Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase.

L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.

Biosynthesis of the antioxidant γ-glutamyl-cysteine with engineered Yarrowia lipolytica.

The dipeptide γ-glutamylcysteine (γ-GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age-related disorders such as Alzheimer and Parkinson diseases. The unusual γ-linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non-conventional yeast Yarrowia lipolytica for efficient γ-GC synthesis. The yeast was first converted into a γ-GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4, CYSE, CYSF, and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ-GC titer of 464 nmol mg-1 protein (93 mg gDCW-1 ) was obtained within 24 h of cell growth.

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3'-hydroxylase/cytochrome P450 reductase (F3'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid ß-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.

A strategy to reduce the byproduct glucose by simultaneously producing levan and single cell oil using an engineered Yarrowia lipolytica strain displaying levansucrase on the surface.

Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C16-C18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis.

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.

Biorefinery-centric ethanol and oleochemical production employing Yarrowia lipolytica and Pichia farinosa.

The research examined the capabilities of Yarrowia lipolytica (YL) and Pichia farinosa (PF) in converting sugars to ethanol and oleochemicals. Lipid, ethanol, protein yield and gene-expressions were analysed at different substrate concentrations (3 to 30 g/L) with glucose, food waste, and fermentation-effluent. Optimal results were obtained at 20 g/L using both synthetic carbon with 4.6 % of total lipid yield. Lauric and Caprylic acid dominance was noted in total lipid fractions. Protein accumulation (6 g/L) was observed in glucose system (20 g/L) indicating yeast strains potential as single-cell proteins (SCP). Fatty-acid desaturase (FAD12) and alcohol dehydrogenase (ADH) expressions were higher at optimum condition of YL (1.15 × 10-1, 3.8 × 10-2) and PF (5.8 × 10-2, 3.8 × 10-2) respectively. Maximum carbon reduction of 87 % depicted at best condition, aligning with metabolic yield. These findings highlights promising role of yeast as biorefinery biocatalyst.

Evaluation of cell disruption methods in the oleaginous yeasts Yarrowia lipolytica QU21 and Meyerozyma guilliermondii BI281A for microbial oil extraction.

The interest for oleaginous yeasts has grown significantly in the last three decades, mainly due to their potential use as a renewable source of microbial oil or single cell oils (SCOs). However, the methodologies for cell disruption to obtain the microbial oil are considered critical and determinant for a large-scale production. Therefore, this work aimed to evaluate different methods for cell wall disruption for the lipid extraction of Yarrowia lipolytica QU21 and Meyerozyma guilliermondii BI281A. The two strains were separately cultivated in 5 L batch fermenters for 120 hours, at 26 ºC and 400 rpm. Three different lipid extraction processes using Turrax homogenizer, Ultrasonicator and Braun homogenizer combined with bead milling were applied in wet, oven-dried, and freeze-dried biomass of both strains. The treatment with the highest percentage of disrupted cells and highest oil yield was the ultrasonication of oven-dried biomass (37-40% lipid content for both strains). The fact that our results point to one best extraction strategy for two different yeast strains, belonging to different species, is a great news towards the development of a unified technique that could be applied at industrial plants.

Cutaneotrichosporon curvatum and Yarrowia lipolytica as key players for green chemistry: efficient oil producers from food waste via the carboxylate platform.

Cutaneotrichosporon curvatum and Yarrowia lipolytica can accumulate microbial oils using short-chain fatty acids (SCFA) as carbon sources. SCFAs-rich media often contain significant amounts of nitrogen that prevent high carbon:nitrogen (C:N) ratios necessary to boost lipid production. This work assessed the intrinsic ability of C. curvatum and Y. lipolytica to produce high amounts of microbial oils from these unusual carbon sources. Results demonstrated that minor differences in SCFA concentration (only 2 g/L) had a significant effect on yeast growth and lipid production. A C:N of 80 promoted yeast growth at all SCFA concentrations and favored SCFA consumption at 19 g/L SCFAs. The different SCFA uptake preferences in C. curvatum and Y. lipolytica highlighted the importance of considering the SCFA profile to select a suitable yeast strain for microbial oils production. At the most challenging SCFA concentration (19 g/L), 57.2% ±1.6% (w/w) and 78.4 ± 0.6% (w/w) lipid content were obtained in C. curvatum and Y. lipolytica, respectively. These values are among the highest reported for wild-type strains. To circumvent the challenges associated with media with high nitrogen content, this report also proved struvite precipitation as an effective method for increasing lipid production (from 17.9 ± 3.9% (w/w) to 41.9 ± 2.6% (w/w)) after nitrogen removal in food waste-derived media.

