Bioreaction network flux analysis for human protein producing Bacillus subtilis based on genome-scale model

Özdamar, Tunçer H.; Şentürk, Birgül; Yilmaz, Özge Deniz; Kocabaş, Pınar; Çalık, Güzide; Çalı́k, Pınar

doi:10.1016/j.ces.2009.06.030

Cited by 4 publications

(3 citation statements)

References 9 publications

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“…The minimum number of active reactions was 162 at =0 h -1 as anticipated since stationary phase implies less maintenance energy and less active metabolism inside the cell. This result was in harmony with the literature [21] where active reactions were calculated to be 322, 318, 43 for Bacillus subtilis (rBsP) in Period I (0<t<4h), Period II (4<t<12h), Periods III-V (12<t<32 h) calculated using GEM of iYO844 using glucose as carbon source, respectively. The number of active reactions at stationary phase calculated with enzyme constrained iYO844 (ec_iYO844-SAP) is 162, while the number of active reactions at stationary phase calculated with iYO844 [21] is 43 since different carbon sources were utilized in both models.…”

Section: Resultssupporting

confidence: 88%

COMPARATIVE FLUX BALANCE ANALYSES OF SERINE ALKALINE PROTEASE OVERPRODUCTION IN Bacillus subtilis AT GENOME AND SMALL SCALE

Kocabaş

2023

Eskişehir Teknik Üniversitesi Bilim Ve Teknoloji Dergisi - C Yaşam Bilimleri Ve Biyoteknoloji

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This work aims to conduct flux balance analysis of serine alkaline protease overproduction in Bacillus subtilis using enzyme-constrained genome scale model and to compare the results with fluxes obtained from a smaller, bioreaction-based model. Fluxes of the enzyme constraint genome scale model were calculated using CobraToolbox v3.0 and compared with those of bioreaction-based model for the specific growth rate of zero. The active reaction number first increased and then remained constant with specific growth rate for enzyme constrained genome scale model. The SAP synthesis flux increased with a decrease in specific growth rate for both models. The TCA cycle was active for both models, but with lower fluxes for enzyme-constrained genome scale model. Anaplerotic reactions were active only for bioreaction-based model. Glycolysis pathway fluxes were active for enzyme-constrained genome scale model, meanwhile gluconeogenesis pathway fluxes were active for bioreaction-based model. Oxidative pentose phosphate pathway was inactive for both models and generally higher pentose phosphate pathway fluxes were obtained using bioreaction-based model. The fluxes toward amino acid synthesis pathways and serine alkaline protease synthesis were higher with bioreaction-based model. Since TCA cycle fluxes were lower with enzyme constrained genome scale model, ATP synthesis was lower with enzyme constrained genome scale model compared to bioreaction-based model. For both models, active pathways were the same for TCA cycle, pentose phosphate pathway, amino acid synthesis pathways except glycolysis pathway. The results showed that bioreaction-based model gave more sound results compared to enzyme constrained genome scale model since gluconeogenesis should be active with the carbon source of citrate.

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Section: Resultssupporting

confidence: 88%

COMPARATIVE FLUX BALANCE ANALYSES OF SERINE ALKALINE PROTEASE OVERPRODUCTION IN Bacillus subtilis AT GENOME AND SMALL SCALE

Kocabaş

2023

Eskişehir Teknik Üniversitesi Bilim Ve Teknoloji Dergisi - C Yaşam Bilimleri Ve Biyoteknoloji

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“…Saccharomyces cerevisiae strains were used to produce rhGH (Gray et al, 1985;Tokunaga et al, 1985;Franchi et al, 1991;Özdamar et al, 2009).…”

Section: List Of Figuresmentioning

confidence: 99%

“…Furthermore, rhGH has been produced intracellularly and specialized secretion vectors are needed for the protein to be secreted to periplasm or culture medium. In addition to E.coli, Bacillus subtilis, Saccharomyces cerevisiae and Pseudomonas strains were used to produce rhGH (Gray et al, 1985;Tokunaga et al, 1985;Franchi et al, 1991;Özdamar et al, 2009).…”

Section: Microorganism Selectionmentioning

confidence: 99%

Effects of carbon sources and feeding strategies on human growth hormone production by metabolically engineered Pichia pastoris

