Expression of Bacterial Hemoglobin in the Yeast, Pichia pastoris, with a Low O2-Induced Promoter

Chien, Liang-Jung; Lee, Cheng‐Kang

doi:10.1007/s10529-005-1324-x

Cited by 22 publications

(11 citation statements)

References 19 publications

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“…The direct transcriptional response of fermentative genes to oxygen limitation has been observed in P. stipitis (Cho and Jeffries, 1999; Passoth et al , 2003). The oxygen‐dependent regulation seems to be characteristic for respiratory yeasts and the promoter of the major P. stipitis ADH was regulated in the same way when transformed into P. pastoris (Chien and Lee, 2005).…”

Section: Discussionmentioning

confidence: 99%

Oxygen and carbon source‐regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala

Fredlund

Beerlage

Melin

et al. 2006

Yeast

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We amplified, sequenced and studied the transcriptional regulation of genes of the alcoholic fermentation pathway in the biocontrol and non-Saccharomyces wine yeast, Pichia anomala. Two ADH isogenes, PaADH1 and PaADH2, and one PDC gene, PaPDC1, were amplified from genomic P. anomala DNA by a two-step PCR approach, using degenerated primers against conserved regions of the respective genes for cloning core regions, and PCR-based gene walking for cloning the respective 5 and 3 -ends. According to sequence analysis, ADH1 and PDC1 are most likely cytoplasmatic proteins, while ADH2 is most probably localized in the mitochondria. PaADH1 was expressed during aerobic growth on glucose, ethanol and succinate, but was ninefold upregulated in response to oxygen limitation when grown on glucose. The gene seems to be involved in both production and consumption of ethanol. Only low expression of PaADH2 was detected during growth on glucose and ethanol, but it was highly expressed during growth on the non-fermentable carbon source succinate and repressed by the addition of glucose. PaPDC1 was expressed during aerobic growth on glucose and was upregulated four-fold in response to oxygen limitation. PaPDC1 expression was lower in cells grown on ethanol and succinate than on glucose and was up-regulated two-and four-fold, respectively, after glucose addition. Our results demonstrate that transcription of genes of the fermentative pathway is regulated by hypoxia and carbon source but posttranscriptional regulation may play a major role in regulating the metabolic flux.

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Section: Discussionmentioning

confidence: 99%

Oxygen and carbon source‐regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala

Fredlund

Beerlage

Melin

et al. 2006

Yeast

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“…Although some control experiments were missing in this paper, such as a comparison to non-inducing conditions and a comparison to a reference promoter from P. pastoris (e.g. GAP dehydrogenase promoter), this is another example of a successful transfer of promoters and their functionality in heterologous hosts [171,172]. …”

Section: Reviewmentioning

confidence: 99%

Untitled

2006

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Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts.

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“…. As an aerobic microorganism, P. pastoris requires continuous oxygen supply and efficient utilization for the expression of recombinant proteins, which is particularly true at the fermentation stage of P. pastoris with high cell density 17 . Vitreoscilla hemoglobin (VHB, coding gene: vhb ) is produced by the gram-negative bacterium Vitreoscilla 18.…”

Section: Introductionmentioning

confidence: 99%

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

Chen

Liang

Wang

et al. 2019

Acta Pharmaceutica Sinica B

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Taxol is a “blockbuster” antitumor drug produced by Taxus species with extremely low amount, while its analogue 7- β -xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1−1 and LXYL-P1−2 can convert 7- β -xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1−2 is twice more active than LXYL-P1−1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1−1 to LXYL-P1−2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1−2 on β -xylosidase and β -glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7- β -xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant E12 , the molecular chaperone gene pdi and the bacterial hemoglobin gene vhb . This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1−2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1−2 for industrial purpose.

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Expression of Bacterial Hemoglobin in the Yeast, Pichia pastoris, with a Low O2-Induced Promoter

Cited by 22 publications

References 19 publications

Oxygen and carbon source‐regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala

Oxygen and carbon source‐regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala

Untitled

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

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