scholarly journals Increased accumulation of squalene in engineered Yarrowia lipolytica through deletion of PEX10 and URE2

2021 ◽  
Author(s):  
Liu-Jing Wei ◽  
Xuan Cao ◽  
Jing-Jing Liu ◽  
Suryang Kwak ◽  
Yong-Su Jin ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Catabolite Repression ◽  
Oleaginous Yeast ◽  
Mevalonate Pathway ◽  
Culture Media ◽  
Yeast Cells ◽  
Nitrogen Catabolite Repression ◽  
Peroxisomal Membrane ◽  
Cell Factories ◽  
Pharmaceutical Industries

Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene as the yeast provides ample storage space for hydrophobic products. Here we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica . First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG, HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 encoding for diacylglycerol acyltransferase and deleting PEX10 for peroxisomal membrane E3 ubiquitin ligase. Third, we deleted URE2 coding for a transcriptional regulator in charge of nitrogen catabolite repression (NCR) to induce lipid accumulation regardless of carbon to nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g DCW) than a parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in S. cerevisiae , and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica . URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells on boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

scholarly journals Biovalorisation of crude glycerol and xylose into xylitol by oleaginous yeast Yarrowia lipolytica

2020 ◽  
Author(s):  
Ashish Prabhu ◽  
Dominic J Thomas ◽  
Rodrigo Ledesma- Amaro ◽  
Gary A Leeke ◽  
Angel Medina Vaya ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Crude Glycerol ◽  
Oleaginous Yeast ◽  
Scale Up ◽  
Microbial Cell ◽  
Department Of Energy ◽  
Cell Factory ◽  
Potential Cost ◽  
Cell Factories ◽  
Pharmaceutical Industries

Abstract Background: Xylitol is a commercially important chemical with multiple applications in the food and pharmaceutical industries. According to the US Department of Energy, xylitol is one of the top twelve platform chemicals that can be produced from biomass. The chemical method for xylitol synthesis is however expensive and energy intensive. In contrast, the biological route using microbial cell factories offers a potential cost-effective alternative process. The bioprocess occurs under ambient conditions and makes use of biocatalysts and biomass which can be sourced from renewable carbon originating from a variety of cheap waste feedstocks. Result: In this study, biotransformation of xylose to xylitol was investigated using Yarrowia lipolytica an oleaginous yeast which was firstly grown on a glycerol/glucose for screening of co-substrate, followed by media optimisation in shake flask, scale up in bioreactor and downstream processing of xylitol. A two-step medium optimization was employed using central composite design and artificial neural network coupled with genetic algorithm. The yeast amassed a concentration of 53.2 g/L xylitol using pure glycerol (PG) and xylose with a bioconversion yield of 0.97 g/g. Similar results were obtained when PG was substituted with crude glycerol (CG) from the biodiesel industry (titer: 50.5 g/L; yield: 0.92 g/g). Even when xylose from sugarcane bagasse hydrolysate was used as opposed to pure xylose, a xylitol yield of 0.54 g/g was achieved. Xylitol was successfully crystallized from PG/xylose and CG /xylose fermentation broths with a recovery of 39.5 and 35.3%, respectively. Conclusion: To the best of the author’s knowledge, this study demonstrates for the first time the potential of using Y. lipolytica as a microbial cell factory for xylitol synthesis from inexpensive feedstocks. The results obtained are competitive with other xylitol producing organisms.

scholarly journals Engineering oleaginous yeast Yarrowia lipolytica for enhanced limonene production from xylose and lignocellulosic hydrolysate

2020 ◽  
Vol 20 (6) ◽  
Author(s):  
Feng Yao ◽  
Shun-Cheng Liu ◽  
Dan-Ni Wang ◽  
Zhi-Jie Liu ◽  
Qiang Hua ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Mevalonate Pathway ◽  
Low Cost ◽  
Lignocellulosic Hydrolysate ◽  
Renewable Biomass ◽  
Lignocellulosic Feedstock ◽  
Pharmaceutical Industries ◽  
The Cost ◽  
Yeast Yarrowia Lipolytica

ABSTRACT Limonene, a valuable cyclic monoterpene, has been broadly studied in recent decades due to its wide application in the food, cosmetics and pharmaceutical industries. Engineering of the yeast Yarrowia lipolytica for fermentation of renewable biomass lignocellulosic hydrolysate may reduce the cost and improve the economics of bioconversion for the production of limonene. The aim of this study was to engineer Y. lipolytica to produce limonene from xylose and low-cost lignocellulosic feedstock. The heterologous genes XR and XDH and native gene XK encoding xylose assimilation enzymes, along with the heterologous genes tNDPS1 and tLS encoding orthogonal limonene biosynthetic enzymes, were introduced into the Po1f strain to facilitate xylose fermentation to limonene. The initially developed strain produced 0.44 mg/L of limonene in 72 h with 20 g/L of xylose. Overexpression of genes from the mevalonate pathway, including HMG1 and ERG12, significantly increased limonene production from xylose to ∼9.00 mg/L in 72 h. Furthermore, limonene production peaked at 20.57 mg/L with 50% hydrolysate after 72 h when detoxified lignocellulosic hydrolysate was used. This study is the first to report limonene production by yeast from lignocellulosic feedstock, and these results indicate the initial steps toward economical and sustainable production of isoprenoids from renewable biomass by engineered Y. lipolytica.

