeast Yarrowia lipolytica is a strong candidate for lipid production, it is currently unable to effectively utilize xylose. By introducing a heterologous oxidoreductase pathway and enabling starvation adaptation, we obtained a Y. lipolytica strain, E26 XUS, that can use xylose as a sole carbon source and produce over 15 g/L of lipid in bioreactor fermentations (29.3% of theoretical yield) with a maximal lipid productivity of 0.19 g/L/h. Genomic sequencing and genetic analysis pointed toward increases in genomic copy number of the pathway and resulting elevated expression levels as the causative mutations underlying this improved phenotype. More broadly, many regions of the genome were duplicated during starvation of Yarrowia. This strain can form the basis for further engineering to enhance xylose catabolic rates and conversion. Finally, this study also reveals the flexibility and dynamic nature of the Y. lipolytica genome, and the means at which starvation can be used to induce genomic duplications., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Itaconic acid is a naturally produced organic acid with diverse applications as a replacement for petroleum derived products. However, its industrial viability as a bio-replacement has been restricted due to limitations with native producers. In this light, Yarrowia lipolytica is an excellent potential candidate for itaconic acid production due to its innate capacity to accumulate citric acid cycle intermediates and tolerance to lower pH. Here, we demonstrate the capacity to produce itaconic acid in Y. lipolytica through heterologous expression of the itaconic acid synthesis enzyme, resulting in an initial titer of 33 mg/L. Further optimizations of this strain via metabolic pathway engineering, enzyme localization, and media optimization strategies enabled 4.6g/L of itaconic acid to be produced in bioreactors, representing a 140-fold improvement over initial titer. Moreover, these fermentation conditions did not require additional nutrient supplementation and utilized a low pH condition that enabled the acid form of itaconic acid to be produced. Overall yields (0.058 g/g yield from glucose) and maximum productivity of 0.045 g/L/h still provide areas for future strain improvement. Nevertheless, this work demonstrates that Y. lipolytica has the potential to serve as an industrially relevant platform for itaconic acid production., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Lipogenic organisms represent great starting points for metabolic engineering of oleochemical production. While previous engineering efforts were able to significantly improve lipid production in Yarrowia lipolytica, the lipogenesis landscape, especially with respect to regulatory elements, has not been fully explored. Through a comparative genomics and transcriptomics approach, we identified and validated a mutant mga2 protein that serves as a regulator of desaturase gene expression and potent lipogenesis factor. The resulting strain is enriched in unsaturated fatty acids. Comparing the underlying mechanism of this mutant to other previously engineered strains suggests that creating an imbalance between glycolysis and the TCA cycle can serve as a driving force for lipogenesis when combined with fatty acid catabolism overexpressions. Further comparative transcriptomics analysis revealed both distinct and convergent rewiring associated with these different genotypes. Finally, by combining metabolic engineering targets, it is possible to further engineer a strain containing the mutant mga2 gene to a lipid production titer of 25g/L., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Lipogenic organisms provide an ideal platform for biodiesel and oleochemical production. Through our previous rational metabolic engineering efforts, lipogenesis titers in Yarrowia lipolytica were significantly enhanced. However, the resulting strain still suffered from decreased biomass generation rates. Here, we employ a rapid evolutionary metabolic engineering approach linked with a floating cell enrichment process to improve lipogenesis rates, titers, and yields. Through this iterative process, we were able to ultimately improve yields from our prior strain by 55% to achieve production titers of 39.1g/L with upwards of 76% of the theoretical maximum yield of conversation. Isolated cells were saturated with up to 87% lipid content. An average specific productivity of 0.56g/L/h was achieved with a maximum instantaneous specific productivity of 0.89g/L/h during the lipid production phase in fermentation. Genomic sequencing of the evolved strains revealed a link between a decrease/loss of function mutation of succinate semialdehyde dehydrogenase, uga2, suggesting the importance of gamma-aminobutyric acid assimilation in lipogenesis. This linkage was validated through gene deletion experiments. This work presents an improved host strain that can serve as a platform for efficient oleochemical production., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Manipulating gene expression using different types of plasmids (centromeric, 2-micron, and autonomously replicating sequence) can expand expression levels and enable a quick characterization of combinations, both of which are highly desired for metabolic engineering applications. However, for the powerful industrial workhorse Yarrowia lipolytica, only a low-copy CEN plasmid is available. In this study, we engineered the CEN plasmid from Y. lipolytica by fusing different promoters upstream of the centromeric region to regulate its function and expand its range. By doing this, we successfully improved the copy number and gene expression at plasmid level by 80% and enabled a dynamic range of nearly 2.7-fold. This improvement was seen to be independent from both the promoter and gene used in the expression cassette. These results present a better starting point for more potent plasmids in Y. lipolytica., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolytica's native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. We further complete direct conversion of lipid content into biodiesel. Tri-level metabolic control results in saturated cells containing upwards of 90% lipid content and titres exceeding 25 g l(-1) lipids, which represents a 60-fold improvement over parental strain and conditions. Through this rewiring effort, we advance fundamental understanding of lipogenesis, demonstrate non-canonical environmental and intracellular stimuli and uncouple lipogenesis from nitrogen starvation. The high titres and carbon-source independent nature of this lipogenesis in Y. lipolytica highlight the potential of this organism as a platform for efficient oleochemical production.

The complete biosynthetic replacement of petroleum transportation fuels requires a metabolic pathway capable of producing short chain n-alkanes. Here, we report and characterize a proof-of-concept pathway that enables microbial production of the C5 n-alkane, pentane. This pathway utilizes a soybean lipoxygenase enzyme to cleave linoleic acid to pentane and a tridecadienoic acid byproduct. Initial expression of the soybean lipoxygenase enzyme within a Yarrowia lipolytica host yielded 1.56 mg/L pentane. Efforts to improve pentane yield by increasing substrate availability and strongly overexpressing the lipoxygenase enzyme successfully increased pentane production three-fold to 4.98 mg/L. This work represents the first-ever microbial production of pentane and demonstrates that short chain n-alkane synthesis is conceivable in model cellular hosts. In this regard, we demonstrate the potential pliability of Y. lipolytica toward the biosynthetic production of value-added molecules from its generous fatty acid reserves., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Both varied and strong promoters are essential for metabolic and pathway engineering applications in any host organism. To enable this capacity, here we demonstrate a generalizable method for the de novo construction of strong, synthetic hybrid promoter libraries. Specifically, we demonstrate how promoter truncation and fragment dissection analysis can be utilized to identify both novel upstream activating sequences (UAS) and core promoters-the two components required to generate hybrid promoters. As a base case, the native TEF promoter in Yarrowia lipolytica was examined to identify putative UAS elements that serve as modular synthetic transcriptional activators. Resulting synthetic promoters containing a core promoter region activated by between one and twelve tandem repeats of the newly isolated, 230 nucleotide UASTEF#2 element showed promoter strengths 3- to 4.5-fold times the native TEF promoter. Further analysis through transcription factor binding site abrogation revealed the GCR1p binding site to be necessary for complete UASTEF#2 function. These various promoters were tested for function in a variety of carbon sources. Finally, by combining disparate UAS elements (in this case, UASTEF and UAS1B), we developed a high-strength promoter with for Y. lipolytica with an expression level of nearly sevenfold higher than that of the strong, constitutive TEF promoter. Thus, the general strategy described here enables the efficient, de novo construction of synthetic promoters to both increase native expression capacity and to produce libraries for tunable gene expression.