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

Synthetic Promoter Library for Modulation of Actinorhodin Production in <i>Streptomyces coelicolor</i> A3(2)

<div><p>The objective of this study was the application of the synthetic promoter library (SPL) technology for modulation of actinorhodin production in <i>Streptomyces coelicolor</i> A3(2). The SPL technology was used to optimize the expression of a pathway specific positive transcriptional regulator ActII orf4, which activates the transcription of the <i>S. coelicolor</i> actinorhodin biosynthetic gene cluster. The native <i>actII orf4</i> promoter was replaced with synthetic promoters, generating a <i>S. coelicolor</i> library with a broad range of expression levels of <i>actII orf4</i>. The resulting library was screened based on the yield of actinorhodin. Selected strains were further physiologically characterized. One of the strains from the library, ScoSPL20, showed considerably higher yield of actinorhodin and final actinorhodin titer, compared to <i>S. coelicolor</i> wild type and <i>S. coelicolor</i> with <i>actII orf4</i> expressed from a strong constitutive promoter. ScoSPL20 demonstrated exceptional productivity despite having a comparatively weak expression from the promoter. Interestingly, the ScoSPL20 promoter was activated at a much earlier stage of growth compared to the wild type, demonstrating the advantage of fine-tuning and temporal tuning of gene expression in metabolic engineering. Transcriptome studies were performed in exponential and actinorhodin-producing phase of growth to compare gene expression between ScoSPL20 and the wild type. To our knowledge, this is the first successful application of the SPL technology for secondary metabolite production in filamentous bacteria.</p></div

Comparative table describing physiological parameters of <b><i>S. coelicolor</i></b><b> WT, </b><b><i>S. coelicolor</i></b><b> oxp-</b><b><i>actII orf4</i></b><b> and ScoSPL20. Results are the mean values with standard deviations from three biological replicates.</b>

†<p>Did not display a defined RED production phase, onset of RED and ACT production was simultaneous and growth associated.</p>‡<p>r_S and Y_SX were calculated in the exponential growth phase.</p