Engineering Yarrowia lipolytica as a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose.

De novo biosynthesis of high-value added food additive p-coumaric acid (p-CA) direct from cellulose/hemicellulose is a more sustainable route compared to the chemical route, considering the abundant cellulose/hemicellulose resources. In this study, a novel factory was constructed for the production of p-CA in Yarrowia lipolytica using cellulose/hemicellulose as the sole carbon source. Based on multicopy integration of the TAL gene and reprogramming the shikimic acid pathway, the engineered strain produced 1035.5 ± 67.8 mg/L p-CA using glucose as a carbon source. The strains with overexpression of cellulases and hemicellulases produced 84.3 ± 2.4 and 65.3 ± 4.6 mg/L p-CA, using cellulose (carboxymethyl-cellulose) or hemicellulose (xylan from bagasse) as the carbon source, respectively. This research demonstrated the feasibility of conversion of cost-effective cellulose/hemicellulose into a value-added product and provided a sustainable cellulolytic cell factory for the utilization of cellulose/hemicellulose.

Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging.

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement.

Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.

Genetical Surface Display of Silicatein on Yarrowia lipolytica Confers Living and Renewable Biosilica-Yeast Hybrid Materials.

In this work, a biological engineering-based biosilica-yeast hybrid material was developed. It was obtained by the aggregation of Yarrowia lipolytica through biosilicification catalyzed using genetically displayed silicatein on its cell surface. With orthosilicate or seawater as the substrate, the silicatein-displayed yeast could aggregate into flocs with a flocculation efficiency of nearly 100%. The resulting floc was found to be a sheetlike biosilica-yeast hybrid material formed by the biosilica-mediated immobilization of yeast cells via cross-linking and embedding, turning the original hydrophilicity of yeast cells into hydrophobicity. In addition, this material was characterized to be porous with an average pore diameter of approximately 10 µm and porosity of over 70%. Because of these properties, this hybrid material could achieve enhanced removal efficiencies for chromium ions and n-hexadecane, which were both above 99%, as compared to the free cells of Y. lipolytica in aqueous environments. Importantly, this hybrid material could be recultivated to generate new batches of yeast cells that maintain parallel properties to the first generation so that the same hybrid material could be reproduced with unchanged highly efficient removal of chromium and n-hexadecane to those of the first generation, demonstrating that this biosilica-yeast hybrid material was living and renewable. This work presented a novel way of harnessing silicatein and Y. lipolytica to achieve biological synthesis of a living inorganic-organic hybrid material that has potential to be applied in water treatment.

Construction of arming Yarrowia lipolytica surface-displaying soybean seed coat peroxidase for use as whole-cell biocatalyst.

Whole-cell biocatalysts could be used in wide-ranging applications. In this study, a new kind of whole-cell biocatalyst was successfully constructed by genetically immobilizing soybean seed coat peroxidase (SBP) on the cell surface of Yarrowia lipolytica Po1h, using a new integrative surface display expression vector (pMIZY05). The coding sequence of SBP was optimized and chemically synthesized, then inserted into pMIZY05 to generate expression plasmid pMIZY05-oEp. A DNA fragment corresponding to SBP and selection marker expression cassettes, without bacterial sequences, was released from pMIZY05-oEp by enzyme digestion and used to transform host yeast cells. A transformant (CM11) with a high recombinant SBP activity of 1571.9 U/mL was obtained, and recombinant SBP was proved to be successfully anchored on cell surface by testing the activities of different cellular fractions. After optimization of culture conditions, the recombinant SBP activity of CM11 was increased to 4187.8 U/mL. Afterwards, biochemical properties of the recombinant SBP were determined: optimum catalytic conditions were 37.5℃ at pH 3.5, and recombinant SBP exhibited high stability during thermal or acidic treatment. Recombinant activity of cell-displayed SBP was re-examined at optimum temperature and pH, which promoted an increase up to 4432.5 U/mL. To our knowledge, this represents the highest activity ever reported for heterologous expression of SBP. This study also provides a useful strategy for heterologous expression of proteins which could be toxic to intracellular content of host cells.

Subcellular engineering of lipase dependent pathways directed towards lipid related organelles for highly effectively compartmentalized biosynthesis of triacylglycerol derived products in Yarrowia lipolytica.

