High-level production and secretion of recombinant proteins by the dimorphic yeast

Wartmann, Thomas; Böer, Erik; Pico, Almudena Huarto; Sieber, Heike; Bartelsen, Oliver; Gellissen, Gerd; Kunze, Gotthard

doi:10.1016/s1567-1356(02)00086-7

Cited by 20 publications

(9 citation statements)

References 30 publications

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“…Transformations of E. coli and A. adeninivorans were performed according to Böer et al ( 2009a ). Plasmid DNA isolation and DNA restriction were carried out as described by Wartmann et al ( 2002 ).…”

Section: Methodsmentioning

confidence: 99%

Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans

et al. 2017

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Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km −0.7 ± 0.2 mM, kcat −42.0 ± 8.2 s−1) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (Km −3.2 ± 0.2 mM, kcat −44.0 ± 3.2 s−1). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be not the only degradation pathway.

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Section: Methodsmentioning

confidence: 99%

Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans

et al. 2017

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“…For instance, a high copy number (eight copies) of an amyA expression vector encoding heterologous alpha-amylase from Bacillus amyloliquefaciens instead of a single copy resulted in strains with superior productivity for a secreted recombinant alpha-amylase (Steinborn et al 2007). The use of green fluorescent protein (GFP)-coding genes from the jellyfish Aequorea victoria as a reporter gene to assess heterologous gene expression was also reported in this yeast (Wartmann et al 2002, Terentiev et al 2004Steinborn et al 2006).…”

Section: Genetic Elements and Vector Systems Developed In Blastobotrys Genusmentioning

confidence: 99%

Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production

Sanya

Onésime

Passoth

et al. 2021

Appl Microbiol Biotechnol

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Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes. Species of this genus are dimorphic, non-pathogenic, thermotolerant, and can assimilate a variety of hydrophilic and hydrophobic substrates. These can constitute a single-cell oil platform in an emerging bio-based economy as oleaginous traits have been discovered recently. However, the regulatory network of lipogenesis in these yeasts is poorly understood. To keep pace with the growing market demands for lipidderived products, it is critical to understand the lipid biosynthesis in these unconventional yeasts to pinpoint what governs the preferential channelling of carbon flux into lipids instead of the competing pathways. This review summarizes information relevant to the regulation of lipid metabolic pathways and prospects of metabolic engineering in Blastobotrys yeasts for their application in food, feed, and beyond, particularly for fatty acid-based fuels and oleochemicals. Key points• The production of biolipids by heterotrophic yeasts is reviewed. • Summary of information concerning lipid metabolism regulation is highlighted.• Special focus on the importance of diacylglycerol acyltransferases encoding genes in improving lipid production is made.

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“…Transformation of E. coli was performed according to Hanahan [ 33 ] and A. adeninivorans cells were transformed according to Böer et al [ 34 ]. Isolation of plasmid and chromosomal DNA and DNA restrictions were carried out as previously described [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

“…Finally, the complete construct covering 1000-bp in front of the AADH2 gene, the ATRP1m selection marker module and 1000-bp behind the AADH2 gene was amplified using the primers AADH2-3 and AADH2-6. The resulting 3196-bp PCR-product was used to transform A. adeninivorans G1212 [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway

Rauter¹,

Kasprzak

Becker³

et al. 2016

Microb Cell Fact

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Background:The non-conventional yeast Arxula adeninivorans uses 1-butanol as a carbon source and has recently attracted attention as a promising organism for 1-butanol production. Alcohol dehydrogenases (adhp) are important catalysts in 1-butanol metabolism, but only Aadh1p from Arxula has been characterized. This enzyme is involved in ethanol synthesis but has a low impact on 1-butanol degradation. Results:In this study, we identified and characterized a second adhp from A. adeninivorans (Aadh2p). Compared to Saccharomyces cerevisiae ADHs' (ScAdh) protein sequences it originates from the same ancestral node as ScAdh6p, 7p and 4p. It is also localized in the cytoplasm and uses NAD(H) as cofactor. The enzyme has its highest activity with medium chain-length alcohols and maximum activity with 1-butanol with the catalytic efficiency of the purified enzyme being 42 and 43,000 times higher than with ethanol and acetaldehyde, respectively. Arxula adeninivorans strain G1212/YRC102-AADH2, which expresses the AADH2 gene under the control of the strong constitutive TEF1 promoter was constructed. It achieved an ADH activity of up to 8000 U/L and 500 U/g dry cell weight (dcw) which is in contrast to the control strain G1212/YRC102 which had an ADH activity of up to 1400 U/L and 200 U/g dcw. Gene expression analysis showed that AADH2 derepression or induction using non-fermentable carbon-sources such as ethanol, pyruvate, glycerol or 1-butanol did occur. Compared to G1212/YRC102 AADH2 knock-out strain had a slower growth rate and lower 1-butanol consumption if 1-butanol was used as sole carbon source and AADH2-transformants did not grow at all in the same conditions. However, addition of the branched-chain amino acids leucine, isoleucine and valine allowed the transformants to use 1-butanol as carbon source. The addition of these amino acids to the control strain and Δaadh2 mutant cultures had the effect of accelerating 1-butanol consumption. Conclusions:Our results confirm that Aadh2p plays a major role in A. adeninivorans 1-butanol metabolism. It is upregulated by up to 60-fold when the cells grow on 1-butanol, whereas only minor changes were found in the relative expression level for Aadh1p. Thus the constitutive overexpression of the AADH2 gene could be useful in the production of 1-butanol by A. adeninivorans, although it is likely that other ADHs will have to be knocked-out to prevent 1-butanol oxidation.

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High-level production and secretion of recombinant proteins by the dimorphic yeast

Cited by 20 publications

References 30 publications

Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans

Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans

Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production

Aadh2p: an Arxula adeninivorans alcohol dehydrogenase involved in the first step of the 1-butanol degradation pathway

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