Engineering Yarrowia lipolytica for Sustainable Production of Fatty

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Engineering Yarrowia lipolytica for Sustainable Production of Fatty Acid Methyl Esters Using in situ Self-cycled Glycerol as a Carbon Source Yangge Qiao, Kaixin Yang, Qinghua Zhou, Zhixin Xu, Yunjun Yan, Li Xu, Catherine Madzak, and Jinyong Yan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00492 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Sustainable Chemistry & Engineering

Engineering Yarrowia lipolytica for Sustainable Production of Fatty Acid Methyl Esters Using in situ Self-cycled Glycerol as a Carbon Source Yangge Qiao1, Kaixin Yang1, Qinghua Zhou1, Zhixin Xu1, Yunjun Yan1, Li Xu1, Catherine Madzak2, Jinyong Yan*1 All authors and their e-mail addresses: Yangge Qiao1, [email protected] Kaixin Yang1, [email protected] Qinghua Zhou1, [email protected] Zhixin Xu1, [email protected] Yunjun Yan1, [email protected] Li Xu1, [email protected] Catherine Madzak2, [email protected] Jinyong Yan*1 (correspondence), [email protected]

Affiliations and mailing addresses: 1

Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science

and Technology, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China 2

GMPA, AgroParisTech, INRA, Université Paris-Saclay, 78850, Thiverval-Grignon,

France

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ABSTRACT Based on Thermomyces lanuginosus lipase functionally displayed on the cell surface of Yarrowia lipolytica yeast, a new green, integrated and sustainable bioprocess for fatty acid methyl esters (FAMEs) production was developed. It couples surface displayed enzyme generation to enzymatic FAMEs production, in combination with simultaneous regeneration of in situ carbon source for yeast utilization. During FAMEs synthesis, in a one-pot fermentation flask containing growing cells displaying lipase, waste cooking oil and methanol, the in situ formed by-product glycerol was used as an alternative carbon source to support further cell growth and lipase generation.

The

partial

consumption

of

glycerol

pushed

the

reversible

reactions (transesterification, esterification, hydrolysis) toward formation of FAMEs. In the growing cells integrated system, the production of FAMEs via hydrolysisesterification or transesterification-esterification reactions was dependent on the time when methanol was added to the culture medium. Compared to the resting cells system with a 81% FAMEs yield after 16 h, the self-cycling of glycerol for cell consumption (allowing cell growth and lipase displaying for further FAMEs forming) in the growing cells system significantly increased FAMEs synthesis with a more than 91% yield obtained in less time (6-10 h). The cells recovered from growing cell systems still exhibited satisfactory reusability, showing more than 80% FAMEs yield during 12 successive batches. KEYWORDS: Fatty acid methyl esters, Glycerol, in situ, Integrated bioprocess, Lipase



INTRODUCTION Fatty acid methyl esters (FAMEs), called biodiesel, are a clean and renewable substitute for fossil diesel since they can be produced in environment-friendly processes from sustainable feedstocks.1 FAMEs are generally produced via transesterification of triacylglycerols (TAGs) or/and esterification of free fatty acids (FFAs) with methanol, by chemical or enzymatic routes.2 Compared to acid/alkali based chemical route, lipase mediated FAMEs production presents obvious advantages such as mild conditions, lesser methanol consuming and higher quality product.3 On the other hand, in vivo biosynthesis of FAMEs through metabolic engineering and systematic regulation of fatty acid biosynthetic pathway on Escherichia coli and Yarrowia lipolytica has emerged.4-6 So far the reported pathways for FAMEs biosynthesis have almost always involved multiple enzymatic steps based on fatty acid biosynthetic pathway and center metabolites fatty acids or their activated forms (acyl-CoAs or acyl-ACPs).7 The inefficient de novo biosynthesis has limited application of these biosynthetic pathways into industry scale production due to complex metabolic network.8 Comparatively, in vitro enzymatic FAMEs production has received ever-increasing attention from both academies and industries considering its easy scale-up from laboratory to industry and environmentally friendly. Regarding enzymatic FAMEs production, many efforts have been made including immobilization of lipase enzymes using various materials, design of whole cell catalysts, or also establishing biocatalytic processes based on solvent-free, ionic liquid systems and so on.9-13 The traditional strategy of enzymatic route is a two-stage process composed of first preparing lipase catalysts and then performing biotransformation. In order to simultaneously combine the preceding step of lipase catalyst preparation and the subsequent step of biotransformation, an integrated 4 ACS Paragon Plus Environment

