Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid.

Punicic acid is a conjugated linolenic acid with various biological activities including antiobesity, antioxidant, anticancer, and anti-inflammatory effects. It is often used as a nutraceutical, dietary additive, and animal feed. Currently, punicic acid is primarily extracted from pomegranate seed oil, but it is restricted due to the extended growth cycle, climatic limitations, and low recovery level. There have also been reports on the chemical synthesis of punicic acid, but it resulted in a mixture of structurally similar isomers, requiring additional purification/separation steps. In this study, a comprehensive strategy for the production of punicic acid in Yarrowia lipolytica was implemented by pushing the supply of linoleic acid precursors in a high-oleic oil strain, expressing multiple copies of the fatty acid conjugase gene from Punica granatum, engineering the acyl-editing pathway to improve the phosphatidylcholine pool, and promoting the assembly of punicic acid in the form of triglycerides. The optimal strain with high oil production capacity and a significantly increased punicic acid ratio accumulated 3072.72 mg/L punicic acid, accounting for 6.19% of total fatty acids in fed-batch fermentation, providing a viable, sustainable, and green approach for punicic acid production to substitute plant extraction and chemical synthesis production.

Revealing the Mechanisms of Enhanced ß-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

ß-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing ß-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of ß-farnesene, the changes in intracellular metabolites in the ß-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the ß-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the ß-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica.

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l-1 betanin and 55 ± 7 mg l-1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr-1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale.

DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting point, we used transcriptomics, metabolomics, and 13C-based metabolic pathway profiling to study the cellular dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster, as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the degradation pathways for the ketogenic amino acids l-lysine, l-leucine, and l-isoleucine were transcriptionally activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted the DHA titer by almost 40%. Isotopic 13C-tracer studies revealed that the supplements were efficiently directed toward intracellular CoA-esters and DHA. Hereby, l-lysine was found to be most efficient, as it enabled long-term activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica, whose synthesis is limited by the availability of CoA ester precursors.

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production.

BACKGROUND: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. RESULTS: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. CONCLUSIONS: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

Isolation, purification, and mass spectrometry identification of the enzyme involved in citrus flavor (+)-valencene biotransformation to (+)-nootkatone by Yarrowia lipolytica.

BACKGROUND: (+)-Nootkatone is a highly valuable sesquiterpene compound that can be used as an aromatic in the food industry because of its grapefruit flavor and low sensory threshold. The unconventional yeast Yarrowia lipolytica has many unique physical and chemical properties, metabolic characteristics, and genetic structure, which has aroused the interest of researchers. Previous research showed that Y. lipolytica possesses the ability to transform the sesquiterpene (+)-valencene to (+)-nootkatone. The aim of this study was to isolate, purify, and identify the enzyme involved in the (+)-valencene bioconversion to (+)-nootkatone by Y. lipolytica. RESULTS: In this study, ultrasonic-assisted extraction, ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography were used to separate and purify the enzyme involved in the (+)-valencene bioconversion by Y. lipolytica. The protein was identified as aldehyde dehydrogenase (ALDH) (gene0658) using sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. The ALDH had the highest activity when the pH value was 6.0 and the temperature was 30 °C. The activity of ALDH was significantly stimulated by ferrous ions and inhibited by barium, calcium, and magnesium ions. CONCLUSION: This is the first time that ALDH was found to participate in (+)-valencene biotransformation by Y. lipolytica. It may be involved in regulating the microbial transformation of (+)-valencene to (+)-nootkatone through redox characteristics. This study provides a theoretical basis and reference for the biological synthesis of citrus flavor (+)-nootkatone. © 2023 Society of Chemical Industry.

Using oils and fats to replace sugars as feedstocks for biomanufacturing: Challenges and opportunities for the yeast Yarrowia lipolytica.

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.

Shifting the distribution: modulation of the lipid profile in Yarrowia lipolytica via iron content.

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.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

Preparation of Sulforaphene from Radish Seed Extracts with Recombinant Food-Grade Yarrowia lipolytica Harboring High Myrosinase Activity.