As an alternative to in vitro lipase dependent biotransformation and to traditional assembly of pathways in cytoplasm, the present study focused on targeting lipase dependent pathways to a subcellular compartment lipid body (LB), in combination with compartmentalization of associated pathways in other lipid relevant organelles including endoplasmic reticulum (ER) and peroxisome for efficient in vivo biosynthesis of fatty acid methyl esters (FAMEs) and hydrocarbons, in the context of improving Yarrowia lipolytica lipid pool. Through knock in and knock out of key genes involved in triacylglycerols (TAGs) biosynthesis and degradation, the TAGs content was increased to 51.5%, from 7.2% in parent strain. Targeting lipase dependent pathway to LB gave a 10-fold higher FAMEs titer (1028.0 mg/L) compared to cytosolic pathway (102.8 mg/L). Furthermore, simultaneously targeting lipase dependent pathway to LB, ER and peroxisome gave rise to the highest FAMEs titer (1644.8 mg/L). The subcellular compartment engineering strategy was extended to other lipase dependent pathways for fatty alkene and alkane biosynthesis, which resulted in a 14-fold titer enhancement compared to traditional cytosolic pathways. We developed yeast subcellular cell factories by directing lipase dependent pathways towards the TAGs storage organelle LB for efficient biosynthesis of TAG derived chemicals for the first time. The successful exploration of targeting metabolic pathways towards LB centered organelles is expected to promote subcellular compartment engineering for other lipid derived product biosynthesis.

Metabolic engineering of Yarrowia lipolytica for the biosynthesis of crotonic acid.

In this study, Y. lipolytica was engineered to produce crotonic acid via the butanol-forming route. Firstly, the crotonase and 3-hydroxybutyryl-CoA dehydrogenase genes from Clostridium beijerinckii, and the thioesterase gene from Bacteroides thetaiotaomicron were heterologously expressed in Y. lipolytica, the engineered strain LZJ001 accumulated 62.3 ±â€¯4.2 mg/L of crotonic acid. Secondly, the acetyl-CoA acetyltransferase from Saccharomyces cerevisiae was overexpressed, the derived recombinant strain LZJ002 produced 123.5 ±â€¯6.8 mg/L of crotonic acid. Finally, the pyruvate dehydrogenase from Escherichia coli was additionally expressed, giving the fully engineered strain LZJ004 that produced 220.0 ±â€¯8.2 mg/L of crotonic acid in shaking-flask culture, which represents a 3.5-fold increase over LZJ001 strain. The approach described here paves the way for environmentally friendly and large-scale industrial production of crotonic acid.

Laccase production from sucrose by recombinant Yarrowia lipolytica and its application to decolorization of environmental pollutant dyes.

Laccases are used in decolorization and biodegradation of synthetic dyes, bioremediation of industrial wastewaters and delignification of lignocellulosic compounds. The aims of the present study were the optimization of a recombinant laccase production in Yarrowia lipolytica yeast using sucrose as a main carbon source, and the application of the resulting enzyme to decolorization of synthetic dyes, which are problematic environmental pollutants. Taguchi's experimental design method was employed to optimize medium compounds. Recombinant laccase production by Y. lipolytica YL4 strain increased to 900 U L-1 after optimization of sucrose, ammonium chloride, yeast extract and thiamine levels in the modified PPB medium. Furthermore, the production rate reached 6760 U L-1 in a 5 L bioreactor which represents 4.5- and 33.5-fold increases compared to cultures that were in shake-flask with optimized and primary media, respectively. The supernatant containing secreted recombinant laccase was applied for decolorization of seven dyes. The effects of pH, the amount of enzyme and incubation period were verified. The effect of incubation time on dye decolorization by recombinant laccase was important, which has an influence of greater extent than 90% after 48 h for all dyes. The Trametes versicolor laccase can be efficiently produced in Y. lipolytica and the recombinant enzyme has a considerable potential in the decolorization of pollutant synthetic dyes.

In silico and in vivo analysis of signal peptides effect on recombinant glucose oxidase production in nonconventional yeast Yarrowia lipolytica.

Signal peptide (SP) is an important factor and biobrick in the production and secretion of recombinant proteins. The aim of this study was in silico and in vivo analysis of SPs effect on the production of recombinant glucose oxidase (GOX) in Yarrowia lipolytica. Several in silico softwares, namely SignalP4, Signal-CF, Phobius, WolfPsort 0.2, SOLpro and ProtParam, were used to analyse the potential of 15 endogenous and exogenous SPs for the secretion of recombinant GOX in Y. lipolytica. According to in silico results, the SP of GOX was predicted as suitable in terms of high secretory potential and of protein solubility and stability which is chosen for in vivo analysis. The recombinant Y. lipolytica strain produced 280 U/L of extracellular GOX after 7 days in YPD medium. The results show that the SP of GOX can be applied to efficient production of extracellular heterologous proteins and metabolic engineering in Y. lipolytica.