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system coupling lipase catalyst generation and lipase catalyzed FAMEs production was explored by Pichia pastoris fermentation in one-pot flask recently.14 Such integrated processes are a promising strategy considering their lower energy consumption, lower overall cost, and the process simplicity compared to traditional two stages processes. There still remains an urgent need to develop efficient and cost-effective bioprocesses for FAMEs production. In the present study, we explored a new integrated growing cells system using a surface displayed lipase catalyst based on Yarrowia lipolytica yeast. Compared to previous FAMEs biocatalytic systems, apart from consolidation of lipase generation and FAMEs biosynthesis, the most innovative aspect of this work is that the FAMEs derived by-product glycerol could be in situ employed as a carbon source to further support engineered yeast growth and lipase expression. The consumption of glycerol by the yeast also drove the reversible reactions (transesterification, esterification and hydrolysis) toward FAMEs formation. The product feedback inhibition effect of glycerol was thus reduced through in situ carbon source utilization by the yeast (Figure 1). The growing cells system was compared to a resting cells system to investigate its consolidated advantages. Although resting cell systems including recombinant P. pastoris, Saccharomyces cerevisiae and Escherichia coli surface displaying lipases were already reported for FAMEs production,15-17 the in situ growing cells system with recycling of by-product glycerol for FAMEs production through surface displayed lipase was developed for the first time in the present study. Crude glycerol derived from FAMEs production was already demonstrated a feasible substrate for industrial applications including various fuels and chemicals production.18-21 However, the used crude glycerol was separated from the FAMEs 5 ACS Paragon Plus Environment


transformation system, in contrast to the in situ utilization of the present study. Our integrated system provides a simplified and integrated, low energy consuming, green and sustainable process which utilizes in situ by-product glycerol to significantly promote FAMEs production through supporting lipase generation and reducing product inhibition effect. Unlike conventional chemical routes and traditional immobilized enzymes for FAMEs production, the sustainability in our new system comes from not only the renewable feedstock waste cooking oil, but also the recycling by-product glycerol for continually manufacturing of FAMEs through a highly efficient and green way. EXPERIMENTAL SECTION Strains, Vectors, Reagents and Media. Y. lipolytica host strain Polh and vector for surface display pINA1317-CWP110 have been described previously.22 All restriction enzymes and molecular genetic manipulation kits were purchased from Solarbio Science and Technology Co., Ltd (China). Immobilized enzyme Lipozyme TLIM was purchased from Novozymes (Denmark). FAME standards were bought from SigmaAldrich (USA). Medium components and other reagents were analytical grade. Waste cooking oil was collected from local restaurants. The oligonucleotide primers were synthesized by Samgong Co., Ltd (China). Plasmid pPICZA-tll harboring Thermomyces lanuginosus lipase gene was previously constructed in our laboratory.23 Y. lipolytica Polh strain was grown in YPD medium (1% yeast extract, 2% peptone and 2% glucose). The yeast transformants were selected on YNBD plates (0.67% yeast nitrogen base without amino acids, 1% glucose, 2% agar). E.coli DH5α strain was used for plasmid maintenance and propagation. The E.coli transformants were grown on LB medium supplemented with 50 µg/ml of kanamycin.


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Recombinant Plasmid and Strain Construction. The gene tll encoding T. lanuginosus lipase was amplified via PCR with primers F1 (5’-TCAAGGCCGTTCTG GCCATGTCACCTATCAGAAGAGAAGTTTCAC-3’,

