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Genetic and bioprocess engineering to improve squalene production in Yarrowia lipolytica.

ABSTRACT: Squalene is the precursor for triterpene-based natural products and steroids-based drugs. It has been widely used as pharmaceutical intermediates and personal care products. The aim of this work is to test the feasibility of engineering Yarrowia lipolytica as a potential host for squalene production. The bottleneck of the pathway was removed by overexpressing native HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase. 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. By optimizing the C/N ratio, controlling the media pH and mitigating acetyl-CoA flux competition from lipogenesis, the engineered strain produced 502.7 mg/L squalene, a 28-fold increase over the parental strain (17.2 mg/L). This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for sustainable production of squalene or terpenoids-based chemicals and natural products.

SUBMITTER: Liu H

PROVIDER: S-EPMC7561614 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Genetic and bioprocess engineering to improve squalene production in Yarrowia lipolytica.

Liu Huan H Wang Fang F Deng Li L Xu Peng P

Bioresource technology 20200810

Squalene is the precursor for triterpene-based natural products and steroids-based drugs. It has been widely used as pharmaceutical intermediates and personal care products. The aim of this work is to test the feasibility of engineering Yarrowia lipolytica as a potential host for squalene production. The bottleneck of the pathway was removed by overexpressing native HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase. With the recycling of NADPH from the mannitol cycle, the engineered strain prod ...[more]

PMID: 32805480

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Engineering ergothioneine production in Yarrowia lipolytica.

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Engineering of <i>Yarrowia lipolytica</i> for terpenoid production.

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Project description:Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.

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Project description:Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA) and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.

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Engineering Yarrowia lipolytica for Campesterol Overproduction.

Project description:Campesterol is an important precursor for many sterol drugs, e.g. progesterone and hydrocortisone. In order to produce campesterol in Yarrowia lipolytica, C-22 desaturase encoding gene ERG5 was disrupted and the heterologous 7-dehydrocholesterol reductase (DHCR7) encoding gene was constitutively expressed. The codon-optimized DHCR7 from Rallus norvegicus, Oryza saliva and Xenapus laevis were explored and the strain with the gene DHCR7 from X. laevis achieved the highest titer of campesterol due to D409 in substrate binding sites. In presence of glucose as the carbon source, higher biomass conversion yield and product yield were achieved in shake flask compared to that using glycerol and sunflower seed oil. Nevertheless, better cell growth rate was observed in medium with sunflower seed oil as the sole carbon source. Through high cell density fed-batch fermentation under carbon source restriction strategy, a titer of 453±24.7 mg/L campesterol was achieved with sunflower seed oil as the carbon source, which is the highest reported microbial titer known. Our study has greatly enhanced campesterol accumulation in Y. lipolytica, providing new insight into producing complex and desired molecules in microbes.

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Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering.

Project description:BACKGROUND:Oleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest. Because medium chain fatty acids (MCFA) are valuable compounds for biokerosene production, we previously generated strains capable of producing MCFA up to 12% of total lipid content (Rigouin et al. in ACS Synth Biol 6:1870-1879, 2017). In order to improve accumulation and content of C14 fatty acid (FA), the elongation, degradation and accumulation of these MCFA in Yarrowia lipolytica were studied. RESULTS:We brought evidence of the role of YALI0F0654 (YlELO1) protein in the elongation of exogenous or de novo synthesized C14 FA into C16 FA and C18 FA. YlELO1 deletion into a ?FAS_I1220W expressing strain leads to the sole production of C14 FA. However, because this strain does not provide the FA essential for its growth, it requires being cultivated with essential fatty acids and C14 FA yield is limited. To promote MCFA accumulation in Y. lipolytica without compromising the growth, we overexpressed a plant diglyceride acyltransferase specific for MCFA and reached an accumulation of MCFA up to 45% of total lipid content. CONCLUSION:We characterized the role of YlELO1 in Y. lipolytica by proving its involvement in Medium chain fatty acids elongation. We showed that MCFA content can be increased in Yarrowia lipolytica by promoting their accumulation into a stable storage form (triacylglycerides) to limit their elongation and their degradation.

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Yarrowia lipolytica fed-batch biolipid production.

Project description:Time-course transcriptomic profilling of the oleaginous yeast Yarrowia lipolytica, during a controlled fed-batch. A nitrogen limitation was applied during the course of the fed-batch to initiate de novo biolipid synthesis.

2011-05-15 | GSE29046 | GEO

Engineering heterologous enzyme secretion in Yarrowia lipolytica.

Project description:BackgroundEukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.ResultsBy engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and [Formula: see text]-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.ConclusionsOverall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

| S-EPMC9252082 | biostudies-literature

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