Sorting of Streptomyces cell pellets using a complex object parametric analyzer and sorter.

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Heterogeneity in the mycelium: implications for the use of fungi as cell factories.

Fungi are widely used as cell factories for the production of pharmaceutical compounds, enzymes and metabolites. Fungi form colonies that consist of a network of hyphae. During the last two decades it has become clear that fungal colonies within a liquid culture are heterogeneous in size and gene expression. Heterogeneity in growth, secretion, and RNA composition can even be found between and within zones of colonies. These findings imply that productivity in a bioreactor may be increased by reducing the heterogeneity within the culture. The results also imply that molecular mechanisms underlying productivity of fungi in bioreactors should not be studied at the culture level but at the level of micro-colony populations or even at zonal or hyphal level.

Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies.

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter-compartmental and inter-hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome-derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild-type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α-glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.

Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach.

Streptomycetes are proficient producers of enzymes and antibiotics. When grown in bioreactors, these filamentous microorganisms form mycelial pellets that consist of interconnected hyphae. We here employed a flow cytometry approach designed for large particles (COPAS) and demonstrate that liquid-grown Streptomyces cultures consist of two distinct populations of pellets. One population consists of mycelia with a constant mean diameter of approximately 260 µm, whereas the other population contains larger mycelia whose diameter depends on the strain, the age of the culture, and medium composition. Quantitative proteomics analysis revealed that 37 proteins differed in abundance between the two populations of pellets. Stress-related proteins and biosynthetic proteins for production of the calcium-dependent antibiotic were more abundant in the population of large mycelia, while proteins involved in DNA topology, modification, or degradation were overrepresented in the population of small mycelia. Deletion of genes for the cellulose synthase-like protein CslA and the chaplins affected the average size of the population of large pellets but not that of small pellets. Considering the fact that the production of enzymes and metabolites depends on pellet size, these results provide new leads toward rational strain design of Streptomyces strains tailored for industrial fermentations.

Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures.

The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 µm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.