SfiI

restriction

site

underlined) and R1 (5’-CGCGGATCCCTACAAGCAAGTTCCGATAAGACC-3’, BamH I restriction site underlined) using previous plasmid pPICZA-tll as a template. The fragment was sub-cloned into the surface display vector pINA1317-CWP110 through Sfi I and BamH I sites to generate a recombinant plasmid pINA1317CWP110-tll. The linearized recombinant plasmid was used to transform competent cells of Y. lipolytica Polh according to the method described by Chen et al.24 Transformants were selected by growth on selective medium YNBD. The host strain harboring an empty vector pINA1317-CWP110 was used as a control. The preliminary detection of recombinant lipase activity displayed on the transformants cell surface was performed on YPD agar plates containing 0.5% (w/v) tributyrin and 0.02% (w/v) rhodamine B, as a halo zone around yeast colonies, due to newly formed FFA-rhodamine B complexes. Cultivation, Lipase Activity and Cell Biomass Assays. A 250 ml flask loaded with 30 ml YPD medium was inoculated by 3 ml pre-culture of the transformant colony showing the largest halo zone, as indicated in preliminary screening. The fermentation was conducted at 28 °C with shaking at 300 rpm for 72 h. The culture supernatant and cell pellets were separated by centrifugation (5,000 rpm, 5 min). The cell pellets harvested from cultures by centrifugation and washed with sterile water were called resting cells and used for determination of lipase activity in the form of surface displayed whole cells and subsequent FAMEs transformation. Dry cell weight (DCW) was measured by oven drying wet cells to constant weight at 115 °C. The activity held by dry cells (U/mg DCW) was determined according to the method described by 7 ACS Paragon Plus Environment


Yan et al using host strain as a negative control.23 The host strain Y. lipolytica Po1h was set as a control for whole cell lipase activity assay. All these experiments were conducted three times, and presented as their means. Conversion of Waste Cooking Oil to FAMEs by Whole Cells with Surface Displayed Lipase. The resting cells and growing cells both came from 72 h culture of the best engineered strain cultivated in 30 ml YPD medium loaded in a 250 ml flask. In the case of resting whole cells mediated FAMEs production, the whole cells separated from 72 h culture were added to the substrate mixture of 2 g oil and 300 µl methanol (molar ratio of 1:4) to initiate transesterification-esterification reactions. The biotransformation was carried out in a 15 ml conical flask with stopper at 30 °C with shaking at 300 rpm. In the case of growing cells catalyzed biotransformation, the cultivation was conducted at 30 °C with shaking at 300 rpm for 72 h. Then, two routes were employed to synthesize FAMEs: transesterification-esterification and hydrolysisesterification. For transesterification-esterification route, 2 g oil and 300 µl methanol were simultaneously added to the growing culture to initiate in situ FAMEs production. For hydrolysis-esterification route, 2 g oil was firstly added to the growing culture followed by supplementation of 300 µl methanol 3 h later. In order to investigate the effects of in situ by-product glycerol on cell biomass and lipase generation, growing cells without addition of any oil and methanol were set as a control. Aliquots were taken from reaction mixtures at different time points, centrifuged and washed to obtain cell pellets and cell-free supernatant. The resulting whole cells were used for determination of DCW and lipase activity, as well as


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recyclability assessment of cells. The supernatant was used for TAGs, FFAs, glycerol and FAMEs determination. Recyclability Assessment. For investigation of reusability of growing cells, whole cells obtained by above mentioned procedure were used as resting cells and applied in fresh reaction mixture for FAMEs production. The recyclability was characterized as the FAMEs yield. Determination of Glycerides, FFAs, Glycerol and FAMEs. At designed time intervals, aliquots were taken from reaction mixture and mixed with solvent containing internal standard as the testing samples for component quantitative and qualitative analysis. The glycerides, FFAs and glycerol contents were determined by gel permeation chromatography on PL-GPC 50 equipped with refractive index detector RI 2000 and a styrene-divinylbenzene copolymer column Shodex GPC KF802 (8.0 mm×300 mm). The column pressure and column temperature were 3.25 MPa and 45 °C, respectively. Tetrahydrofuran was used as mobile phase with a flow rate of 1.0 ml/min. FAMEs were quantified by gas chromatography, using a flame-ionization detector and a capillary column (INNOWAX, Agilent, 30 m×0.25 mm×0.25µm). The column temperature was raised from 180 to 250 °C at 5 °C/min, and the temperatures of injector and detector were set at 240 and 280 °C, respectively. Identification of FAMEs and FAMEs yield calculation based on GC analysis were performed according to the method described in our previous study.13 The detailed information regarding calculation of FAMEs yield is described in the section of Supporting Information “Process for preparation of FAMEs standard as theoretical yield from waste cooking oil for experimental FAMEs yield calculation”.