Slight variations in SCFA concentrations have a relevant effect on yeast lipid productionHigh nitrogen availability is crucial to promote cell growth at very high SCFA concentrationsC:N effect on cell growth and lipid production is specie-specific and may depend on yeast robustnessYeast strains have diverse SCFA preferences and differently metabolize these acidsStruvite precipitation effectively removes nitrogen from real digestates increasing C:N.

Combination of microbial and chemical synthesis for the sustainable production of ß-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

Production of perillic acid from orange essential oil by Yarrowia lipolytica using a top-aerated bioreactor.

R-(+)-Perillic acid, a promising anticancer and immunomodulatory agent, is the major product from the biotransformation of R-(+)-limonene-rich orange essential oil by the yeast Yarrowia lipolytica. Due to the abundance and low cost of orange essential oil, which is a byproduct of the citrus industry, we attempted to improve the biotransformation process by optimizing yeast cell mass production. Then, the whole process was transposed and adapted to a 2-L instrumented bioreactor. Cell mass production was optimized in shaker flasks using a statistical experimental design. The optimized medium (g·L-1: 22.9 glucose, 7.7 peptone, 4.1 yeast extract and 1.0 malt extract) resulted in a 13.0 g·L-1 final cell concentration and 0.18 g cell·L-1·h-1 productivity. A further increase to 18.0 g·L-1 was achieved in a 2-L bioreactor upon fed-batch culture. High-purity limonene bioconversion was performed in the same bioreactor utilizing top aeration to diminish terpene volatilization; as a result, 839.6 mg·L-1 perillic acid accumulated after 48 h. Under the same conditions, industrial orange essential oil afforded 806.4 mg·L-1 perillic acid. The yeast growth medium optimization resulted in a twofold increase in biomass accumulation and a reduction in growth medium nitrogen sources, which lowered the catalytic biomass production cost. Compared with conventional bottom aeration, the bioreactor top aeration strategy resulted in higher bioconversion rates. The conditions developed for high-purity limonene bioconversion were successfully applied to low-cost orange essential oil, showing the robustness of Y. lipolytica yeast.

Metabolic Engineering of Yarrowia lipolytica for Zeaxanthin Production.

Zeaxanthin is a carotenoid, a dihydroxy derivative of ß-carotene. Zeaxanthin has antioxidant, anti-inflammatory, anticancer, and neuroprotective properties. In this study, Yarrowia lipolytica was used as a host for the efficient production of zeaxanthin. The strain Y. lipolytica PO1h was used to construct the following engineered strains for carotenoid production since it produced the highest ß-carotene among the Y. lipolytica PO1h- and Y. lipolytica PEX17-HA-derived strains. By regulating the key nodes on the carotenoid pathway through wild and mutant enzyme comparison and successive modular assembly, the ß-carotene concentration was improved from 19.9 to 422.0 mg/L. To provide more precursor mevalonate, heterologous genes mvaE and mvaSMT were introduced to increase the production of ß-carotene by 27.2% to the yield of 536.8 mg/L. The ß-carotene hydroxylase gene crtZ was then transferred, resulting in a yield of zeaxanthin of 326.5 mg/L. The oxidoreductase RFNR1 and CrtZ were then used to further enhance zeaxanthin production, and the yield of zeaxanthin was up to 775.3 mg/L in YPD shake flask.

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol.

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Combining orthogonal plant and non-plant fatty acid biosynthesis pathways for efficient production of microbial oil enriched in nervonic acid in Yarrowia lipolytica.

Nervonic acid has proven efficacy in brain development and the prevention of neurodegenerative diseases. Here, an alternative and sustainable strategy for nervonic acid-enriched plant oil production was established. Different ß-ketoacyl-CoA synthases and heterologous Δ15 desaturase were co-expressed, combined with the deletion of the ß-oxidation pathway to construct orthogonal plant and non-plant nervonic acid biosynthesis pathways in Yarrowia lipolytica. A "block-pull-restrain" strategy was further applied to improve the supply of stearic acid as the precursor of the non-plant pathway. Then, lysophosphatidic acid acyltransferase from Malania oleifera (MoLpaat) was identified, which showed specificity for nervonic acid. Endogenous LPAAT was exchanged by MoLPAAT resulted in 17.10 % nervonic acid accumulation. Finally, lipid metabolism was engineered and cofactor supply was increased to boost the lipid accumulation in a stable null-hyphal strain. The final strain produced 57.84 g/L oils with 23.44 % nervonic acid in fed-batch fermentation, which has the potential to substitute nervonic acid-enriched plant oil.

Enhanced ß-carotene production in Yarrowia lipolytica through the metabolic and fermentation engineering.

ß-Carotene is a kind of high-value tetraterpene compound, which shows various applications in medical, agricultural, and industrial areas owing to its antioxidant, antitumor, and anti-inflammatory activities. In this study, Yarrowia lipolytica was successfully metabolically modified through the construction and optimization of ß-carotene biosynthetic pathway for ß-carotene production. The ß-carotene titer in the engineered strain Yli-C with the introduction of the carotenogenesis genes crtI, crtE, and crtYB can reach 34.5 mg/L. With the overexpression of key gene in the mevalonate pathway and the enhanced expression of the fatty acid synthesis pathway, the ß-carotene titer of the engineered strain Yli-CAH reached 87 mg/L, which was 152% higher than that of the strain Yli-C. Through the further expression of the rate-limiting enzyme tHMGR and the copy number of ß-carotene synthesis related genes, the ß-carotene production of Yli-C2AH2 strain reached 117.5 mg/L. The final strain Yli-C2AH2 produced 2.7 g/L ß-carotene titer by fed-batch fermentation in a 5.0-L fermenter. This research will greatly speed up the process of developing microbial cell factories for the commercial production of ß-carotene. ONE-SENTENCE SUMMARY: In this study, the ß-carotene synthesis pathway in engineered Yarrowia lipolytica was enhanced, and the fermentation conditions were optimized for high ß-carotene production.

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies.

Terpenoids are a diverse group of compounds with isoprene units as basic building blocks. They are widely used in the food, feed, pharmaceutical, and cosmetic industries due to their diverse biological functions such as antioxidant, anticancer, and immune enhancement. With an increase in understanding the biosynthetic pathways of terpenoids and advances in synthetic biology techniques, microbial cell factories have been built for the heterologous production of terpenoids, with the oleaginous yeast Yarrowia lipolytica emerging as an outstanding chassis. In this paper, recent progress in the development of Y. lipolytica cell factories for terpenoid production with a focus on the advances in novel synbio tools and metabolic engineering strategies toward enhanced terpenoid biosynthesis is reviewed.

Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: A route for poly(ethylene terephthalate) (PET) upcycling.

Biological recycling of PET waste has been extensively investigated recently to tackle plastic waste pollution, and ethylene glycol (EG) is one of the main building blocks recovered from this process. Wild-type Yarrowia lipolytica IMUFRJ 50682 can be a biocatalyst to biodepolymerize PET. Herein, we report its ability to perform oxidative biotransformation of EG into glycolic acid (GA): a higher value-added chemical with varied industrial applications. We found that this yeast tolerates high EG concentrations (up to 2 M) based on maximum non-inhibitory concentration (MNIC) tests. Whole-cell biotransformation assays using resting yeast cells showed GA production uncoupled to cell growth metabolism, and 13 C nuclear magnetic resonance (NMR) analysis confirmed GA production. Moreover, higher agitation speed (450 vs. 350 rpm) resulted in a 1.12-fold GA production improvement (from 352 to 429.5 mM) during Y. lipolytica cultivation in bioreactors after 72 h. GA was constantly accumulated in the medium, suggesting that this yeast may also share an incomplete oxidation pathway (i.e., it is not metabolized to carbon dioxide) as seen in acetic acid bacterial group. Additional assays using higher chain-length diols (1,3-propanediol, 1,4-butanediol, and 1,6-hexanediol) revealed that C4 and C6 diols were more cytotoxic, suggesting that they underwent different pathways in the cells. We found that this yeast consumed extensively all these diols, however, 13 C NMR analysis from supernatant identified solely the presence of 4-hydroxybutanoic acid from 1,4-butanediol, along with GA from EG oxidation. Findings reported herein reveal a potential route for PET upcycling to a higher value-added product.

Increased Cordycepin Production in Yarrowia lipolytica Using Combinatorial Metabolic Engineering Strategies.

As the first nucleoside antibiotic discovered in fungi, cordycepin, with its various biological activities, has wide applications. At present, cordycepin is mainly obtained from the natural fruiting bodies of Cordyceps militaris. However, due to long production periods, low yields, and low extraction efficiency, harvesting cordycepin from natural C. militaris is not ideal, making it difficult to meet market demands. In this study, an engineered Yarrowia lipolytica YlCor-18 strain, constructed by combining metabolic engineering strategies, achieved efficient de novo cordycepin production from glucose. First, the cordycepin biosynthetic pathway derived from C. militaris was introduced into Y. lipolytica. Furthermore, metabolic engineering strategies including promoter, protein, adenosine triphosphate, and precursor engineering were combined to enhance the synthetic ability of engineered strains of cordycepin. Fermentation conditions were also optimized, after which, the production titer and yields of cordycepin in the engineered strain YlCor-18 under fed-batch fermentation were improved to 4362.54 mg/L and 213.85 mg/g, respectively, after 168 h. This study demonstrates the potential of Y. lipolytica as a cell factory for cordycepin synthesis, which will serve as the model for the green biomanufacturing of other nucleoside antibiotics using artificial cell factories.

