Metabolic engineering of Yarrowia lipolytica for the biosynthesis of crotonic acid

Wang, Lei; Zong, Zhen; Liu, Yuanlin; Zheng, Menglin; Li, Dongsheng; Wang, Chao; Zheng, Fang; Madzak, Catherine; Liu, Zhijie

doi:10.1016/j.biortech.2019.121484

Cited by 27 publications

(17 citation statements)

References 39 publications

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“…Moreover, Y. lipolytica can utilize a variety of inexpensive renewable substrates as carbon sources and can accommodate high flux of acetyl-CoA (Magdouli et al, 2017; Ledesma-Amaro and Nicaud, 2016a; Nambou et al, 2014;Poli et al, 2014). These characteristics make Y. lipolytica a remarkable industrial host for the production of many products, including lipid-derived biodiesel (Ledesma-Amaro and Nicaud, 2016b; Ledesma-Amaro, 2015); alkanes (Bruder et al, 2019); odd-chain fatty acids (Park et al, 2020;Park et al, 2018); plant-derived terpenoids, such as α-farnesene (Liu et al, 2019c;Yang et al, 2016), carotenoids (Jacobsen et al, 2020;Larroude et al, 2018a), linalool (Cao et al, 2017), and others (Ma et al, 2019); organic acids, such as citric acid (Kamzolova and Morgunov, 2017), isocitric acid (Rzechonek et al, 2019), succinic acid (Li et al, 2018;Li et al, 2017), α-ketoglutarate (Lei et al, 2019;Zeng et al, 2017), pyruvic acid (Kamzolova and Morgunov, 2018) and crotonic acid (Wang et al, 2019); as well as sugar alcohols, such as erythritol (Liu et al, 2019b;Liu et al, 2017c), erythrulose (Carly et al, 2018), isomaltulose (Zhang et al, 2018). Even more value-added functional polyunsaturated fatty acids (PUFAs), such as γ-linolenic acid (Sun et al, 2017), arachidonic acid (Liu et al, 2019a;Liu et al, 2017a;Liu et al, 2017b), eicosapentaenoic acid (Xue et al, 2013), and docosahexaenoic acid (Gemperlein et al, 2019) were produced in engineered Y. lipolytica strains.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica – A Review

Wang

Ledesma-Amaro

Wei

et al. 2020

Bioresource Technology

159

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Section: Introductionmentioning

confidence: 99%

Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica – A Review

Wang

Ledesma-Amaro

Wei

et al. 2020

Bioresource Technology

159

View full text Add to dashboard Cite

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“…Y. lipolytica , as a promising oleaginous yeast cell factory, has been extensively engineered for the production of lipids (Qiao et al., 2017; Xu et al., 2017b, Xu et al., 2017a), oleochemicals (Xu et al., 2016), drop-in transportation fuels (Xu et al., 2016) and commodity chemicals (Blazeck et al., 2015; Wang et al., 2019) recently. It is known as a ‘generally regarded as safe’ (GRAS) organism for the production of organic acids and polyunsaturated fatty acids (PUFAs) (Bailey et al., 2010; Sharpe et al., 2014) in the food and nutraceutical industry.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica

Yang

Edwards

2020

Metabolic Engineering Communications

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CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5′-phosphate decarboxylase (~96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%–75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3′ polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%–83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.

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“…At present, microbial fermentation provides an alternative way to manufacture chemicals from renewable sources such as biomass feedstock, which is of great significance for sustainable development [2,4,14,15]. Natural AAP production was first reported in the liquid culture of strain Pseudomonas fluorescens 2-79, but the maximum titer of AAP was only 50 mg/L [9], that is too low to meet the current demand for industrial production.…”

Section: Introductionmentioning

confidence: 99%

Identification of new arylamine N-acetyltransferases and enhancing 2-acetamidophenol production in Pseudomonas chlororaphis HT66

Guo

Wang

et al. 2020

Microb Cell Fact

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Background: 2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway. Result: In this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66ΔphzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66ΔphzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe 3+ to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3ΔphzBΔlonΔpykAΔrsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date. Conclusion: In conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.

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Metabolic engineering of Yarrowia lipolytica for the biosynthesis of crotonic acid

Cited by 27 publications

References 39 publications

Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica – A Review

Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica – A Review

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica

Identification of new arylamine N-acetyltransferases and enhancing 2-acetamidophenol production in Pseudomonas chlororaphis HT66

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