RESULTS AND DISCUSSION Surface Display of Lipase. The cell wall protein GPI anchoring sequence present in pINA1317-CWP110 vector is able to direct lipase to the cell surface of Y. lipolytica by fusion expression of target protein and GPI anchor. Surface display of functional lipase on the cell surface of Y. lipolytica was preliminarily confirmed on YPDtributyrin-rhodamine B plates using halo zone formation as an indicator (Figure S1). As described in Figure 2, the resting whole cells with surface displayed lipase exhibited 3.2-fold and 1.6-fold higher activity, compared to respectively commercial counterpart immobilized Lipozyme TLIM and previously constructed recombinant P. pastoris yeast whole cells with intracellular overexpression of the same lipase.23 The lipase displayed on the cell surface of Y. lipolytica was exposed outside the cells, with direct access to substrate, thus optimizing bioconversion. In contrast, the intracellularly expressed lipase from recombinant P. pastoris suffered from substrate transfer limitation due to cell membrane and cell wall barriers. Obtaining a higher lipase activity than with Lipozyme TLIM demonstrated that tll heterologous gene was efficiently expressed and its functional product efficiently displayed on Y. lipolytica cell surface. In our experiment, original Y. lipolytica host strain just secreted very low level of extracellular lipase to medium (data not shown), and harbored even low intracellualr lipase (data not shown) inside the cell. The very low level of extracellualr lipase is obviously not adhered to the cell, and do not related to the specific activity associated with cell. Also, the even low intracellular lipase is difficult to contact the substrate olive oil due to the hamper of cell membrane and cell wall. Thus both the extracellular and intracellular lipases do not make contribution to the whole cell lipase activity. Compared to Y. lipolytica Po1h, the surface displayed yeast showed three orders of magnitude higher specific activity.


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Conversion of Waste Cooking Oil to FAMEs in a Resting Cells System. The time course of FAMEs production in the resting cells system is presented in Figure 3: a 81% yield was achieved after 16 h. In the resting cells system, glycerol concentration gradually rose during FAMEs production process, and reached the highest concentration of 14.6% (weight relative to oil). The concentrations of glycerides and FFAs decreased with reaction time due to transesterification of TAGs and esterification of FFAs with methanol to form FAMEs. The cell biomass together with its lipase activity did not vary significantly during the resting cells catalyzed FAMEs production process (data not shown). Transesterification-esterification and hydrolysisesterification reactions did not affect FAMEs yield in the resting cells system (data not shown). Conversion of Waste Cooking Oil to FAMEs in a Growing Cells System through Hydrolysis-esterification Route. In the growing cells system, FAMEs production via either hydrolysis-esterification reactions or transesterification-esterification reactions depended on the time of methanol addition. In order to allow displaying functional lipase on the cell surface of the yeast, 72 h of substrate-free cultivation was performed. After then, waste cooking oil was added to the culture medium. This point corresponds to the start of the bioconversion process described in Figure 4. The glycerides present in waste cooking oil were hydrolyzed to FFAs through the displayed lipase mediated hydrolysis reaction, as reflected by the glycerides content dramatic decrease and FFAs level significant increase. Glycerides were hardly detected and FFAs reached the maximum amount after a reaction time of around 2 h. During the period of time of 73-74 h cultivation (1-2 h after waste cooking oil addition) (Figure 4 A), cell biomass and lipase activity exhibited almost no change (Figure 4 B). After 74 h cultivation (reaction time of 2 h), methanol was



added to start the esterification reaction for FAMEs synthesis. FAMEs achieved the highest yield of 96% after a 4 h esterification time (reaction time of 6 h). FFAs were gradually decreased and FAMEs were increased to the highest yield (96%) from 75 h to 78 h (reaction time of 2-6 h). However, in contrast to FAMEs level, the final concentration of glycerol (the FAMEs derived by-product) was only of 3% (weight relative to oil), hence much lower than in the resting cells system. At the same time, cell biomass and lipase activity were further enhanced compared to a growing cells control (Figure 2 A, Figure 4 B). Considering that Y. lipolytica can easily utilize glycerol as a carbon source,25-27 it could be referred that the in situ formed glycerol was metabolized by Y. lipolytica for further cell growth and lipase production. The cycles of “glycerol consuming-lipase regeneration” resulted in a shift towards FAMEs production in the reversible esterification reaction, reflected as higher FAMEs yield and productivity than in the resting cells system. Conversion of Waste Cooking Oil to FAMEs in a Growing Cells System through Transesterification-esterification Route. Through controlling the methanol addition time, transesterification-esterification reaction could replace hydrolysis-esterification reaction and dominate FAMEs production. Cultivation was performed for 72 h as described above, then waste cooking oil and methanol were simultaneously added to the culture medium and transesterification-esterification reactions occurred. As presented in Figure 5 A, the highest FAMEs yield of 91% was obtained after 10 h of bioconversion. The cell biomass and lipase activity increased during the reaction time, between 73 and 78 h of cultivation (1-6 h in Figure 5 B). In contrast, the by-product glycerol did not accumulate during FAMEs formation process. In a similar observation as for the hydrolysis-esterification route, the final glycerol concentration was much lower than for the resting cells system. It can be concluded that the by-


