Production and characterization of two medium-chain-length polydroxyalkanoates by engineered strains of Yarrowia lipolytica

Background: The oleaginous yeast Yarrowia lipolytica is an organism of choice for the tailored production of various compounds such as biofuels or biopolymers. When properly engineered, it is capable of producing medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biobased and biodegradable polymer that can be used as bioplastics or biopolymers for environmental and biomedical applications.Results: This study describes the bioproduction and the main properties of two different mcl-PHA polymers. We generated by metabolic engineering, strains of Y. lipolytica capable of accumulating more than 25% (g/g) of mcl-PHA polymers. Depending of the strain genetic background and the culture conditions, we produced (i) a mcl-PHA homopolymer of 3-hydroxydodecanoic acids, with a mass-average molar mass (M-w) of 316,000 g/mol, showing soft thermoplastic properties with potential applications in packaging and (ii) a mcl-PHA copolymer made of 3-hydroxyoctanoic (3HO), decanoic (3HD), dodecanoic (3HDD) and tetradecanoic (3TD) acids with a M-w of 128,000 g/mol, behaving like a thermoplastic elastomer with potential applications in biomedical material.Conclusion: The ability to engineer Y. lipolytica to produce tailored PHAs together with the range of possible applications regarding their biophysical and mechanical properties opens new perspectives in the field of PHA bioproduction

Production of medium chain fatty acid by Yarrowia lipolytica: Combining molecular design and TALEN to engineer the fatty acid synthase

Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered lipid biosynthesis through rational engineering of the giant multifunctional Fatty Acid Synthase (FAS) enzyme to modulate fatty acid chain length and produce shorter fatty acids. Based on the hypothesis that the Ketoacyl Synthase (KS) domain, responsible for chain elongation in Yarrowia lipolytica, is directly involved in chain length specificity, a computer-based strategy was undertaken to re-design mutants of the Ketoacyl Synthase. Molecular modelling of this domain in interaction with a C16-acyl substrate enabled identification of a key residue from the fatty acid binding site. This site was then targeted by mutagenesis in order to modify KS fatty acid chain length specificity. To introduce point mutations in this essential gene, we applied, for the first time, the TALEN technology to Yarrowia lipolytica and demonstrated the efficiency of the technique to perform site-directed mutagenesis at a specific genomic locus. Some mutants led to a significant increase of C14 fatty acid. Thanks to the use of an elegant combination of genome editing technology and molecular modelling, this study provides for the first time, evidences that the KS domain of the fungal FASI system is directly involved in fatty acid chain length specificity

Genome Editing in Y. lipolytica Using TALENs

International audienceTALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied. Here we introduce how to use TALENs in the oleaginous yeast Yarrowia lipolytica by presenting a step-by-step method allowing to knock out or to introduce in vivo a point mutation in a gene of Yarrowia lipolytica. This chapter describes the material required, the transformation procedure, and the screening process

Multiple parameters drive the efficiency of CRISPR/Cas9-induced gene modifications in Yarrowia lipolytica

Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide

Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering

Background: Oleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest. Because medium chain fatty acids (MCFA) are valuable compounds for biokerosene production, we previously generated strains capable of producing MCFA up to 12% of total lipid content (Rigouin et al. in ACS Synth Biol 6:1870-1879, 2017). In order to improve accumulation and content of C14 fatty acid (FA), the elongation, degradation and accumulation of these MCFA in Yarrowia lipolytica were studied. Results: We brought evidence of the role of YALI0F0654 (YlELO1) protein in the elongation of exogenous or de novo synthesized C14 FA into C16 FA and C18 FA. YlELO1 deletion into a alpha FAS_I1220W expressing strain leads to the sole production of C14 FA. However, because this strain does not provide the FA essential for its growth, it requires being cultivated with essential fatty acids and C14 FA yield is limited. To promote MCFA accumulation in Y. lipolytica without compromising the growth, we overexpressed a plant diglyceride acyltransferase specific for MCFA and reached an accumulation of MCFA up to 45% of total lipid content. Conclusion: We characterized the role of YlELO1 in Y. lipolytica by proving its involvement in Medium chain fatty acids elongation. We showed that MCFA content can be increased in Yarrowia lipolytica by promoting their accumulation into a stable storage form (triacylglycerides) to limit their elongation and their degradation

Real-time RT-PCR analysis of PCS in response to various stresses.

<p><b>A</b>. Adult worms were exposed to different stressors (Fe: 100 µM FeCl₃; Cd: 100 µM CdCl₂; PZQ: 1 µg/mL praziquantel; mBrB: 30 µM monobromobimane; EA: 10 µM ethacrynic acid; H₂O₂: 100 µM hydrogen peroxide) for 6 h. SmPCS mRNA abundance was determined by real-time RT-PCR. <b>B</b>. Real-time RT-PCR analysis of PCS in response to metal with time. Adult worms were exposed to 100 µM FeCl₃ (dark grey) or 100 µM CdCl₂ (light grey) during 2, 4 and 6 h. SmPCS mRNA abundance was determined by real-time RT-PCR.</p

List of the primers used for RT-PCR analysis.

<p>Smp = Schistosoma mansoni GeneDB accession number (<a href="http://www.genedb.org/Homepage/Smansoni" target="_blank">http://www.genedb.org/Homepage/Smansoni</a>).</p

Relative activity of SmPCS_66–591 and SmPCS_66–300 with different metals.

<p>Values are given as the percentage of activity (PC₂ synthesized µmol min⁻¹ mg⁻¹ enzyme) of respective enzyme in the absence of added metal in the presence of DTPA (6.3 mM). Mean value of three separate assays (± SE) are given (N = 3). The incubation mixtures contained 200 mM Tris-HCl (pH 8), 10 mM β-Me, 10 mM GSH, 3 µg of enzyme and the metal ion.</p>*<p>, p<0.05 by Student's t test.</p