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

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.

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

Microorganisms ◽

10.3390/microorganisms7100472 ◽

2019 ◽

Vol 7 (10) ◽

pp. 472 ◽

Cited By ~ 9

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.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.627150 ◽

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.

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Yarrowia lipolytica as a Cell Factory for Oleochemical Biotechnology

10.1007/springerreference_168221 ◽

2012 ◽

Keyword(s):

Yarrowia Lipolytica ◽

Cell Factory ◽

A Cell

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Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions

Antibiotics ◽

10.3390/antibiotics10111349 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1349

Author(s):

Nina Patel ◽

Jeremy C. Curtis ◽

Balbina J. Plotkin

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Fold Increase ◽

Growth Conditions ◽

Yeast Nitrogen Base ◽

Nitrogen Base ◽

E Coli ◽

Biofilm Production ◽

Mueller Hinton ◽

Static Conditions

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 μU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

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Biosynthesis of cannabinoid precursor olivetolic acid by overcoming rate-limiting steps in genetically engineered Yarrowia lipolytica

10.1101/2021.06.10.447928 ◽

2021 ◽

Author(s):

Jingbo Ma ◽

Yang Gu ◽

Peng Xu

Keyword(s):

Yarrowia Lipolytica ◽

Fold Increase ◽

Genetically Engineered ◽

Manufacturing Cost ◽

Market Demand ◽

Malonyl Coa ◽

Central Nervous Systems ◽

Anxiety Depression ◽

Rate Limiting ◽

Cost Limit

Natural products acting on our central nervous systems are in utmost demand to fight against pain and mental disorders. Cannabinoids (CBDs) are proven neuroactive agents to treat anxiety, depression, chronic pain diseases, seizure, strokes and neurological disorders. The scarcity of the hemp-sourced CBD products and the prohibitive manufacturing cost limit the wide application of CBDs. Yeast metabolic engineering offers the flexibility to meet the ever-increasing market demand. In this work, we took a retrosynthetic approach and sequentially identified the rate-limiting steps to improve the biosynthesis of the CBD precursor olivetolic acid (OLA) in Yarrowia lipolytica. We debottlenecked the critical enzymatic steps to overcome the supply of hexanoyl-CoA, malonyl-CoA, acetyl-CoA, NADPH and ATPs to redirect carbon flux toward OLA. Implementation of these strategies led to an 83-fold increase in OLA titer in shaking flask experiment. This work may serve as a baseline for engineering CBD biosynthesis in oleaginous yeast species.

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