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product glycerol was used as an in situ carbon source for supporting further cell growth and lipase production. The continual removal of glycerol and the additionally generated lipase obviously contributed to FAMEs production with higher yield and productivity. Crude glycerol was also a carbon source candidate for recombinant P. pastoris yeast.28 We also attempted the recycling of glycerol as an in situ carbon source in the P. pastoris system. However, the in situ formed glycerol benefited only to cell growth, and was not favorable for lipase expression (data not shown). The Cell Metabolism Reflected as enhancements of Cell biomass and Lipase Activity in Growing Cells Systems. In growing cells mediated FAMEs production systems via hydrolysis-esterification reactions and transesterification-esterification reactions, the in situ FAMEs derived by-product glycerol was partially utilized as a carbon source by the yeast, in support of cell growth and lipase expression. Thus, more lipase was surface displayed, enhancing catalyst amount and increasing FAMEs production. In addition, reducing product feedback inhibition effect by the continual removal of by-product glycerol pulled the reversible reactions (transesterification, esterification, hydrolysis) toward FAMEs formation. For the resting cell system, the cell biomass and specific activity did not altered during FAMEs formation. However, both of them were significantly increased in the case of growing cell systems. The decrease of by-product glycerol in growing cell systems indicated that the yeast assimilated the in situ carbon source glycerol to restart cell metabolism, reflected as enhancements of cell biomass and lipase production. The facts suggest resting status of cell metabolism in resting cell system, while active state in growing cell systems.



Our present results with Y. lipolytica surface displaying tll lipase in situ growing cells system show improved FAMEs yield (96%) and productivity ( 21.07 g FAMEs L-1 h-1) compared to previous works from our laboratory with two P. pastoris systems (in situ growing cells or resting cells) expressing the same lipase in similar reaction conditions.14,

23

There could be several reasons for the superiority of the

recombinant Y. lipolytica system, especially the fact that lipase displayed on the cell surface optimized bioconversion. Another probably important reason is that the efficient recycling of glycerol into cell growth and lipase displaying had a crucial effect in promoting FAMEs synthesis. Reusability of Whole Cells from Growing Cells Systems. For both growing cells systems based on hydrolysis-esterification reactions and transesterificationesterification reactions, after biotransformation completion, the whole cells separated from the growing cells systems were employed on fresh substrates as resting cells system for FAMEs production. As shown in Figure 6, growing cells systems demonstrated satisfactory recyclability after 12 cycles of batches. Yields of 82%-86% were achieved after 12 successive batches. The fact that slightly higher FAMEs yields were obtained compared to a resting cells system (81%) demonstrated further the benefit of in situ carbon source utilization in a growing cells system for the display of more functional lipases. Proof of concept of the new growing cell systems for efficient FAMEs production was experimental verified at small scale. However, further work including larger scale production together with characterization of final FAMEs product are still needed, like previously well established processes for biodiesel production by whole cell based systems. 29-32


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In conclusion, the generation of lipase-displaying cell biocatalysts and the subsequent whole cell mediated FAMEs production were integrated into a one-pot simultaneous fermentation and biotransformation process, during which by-product glycerol was used as an in situ carbon source. The self-cycling “glycerol consumption-cell growth and lipase displaying-FAMEs forming” contributed to high FAMEs yield and productivity. Overall, we demonstrated that a new integrated process with surface displayed lipase could be used in a streamline procedure for FAMEs production. ASSOCIATED CONTENT Supporting Information: Process for preparation of FAMEs standard as theoretical yield from waste cooking oil for experimental FAMEs yield calculation; Scheme and preliminary confirmation of surface displaying functional lipase on the cell surface of Y. lipolytica. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by funding from the National Natural Science Foundation of China (Grant Number: NSFC31570793), the Innovation Program of Huazhong University of Science and Technology, Wuhan Morning Light Plan of Youth Science and Technology (Grant Number: 2017050304010292), and the Startup Fund for Talent Scholars of Huazhong University of Science and Technology. REFERENCES



