Synthetic biology tools for engineering Yarrowia lipolytica

Larroude, Macarena; Rossignol, Tristan; Nicaud, Jean‐Marc; Ledesma-Amaro, Rodrigo

doi:10.1016/j.biotechadv.2018.10.004

Cited by 135 publications

(96 citation statements)

References 175 publications

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“…For these reasons, Y. lipolytica, a GRAS strain, has attracted great attention in recent years, especially for the production of terpenoids. Several useful synthetic biology tools have been aiding the creation of new and better performing Y. lipolytica strains [42,43]. To date, the highest yields of lipid, lycopene and carotene produced via microbial cell factories have been achieved by using engineered Y. lipolytica strains [7,12].…”

Section: Discussionmentioning

confidence: 99%

A modular pathway engineering strategy for the high-level production of β-ionone in Yarrowia lipolytica

Yang

Lin

et al. 2020

Microb Cell Fact

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Background: The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. Results: In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. Conclusions: An efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.

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Section: Discussionmentioning

confidence: 99%

A modular pathway engineering strategy for the high-level production of β-ionone in Yarrowia lipolytica

Yang

Lin

et al. 2020

Microb Cell Fact

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“…Considering this point, we plan to construct a series of artificial core promoter sequences and characterize their performances to get some kind of rule. In the present study, we constructed artificial core promoters in Y. lipolytica, which was engineered for producing valuable chemicals as fatty acid derivatives, organic acids, terpenoids and sterols [20][21][22]. We chose an existing object that the artificial promoters might enhance the expression levels of CrtY enzyme to get higher beta-carotene production form the substrate lycopene ( Fig.…”

Section: Open Accessmentioning

confidence: 99%

Engineering yeast artificial core promoter with designated base motifs

Liu

et al. 2020

Microb Cell Fact

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Background: Synthetic biology requires toolbox of promoters to finely tune gene expression levels for building up efficient cell factories. Yeast promoters owned variable core promoter regions between the TATA-box and transcriptional starting site (TSS) at the length mostly around 20-80 bases. This region allowed flexible design of artificial promoter but potentially demand special base motifs to maintain or enhance the promoter's strength.Results: Here, we designed and screened the base motifs and tested the activities of yeast artificial core promoters. Different 30 bases of artificial sequences led to variable expression levels of CrtY enzyme which determined the lycopene-carotene compositions, represented in the colony-color spectrum of red-orange-yellow. The upstream sequences of two strong promoter P EXP1 and P GPD and two starting strains with distinguishable lycopene production levels were utilized to characterize the promoter sequences. Different partition designs of T-rich or G/C-rich base motifs led to distinguishable colony-color distributions. Finally, we screened a champion promoter with a highest 5.5-fold enhancement of lycopene-carotene transformation. Another selected promoter generated a highest betacarotene production as 7.4 mg/g DCW. Conclusions:This work offered an approach to redesign promoter with artificial sequences. We concluded that the core promoter region could be designated as 30 bases and different base motifs would enhance or weaken the promoter's strength. Generally, more T-rich elements, higher %T and lower G/C percentage were beneficial to enhance the strength of artificial core promoter.

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“…This methodology combined with the multigene overexpression system for heterologous genes (Ledesma‐Amaro et al ., ) will allow to avoid the use of selectable markers for metabolic engineering applications in A. gossypii . Similar toolkits have been described in other biotechnological microorganisms such as Yarrowia lypolytica , thus enabling the rapid engineering of metabolic pathways in that oleaginous yeast (Wong et al ., ; Holkenbrink et al ., ; Larroude et al ., ).…”

Section: Resultsmentioning

confidence: 99%

One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

Jiménez

Muñoz‐Fernández

Ledesma‐Amaro

et al. 2019

Microbial Biotechnology

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Summary The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low‐cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high‐added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one‐vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker‐less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker‐less manner.

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Synthetic biology tools for engineering Yarrowia lipolytica

Cited by 135 publications

References 175 publications

A modular pathway engineering strategy for the high-level production of β-ionone in Yarrowia lipolytica

A modular pathway engineering strategy for the high-level production of β-ionone in Yarrowia lipolytica

Engineering yeast artificial core promoter with designated base motifs

One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

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