Insights into Streptomyces coelicolor A3(2) growth and pigment formation with high‐throughput online monitoring

Abstract

Streptomyces species are intensively studied for their ability to produce a variety of natural products. However, conditions influencing and leading to product formation are often not completely recognized. Therefore, in this study, high-throughput online monitoring is presented as a powerful tool to gain indepth understanding of the cultivation of the model organism Streptomyces coelicolor A3(2). Through online measurements of oxygen transfer rate and autofluorescence, valuable information about availability of nutrients and product formation patterns of the pigments actinorhodin and undecylprodigiosin can be obtained and explained. Therefore, it is possible to determine the onset of pigmentation and to study in detail the influencing factors thereof. One factor identified in this study is the filling volume of the cultivation vessel. Slight variations led to varying pigmentation levels. By combining optical and metabolic online monitoring techniques, the correlation of the filling volume with pigmentation could be explained as a result of different growth trajectories caused by varying specific power inputs and their influence on the pellet formation of the filamentous system. Finally, experiments with the addition of supernatant from unpigmented and pigmented cultures could highlight the applicability of the presented approach to study quorum sensing and cell-cell interaction.

Tunable population dynamics in a synthetic filamentous coculture

“…In contrast, for the cocultures with the two highest T. reesei to S. coelicolor ratios, which are highly pigmented, a drop in the unmixed signal is visible at the end of the culture. We suggest that the onset of pigment formation is the main cause of this drop, as already reported by Finger et al ( 2022 ) and Palacio‐Barrera et al ( 2022 ). This exemplary process of unmixing the mNG fluorescence signal as a specific biomass measure for S. coelicolor from the green autofluorescence signal shows for this coculture that the overall green autofluorescence signal also gives a good relative account for the biomass of S. coelicolor (compare Figure A2b with Figure 3c ).…”

“…Further, we have developed a fluorescence‐based procedure for online monitoring of population dynamics in filamentous cocultures, which is applied here (Palacio‐Barrera et al, 2022 ). Additionally, we previously could show that fluorescence monitoring can help estimate the onset of pigment biosynthesis for S. coelicolor (Finger et al, 2022 ).…”

“…Unless stated otherwise, cultivations were inoculated with 10 6 spores/ml. S. coelicolor spores were prepared similarly to Hobbs et al ( 1989 ) and as mentioned in previous works (Finger et al, 2022 ; Hobbs et al, 1989 ; Palacio‐Barrera et al, 2022 ). Briefly, a spore suspension was spread on soy‐flour mannitol agar plates, which consisted of 20 g/L mannitol, 20 g/L soy flour, and 20 g/L agar.…”

“…The medium for all cultivations was an LNP minimal medium, which is described also in previous works (Finger et al, 2022 ; Palacio‐Barrera et al, 2022 ). For the carbon source, 5 g/L glucose and 30 g/L α‐cellulose were added.…”

Reliable online measurement of population dynamics for filamentous co‐cultures

“…A phosphate limitation occurs after approx. Seventy-two hour marked by a stress signal in the fluorescence of CH2 (Figure 2C, dotted lines) (Finger et al, 2022), which results in a slowdown of growth. This leads to a plateau in the unmixed signal (Figure 2D), while both raw fluorescence signals continue to increase until or beyond 96 h. This increase in autofluorescence is clearly not related to growth, as the autofluorescence still increases, while the scattered light value levels off.…”