Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism

Huang, Yuying; Jian, Xingxing; Lv, Yu-Bei; Nian, Ke-Qing; Gao, Qi; Chen, Jun; Wei, Liujing; Hua, Qiang

doi:10.1016/j.jbiotec.2018.07.001

Cited by 88 publications

(68 citation statements)

References 35 publications

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“…Our study confirmed that the introduction of PK-PTA pathway is a useful route to increase terpenoids production by increasing the production of acetyl-CoA directly in the cytosol. As Y. lipolytica is recognized to have a unique propensity for high flux through acetyl-CoA, several groups have focused on using the PK-PTA pathway for the improvement of the synthesis of acetyl-CoA derivatives such as lipid and triacetic acid lactone [4,30,32,45]. To the best of our knowledge, this work is the first that uses the PK-PTA pathway to increase terpenoids production in Y. lipolytica.…”

Section: Discussionmentioning

confidence: 99%

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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%

“…The overall pathway from glucose to β-ionone was organized into the following three modules, considering the two most important intermediates: acetyl-CoA and geranylgeranyl diphosphate (GGPP) [31][32][33] (Fig. 1a, b).…”

Section: Re-casting the β-Ionone Biosynthesis Into Three Modules And mentioning

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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“…It was consistent with our findings that medium C/N ratio was beneficial for squalene synthesis. By applying these engineering strategies, we have obtained an oleaginous yeast strain with a similar squalene level to the strain S. cerevisiae (Han, Seo, Song, Lee, & Choi, 2018;Huang et al, 2018). This work highlights the potential of engineering Y. lipolytica as a promising microbial platform for efficient synthesis of squalene and terpene-related compounds.…”

Section: Shake Flask Cultivation Of Engineered Strain With Ph and Carmentioning

confidence: 81%

Optimizing mevalonate pathway for squalene production inYarrowia lipolytica

Liu

Wang

Deng

et al. 2020

Preprint

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Li Deng) and pengxu@umbc.edu (PX). AbstractSqualene is the gateway molecule for triterpene-based natural products and steroidsbased pharmaceuticals. As a super lubricant, it has been used widely in health care industry due to its skin compatibility and thermostability. Squalene is traditionally sourced from sharkhunting or oil plant extraction, which is cost-prohibitive and not sustainable. Reconstitution of squalene biosynthetic pathway in microbial hosts is considered as a promising alternative for cost-efficient and scalable synthesis of squalene. In this work, we reported the engineering of the oleaginous yeast, Y. lipolytica, as a potential host for squalene production. We systematically identified the bottleneck of the pathway and discovered that the native HMG-CoA reductase led to the highest squalene improvement. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/l and 188.2 mg/L squalene from glucose or acetate minimal media, respectively. By optimizing the C/N ratio, controlling the media pH and mitigating the acetyl-CoA flux competition from lipogenesis, the engineered strain produced about 502.7 mg/L squalene in shake flaks, a 28-fold increase compared to the parental strain (17.2 mg/L). We also profiled the metabolic byproducts citric acid and mannitol level and observed that they are reincorporated into cell metabolism at the late stage of fermentation. This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for production of squalene or terpene-based chemicals.

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“…In order to improve lupeol production, we first up-regulated the mevalonate pathway by overexpressing the rate-limiting enzyme HMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase) [29] in strain LU-9, generating strain LU-10. It has been reported that synchronous overexpression of ERG1 (squalene synthase) and ERG9 (squalene monooxygenase) [30] exhibited cooperativity towards enhancing terpenes synthesis [22].…”

Section: Engineering Of Mva and Lipid Metabolism To Improve Lupeol Prmentioning

confidence: 99%

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components

Zhang

Bai

Peng

et al. 2020

Preprint

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BackgroundLupeol exhibits novel physiological and pharmacological activities, such as anti-cancer and immunity enhancement. However, cytotoxicity is still a challenge for triterpenoids overproduction in microbial cell factories. As lipophilic and relatively small-molecular compounds, triterpenes are generally secreted to the extracellular space. The effect of increasing triterpenes efflux on the synthesis capacity remains unknown.ResultsIn this study, we developed a strategy to enhance the triterpenes efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme of Δ9-fatty acid desaturase (OLE1) and disturbing the phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). As a result, we obtained a high-yield lupeol strain with the highest lupeol production of 411.72 mg/L in shake flasks reported to date, reaching a 33.2-fold improvement over the initial strain. The lipid manipulation led to a two-fold increase of unsaturated fatty acids (UFAs) content, up to 61%~73%, and an exceptionally elongated cell morphology, which might be caused by enhanced membrane phospholipids biosynthesis flux. Both of the phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity of triterpenoids synthesis, which were proved by 5.11-fold higher ratio of extra-/intra-cellular lupeol concentrations, 2.7-fold higher biomass accumulation and 2.60-fold higher lupeol productivity per unit OD in modified strains. This strategy was also highly efficient for biosynthesis of other triterpenes and sesquiterpenes, including α-, β-arymin, longifolene, longipinene and longicyclene.ConclusionsTo conclude, we successfully created a high-yield lupeol strain via lipid manipulation. And we demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased unsaturated fatty acids content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

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Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism

Cited by 88 publications

References 35 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

Optimizing mevalonate pathway for squalene production inYarrowia lipolytica

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components

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