On the Uniform Random Generation of Non Deterministic Automata Up to Isomorphism

In this paper we address the problem of the uniform random generation of non deterministic automata (NFA) up to isomorphism. First, we show how to use a Monte-Carlo approach to uniformly sample a NFA. Secondly, we show how to use the Metropolis-Hastings Algorithm to uniformly generate NFAs up to isomorphism. Using labeling techniques, we show that in practice it is possible to move into the modified Markov Chain efficiently, allowing the random generation of NFAs up to isomorphism with dozens of states. This general approach is also applied to several interesting subclasses of NFAs (up to isomorphism), such as NFAs having a unique initial states and a bounded output degree. Finally, we prove that for these interesting subclasses of NFAs, moving into the Metropolis Markov chain can be done in polynomial time. Promising experimental results constitute a practical contribution.Comment: Frank Drewes. CIAA 2015, Aug 2015, Umea, Sweden. Springer, 9223, pp.12, 2015, Implementation and Application of Automata - 20th International Conferenc

Synthetic biology tools for engineering Yarrowia lipolytica

The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools

Engineering Yarrowia lipolytica to enhance lipid production from lignocellulosic materials

Background: Yarrowia lipolytica is a common biotechnological chassis for the production of lipids, which are the pre‑ ferred feedstock for the production of fuels and chemicals. To reduce the cost of microbial lipid production, inexpen‑ sive carbon sources must be used, such as lignocellulosic hydrolysates. Unfortunately, lignocellulosic materials often contain toxic compounds and a large amount of xylose, which cannot be used by Y. lipolytica. Results: In this work, we engineered this yeast to efciently use xylose as a carbon source for the production of lipids by overexpressing native genes. We further increased the lipid content by overexpressing heterologous genes to facilitate the conversion of xylose-derived metabolites into lipid precursors. Finally, we showed that these engineered strains were able to grow and produce lipids in a very high yield (lipid content = 67%, titer = 16.5 g/L, yield = 3.44 g/g sugars, productivity 1.85 g/L/h) on a xylose-rich agave bagasse hydrolysate in spite of toxic compounds. Conclusions: This work demonstrates the potential of metabolic engineering to reduce the costs of lipid production from inexpensive substrates as source of fuels and chemicals

Genetic engineering of the β-oxidation pathway in the yeast Yarrowia lipolytica to increase the production of aroma compounds

peer reviewedThe yeast Yarrowia lipolytica possesses five acyl-CoA oxidases (Aox1p to 5), the enzyme catalysing the first reaction of β-oxidation. The understanding of the specific role of each acyl-CoA oxidase is important to construct a yeast strain growing at a good rate and able to produce without degrading the aroma compound γ-decalactone. In this study we observed that Aox4p exhibits a slight activity on a broad spectrum of substrates and that it is involved in lactone degradation. We constructed a strain lacking this activity. Its growth was only slightly altered and it produced 10 times more lactone than the wild type in 48h. © 2004 Elsevier B.V. All rights reserved

De novo biosynthesis of odd-chain fatty acids in yarrowia lipolytica enabled by modular pathway engineering

© 2020 Park, Ledesma-Amaro and Nicaud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) (http://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms

A yeast-based tool for screening mammalian diacylglycerol acyltransferase inhibitors

Dysregulation of lipid metabolism is associated with obesity and metabolic diseases but there is also increasing evidence of a relationship between lipid body excess and cancer. Lipid body synthesis requires diacylglycerol acyltransferases (DGATs) which catalyze the last step of triacylglycerol synthesis from diacylglycerol and acyl-coenzyme A. The DGATs and in particular DGAT2, are therefore considered potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGAT activities, to evaluate the functionality of the enzymes in this heterologous host and DGAT activity inhibitors. This work provides evidence that mammalian DGATs expressed in Y. lipolytica are a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest

Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica

Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with a chain of four or more carbon atoms. Besides potential industrial applications, fatty alcohols have important biological functions as well. In nature, fatty alcohols are produced as a part of a mixture of pheromones in several insect species, such as moths, termites, bees, wasps, etc. In addition, FA-OHs have a potential for agricultural applications, for example, they may be used as a suitable substitute for commercial insecticides. The insecticides have several drawbacks associated with their preparation, and they exert a negative impact on the environment. Currently, pheromone components are prepared mainly through the catalytic hydrogenation of plant oils and petrochemicals, which is an unsustainable, ecologically unfriendly, and highly expensive process. The biotechnological production of the pheromone components using engineered microbial strains and through the expression of the enzymes participating in the biosynthesis of these components is a promising approach that ensures ecological sustenance as well. The present study was aimed at evaluating the production of FA-OHs in the oleaginous yeast, Yarrowia lipolytica, with different lengths of fatty-acyl chains by expressing the fatty acyl-CoA reductase (FAR) BlapFAR4 from B. lapidarius, producing C16:0-OH, C16:1Δ9-OH, and lower quantities of both C14:0-OH and C18:1Δ9-OH, and BlucFAR1 from B. lucorum, producing FA-OHs with a chain length of 18–26 carbon atoms, in this yeast. Among the different novel Y. lipolytica strains used in the present study, the best results were obtained with JMY7086, which carried several lipid metabolism modifications and expressed the BlucFAR1 gene under the control of a strong constitutive promoter 8UAS-pTEF. JMY7086 produced only saturated fatty alcohols with chain lengths from 18 to 24 carbon atoms. The highest titer and accumulation achieved were 166.6 mg/L and 15.6 mg/g DCW of fatty alcohols, respectively. Unlike JMY7086, the BlapFAR4-expressing strain JMY7090 produced only 16 carbon atom-long FA-OHs with a titer of 14.6 mg/L

Metabolic engineeringof Yarrowia lipolytica to produce chemicals and fuels from xylose

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials