Comparison of the Efficiency of Various Promoters for the Production of Secreted β-Mannanase by Bacillus subtilis by Cells of the Methylotrophic Yeast Ogataea haglerorum

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Abstract

In this article, strong promoters of thermotolerant methylotrophic yeast Ogataea haglerorum have been characterized. Promoters play a key role in the regulation of gene expression; therefore, they are the important element of expression vectors. Strong and strictly regulated promoters are a powerful tool for creating highly productive strains — producers of recombinant proteins. To expand the potential of the O. haglerorum expression system natural methanol-induced promoters of the OhMOX and OhFMD genes and the constitutive promoter of the OhGAP gene were studied in comparison with the promoter of the MOX gene from O. polymorpha yeast. A gene encoding recombinant β-mannanase was used as a reporter gene. It has been shown that in O. haglerorum yeast cells, the expression level (strength) of the pOhMOX promoter is about 1.4–1.9 times higher relative to the pOpMOX promoter from O. polymorpha yeast. The obtained data on the strength of promoters from yeast O. haglerorum can be useful in designing producers of recombinant proteins and optimizing metabolic pathways in methylotrophic yeast O. haglerorum.

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About the authors

D. A. Podpletnev

NRC «Kurchatov Institute»

Email: m_tarutina@mail.ru
Russian Federation, Moscow, 123182

A. R. Lapteva

NRC «Kurchatov Institute»

Email: m_tarutina@mail.ru
Russian Federation, Moscow, 123182

S. P. Sineoky

NRC «Kurchatov Institute»

Email: m_tarutina@mail.ru
Russian Federation, Moscow, 123182

M. G. Tarutina

NRC «Kurchatov Institute»

Author for correspondence.
Email: m_tarutina@mail.ru

Kurchatov Genomic Center

Russian Federation, Moscow, 123182

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2. Fig. 1. The scheme of the expression vector pOpMOX-Km (a) and the plasmid pOhMOX-MANS-HARS (b). The promoters of pOhFMD, pOhGAP and pOpMOX contain the plasmids pOhFMD-MANS-HARS (7986 bp), pOhGAP-MANS-HARS (8091 bp) and pOpMOX-MANS-HARS (7993 bp), respectively.

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3. Fig. 2. The sequence localized before the ATG codon of the OhMOX gene. The region between the open reading frames of adjacent genes is underlined. The putative sequences of URS (upstream repressing sequence), UAS1, UAS2 (upstream activator sequences) and TATA-box are highlighted in bold. The putative binding site for the CYCC core sequence of the Mrx1 homologue is shown in bold and italics. The start codon of the open reading frame for methanol oxidase (ATG) is shown in bold. The location of the missing 22 bp region is shown as a broken line.

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4. Fig. 3. Mannanase activity levels (U/ml) in samples with the MANS gene under the control of the promoters: 1 — pOpMOX; 2 — pOhGAP; 3 — pOhFMD; 4 — pOhMOX. Error bars show the standard deviation of the mean activity of 12 transformants. p < 0.05, ns — not significant difference.

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5. Fig. 4. SDS-PAGE electrophoresis of recombinant β-mannanase secreted by O. haglerorum transformants containing plasmids with pOhMOX, pOpMOX, pOhFMD, and pOhGAP promoters. 1, 7 — molecular weight marker, kDa, 2 — pOhMOX, 3 — pOpMOX, 4 — pOhFMD; 5 — pOhGAP; 6 — sample with pOpMOX promoter under conditions without induction.

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6. Fig. 5. Relative transcription level of the MANS gene (%) under the control of the pOhMOX (II) or pOpMOX (I) promoters. The average transcription level of the MANS gene in 6 transformants with each promoter during cultivation in a medium with methanol for 12 (1) and 20 h (2) is shown. The transcription level of the MANS gene under the control of pOpMOX after 12 h of induction is taken as 100%. Error bars show the standard deviation for three independent experiments, p < 0.05.

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