Dataset Information

GAS1 Deficient Enhances UPR Activity in Saccharomyces cerevisiae.

ABSTRACT: Beta-1,3-glucanosyltransferase (Gas1p) plays important roles in cell wall biosynthesis and morphogenesis and has been implicated in DNA damage responses and cell cycle regulation in fungi. Yeast Gas1p has also been reported to participate in endoplasmic reticulum (ER) stress responses. However, the precise roles and molecular mechanisms through which Gas1p affects these responses have yet to be elucidated. In this study, we constructed GAS1-deficient (gas1?) and GAS1-overexpressing (GAS1 OE) yeast strains and observed that the gas1? strain exhibited a decreased proliferation ability and a shorter replicative lifespan (RLS), as well as enhanced activity of the unfolded protein response (UPR) in the absence of stress. However, under the high-tunicamycin-concentration (an ER stress-inducing agent; 1.0??g/mL) stress, the gas1? yeast cells exhibited an increased proliferation ability compared with the wild-type yeast strain. In addition, our findings demonstrated that IRE1 and HAC1 (two upstream modulators of the UPR) are required for the survival of gas1? yeast cells under the tunicamycin stress. On the other hand, we provided evidence that the GAS1 overexpression caused an obvious sensitivity to the low-tunicamycin-concentration (0.25??g/mL). Collectively, our results indicate that Gas1p plays an important role in the ageing and ER stress responses in yeast.

SUBMITTER: Cui HJ

PROVIDER: S-EPMC6582843 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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GAS1 Deficient Enhances UPR Activity in Saccharomyces cerevisiae.

Cui Hong-Jing HJ Cui Xin-Gang XG Jing Xia X Yuan Yuan Y Chen Ya-Qin YQ Sun Ya-Xin YX Zhao Wei W Liu Xin-Guang XG

BioMed research international 20190602

Beta-1,3-glucanosyltransferase (Gas1p) plays important roles in cell wall biosynthesis and morphogenesis and has been implicated in DNA damage responses and cell cycle regulation in fungi. Yeast Gas1p has also been reported to participate in endoplasmic reticulum (ER) stress responses. However, the precise roles and molecular mechanisms through which Gas1p affects these responses have yet to be elucidated. In this study, we constructed GAS1-deficient (gas1Δ) and GAS1-overexp ...[more]

PMID: 31275960

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Project description:BackgroundCYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae.ResultsInvestigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses.ConclusionsThis study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains.

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GAS1 Deficient Enhances UPR Activity in Saccharomyces cerevisiae.

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<i>GAS1</i> Deficient Enhances UPR Activity in <i>Saccharomyces cerevisiae</i>.

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