Profiling the composition and metabolic functions of microbial community in pellicle-forming radish paocai.

Pellicle formation is an obvious indicator of spoilage and is followed by a loss of flavor in a variety of fermented vegetables. In this study, the pellicle-forming microorganisms were isolated using culture-dependent approaches, then a comparative analysis between the pellicle-forming (PF) radish paocai and normal fermented paocai in the diversity and function of microbial community was conducted by metagenome sequencing. Based on a pairwise t-test and OPLS-DA analysis, diallyl sulfide, (z)-1-allyl-2-(prop-1-en-1-yl) disulfane, and terpineol were considered to be the main components responsible for the unpleasant flavor of PF paocai. Yarrowia spp., Enterobacter spp., and Pichia spp. were the main pellicle-forming microorganisms. All 17 isolated Enterobacter strains showed pectinase-producing and cellulase-producing abilities, and 3 isolated Pichia strains showed gas-producing capacity. According to LEfSe analysis based on metagenomes, unclassified_g__Citrobacter and Yarrowia lipolytica were the uppermost biomarkers that distinguished the PF paocai from normal paocai. Unclassified_g__Lactobacillus and Lactobacillus plantarum were found to be actively engaged in starch and sucrose metabolism, cysteine and methionine metabolism, galactose metabolism, fructose and mannose metabolism, lysine biosynthesis, fatty acid biosynthesis, and arginine biosynthesis, all of which contributed to the flavor formation of paocai. Combining the results of metagenome sequencing with the data obtained based on the culture-dependent method, we could deduce that the growth of Yarrowia lipolytica first promoted the increase of pH and the formation of pellicle, which provided a suitable niche for the growth of some harmful bacteria such as Enterobacter, Citrobacter, and Serratia. These hazardous bacteria then worked in concert to induce the odorous stench and texture softening of paocai, as well as more pellicle formation.

Sophorolipids produced by Yarrowia lipolytica grown on Moringa oleifera oil cake protect against acetic acid-induced colitis in rats: impact on TLR-4/p-JNK/NFκB-p65 pathway.

OBJECTIVES: Toll-like receptor-4 (TLR-4) activation plays a major role in triggering oxidative stress (OS) and inflammation implicated in the pathogenesis of ulcerative colitis (UC). Due to sophorolipids (SLs) antioxidant and anti-inflammatory properties, they are interestingly becoming more valued for their potential effectiveness in treating a variety of diseases. This study was designed to explore the effect of SLs produced by microbial conversion of Moringa oleifera oil cake using isolated yeast Yarrowia lipolytica against UC induced by acetic acid (AA) in rats. METHODS: The produced SLs were identified by FTIR, 1H NMR and LC-MS/MS spectra, and administered orally for 7 days (200 mg/kg/day) before AA (2 ml, 4% v/v) to induce UC intrarectally on day eight. Biochemically, the levels of TLR-4, c-Jun N-terminal kinase (JNK), nuclear factor kappa B-p65 (NFκB-p65), interleukin-1beta (IL-1ß), malondialdehyd, glutathione, Bax/Bcl2 ratio and the immunohistochemical evaluation of inducible nitric oxide synthase and caspase-3 were assayed. KEY FINDINGS: SLs significantly reduced OS, inflammatory and apoptotic markers in AA-treated rats, almost like the reference sulfasalazine. CONCLUSIONS: This study provided a novel impact for SLs produced by microbial conversion of M. oleifera oil cake against AA-induced UC in rats through hampering the TLR-4/p-JNK/NFκB-p65 signalling pathway.

High-yield α-humulene production in Yarrowia lipolytica from waste cooking oil based on transcriptome analysis and metabolic engineering.

BACKGROUND: α-Humulene is an important biologically active sesquiterpene, whose heterologous production in microorganisms is a promising alternative biotechnological process to plant extraction and chemical synthesis. In addition, the reduction of production expenses is also an extremely critical factor in the sustainable and industrial production of α-humulene. In order to meet the requirements of industrialization, finding renewable substitute feedstocks such as low cost or waste substrates for terpenoids production remains an area of active research. RESULTS: In this study, we investigated the feasibility of peroxisome-engineering strain to utilize waste cooking oil (WCO) for high production of α-humulene while reducing the cost. Subsequently, transcriptome analysis revealed differences in gene expression levels with different carbon sources. The results showed that single or combination regulations of target genes identified by transcriptome were effective to enhance the α-humulene titer. Finally, the engineered strain could produce 5.9 g/L α-humulene in a 5-L bioreactor. CONCLUSION: To the best of our knowledge, this is the first report that converted WCO to α-humulene in peroxisome-engineering strain. These findings provide valuable insights into the high-level production of α-humulene in Y. lipolytica and its utilization in WCO bioconversion.