Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: effects of the oxygen transfer rate.
The metabolic fluxes through the central carbon pathways in the bioprocess for serine alkaline protease (SAP) production by Bacillus licheniformis were calculated by the metabolic flux-based stoichiometric model based on the proposed metabolic network that contains 102 metabolites and 133 reaction fluxes using the time profiles of citrate, dry cell, organic acids, amino acids, and SAP as the constraints. The model was solved by minimizing the SAP accumulation rate in the cell. The effects of the oxygen-transfer rate (OTR) on the metabolic fluxes were investigated in a defined medium where citrate was used as the sole carbon source. The central pathways were active for the growth and the SAP synthesis in all the periods of the bioprocess at low (LOT), medium (MOT), and high (HOT) oxygen-transfer conditions. The flux partitioning in the TCA cycle at alpha-ketoglutarate towards glutamate group and at oxalacetate (OA) toward aspartic acid group amino acids were dependent on the OTR. The flux of the anaplerotic reaction that connects the TCA cycle either from malate or OA to the gluconeogenesis pathway via the main branch point pyruvate (Pyr) was also influenced by the OTR. With the decrease in the OTR, the intracellular flux values after glycerate 3-phosphate (PG3) in the gluconeogenesis pathway and the specific growth rate decreased. The total ATP-generation rate increased with the increase in OTR. The pathway towards the aspartic acid family amino acids which is important for sporulation that precedes the SAP synthesis were all active throughout the bioprocess. Metabolic flux analysis results at LOT, MOT, and HOT conditions encourage the design of an oxygen-transfer strategy in the bioreactor; moreover, asparagine synthetase or aspartate kinase could be the potential metabolic engineering sites due to the low value of the flux from the branch point aspartate toward asparagine.
Department of Chemical Engineering, and Industrial Biotechnology Department of Biotechnology Research Center, Ankara University, 06100 Tandogan, Ankara, Turkey.
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D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000596:Amino Acids / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D005285:Fermentation; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013997:Time Factors
D000596:Amino Acids; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D010100:Oxygen