Sulforaphene prepared from glucoraphenin by myrosinase is one of the main active ingredients of radish, which has various biological activities and brilliant potential for food and pharmaceutical applications. In this paper, a recombinant food-grade yeast transformant 20-8 with high-level myrosinase activity was constructed by over-expressing a myrosinase gene from Arabidopsis thaliana in Yarrowia lipolytica. The highest myrosinase activity produced by the transformant 20-8 reached 44.84 U/g dry cell weight when it was cultivated in a 10 L fermentor within 108 h. Under the optimal reaction conditions, 6.1 mg of sulforaphene was yielded from 1 g of radish seeds under the catalysis of the crude myrosinase preparation (4.95 U) at room temperature within 1.5 h. What is more is that when the whole yeast cells harboring myrosinase activity were reused 10 times, the sulforaphene yield still reached 92.53% of the initial level. Therefore, this efficient approach has broad application prospects in recyclable and large-scale preparation of sulforaphene.

Transcriptome Response of the Tropical Marine Yeast Yarrowia lipolytica on Exposure to Uranium.

In our earlier investigation, we reported the consequences of uranium (U)-induced oxidative stress and cellular defense mechanisms alleviating uranium toxicity in the marine yeast Yarrowia lipolytica NCIM 3589. However, there is lack of information on stress response towards uranium toxicity at molecular level in this organism. To gain an insight on this, transcriptional response of Y. lipolytica after exposure to 50 µM uranium was investigated by RNA sequencing at the global level in this study. The de novo transcriptome analysis (in triplicates) revealed 56 differentially expressed genes with significant up-regulation and down-regulation of 33 and 23 transcripts, respectively, in U-exposed yeast cells as compared to the control, U-unexposed cells. Highly up-regulated genes under U-treated condition were identified to be primarily involved in transport, DNA damage repair and oxidative stress. The major reaction of Y. lipolytica to uranium exposure was the activation of oxidative stress response mechanisms to protect the important biomolecules of the cells. On the other hand, genes involved in cell wall and cell cycle regulation were significantly down-regulated. Overall, the transcriptional profiling by RNA sequencing to stress-inducing concentration of uranium sheds light on the various responses of Y. lipolytica for coping with uranium toxicity, providing a foundation for understanding the molecular interactions between uranium and this marine yeast.

A CRISPR/Cas9-Mediated, Homology-Independent Tool Developed for Targeted Genome Integration in Yarrowia lipolytica.

Yarrowia lipolytica has been extensively used to produce essential chemicals and enzymes. As in most other eukaryotes, nonhomologous end joining (NHEJ) is the major repair pathway for DNA double-strand breaks in Y. lipolytica Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica Through optimization of the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with 12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, the cell cycle, and the integration sites. Thus, the targeted integration rate reached 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes in the pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers and achieves one-step targeted genome integration of the 8,417-bp DNA fragment, potentially replacing current HR-dependent genome-editing methods for Y. lipolyticaIMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica This tool does not require the construction of homologous templates and can be used to rapidly verify genetic elements and to iteratively integrate multiple-gene pathways in Y. lipolytica This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.

Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates.

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

Yarrowia lipolytica as an emerging biotechnological chassis for functional sugars biosynthesis.

