Enhanced β-carotene production by overexpressing the DID2 gene, a subunit of ESCRT complex, in engineered Yarrowia lipolytica

2021 ◽  
Author(s):  
Fan Yang ◽  
Liang Liu ◽  
Shan Qiang ◽  
Ching Yuan Hu ◽  
Ying Li ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
A Subunit ◽  
Escrt Complex ◽  
Β Carotene

scholarly journals Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuxiao Xie ◽  
Shulin Chen ◽  
Xiaochao Xiong
Keyword(s):  
Yarrowia Lipolytica ◽  
Fold Increase ◽  
Genetically Engineered ◽  
Cell Factory ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Yeast Extract Peptone Dextrose ◽  
Dry Cell Weight ◽  
A Cell ◽  
Β Carotene

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica

2020 ◽  
Vol 42 (6) ◽  
pp. 945-956 ◽  
Author(s):  
Xin-Kai Zhang ◽  
Dan-Ni Wang ◽  
Jun Chen ◽  
Zhi-Jie Liu ◽  
Liu-Jing Wei ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
Β Carotene

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 Elevated β-Carotene Production Using Codon-Adapted CarRA&B and Metabolic Balance in Engineered Yarrowia lipolytica

2021 ◽  
Vol 12 ◽  
Author(s):  
Liang Liu ◽  
Yu Ling Qu ◽  
Gui Ru Dong ◽  
Jing Wang ◽  
Ching Yuan Hu ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Large Scale ◽  
Blakeslea Trispora ◽  
Scale Production ◽  
Total Production ◽  
Metabolic Balance ◽  
Carotene Content ◽  
Large Scale Production ◽  
Dry Cell Weight ◽  
Β Carotene

β-carotene is a precursor of vitamin A and has multiple physiological functions. Producing β-carotene by microbial fermentation has attracted much attention to consumers’ preference for natural products. This study focused on improving β-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing β-carotene. Thereafter, the β-carotene biosynthetic pathway’s metabolic balance was accurately regulated to reduce the intermediates’ accumulation. Notably, the β-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of β-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing β-carotene. The β-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

Impact of culture conditions on β-carotene encapsulation using Yarrowia lipolytica cells

2017 ◽  
Author(s):  
Tran Hai Dang ◽  
Ho Thi Thu Minh ◽  
Tran Nguyen Van Nhi ◽  
Ta Thi Minh Ngoc
Keyword(s):  
Yarrowia Lipolytica ◽  
Culture Conditions ◽  
Β Carotene

The safety of β-carotene from Yarrowia lipolytica

2014 ◽  
Vol 65 ◽  
pp. 1-11 ◽  
Author(s):  
Daniel Grenfell-Lee ◽  
Samuel Zeller ◽  
Renato Cardoso ◽  
Kresimir Pucaj
Keyword(s):  
Yarrowia Lipolytica ◽  
Β Carotene

scholarly journals A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica

2019 ◽  
Author(s):  
N Milne ◽  
L R R Tramontin ◽  
I Borodina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
Yeast Species ◽  
Production Capacity ◽  
Value Added ◽  
Phenotypic Screen ◽  
Rate Limiting ◽  
Β Carotene

ABSTRACT We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used ‘as is’ for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.

scholarly journals Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Peng Jun Lv ◽  
Shan Qiang ◽  
Liang Liu ◽  
Ching Yuan Hu ◽  
Yong Hong Meng
Keyword(s):  
Dissolved Oxygen ◽  
Yarrowia Lipolytica ◽  
Batch Fermentation ◽  
Utilization Rate ◽  
Crabtree Effect ◽  
Fed Batch ◽  
Promising Strategy ◽  
Fed Batch Fermentation ◽  
Β Carotene ◽  
Gene Expression Levels

Abstract The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of β-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L β-carotene. Both biomass and β-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP+/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the kinetic parameters in DO-stat fed-batch fermentation, μm′, Yx/s′, and Yp/s′ was 0.527, 0.353, and 0.158, respectively. The μm′ was elevated 4.66-fold than that in fed-batch fermentation. These data illustrate that more dissolved oxygen increased the biomass. The Yx/s′ and Yp/s′ were increased 1.15 and 22.57-fold, which suggest that the DO-stat fed-batch fermentation reduced the Crabtree effect and improved the utilization rate of glucose. Therefore, DO-stat fed-batch fermentation is a promising strategy in the industrialized production of β-carotene.

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