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

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