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(22) Madzak, C. Yarrowia lipolytica: Recent Achievements in Heterologous Protein Expression and Pathway Engineering. Appl. Microbiol. Biotechnol. 2015, 99, 45594577. (23) Yan, JY.; Zheng, XL.; Li, SY. A Novel and Robust Recombinant Pichia pastoris Yeast Whole Cell Biocatalyst with Intracellular Overexpression of a Thermomyces lanuginosus Lipase: Preparation, Characterization and Application in Biodiesel Production. Bioresour. Technol. 2014, 151, 43-48. (24) Chen, DC.; Beckerich, JM.; Gaillardin, C. One-Step Transformation of the Dimorphic Yeast Yarrowia lipolytica. Appl. Microbiol. Biotechnol.1997, 48, 232-235. (25) Juszczyk, P.; Tomaszewska, L.; Kita, A.; Rymowicz, W. Biomass Production by Novel Strains of Yarrowia lipolytica Using Raw Glycerol, Derived from Biodiesel Production. Bioresour. Technol. 2013, 137, 124-131. (26) Makri, A.; Fakas, S.; Aggelis, G. Metabolic Activities of Biotechnological Interest in Yarrowia lipolytica Grown on Glycerol in Repeated Batch Cultures. Bioresour. Technol. 2010, 101, 2351-2358. (27) Rywińska, A.; Juszczyk, P.; Wojtatowicz, M.; Robak, M.; Lazar, Z, Tomaszewska, L.; Rymowicz , W. Glycerol as a Promising Substrate for Yarrowia lipolytica Biotechnological Applications. Biomass. Bioenerg. 2013, 48, 148-166. (28) Anastácio, GS.; Santos, KO.; Suarez, PAZ.; Torres, FAG.; Marco, JLD.; Parachin, NS. Utilization of Glycerin Byproduct Derived from Soybean Oil Biodiesel as a Carbon Source for Heterologous Protein Production in Pichia pastoris. Bioresour. Technol. 2014, 152, 505-510.



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Figure captions: Figure 1. Scheme of the two routes, transesterification-esterification (A) and hydrolysis-esterification (B), for FAMEs synthesis based on surface display of a lipase on Y. lipolytica cells, using by-product glycerol as in situ carbon source in a growing cells system. Figure 2. Time course of cell density and specific activity for lipase-displaying Y. lipolytica strain (A). Comparison of specific activities of the engineered Y. lipolytica surface displaying T. lanuginosus lipase, of engineered P. pastoris overexpressing of the same lipase gene, and of immobilized T. lanuginosus lipase (Lipozyme TLIM) (B). Figure 3. Time course of various components during FAMEs production in a resting cells system. Figure 4. Time course of contents of various components including FAMEs, FFAs, glycerides, glycerol during FAMEs production via hydrolysis-esterification route in a growing cells system (A). Time course of cell density and specific activity for lipasedisplaying Y. lipolytica strain during FAMEs production via hydrolysis-esterification route in a growing cells system (B). Figure 5. Time course of contents of various components including FAMEs, FFAs, glycerides, glycerol during FAMEs production via transesterification-esterification route in a growing cells system (A). Time course of cell density and specific activity for

lipase-displaying

Y.

lipolytica

strain

during

FAMEs

transesterification-esterification route in a growing cells system (B).


production

via


Figure 6. Recyclability during 12 successive cycles of batches using harvested cells from growing cell system on fresh substrates for FAMEs production.


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Figure 1.



Figure 2.


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Figure 3.



Figure 4.


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Figure 5.



Figure 6.


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Abstract graphic

Synopsis: Innovative system based on an engineered microorganism is developed for green manufacturing of alternative energy (biodiesel) using waste cooking oil as a resource.


Engineering Yarrowia lipolytica for Sustainable Production of Fatty

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