A modular Golden Gate toolkit for <i>Yarrowia lipolytica</i> synthetic biology

Larroude, Macarena; Park, Young Kyoung; Soudier, Paul; Kubiak, Monika; Nicaud, Jean‐Marc; Rossignol, Tristan

doi:10.1111/1751-7915.13427

Cited by 69 publications

(93 citation statements)

References 34 publications

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“…Golden Gate cloning is also frequently used for assembly of CRISPR gRNA expression arrays for site‐specific mutagenesis in a variety of organisms (McCarty, Shaw, Ellis, & Ledesma‐Amaro, 2019; Vad‐Nielsen, Lin, Bolund, Nielsen, & Luo, 2016). Finally, Golden Gate cloning has led to the development of the modular cloning system MoClo (Engler et al., 2014; Kowarschik, Hoehenwarter, Marillonnet, & Trujillo, 2018; Weber, Engler, et al., 2011; Werner et al., 2012) and of several other related modular cloning systems (Agmon et al., 2015; Andreou & Nakayama, 2018; Binder et al., 2014; Larroude et al., 2019; Lee, DeLoache, Cervantes, & Dueber, 2015; Martella, Matjusaitis, Auxillos, Pollard, & Cai, 2017; Moore et al., 2016; Occhialini et al., 2019; Pollak et al., 2019; Prielhofer et al., 2017; Sarrion‐Perdigones et al., 2011, 2013).…”

Section: Commentarymentioning

confidence: 99%

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Marillonnet

Grützner

2020

CP Molecular Biology

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Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one‐pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline. © 2020 The Authors. Basic Protocol 1: Performing a typical Golden Gate cloning reaction Basic Protocol 2: Accommodating a vector to Golden Gate cloning Basic Protocol 3: Accommodating an insert to Golden Gate cloning Basic Protocol 4: Generating small standardized parts compatible with hierarchical modular cloning (MoClo) using level 0 vectors Alternate Protocol: Generating large standardized parts compatible with hierarchical modular cloning (MoClo) using level –1 vectors Basic Protocol 5: Assembling transcription units and multigene constructs using level 1, M, and P MoClo vectors

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Section: Commentarymentioning

confidence: 99%

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Marillonnet

Grützner

2020

CP Molecular Biology

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“…The strain was cultivated in the lysogeny broth (LB) liquid medium at 37 °C. The gene encoding xylose reductase (XR) (YALI0D07634), xylitol dehydrogenase (XDH) (YALI0E12463) and xylulokinase (XK) (YALIF10923) were extracted from the genome of Po1d using the appropriate primers (Additional file 1: Table S1) and the Golden Gateway (GG) assembly was constructed according to former studies [39,40]. The GG was constructed with a scaffold of three genes comprising three transcription units and selection marker, flanked with integration targeting sequences, constructed on a destination vector backbone.…”

Section: Cloning and Expression Of Heterologous Xylose Assimilation Gmentioning

confidence: 99%

Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

Prabhu

Ledesma-Amaro

Lin

et al. 2020

Biotechnol Biofuels

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Background: Xylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. Results: In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of the recombinant strain in a bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in terms of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD 600 : 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main by-product in all the fermentations. Conclusion: The recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

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“…The current study made use of strain originated from adaptive evolution of engineered Y. lipolytica extracted from the genome of Po1d using the appropriate primers (Table S2), the golden gateway (GG) assembly was constructed according to former studies [51,52]. The GG was constructed with Scaffold of three genes comprising three transcription units and selection marker, flanked with integration targeting sequences, constructed on a destination vector backbone.…”

Section: Microorganism Culture Maintenance and Inoculum Preparationmentioning

confidence: 99%

Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

Prabhu

Amaro

Lin

et al. 2020

Preprint

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Background Xylose is a most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast to produce industrially important metabolites, and it is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economically feasible of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. Results In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising of xylose reductase ( XR ), xylitol dehydrogenase ( XDH ) and xylulose kinase ( XK ) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and accumulated SA (3.8 g/L) with a yield of 0.19 g/g in shake flask studies. Substrate inhibition studies revealed a marked negative impact on cell growth and product formation above 60 g/L xylose concentration. The modelling based on inhibition kinetics revealed that Aiba model showed better fit with experimental data, which resulted the correlation coefficient (R 2 ) of 0.82 and inhibition constant (K I ) 88.9 g/L. The batch cultivation of recombinant strain in bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.18 g/g. Similar results in term of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD 600 : 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main byproduct in all the fermentations. Conclusion The recombinant strain displayed potential bioconversion of xylose to succinic acid. Further this study provided a new insight on conversion of LCB into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

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A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

Cited by 69 publications

References 34 publications

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

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