Functional sugars have unique structural and physiological characteristics with applied perspectives for modern biomedical and biotechnological sectors, such as biomedicine, pharmaceutical, cosmeceuticals, green chemistry, and agro-food. They can also be used as starting matrices to produce biologically active metabolites of interests. Though numerous chemical synthesis routes have been proposed and deployed for the synthesis of rare sugars, however, many of them are limited and economically incompetent because of expensive raw starting feedstocks. Whereas, the biosynthesis by enzymatic means are often associated with high catalyst costs and low space-time yields. Microbial production of rare sugars via green routes using bio-renewable resources offers noteworthy solutions to overcome the aforementioned limitations of synthetic and enzymatic synthesis routes. From the microbial-based synthesis perspective, the lipogenic yeast Yarrowia lipolytica is rapidly evolving as the most prevalent and unique "non-model organism" in the bio-production arena. Due to high flux tendency through the tri-carboxylic acid cycle intermediates and precursors such as acetyl-CoA and malonyl-CoA, this yeast has been widely investigated to meet the increasing demand of industrially relevant fine chemicals, including functional sugars. Incredible interest in Y. lipolytica originates from its robust tolerance to unstable pH, salt levels, and organic compounds, which subsequently enable easy bioprocess optimization. Meaningfully, GRAS (generally recognized as safe) status creates Y. lipolytica as an attractive and environmentally friendly microbial host for the manufacturing of nutraceuticals, fermented food, and dietary supplements. In this review, we highlight the recent and state-of-the-art research progress on Y. lipolytica as a host to synthesize bio-based compounds of interest beyond the realm of well-known fatty acid production. The unique physicochemical properties, biotechnological applications, and biosynthesis of an array of value-added functional sugars including erythritol, threitol, fructooligosaccharides, galactooligosaccharides, isomalto-oligosaccharides, isomaltulose, trehalose, erythrulose, xylitol, and mannitol using sustainable carbon sources are thoroughly vetted. Finally, we conclude with perspectives that would be helpful to engineer Y. lipolytica in greening the twenty-first century biomedical and biotechnological sectors of the modern world.

Production and excretion of astaxanthin by engineered Yarrowia lipolytica using plant oil as both the carbon source and the biocompatible extractant.

This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones.Key Points• Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production.• Astaxanthin is excreted and accumulated in the extracellular oil phase.• Astaxanthin is the predominant carotenoid in the extracellular oil phase.• Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction. Graphical abstract.

Pathway engineering and medium optimization for α-farnesene biosynthesis in oleaginous yeast Yarrowia lipolytica.

Farnesene is a typical sesquiterpene with applications as fragrance, flavor and precursor for the synthesis of vitamin E/K1. In this study, a series of strategies were employed to facilitate α-farnesene accumulation in Yarrowia lipolytica. Among them, the promoter optimization of OptFSLERG20, Sc-tHMG1 and IDI resulted in more than 62 % increase in α-farnesene production. Together with the overexpression of Yl-HMGR and ERG19, α-farnesene content was significantly improved by more than 3.5 times. The best metabolic engineered strain obtained was therefore used for a uniform design in shake flasks to determine the optimal medium compositions. Furthermore, a maximum α-farnesene production of approximately 2.57 g/L (34 mg/g DCW) was obtained in fed-batch fermentation where glycerol was supplemented as the feeding carbon source when initial glucose was depleted. This study has laid a good foundation for the development of Y. lipolytica as a promising chassis microbial cell for heterologous biosynthesis of α-farnesene and other sesquiterpenes.

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach.

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.

Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil.

Fatty acid ethyl esters (FAEEs) can potentially be used as biodiesel, which provides a renewable alternative to petroleum-derived diesel. FAEEs are primarily produced via transesterification of vegetable oil with an alcohol catalyzed by a strong base, which raises safety concerns. Microbial production presents a more environmentally sustainable method for FAEE production, and by harnessing the ability of oleaginous yeast Yarrowia lipolytica to degrade and assimilate hydrophobic substrates, FAEE production could be coupled to food waste bioremediation. In this study, we engineered Y. lipolytica to produce FAEEs from dextrose as well as from vegetable cooking oil as a model food waste. Firstly, we introduced pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis to reconstitute the heterologous pathway for ethanol production. Second, we introduced and compared two heterologous wax ester synthases ws2 and maqu_0168 from Marinobacter sp. for FAEE biosynthesis. Next, we disrupted competitive pathways to increase fatty acyl-CoA pool, and optimized carbon sources and cell density for shake-flask fermentation. The engineered strain showed a 24-fold improvement in FAEE production titer over the starting strain. Moreover, we explored the potential of the engineered strain for FAEE production from the model food waste by supplementing vegetable cooking oil to the culture medium. To the best of our knowledge, this is the first report on FAEE production with the supplementation of vegetable cooking oil in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for high-level production of FAEEs and its utilization in food waste bioremediation.

Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.