Metabolic engineering of Yarrowia lipolytica for industrial applications

Zhu, Quinn; Jackson, Evelyn M.

doi:10.1016/j.copbio.2015.08.010

Cited by 165 publications

(104 citation statements)

References 56 publications

(80 reference statements)

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“…Acetyl-CoA is a key terpenoid precursor needed to push flux through the over-expressed MVA pathway. While Y. lipolytica has large quantities of cytosolic acetyl-CoA due to its ACL [20, 21], many important competing pathways, including fatty acids and lipids synthesis, can quickly drain the available resources. Interestingly, the supplementation of citrate and acetate (direct carbon contributors for cytosolic acetyl-CoA) did not show significant improvement in the β-carotene producing strain’s titer (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…This GRAS species offers the potential to become a new production platform for β-ionone due to its innate metabolic capabilities [19]. 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].…”

Section: Introductionmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…These non-conventional microbes naturally accumulate high amounts of lipids ranging from 20 to 80% of the dry cell weight (DCW) (Sitepu et al, 2014) but often their metabolic knowledge and genetic tools are scarce. Yarrowia lipolytica is by far the most studied oleaginous yeast with a vast array of metabolic engineering techniques available (Beopoulos et al, 2009a;Liu et al, 2015a;Zhu and Jackson, 2015). Although Y. lipolytica present two main drawbacks since it accumulates limited amounts of lipids (Beopoulos et al, 2009a) and it cannot consume some preferred substrates such as lignocellulosic biomass or starch, it has been metabolically engineered to overcome these issues.…”

Section: Introductionmentioning

confidence: 99%

Combining metabolic engineering and process optimization to improve production and secretion of fatty acids

Ledesma-Amaro

Dulermo

Niehus

2016

Metabolic Engineering

145

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Microbial oils are sustainable alternatives to petroleum for the production of chemicals and fuels. Oleaginous yeasts are promising source of oils and Yarrowia lipolytica is the most studied and engineered one. Nonetheless the commercial production of biolipids is so far limited to high value products due to the elevated production and extraction costs. In order to contribute to overcoming these limitations we exploited the possibility of secreting lipids to the culture broth, uncoupling production and biomass formation and facilitating the extraction. We therefore considered two synthetic approaches, Strategy I where fatty acids are produced by enhancing the flux through neutral lipid formation, as typically occurs in eukaryotic systems and Strategy II where the bacterial system to produce free fatty acids is mimicked. The engineered strains, in a coupled fermentation and extraction process using alkanes, secreted the highest titer of lipids described so far, with a content of 120% of DCW.

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“…Even though engineering the TAG biosynthesis pathway has been the focus of Y. lipolytica studies [17][18][19]60,61], this pathway is not well understood in T. reesei. We have recently reported the genome re-annotation and reconstruction of glycolysis and fermentation; as well as terpenoid biosynthesis pathways in Trichoderma reesei [62].…”

Section: Reconstruction Of Tag Biosynthesis Pathway In Trichoderma Rementioning

confidence: 99%

Comparison of Nitrogen Depletion and Repletion on Lipid Production in Yeast and Fungal Species

et al. 2016

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Abstract:Although it is well known that low nitrogen stimulates lipid accumulation, especially for algae and some oleaginous yeast, few studies have been conducted in fungal species, especially on the impact of different nitrogen deficiency strategies. In this study, we use two promising consolidated bioprocessing (CBP) candidates to examine the impact of two nitrogen deficiency strategies on lipid production, which are the extensively investigated oleaginous yeast Yarrowia lipolytica, and the commercial cellulase producer Trichoderma reesei. We first utilized bioinformatics approaches to reconstruct the fatty acid metabolic pathway and demonstrated the presence of a triacylglycerol (TAG) biosynthesis pathway in Trichoderma reesei. We then examined the lipid production of Trichoderma reesei and Y. lipomyces in different media using two nitrogen deficiency strategies of nitrogen natural repletion and nitrogen depletion through centrifugation. Our results demonstrated that nitrogen depletion was better than nitrogen repletion with about 30% lipid increase for Trichoderma reesei and Y. lipomyces, and could be an option to improve lipid production in both oleaginous yeast and filamentous fungal species. The resulting distinctive lipid composition profiles indicated that the impacts of nitrogen depletion on yeast were different from those for fungal species. Under three types of C/N ratio conditions, C16 and C18 fatty acids were the predominant forms of lipids for both Trichoderma reesei and Y. lipolytica. While the overall fatty acid methyl ester (FAME) profiles of Trichoderma reesei were similar, the overall FAME profiles of Y. lipolytica observed a shift. The fatty acid metabolic pathway reconstructed in this work supports previous reports of lipid production in T. reesei, and provides a pathway for future omics studies and metabolic engineering efforts. Further investigation to identify the genetic targets responsible for the effect of nitrogen depletion on lipid production improvement will facilitate strain engineering to boost lipid production under more optimal conditions for productivity than those required for nitrogen depletion.

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Metabolic engineering of Yarrowia lipolytica for industrial applications

Cited by 165 publications

References 56 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

Combining metabolic engineering and process optimization to improve production and secretion of fatty acids

Comparison of Nitrogen Depletion and Repletion on Lipid Production in Yeast and Fungal Species

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