Robust, small-scale cultivation platform for Streptomyces coelicolor

<p>Abstract</p> <p>Background</p> <p>For fermentation process and strain improvement, where one wants to screen a large number of conditions and strains, robust and scalable high-throughput cultivation systems are crucial. Often, the time lag between bench-scale cultivations to production largely depends on approximate estimation of scalable physiological traits. Microtiter plate (MTP) based screening platforms have lately become an attractive alternative to shake flasks mainly because of the ease of automation. However, there are very few reports on applications for filamentous organisms; as well as efforts towards systematic validation of physiological behavior compared to larger scale are sparse. Moreover, available small-scale screening approaches are typically constrained by evaluating only an end point snapshot of phenotypes.</p> <p>Results</p> <p>To address these issues, we devised a robust, small-scale cultivation platform in the form of MTPs (24-square deepwell) for the filamentous bacterium <it>Streptomyces coelicolor </it>and compared its performance to that of shake flasks and bench-scale reactors. We observed that re-designing of medium and inoculum preparation recipes resulted in improved reproducibility. Process turnaround time was significantly reduced due to the reduction in number of unit operations from inoculum to cultivation. The incorporation of glass beads (ø 3 mm) in MTPs not only improved the process performance in terms of improved oxygen transfer improving secondary metabolite production, but also helped to transform morphology from pellet to disperse, resulting in enhanced reproducibility. Addition of MOPS into the medium resulted in pH maintenance above 6.50, a crucial parameter towards reproducibility. Moreover, the entire trajectory of the process was analyzed for compatibility with bench-scale reactors. The MTP cultivations were found to behave similar to bench-scale in terms of growth rate, productivity and substrate uptake rate and so was the onset of antibiotic synthesis. Shake flask cultivations however, showed discrepancy with respect to morphology and had considerably reduced volumetric production rates of antibiotics.</p> <p>Conclusion</p> <p>We observed good agreement of the physiological data obtained in the developed MTP platform with bench-scale. Hence, the described MTP-based screening platform has a high potential for investigation of secondary metabolite biosynthesis in <it>Streptomycetes </it>and other filamentous bacteria and the use may significantly reduce the workload and costs.</p

Hierarchical amino acid utilization and its influence on fermentation dynamics: rifamycin B fermentation using Amycolatopsis mediterranei S699, a case study

BACKGROUND: Industrial fermentation typically uses complex nitrogen substrates which consist of mixture of amino acids. The uptake of amino acids is known to be mediated by several amino acid transporters with certain preferences. However, models to predict this preferential uptake are not available. We present the stoichiometry for the utilization of amino acids as a sole carbon and nitrogen substrate or along with glucose as an additional carbon source. In the former case, the excess nitrogen provided by the amino acids is excreted by the organism in the form of ammonia. We have developed a cybernetic model to predict the sequence and kinetics of uptake of amino acids. The model is based on the assumption that the growth on a specific substrate is dependent on key enzyme(s) responsible for the uptake and assimilation of the substrates. These enzymes may be regulated by mechanisms of nitrogen catabolite repression. The model hypothesizes that the organism is an optimal strategist and invests resources for the uptake of a substrate that are proportional to the returns. RESULTS: Stoichiometric coefficients and kinetic parameters of the model were estimated experimentally for Amycolatopsis mediterranei S699, a rifamycin B overproducer. The model was then used to predict the uptake kinetics in a medium containing cas amino acids. In contrast to the other amino acids, the uptake of proline was not affected by the carbon or nitrogen catabolite repression in this strain. The model accurately predicted simultaneous uptake of amino acids at low cas concentrations and sequential uptake at high cas concentrations. The simulated profile of the key enzymes implies the presence of specific transporters for small groups of amino acids. CONCLUSION: The work demonstrates utility of the cybernetic model in predicting the sequence and kinetics of amino acid uptake in a case study involving Amycolatopsis mediterranei, an industrially important organism. This work also throws some light on amino acid transporters and their regulation in A. mediterranei .Further, cybernetic model based experimental strategy unravels formation and utilization of ammonia as well as its inhibitory role during amino acid uptake. Our results have implications for model based optimization and monitoring of other industrial fermentation processes involving complex nitrogen substrate