scholarly journals Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway

2019 ◽  
Vol 7 (10) ◽  
pp. 472 ◽  
Author(s):  
Larissa Ribeiro Ramos Tramontin ◽  
Kanchana Rueksomtawin Kildegaard ◽  
Suresh Sudarsan ◽  
Irina Borodina
Keyword(s):  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Small Scale ◽  
Cell Factory ◽  
Standard Equipment ◽  
Cell Factories ◽  
Dry Cell Weight ◽  
Astaxanthin Production ◽  
Yeast Yarrowia Lipolytica ◽  
Β Carotene

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

scholarly journals Sugar Alcohols and Organic Acids Synthesis in Yarrowia lipolytica: Where Are We?

2020 ◽  
Vol 8 (4) ◽  
pp. 574 ◽  
Author(s):  
Patrick Fickers ◽  
Hairong Cheng ◽  
Carol Sze Ki Lin
Keyword(s):  
Organic Acids ◽  
Yarrowia Lipolytica ◽  
Current Knowledge ◽  
Raw Materials ◽  
Low Cost ◽  
Industrial Wastes ◽  
Sugar Alcohols ◽  
Cell Factories ◽  
Main Challenge ◽  
Pharmaceutical Industries

Sugar alcohols and organic acids that derive from the metabolism of certain microorganisms have a panoply of applications in agro-food, chemical and pharmaceutical industries. The main challenge in their production is to reach a productivity threshold that allow the process to be profitable. This relies on the construction of efficient cell factories by metabolic engineering and on the development of low-cost production processes by using industrial wastes or cheap and widely available raw materials as feedstock. The non-conventional yeast Yarrowia lipolytica has emerged recently as a potential producer of such metabolites owing its low nutritive requirements, its ability to grow at high cell densities in a bioreactor and ease of genome edition. This review will focus on current knowledge on the synthesis of the most important sugar alcohols and organic acids in Y. lipolytica.

scholarly journals Analysis of lipid composition reveals mechanisms of ethanol tolerance in the model yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
M Lairón-Peris ◽  
S. J. Routledge ◽  
J. A. Linney ◽  
J Alonso-del-Real ◽  
C.M. Spickett ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Responses ◽  
Ethanol Tolerance ◽  
Yeast Species ◽  
Culture Media ◽  
Sugar Content ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Tolerant Strain ◽  
Cell Factories

Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyse five representatives with varying ethanol tolerances. The most tolerant strain, AJ4, was dominant in co-culture at 0% and 10% ethanol. Unexpectedly, although it does not have the highest NIC or MIC, MY29 was the dominant strain in co-culture at 6% ethanol, which may be linked to differences in its basal lipidome. Whilst relatively few lipidomic differences were observed between strains, a significantly higher PE concentration was observed in the least tolerant strain, MY26, at 0% and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggests a limited set of membrane compositions that diverse yeast species use to achieve this. Importance Many microbial processes are not implemented at the industrial level because the product yield is poorer and more expensive than can be achieved by chemical synthesis. It is well established that microbes show stress responses during bioprocessing, and one reason for poor product output from cell factories is production conditions that are ultimately toxic to the cells. During fermentative processes, yeast cells encounter culture media with high sugar content, which is later transformed into high ethanol concentrations. Thus, ethanol toxicity is one of the major stresses in traditional and more recent biotechnological processes. We have performed a multilayer phenotypic and lipidomic characterization of a large number of industrial and environmental strains of Saccharomyces to identify key resistant and non-resistant isolates for future applications.

scholarly journals Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Chuanbo Zhang ◽  
Man Li ◽  
Guang-Rong Zhao ◽  
Wenyu Lu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mevalonate Pathway ◽  
Cell Factory ◽  
Fed Batch ◽  
Cell Factories ◽  
Fed Batch Fermentation ◽  
Pharmaceutical Industries ◽  
Engineered Saccharomyces Cerevisiae ◽  
Rate Limiting ◽  
Production Cell

Abstract Background Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. Results α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. Conclusions An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

RTG-dependent Mitochondria-to-Nucleus Signaling Is Regulated byMKS1and Is Linked to Formation of Yeast Prion [URE3]

2002 ◽  
Vol 13 (3) ◽  
pp. 795-804 ◽  
Author(s):  
Takayuki Sekito ◽  
Zhengchang Liu ◽  
Janet Thornton ◽  
Ronald A. Butow
Keyword(s):  
Gene Expression ◽  
Catabolite Repression ◽  
Target Gene ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Nitrogen Catabolite Repression ◽  
Yeast Prion ◽  
Phosphorylation Pattern ◽  
Bhlh Transcription Factors ◽  
Do So