Acik¹,

Çalı́k²,

Özdamar

2009

New Biotechnology

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Azadirachtin has been recognized as an efficient bio-pesticide worldwide. The current protocol for azadirachtin production is by solvent extraction of the seeds of Azadirachta indica which features low variable yield and contamination problems. Bioreactor plant cell cultivation could be an attractive alternative technology for commercial production of azadirachtin. Callus cultures were initiated from the seeds of elite variety of Azadirachta indica tree having high azadirachtin content. Suspension culture was developed in the shake flask from the selected cell line under statistically optimized medium composition and environmental conditions. Addition of suitable elicitors and precursors (and their addition time) was optimized in shake flask culture for yield enhancement of azadirachtin. Studies on addition of various permeabilizing agents in optimized concentrations resulted in appreciable azadirachtin release in fermentation broth with minimum reduction in cell viability. Batch kinetics of the cell suspension culture was then established in the stirred tank reactor under optimized cultivation conditions. In order to facilitate the design of appropriate feeding strategies in fed-batch and continuous mode of cultivation, a mathematical model was developed using the batch kinetics. This was eventually used to design a suitable fed-batch cultivation strategy. The mathematical Model was also used to simulate best off-line continuous cultivation strategy with cell retention for maximum biomass and azadirachtin production which when experimentally implemented yielded significant improvement in product formation and productivity.Recombinant human growth hormone (rhGH) is one of the crucial industrial proteins produced for pharmaceutical applications. It has many therapeutic uses such as treatments of Turner syndrome, chronic renal failure, Prader-Willi syndrome, intrauterine growth retardation. RhGH can be produced using different microorganisms. Among the host microorganisms, Pichia pastoris, yeast that can utilize methanol as a carbon and energy source, is a good candidate for the production of mature rhGH. In the methanol utilization pathway of P. pastoris, alcohol oxidase (AOX) promot-ers, induced by methanol and repressed by multi-carbon sources such as glucose and glycerol, play an important role. Thus, usually the gene for the desired protein is introduced under the control of the AOX promoters. Therefore, the plasmid, pPIC␣A::hGH was integrated to the AOX1 locus of P. pastoris. In order to increase the product yield, and control and scale-up the fermentation process easily, investigation of carbon sources that support growth but do not repress the methanol utilization pathway and their feeding strategies is essential.In this study, by using P. pastoris Mut + strain, effects of different carbon sources on growth and production were investigated in laboratory scale air filtered shake bioreactor. Then effects of mixed feed of the selected carbon sources and methanol on rhGH and cell concentration were investigated...

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Protein Secretion in Gram-Positive Bacteria: From Multiple Pathways to Biotechnology

Anné

Economou

Bernaerts

2016

Current Topics in Microbiology and Immunology

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A number of Gram-positive bacteria are important players in industry as producers of a diverse array of economically interesting metabolites and proteins. As discussed in this overview, several Gram-positive bacteria are valuable hosts for the production of heterologous proteins. In contrast to Gram-negative bacteria, proteins secreted by Gram-positive bacteria are released into the culture medium where conditions for correct folding are more appropriate, thus facilitating the isolation and purification of active proteins. Although seven different protein secretion pathways have been identified in Gram-positive bacteria, the majority of heterologous proteins are produced via the general secretion or Sec pathway. Not all proteins are equally well secreted, because heterologous protein production often faces bottlenecks including hampered secretion, susceptibility to proteases, secretion stress, and metabolic burden. These bottlenecks are associated with reduced yields leading to non-marketable products. In this chapter, besides a general overview of the different protein secretion pathways, possible hurdles that may hinder efficient protein secretion are described and attempts to improve yield are discussed including modification of components of the Sec pathway. Attention is also paid to omics-based approaches that may offer a more rational approach to optimize production of heterologous proteins.

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Bioreaction network flux analysis for human protein producing Bacillus subtilis based on genome-scale model

Cited by 4 publications

References 9 publications

COMPARATIVE FLUX BALANCE ANALYSES OF SERINE ALKALINE PROTEASE OVERPRODUCTION IN Bacillus subtilis AT GENOME AND SMALL SCALE

COMPARATIVE FLUX BALANCE ANALYSES OF SERINE ALKALINE PROTEASE OVERPRODUCTION IN Bacillus subtilis AT GENOME AND SMALL SCALE

Effects of carbon sources and feeding strategies on human growth hormone production by metabolically engineered Pichia pastoris

Protein Secretion in Gram-Positive Bacteria: From Multiple Pathways to Biotechnology

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