Improving an Alternative Glycerol Catabolism Pathway in Yarrowia lipolytica to Enhance Erythritol Production.

Engineering the glycerol-3-phosphate pathway could enhance erythritol production by accelerating glycerol uptake. However, little work has been conducted on the alternative dihydroxyacetone (DHA) pathway in Yarrowia lipolytica. Herein, this route was identified and characterized in Y. lipolytica by metabolomic and transcriptomic analysis. Moreover, the reaction catalyzed by dihydroxyacetone kinase encoded by dak2 was identified as the rate-limiting step. By combining NHEJ-mediated insertion mutagenesis with a push-and-pull strategy, Y. lipolytica strains with high-yield erythritol synthesis from glycerol were obtained. Screening of a library of insertion mutants allows the identification of a mutant with fourfold increased erythritol production. Overexpression of DAK2 and glycerol dehydrogenase GCY3 together with gene encoding transketolase and transaldolase from the nonoxidative part of the pentose phosphate pathway led to a strain with further increased productivity with a titer of 53.1 g/L and a yield 0.56 g/g glycerol, which were 8.1- and 4.2-fold of starting strain.

Effect of low frequency alternating magnetic field for erythritol production in Yarrowia lipolytica.

Numerous works have reported that magnetic fields serve as signals capable of influencing microbial metabolism. However, little is known about the effect of magnetic field on erythritol production by the model microorganism Yarrowia lipolytica (Y. lipolytica). Therefore, we investigated the effect of low-frequency alternating magnetic fields (LF-AMF) with different magnetic field intensities (0-1.5 mT) and different magnetic field treatment times (1-10 days) on the production of erythritol by Y. lipolytica -JZ204. The optimal treatment condition was 0.5 mT for 8 days. As a result, a maximal erythritol yield was achieved 63.74 g/L, the biomass was reached 37 g/L, and the specific erythritol yield per unit of biomass was 1.7227 g/g, which were 60.72%, 32.09%, and 24.85% higher than the control, respectively. We investigated the internal mechanism of magnetic fields impact by using transcriptomics and RT-qPCR technology. This study demonstrated the effectiveness of LF-AMF in enhancing erythritol production by Y. lipolytica JZ-204, providing insights for the application of magnetic field in assisting microbial fermentation and improving the synthesis of beneficial products.

Phosphatidylserine synthase plays a critical role in the utilization of n-alkanes in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica can assimilate n-alkane as a carbon and energy source. To elucidate the significance of phosphatidylserine (PS) in the utilization of n-alkane in Y. lipolytica, we investigated the role of the Y. lipolytica ortholog (PSS1) of Saccharomyces cerevisiae PSS1/CHO1, which encodes a PS synthase. The PSS1 deletion mutant (pss1Δ) of Y. lipolytica could not grow on minimal medium in the absence of ethanolamine and choline but grew when either ethanolamine or choline was supplied to synthesize phosphatidylethanolamine and phosphatidylcholine. The pss1Δ strain exhibited severe growth defects on media containing n-alkanes even in the presence of ethanolamine and choline. In the pss1Δ strain, the transcription of ALK1, which encodes a primary cytochrome P450 that catalyzes the hydroxylation of n-alkanes in the endoplasmic reticulum, was upregulated by n-alkane as in the wild-type strain. However, the production of functional P450 was not detected, as indicated by the absence of reduced CO-difference spectra in the pss1Δ strain. PS was undetectable in the lipid extracts of the pss1Δ strain. These results underscore the critical role of PSS1 in the biosynthesis of PS, which is essential for the production of functional P450 enzymes involved in n-alkane hydroxylation in Y. lipolytica.

Engineering Yarrowia lipolytica for Efficient Synthesis of Geranylgeraniol.

Geranylgeraniol (GGOH) is a crucial component in fragrances and essential oils, and a valuable precursor of vitamin E. It is primarily extracted from the oleoresin of Bixa orellana, but is challenged by long plant growth cycles, severe environmental pollution, and low extraction efficiency. Chemically synthesized GGOH typically comprises a mix of isomers, making the separation process both challenging and costly. Advancements in synthetic biology have enabled the construction of microbial cell factories for GGOH production. In this study, Yarrowia lipolytica was engineered to efficiently synthesize GGOH by expressing heterologous phosphatase genes, enhancing precursor supplies of farnesyl diphosphate, geranylgeranyl pyrophosphate, and acetyl-CoA, and downregulating the squalene synthesis pathway by promoter engineering. Additionally, optimizing fermentation conditions and reducing reactive oxygen species significantly increased the GGOH titer to 3346.47 mg/L in a shake flask. To the best of our knowledge, this is the highest reported GGOH titer in shaking flasks to date, setting a new benchmark for terpenoid production.

Yarrowia lipolytica growth, lipids, and protease production in medium with higher alkanes and alkenes.

Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L-1 of each alkane (dodecane and hexadecane), and 2 g·L-1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L-1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L-1) and lipids concentrations of 3.4 g·L-1 and 4.3 g·L-1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica.

Efficient expression of OUC-Sb-lip2 in Yarrowia lipolytica and its comprehensive utilization in the enrichment of DHA and EPA from fish oil.

Lipases are widely used in the modification of functional lipids, particularly in the enrichment of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). In this study, a lipase named OUC-Sb-lip2 was expressed in Yarrowia lipolytica, achieving a promising enzyme activity of 472.6 U/mL by optimizing the culture medium, notably through olive oil supplementation. A significant proportion (58.8%) of the lipase activity was located in the cells, whereas 41.2% was secreted into the supernatant. Both whole-cell and immobilized OUC-Sb-lip2 were used to enrich DHA and EPA from fish oil. The whole-cell approach increased the DHA and EPA contents to 2.59 and 2.55 times that of the original oil, respectively. Similarly, the immobilized OUC-Sb-lip2 resulted in a 2.00-fold increase in DHA and an 1.99-fold increase in EPA after a 6-h hydrolysis period. Whole cell and the immobilized OUC-Sb-lip2 retained 48.7% and 52.7% of their activity after six cycles of reuse, respectively.

Advances in Flavonoid and Derivative Biosynthesis: Systematic Strategies for the Construction of Yeast Cell Factories.

Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.

Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable ß-caryophyllene production.

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce ß-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum ß-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO2 and light through co-culture systems.

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica for 2-phenylethanol overproduction.

The rose fragrance molecule 2-phenylethanol (2-PE) has huge market demand in the cosmetics, food and pharmaceutical industries. However, current 2-PE synthesis methods do not meet the efficiency market requirement. In this study, CRISPR-Cas9-related metabolic engineering strategies were applied to Yarrowia lipolytica for the de novo biosynthesis of 2-PE. Initially, overexpressing exogenous feedback-resistant EcAROGfbr and EcPheAfbr increased 2-PE production to 276.3 mg/L. Subsequently, the ylARO10 and ylPAR4 from endogenous genes were enhanced with the multi-copies to increase the titer to 605 mg/L. Knockout of ylTYR1 and enhancement of shikimate pathway by removing the precursor metabolic bottleneck and overexpressing the genes ylTKT, ylARO1, and ylPHA2 resulted in a significant increase of the 2-PE titer to 2.4 g/L at 84 h, with the yield of 0.06 g/gglu, which is the highest yield for de novo synthesis in yeast. This study provides a valuable precedent for the efficient biosynthesis of shikimate pathway derivatives.

Optimization and mechanism studies for the biosorption of rare earth ions by Yarrowia lipolytica.

Research on the recovery of rare earth elements from wastewater has attracted increasing attention. Compared with other methods, biosorption is a simple, efficient, and environmentally friendly method for rare earth wastewater treatment, which has greater prospects for development. The objective of this study was to investigate the biosorption behavior and mechanism of Yarrowia lipolytica for five rare earth ions (La3⁺, Nd3⁺, Er3⁺, Y3⁺, and Sm3⁺) with a particular focus on biosorption behavior, biosorption kinetics, and biosorption isotherm. It was demonstrated that the biosorption capacity of Y. lipolytica at optimal conditions was 76.80 mg/g. It was discovered that the biosorption process complied with the pseudo-second-order kinetic model and the Langmuir biosorption isotherm, indicating that Y. lipolytica employed a monolayer chemical biosorption process to biosorb rare earth ions. Characterization analysis demonstrated that the primary functional groups involved in rare earth ion biosorption were amino, carboxyl, and hydroxyl groups. The cooperative biosorption of rare earth ions by Y. lipolytica was facilitated by means of surface complexation, ion exchange, and electrostatic interactions. These findings suggest that Y. lipolytica has the potential to be an effective biosorbent for the removal of rare earth elements from wastewater.

Production of γ-aminobutyric acid by immobilization of two Yarrowia lipolytica glutamate decarboxylases on electrospun nanofibrous membrane.

This study harnesses glutamate decarboxylase (GAD) from Yarrowia lipolytica to improve the biosynthesis of γ-aminobutyric acid (GABA), focusing on boosting the enzyme's catalytic efficiency and stability by immobilizing it on nanofibrous membranes. Through recombinant DNA techniques, two GAD genes, YlGAD1 and YlGAD2, were cloned from Yarrowia lipolytica and then expressed in Escherichia coli. Compared to their soluble forms, the immobilized enzymes exhibited significant improvements in thermal and pH stability and increased resistance to chemical denaturants. The immobilization notably enhanced substrate affinity, as evidenced by reduced Km values and increased kcat values, indicating heightened catalytic efficiency. Additionally, the immobilized YlGAD1 and YlGAD2 enzymes showed substantial reusability, maintaining 50% and 40% of their activity, respectively, after six consecutive cycles. These results underscore the feasibility of employing immobilized YlGAD enzymes for cost-effective and environmentally sustainable GABA production. This investigation not only affirms the utility of YlGADs in GABA synthesis but also underscores the advantages of enzyme immobilization in industrial settings, paving the way for scalable biotechnological processes.

Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

Non-sterile cultivation of Yarrowia lipolytica in fed-batch mode for the production of lipids and biomass.

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry.

SNF1 plays a crucial role in the utilization of n-alkane and transcriptional regulation of the genes involved in it in the yeast Yarrowia lipolytica.

Yarrowia lipolytica is an ascomycetous yeast that can assimilate hydrophobic carbon sources including oil and n-alkane. The sucrose non-fermenting 1/AMP-activated protein kinase (Snf1/AMPK) complex is involved in the assimilation of non-fermentable carbon sources in various yeasts. However, the role of the Snf1/AMPK complex in n-alkane assimilation in Y. lipolytica has not yet been elucidated. This study aimed to clarify the role of Y. lipolytica SNF1 (YlSNF1) in the utilization of n-alkane. The deletion mutant of YlSNF1 (ΔYlsnf1) exhibited substantial growth defects on n-alkanes of various lengths (C10, C12, C14, and C16), and its growth was restored through the introduction of YlSNF1. Microscopic observations revealed that YlSnf1 tagged with enhanced green fluorescence protein showed dot-like distribution patterns in some cells cultured in the medium containing n-decane, which were not observed in cells cultured in the medium containing glucose or glycerol. The RNA sequencing analysis of ΔYlsnf1 cultured in the medium containing n-decane exhibited 302 downregulated and 131 upregulated genes compared with the wild-type strain cultured in the same medium. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses suggested that a significant fraction of the downregulated genes functioned in peroxisomes or were involved in the metabolism of n-alkane and fatty acids. Quantitative real-time PCR analysis confirmed the downregulation of 12 genes involved in the metabolism of n-alkane and fatty acid, ALK1-ALK3, ALK5, ADH7, PAT1, POT1, POX2, PEX3, PEX11, YAS1, and HFD3. Furthermore, ΔYlsnf1 exhibited growth defects on the medium containing the metabolites of n-alkane (fatty alcohol and fatty aldehyde). These findings suggest that YlSNF1 plays a crucial role in the utilization of n-alkane in Y. lipolytica. This study provides important insights into the advanced biotechnological applications of this yeast, including the bioconversion of n-alkane to useful chemicals and the bioremediation of petroleum-contaminated environments.

Bioreduction of 4'-Hydroxychalcone in Deep Eutectic Solvents: Optimization and Efficacy with Various Yeast Strains.

4'-dihydrochalcones are secondary metabolites isolated from many medicinal plants and from the resin known as 'dragon's blood'. Due to their biological potential, our research objective was to determine the possibilities of using biocatalysis processes carried out in deep eutectic solvents (DESs) to obtain 4'-dihydrochalcones as a model compound. The processes were carried out in a culture of the yeast Yarrowia lipolytica KCh 71 and also in cultures of strains of the genera Rhodotorula and Debaryomyces. Based on the experiments carried out, an optimum process temperature of 35 °C was chosen, and the most suitable DES contained glycerol as a hydrogen bond donor (HBD). For a medium with 30% water content (DES 11), the conversion observed after 24 h exceeded 70%, while increasing the amount of water to 50% resulted in a similar level of conversion after just 1 h. A fivefold increase in the amount of added substrate resulted in a reduction in conversion, which reached 30.3%. Of the other yeast strains tested, Rhodotorula marina KCh 77 and Rhodotorula rubra KCh 4 also proved to be good biocatalysts for the bioreduction process. For these strains, the conversion reached 95.4% and 95.1%, respectively. These findings highlight the potential of yeast as a biocatalyst for the selective reduction of α,ß-unsaturated ketones and the possibility of using a DESs as a reaction medium in this process.

Enhanced production of isobutyl and isoamyl acetate using Yarrowia lipolytica.

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.

Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies.

BACKGROUND: Limonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene. RESULTS: In this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce D- and L-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of L-limonene and 24.8 mg/L of D-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of D-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased D-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, D-limonene production titer reached 69.3 mg/L. CONCLUSIONS: In this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Sustainable succinic acid production from lignocellulosic hydrolysates by engineered strains of Yarrowia lipolytica at low pH.

Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2, the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation.

Protein rational design and modification of erythrose reductase for the improvement of erythritol production in Yarrowia lipolytica.

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ERK26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.

Elucidation of triacylglycerol catabolism in Yarrowia lipolytica: How cells balance acetyl-CoA and excess reducing equivalents.

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from ß-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.