A synthetic biology approach to transform <i>Yarrowia lipolytica</i> into a competitive biotechnological producer of β‐carotene

Larroude, Macarena; Celińska, Ewelina; Back, Alexandre; Stephan, T.; Nicaud, Jean‐Marc; Ledesma-Amaro, Rodrigo

doi:10.1002/bit.26473

Cited by 257 publications

(242 citation statements)

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“…Particularly, Y. lipolytica has a rich acetyl-CoA pool due to its cytosolic ATP citrate lyase (ACL) [20, 21]. Genetic tools have been established in Y. lipolytica [22], and several groups have successfully engineered the species to produce diverse carotenoids [23–25]. Another industrial advantage of utilizing Y. lipolytica is its capacity to grow on a large range of carbon sources (including but not limited to: glucose, glycerol, alcohols, and fatty acids), allowing for the use of diverse and cheap feedstock [26].…”

Section: Introductionmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push–pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0984-x) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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“…The capacity and efficacy of our system to express three TUs on a same construct has been validated previously (Celinska et al ., ; Larroude et al ., ). However, the expression levels of the three TUs have not been evaluated.…”

Section: Resultsmentioning

confidence: 97%

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

Larroude

Park

Soudier

et al. 2019

Microbial Biotechnology

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Summary The oleaginous yeast Yarrowia lipolytica is an established host for the bio‐based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one‐step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine‐tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic‐resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three‐gene pathway allowing xylose utilization by Y. lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. lipolytica strains and is available to the community through Addgene.

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“…The pTEF promoter demonstrated the strongest expression of the downstream target gene as compared to the pGPM and pGAPDH promoters . Additionally, the engineered Y. lipolytica strain with an extra three heterologous β‐carotene expression cassettes under the control of constitutive pTEF promoter, with each cassette containing the native geranylgeranyl diphosphate synthase ( GGS1 ) gene and two pytoene synthase/lycopene cyclase ( CarRP ) and phytoene dehydrogenase ( CarB ) genes from Mucor circinelloides , produced 6.5 g/L β‐carotene in the fed‐batch fermentation . Moreover, the coproduction of the omega3‐fatty acids and carotenoids by engineered Y. lipolytica was developed via the integration of the heterologous codon‐optimized carotenoid synthesis genes (i.e.…”

Section: Y Lipolytica As the Biomanufacturing Platformmentioning

confidence: 99%

Cellular and metabolic engineering of oleaginous yeast Yarrowia lipolytica for bioconversion of hydrophobic substrates into high‐value products

Soong

Liu

Yoon

et al. 2019

Engineering in Life Sciences

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The non‐conventional oleaginous yeast Yarrowia lipolytica is able to utilize both hydrophilic and hydrophobic carbon sources as substrates and convert them into value‐added bioproducts such as organic acids, extracellular proteins, wax esters, long‐chain diacids, fatty acid ethyl esters, carotenoids and omega‐3 fatty acids. Metabolic pathway analysis and previous research results show that hydrophobic substrates are potentially more preferred by Y. lipolytica than hydrophilic substrates to make high‐value products at higher productivity, titer, rate, and yield. Hence, Y. lipolytica is becoming an efficient and promising biomanufacturing platform due to its capabilities in biosynthesis of extracellular lipases and directly converting the extracellular triacylglycerol oils and fats into high‐value products. It is believed that the cell size and morphology of the Y. lipolytica is related to the cell growth, nutrient uptake, and product formation. Dimorphic Y. lipolytica demonstrates the yeast‐to‐hypha transition in response to the extracellular environments and genetic background. Yeast‐to‐hyphal transition regulating genes, such as YlBEM1, YlMHY1 and YlZNC1 and so forth, have been identified to involve as major transcriptional factors that control morphology transition in Y. lipolytica. The connection of the cell polarization including cell cycle and the dimorphic transition with the cell size and morphology in Y. lipolytica adapting to new growth are reviewed and discussed. This review also summarizes the general and advanced genetic tools that are used to build a Y. lipolytica biomanufacturing platform.

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A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β‐carotene

Cited by 257 publications

References 40 publications

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

Cellular and metabolic engineering of oleaginous yeast Yarrowia lipolytica for bioconversion of hydrophobic substrates into high‐value products

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