Engineering Yarrowia lipolytica for Use in Biotechnological Applications: A Review of Major Achievements and Recent Innovations.

Yarrowia lipolytica is an oleaginous saccharomycetous yeast with a long history of industrial use. It aroused interest several decades ago as host for heterologous protein production. Thanks to the development of numerous molecular and genetic tools, Y. lipolytica is now a recognized system for expressing heterologous genes and secreting the corresponding proteins of interest. As genomic and transcriptomic tools increased our basic knowledge on this yeast, we can now envision engineering its metabolic pathways for use as whole-cell factory in various bioconversion processes. Y. lipolytica is currently being developed as a workhorse for biotechnology, notably for single-cell oil production and upgrading of industrial wastes into valuable products. As it becomes more and more difficult to keep up with an ever-increasing literature on Y. lipolytica engineering technology, this article aims to provide basic and actualized knowledge on this research area. The most useful reviews on Y. lipolytica biology, use, and safety will be evoked, together with a resume of the engineering tools available in this yeast. This mini-review will then focus on recently developed tools and engineering strategies, with a particular emphasis on promoter tuning, metabolic pathways assembly, and genome editing technologies.

Effect of Bulk MoS2 on the Metabolic Profile of Yeast.

MoS2, a kind of two-dimensional material with unique performances, has been widely used in many fields. However, an in-depth understanding of its toxicity is still needed, let alone its effects on the environmental microorganism. Herein, we used different methods, including metabolomics technology, to investigate the influence of bulk MoS2 (BMS) on yeast cells. The results indicated that high concentrations (1 mg/L and more) of BMS could destroy cell membrane and induce ROS accumulation. When exposed to a low concentration of BMS (0.1 mg/L), the intracellular concentrations of many metabolites (e.g., fumaric acid, lysine) increased. However, most of their concentrations descended significantly as the yeast cells were treated with BMS of high concentrations (1 mg/L and more). Metabolomics analysis further revealed that exposure to high concentrations of BMS could significantly affect some metabolic pathways such as amino acid and citrate cycle related metabolism. These findings will be beneficial for MoS2 toxicity assessment and further applications.

Engineering Yarrowia lipolytica to Simultaneously Produce Lipase and Single Cell Protein from Agro-industrial Wastes for Feed.

Lipases are scarcely exploited as feed enzymes in hydrolysis of lipids for increasing energy supply and improving nutrient use efficiency. In this work, we performed homologous overexpression, in vitro characterization and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose. Simultaneously, a large amount of yeast cell biomass was produced, for use as single cell protein, a potential protein-rich feed resource. Three kinds of low cost agro-industrial wastes were tested as substrates for simultaneous production of lipase and single cell protein (SCP) as feed additives: sugarcane molasses, waste cooking oil and crude glycerol from biodiesel production. Sugarcane molasses appeared as the most effective cheap medium, allowing production of 16420 U/ml of lipase and 151.2 g/L of single cell protein at 10 liter fermentation scale. In vitro characterization by mimicking a gastro-intestinal environment and determination of essential amino acids of the SCP, and in vivo oral feeding test on fish all revealed that lipase, SCP and their combination were excellent feed additives. Such simultaneous production of this lipase and SCP could address two main concerns of feed industry, poor utilization of lipid and shortage of protein resource at the same time.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

Production of Laccase by Recombinant Yarrowia lipolytica from Molasses: Bioprocess Development Using Statistical Modeling and Increase Productivity in Shake-Flask and Bioreactor Cultures.

Laccases are used in numerous applications, from green degradation of various xenobiotic compounds, waste detoxification, textile dye bleaching, and delignification of lignocellulose materials to biofuel production. In this study, the recombinant Yarrowia lipolytica YL4 strain carrying the white-rot fungus Trametes versicolor laccase IIIb gene was used for laccase production from beet molasses as an agro-industrial residue. Response surface methodology was used to statistical optimization of the production of laccase by Y. lipolytica using an industrial medium containing molasses which allows a six times increase in laccase activity compared to primary medium contains glucose after 144 h. In bioreactor cultivation after 48 h, laccase production reached to 3.7- and 22.5-fold more than optimized and primary media in shake-flask cultures, respectively. Laccase productivity in bioreactor (0.0937 U/h) was higher than shake-flask culture (0.0084 U/h). The present study provides valuable information about statistical optimization of bioprocess development for cost-effective production of laccase and other heterologous proteins in Y. lipolytica from beet molasses as sole carbon source, thus allowing the valorization and decreasing environmental pollution of this agro-industrial waste.

