Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance

Date

2015-05-01

Author

Ata, Ozge
Boy, Erdem
Gunes, Hande
Çalık, Pınar

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

222
views

0
downloads

The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L-1; and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L-1 and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L-1 at t = 66 h and 32530 U L-1 at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy.

Subject Keywords

Biotechnology, Bioengineering, General Medicine

URI

https://hdl.handle.net/11511/47945

Journal

BIOPROCESS AND BIOSYSTEMS ENGINEERING

DOI

https://doi.org/10.1007/s00449-014-1333-z

Collections

Test and Measurement Center In advanced Technologies (MERKEZ LABORATUVARI), Article

Suggestions

OpenMETU
Core

Beet molasses-based feeding strategy enhances recombinant thermostable glucose isomerase production by Escherichia coli BL21 (DE3) Yaman, Sena; Çalık, Pınar (Wiley, 2017-11-01) The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity...
Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: Effects on cephamycin C production Özcengiz, Gülay; Ünsaldi, Eser; Taşkin, Bilgin; Liras, Paloma; Piret, Jacqueline (Informa UK Limited, 2010-05-01) Abstract In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the w...
Exponential feeding strategy development for benzaldehyde lyase production by recombinant "Escherichia coli" Taşpınar, Hatice; Çalık, Pınar; Özdamar, Tunçer H.; Department of Biotechnology (2010) In this study, the aim was to investigate the effects of exponential feeding strategy on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21. For this purpose, the effects of medium components were investigated to optimize the initial medium composition of the fed-batch fermentations. For the batch bioreactor operations, the highest cell concentration and BAL activity were achieved in a media containing 30 g L-1 pretreated molasses, and 5 g L-1 (NH4)2HPO4 as 5.07 g L-1, and 1611 U ml-1 ...
Phosphate enrichment and fed-batch operation for prolonged beta-lactamase production by Bacillus licheniformis Ileri, N.; Çalık, Pınar; Sengul, A. (Wiley, 2007-05-01) Aims: Investigation of the phosphate effect and feeding strategy, i.e. linear and exponential feeding, to improve beta-lactamase production by Bacillus licheniformis considering the viability of the cells.
Determination and metabolic engineering of rate limiting reactions in aromatic amino acid pathway in Bacillus subtilis for L-phenylaianine production Guzide, Calik; Yasemin, Demirci; Pinar, Calik; Ozdamar, Tuncer H. (2007-09-01) Rate limiting reactions in the aromatic-group amino acid pathway (AAAP) in Bacillus subtilis for l-phenylalanine (Phe) production were determined as reported elsewhere (Özçelik-Şenver et al., 2004). The biochemical reactions in the AAAP start with the reaction using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) synthesised in the glycolysis pathway and the PPP, respectively, leading to the formation of Phe via chorismate by branching at prephenate. The branch-point metabolites E4P supplied in vi...

Citation Formats

O. Ata, E. Boy, H. Gunes, and P. Çalık, “Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance,” BIOPROCESS AND BIOSYSTEMS ENGINEERING, pp. 889–903, 2015, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/47945.