An important function of the RTG signaling pathway is maintenance of intracellular glutamate supplies in yeast cells with dysfunctional mitochondria. Herein, we report that MKS1is a negative regulator of the RTG pathway, acting between Rtg2p, a proximal sensor of mitochondrial function, and the bHLH transcription factors Rtg1p and Rtg3p. In mks1Δcells, RTG target gene expression is constitutive, bypassing the requirement for Rtg2p, and is no longer repressible by glutamate. We show further that Mks1p is a phosphoprotein whose phosphorylation pattern parallels that of Rtg3p in response to activation of the RTG pathway, and that Mks1p is in a complex with Rtg2p. MKS1 was previously implicated in the formation of [URE3], an inactive prion form of a negative regulator of the nitrogen catabolite repression pathway, Ure2p.rtgΔ mutations induce [URE3] and can do so independently of MKS1. We find that glutamate suppresses [URE3] formation, suggesting that the Mks1p effect on the formation of [URE3] can occur indirectly via regulation of theRTG pathway.

scholarly journals Biovalorisation of crude glycerol and xylose into xylitol by oleaginous yeast Yarrowia lipolytica

2020 ◽  
Author(s):  
Ashish Prabhu ◽  
Dominic J Thomas ◽  
Rodrigo Ledesma- Amaro ◽  
Gary A Leeke ◽  
Angel Medina Vaya ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Crude Glycerol ◽  
Oleaginous Yeast ◽  
Scale Up ◽  
Microbial Cell ◽  
Department Of Energy ◽  
Cell Factory ◽  
Potential Cost ◽  
Pure Glycerol ◽  
Pharmaceutical Industries

Abstract Background: Xylitol is a commercially important chemical with multiple applications in food and pharmaceutical industries. According to the US Department of Energy, xylitol is among the twelve platform chemicals that can be produced from biomass. The chemical method for xylitol synthesis is however expensive and energy intensive. In contrast, the biological route involving microbial cell factories offers a potential cost-effective alternative process. The bioprocess occurs under ambient conditions and makes use of biocatalysts which can be sourced from renewable carbon coming from a variety of cheap feedstocks classified as wastes. Result: In this study, biotransformation of xylose to xylitol was investigated using Yarrowia lipolytica an oleaginous yeast grown on glycerol/glucose screening of primary carbon source, media optimisation in shake flask, scale up in bioreactor and downstream processing of xylitol were carried out. With the two step medium optimization involving central composite design and artificial neural network coupled with genetic algorithm, the yeast amassed 53.2 g/L of xylitol using glycerol and xylose with a bioconversion yield of 0.97 g/g. Similar results were obtained when pure glycerol was substituted with crude glycerol from biodiesel industry (titer: 50.5 g/L; yield: 0.92 g/g). Even when xylose from lignocellulosic hydrolysate was used as opposed to pure xylose, a xylitol yield of 0.54 g/g was achieved. Xylitol was successfully crystallized from fermented broth using pure glycerol/xylose and crude glycerol /xylose with a recovery of 35 and 39.45%, respectively. Conclusion: To the best of the author’s knowledge, this is the first study demonstrating the potential of Y. lipolytica as microbial cell factory for xylitol synthesis from inexpensive feedstocks. The results obtained are competitive with other xylitol producing organisms.

scholarly journals Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yaru Pang ◽  
Yakun Zhao ◽  
Shenglong Li ◽  
Yu Zhao ◽  
Jian Li ◽  
...  
Keyword(s):  
Carbon Source ◽  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Waste Cooking Oil ◽  
Biologically Active ◽  
Citrus Limon ◽  
Mentha Spicata ◽  
Cooking Oil ◽  
Limonene Synthase ◽  
Pharmaceutical Industries

Abstract Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

scholarly journals High-Efficiency Production of the Bisabolene from Waste Cooking Oil By Metabolically Engineered Yarrowia Lipolytica

2020 ◽  
Author(s):  
Yakun Zhao ◽  
Kun Zhu ◽  
Jian Li ◽  
Yu Zhao ◽  
Shenglong Li ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
High Efficiency ◽  
De Novo ◽  
Mevalonate Pathway ◽  
Zingiber Officinale ◽  
Waste Cooking Oil ◽  
Cooking Oil ◽  
Cell Factories ◽  
Wide Range

Abstract Background: The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders their isolation from plant sources economically inviable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene.Results: In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, β-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the β-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus, respectively. Subsequently, metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize de novo bisabolene production by the engineered Y. lipolytica strains from glucose. Our engineering strategies have led to engineered Y. lipolytica strains that produce 282.6 mg/L α-bisabolene, 48.3 mg/L β-bisabolene and 5.3 mg/L γ-bisabolene. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from waste cooking oil. The results showed that α-bisabolene, β-bisabolene and γ-bisabolene could be produced at the respective titers of 973.0 mg/L, 68.2 mg/L, 20.2 mg/L in shake flasks. These titers correspond to 2433-fold, 340-fold and 404-fold enhancement in bisabolene production, respectively, over the parent strain.Conclusions: To our knowledge, this is the first report of bisabolene production in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.

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