Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network

Çalık, Pınar
Oliver, SG
Ozdamar, TH
This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes were observed in the recombinant B. licheniformis in response to altered bioreactor-operation conditions, i.e. initial glucose concentration, The growth kinetics of microbial cells were investigated prior to the investigation of intracellular reactions and rates within the cell; the unstructured substrate inhibition and Monod models were found valid for the wild-type and recombinant B. licheniformis, respectively. Optimum initial glucose concentration for maximum SAP production and the corresponding cultivation time of the recombinant B. licheniformis shifted respectively from C-G0 = 6 to 8 kg m(-3) and from t = 43 to 67 h. The maximum SAP activity was obtained as 950 U cm(-3) with the recombinant B. licheniformis, which was ca. 2.5-fold higher than that of the wild-type. Carbon fluxes through the central metabolic pathways in the wild-type and recombinant B. licheniformis were calculated, using a mass balance-based mathematical model that contains 105 metabolites and 148 reaction fluxes and the time profiles of glucose. dry cell weight, organic acids, amino acids and SAP obtained in 3.5 dm(3) bioreactor systems at C-G0 = 6 kg m(-3) for the exponential growth phase and the SAP production phase. The bioreaction network flux analyses were first accomplished by using the theoretical data-based approach, and then by using the theoretical data-based capacity analysis approach. During the SAP synthesis period, the actual fluxes of the glycolysis pathway. the pentose phosphate pathway, the tricarboxylic acid cycle, the amino acids biosynthetic pathways (and, consequently, SAP synthesis) are higher in the recombinant B. licheniformis strain than in the wild-type. Further, the normalised relative flux values of all the pathways, except the glycolysis pathway, change considerably in the recombinant bacteria. The effectiveness factor, defined as the SAP synthesis rate per maximum possible SAP synthesis rate was eta = 0.20 for the recombinant B. licheniformis. This indicates the possibility of a further increase in SAP production through metabolic engineering, and potential strategies to achieve this are. also discussed. (C) 2003 Elsevier Science Inc. All rights reserved.


Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses
Çalık, Pınar; Ozdamar, TH (Elsevier BV, 2003-12-02)
Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli-Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP product...
Role of the cmcH-ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus
Kurt, Aslihan; Alvarez-Alvarez, Ruben; Liras, Paloma; Özcengiz, Gülay (Springer Science and Business Media LLC, 2013-07-01)
The effect of the CcaR regulatory protein on expression of the cephamycin C gene cluster is studied. Quantitative reverse transcription PCR (qRT-PCR) expression analysis of the cephamycin biosynthesis genes in the ccaR-disrupted strain, S. clavuligerus ccaR::aph, revealed that in the absence of CcaR, the lat and cmcI genes expression was reduced 2,200-and 1,087-fold compared with the wild type. Expression of pcbAB-pcbC-cefD-cefE-cmcJ-cmcH and blp was 225- to 359-fold lower, while expression of pcbR-pbpA-bla...
Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis
Okay, S.; Tefon, B. E.; ÖZKAN, MELEK; Özcengiz, Gülay (Wiley, 2008-01-01)
Aims: The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs.
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...
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
P. Çalık, S. Oliver, and T. Ozdamar, “Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network,” ENZYME AND MICROBIAL TECHNOLOGY, pp. 706–720, 2003, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/46042.