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

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 in-depth 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.

Microbial cocultures are used as a tool to stimulate natural product biosynthesis. However, studies often empirically combine different organisms without a deeper understanding of the population dynamics. As filamentous organisms offer a vast metabolic diversity, we developed a model filamentous coculture of the cellulolytic fungus Trichoderma reesei RUT-C30 and the noncellulolytic bacterium Streptomyces coelicolor A3(2). The coculture was set up to use α-cellulose as a carbon source. This established a dependency of S. coelicolor on hydrolysate sugars released by T. reesei cellulases. To provide detailed insight into coculture dynamics, we applied high-throughput online monitoring of the respiration rate and fluorescence of the tagged strains. The respiration rate allowed us to distinguish the conditions of successful cellulase formation. Furthermore, to dissect the individual strain contributions, T. reesei and S. coelicolor were tagged with mCherry and mNeonGreen (mNG) fluorescence proteins, respectively. When evaluating varying inoculation ratios, it was observed that both partners outcompete the other when given a high inoculation advantage. Nonetheless, adequate proportions for simultaneous growth of both partners, cellulase, and pigment production could be determined. Finally, population dynamics were also tuned by modulating abiotic factors. Increased osmolality provided a growth advantage to S. coelicolor. In contrast, an increase in shaking frequency had a negative effect on S. coelicolor biomass formation, promoting T. reesei. This comprehensive analysis fills important knowledge gaps in the control of complex cocultures and accelerates the setup of other tailor-made coculture bioprocesses.

Reliable online measurement of population dynamics for filamentous co-cultures.

Understanding population dynamics is a key factor for optimizing co-culture processes to produce valuable compounds. However, the measurement of independent population dynamics is difficult, especially for filamentous organisms and in presence of insoluble substrates like cellulose. We propose a workflow for fluorescence-based online monitoring of individual population dynamics of two filamentous microorganisms. The fluorescent tagged target co-culture is composed of the cellulolytic fungus Trichoderma reesei RUT-C30-mCherry and the pigment-producing bacterium Streptomyces coelicolor A3(2)-mNeonGreen (mNG) growing on insoluble cellulose as a substrate. To validate the system, the fluorescence-to-biomass and fluorescence-to-scattered-light correlation of the two strains was characterized in depth under various conditions. Thereby, especially for complex filamentous microorganisms, microbial morphologies have to be considered. Another bias can arise from autofluorescence or pigments that can spectrally interfere with the fluorescence measurement. Green autofluorescence of both strains was uncoupled from different green fluorescent protein signals through a spectral unmixing approach, resulting in a specific signal only linked to the abundance of S. coelicolor A3(2)-mNG. As proof of principle, the population dynamics of the target co-culture were measured at varying inoculation ratios in presence of insoluble cellulose particles. Thereby, the respective fluorescence signals reliably described the abundance of each partner, according to the variations in the inocula. With this method, conditions can be fine-tuned for optimal growth of both partners along with natural product formation by the bacterium.

Measurement Techniques to Resolve and Control Population Dynamics of Mixed-Culture Processes.

Microbial mixed cultures are gaining increasing attention as biotechnological production systems, since they offer a large but untapped potential for future bioprocesses. Effects of secondary metabolite induction and advantages of labor division for the degradation of complex substrates offer new possibilities for process intensification. However, mixed cultures are highly complex, and, consequently, many biotic and abiotic parameters are required to be identified, characterized, and ideally controlled to establish a stable bioprocess. In this review, we discuss the advantages and disadvantages of existing measurement techniques for identifying, characterizing, monitoring, and controlling mixed cultures and highlight promising examples. Moreover, existing challenges and emerging technologies are discussed, which lay the foundation for novel analytical workflows to monitor mixed-culture bioprocesses.

Impact of Oxygen Supply and Scale Up on Mycobacterium smegmatis Cultivation and Mycofactocin Formation.