Applying pathway engineering to enhance production of alpha-ketoglutarate in Yarrowia lipolytica.

α-Ketoglutarate (α-KG), one of short-chain carboxylates of high commercial relevance, has been widely used in food, medicine, chemical, and cosmetic fields. Compared to other carboxylates, α-KG occupies key positions in the tricarboxylate cycle (TCA cycle) and amino acid metabolic pathway, the over-accumulation of α-KG is restricted both by tighter carbon and nitrogen regulation process. Biotechnology production of α-KG on large industrial level has been impeded by many obstacles. This review aims at highlighting and stating recent efforts toward improving the yield and titer of α-KG in the strains of Yarrowia lipolytica to reach industrial relevance. Fermentation process optimization concerning feedstock utilization, dissolved oxygen controlling, pH manipulation and establishment of fed-batch process, have been assessed and evaluated. Moreover, pathway engineering routes have been applied for enhancing carbon commitment to α-KG, blocking competing pathways, regenerating of co-factors and regulating of carboxylate transporters to facilitate production and accumulation of α-KG. Although no engineered strain can satisfy the requirements of industrial production relevance to date, these strategies provide many clues for accelerating strain development for α-KG production.

Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation of α-ketoglutarate in Yarrowia lipolytica.

α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle and has broad applications. The mitochondrial ketoglutarate dehydrogenase (KGDH) complex catalyzes the oxidation of α-KG to succinyl-CoA. Disruption of KGDH, which may enhance the accumulation of α-KG theoretically, was found to be lethal to obligate aerobic cells. In this study, individual overexpression of dihydrolipoamide succinyltransferase (DLST), which serves as the inner core of KGDH, decreased overall activity of the enzyme complex. Furthermore, two conserved active site residues of DLST, His419, and Asp423 were identified. In order to determine whether these residues are engaged in enzyme reaction or not, these two conserved residues were individually mutated. Analysis of the kinetic parameters of the enzyme variants provided evidence that the catalytic reaction of DLST depended on residues His419 and Asp423. Overexpression of mutated DLST not only impaired balanced assembly of KGDH, but also disrupted the catalytic integrity of the enzyme complex. Replacement of the Asp423 residue by glutamate increased extracellular α-KG by 40 % to 50 g L(-1) in mutant strain. These observations uncovered catalytic roles of two conserved active site residues of DLST and provided clues for effective metabolic strategies for rational carbon flux control for the enhanced production of α-KG and related bioproducts.

Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica.

Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA) and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.

Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization.

BACKGROUND: Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers. RESULTS: The pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the K m and k cat values of the variants decreased accordingly. CONCLUSIONS: This study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing.

Exploring medium-chain-length polyhydroxyalkanoates production in the engineered yeast Yarrowia lipolytica.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are a large class of biopolymers that have attracted extensive attention as renewable and biodegradable bio-plastics. They are naturally synthesized via fatty acid de novo biosynthesis pathway or ß-oxidation pathway from Pseudomonads. The unconventional yeast Yarrowia lipolytica has excellent lipid/fatty acid catabolism and anabolism capacity depending of the mode of culture. Nevertheless, it cannot naturally synthesize PHA, as it does not express an intrinsic PHA synthase. Here, we constructed a genetically modified strain of Y. lipolytica by heterologously expressing PhaC1 gene from P. aeruginosa PAO1 with a PTS1 peroxisomal signal. When in single copy, the codon optimized PhaC1 allowed the synthesis of 0.205 % DCW of PHA after 72 h cultivation in YNBD medium containing 0.1 % oleic acid. By using a multi-copy integration strategy, PHA content increased to 2.84 % DCW when the concentration of oleic acid in YNBD was 1.0 %. Furthermore, when the recombinant yeast was grown in the medium containing triolein, PHA accumulated up to 5.0 % DCW with as high as 21.9 g/L DCW, which represented 1.11 g/L in the culture. Our results demonstrated the potential use of Y. lipolytica as a promising microbial cell factory for PHA production using food waste, which contains lipids and other essential nutrients.