Mycofactocin (MFT) is a recently discovered glycosylated redox cofactor, which has been associated with the detoxification of antibiotics in pathogenic mycobacteria, and, therefore, of potential medical interest. The MFT biosynthetic gene cluster is commonly found in mycobacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the MFT molecule is highly interesting for basic research and could even serve as a potential drug target, large-scale production of the molecule is highly desired. However, conventional shake flask cultivations failed to produce enough MFT for further biochemical characterization like kinetic studies and structure elucidation, and a more comprehensive study of cultivation parameters is urgently needed. Being a redox cofactor, it can be hypothesized that the oxygen transfer rate (OTR) is a critical parameter for MFT formation. Using the non-pathogenic strain Mycobacterium smegmatis mc2 155, shake flask experiments with online measurement of the oxygen uptake and the carbon dioxide formation, were conducted under different levels of oxygen supply. Using liquid chromatography and high-resolution mass spectrometry, a 4-8 times increase of MFT production was identified under oxygen-limited conditions, in both complex and mineral medium. Moreover, the level of oxygen supply modulates not only the overall MFT formation but also the length of the glycosidic chain. Finally, all results were scaled up into a 7 L stirred tank reactor to elucidate the kinetics of MFT formation. Ultimately, this study enables the production of high amounts of these redox cofactors, to perform further investigations into the role and importance of MFTs.

Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis.

BACKGROUND: Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation. RESULTS: The efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation. CONCLUSION: This study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Yap1p, the central regulator of the S. cerevisiae oxidative stress response, is activated by allicin, a natural oxidant and defence substance of garlic.

Allicin is a thiol-reactive sulfur-containing natural product from garlic with a broad range of antimicrobial effects against prokaryotes and eukaryotes. Previous work showed that the S. cerevisiae OSI1 gene is highly induced by allicin and other thiol-reactive compounds, and in silico analysis revealed multiple Yap1p binding motifs in the OSI1 promoter sequence. An OSI1-promoter::luciferase reporter construct expressed in Wt and Δyap1 cells showed absolute Yap1p-dependence for allicin-induced OSI1-expression. A GFP: Yap1p fusion protein accumulated in the nucleus within 10min of allicin treatment and a Δyap1 mutant was highly sensitive to allicin. Yap1p regulates glutathione (GSH) metabolism genes, and Δgsh1, Δgsh2 and Δglr1 mutants showed increased sensitivity to allicin. Allicin activated the OSI1-promoter::luciferase reporter construct in Δgpx3 and Δybp1 cells, indicating that allicin activates Yap1p directly rather than via H2O2 production. A systematic series of C-to-A Yap1p exchange mutants showed that the C-term C598 and C620 residues were necessary for allicin activation. These data suggest that Yap1p is an important transcriptional regulator for the resistance of yeast cells to allicin, and that activation occurs by direct modification of C-term cysteines as shown for other electrophiles.

Process relevant screening of cellulolytic organisms for consolidated bioprocessing.

BACKGROUND: Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics. RESULTS: The screening of five different cellulase producers with the freeze assay identified Trichoderma reesei and Penicillium verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P. verruculosum but a similar cellulase production rate afterwards, compared to T. reesei. The freeze assay measurement depicted that P. verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches. CONCLUSIONS: With two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T. reesei and P. verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Efficient evaluation of cellulose digestibility by Trichoderma reesei Rut-C30 cultures in online monitored shake flasks.

BACKGROUND: Pretreated lignocellulosic biomass is considered as a suitable feedstock for the sustainable production of chemicals. However, the recalcitrant nature of cellulose often results in very cost-intensive overall production processes. A promising concept to reduce the costs is consolidated bioprocessing, which integrates in a single step cellulase production, cellulose hydrolysis, and fermentative conversion of produced sugars into a valuable product. This approach, however, requires assessing the digestibility of the applied celluloses and, thus, the released sugar amount during the fermentation. Since the released sugars are completely taken up by Trichoderma reesei Rut-C30 and the sugar consumption is stoichiometrically coupled to oxygen uptake, the respiration activity was measured to evaluate the digestibility of cellulose. RESULTS: The method was successfully tested on commercial cellulosic substrates identifying a correlation between the respiration activity and the crystallinity of the substrate. Pulse experiments with cellulose and cellulases suggested that the respiration activity of T. reesei on cellulose can be divided into two distinct phases, one limited by enzyme activity and one by cellulose-binding-sites. The impact of known (cellobiose, sophorose, urea, tween 80, peptone) and new (miscanthus steepwater) compounds enhancing cellulase production was evaluated. Furthermore, the influence of two different pretreatment methods, the OrganoCat and OrganoSolv process, on the digestibility of beech wood saw dust was tested. CONCLUSIONS: The introduced method allows an online evaluation of cellulose digestibility in complex and non-complex cultivation media. As the measurements are performed under fermentation conditions, it is a valuable tool to test different types of cellulose for consolidated bioprocessing applications. Furthermore, the method can be applied to identify new compounds, which influence cellulase production.