Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)

Background: Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2). Results: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism. Conclusions: The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA

Mycelium Differentiation and Development of <em>Streptomyces</em> in Liquid Nonsporulating Cultures: Programmed Cell Death, Differentiation, and Lysis Condition Secondary Metabolite Production

Streptomycetes are mycelium-forming sporulating bacteria that produce two thirds of clinically relevant secondary metabolites. Secondary metabolite production is activated at specific developmental stages of Streptomyces life cycle. Despite this, Streptomyces differentiation in liquid nonsporulating cultures (flasks and industrial bioreactors) tends to be underestimated and the most important parameters managed are only indirectly related to differentiation: modifications to the culture media, optimization of productive strains by random or directed mutagenesis, analysis of biophysical parameters, etc. In this chapter, we review the relationship between differentiation and antibiotic production in liquid cultures. Morphological differentiation in liquid cultures is comparable to that occurring during pre-sporulation stages in solid cultures: an initial compartmentalized mycelium suffers a programmed cell death, and remaining viable segments then differentiate to a second multinucleated antibiotic-producing mycelium. Differentiation is one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in liquid cultures

Study of Glucose-6-Phosphate dehydrogenase activity assay in mangrove streptomyces for actinohordin and undercylprodigiosin production

This study evaluates the potential of using glucose-6-phosphate dehydrogenase activity assay for Actinohordin and Undecylprodigiosin productions from mangrove Streptomyces. Previously, there were several methods used to screen antimicrobial activities such as agar spot test and disc diffusion assay, but those are lengthy screening methods and time consuming. Thus, to overcome the limitations plate-based assay is suggested to enable rapid screening on secondary metabolite production of numerous samples at one time. The development of plate-based assay was performed by optimizing glucose-6-phosphate dehydrogenase activity assay. This coupled assay was based on the production of dihydronicotinamideadenine dinucleotide phosphate (NADPH) whereby a right combination of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) were refined. The production of NADPH was measured at absorbance of 340 nm where reduced cofactor NADPH are absorbed readily at this wavelength. Sample with different concentrations of crude lysate was subjected to various substrates concentration to obtain the best activity curve. Even though elucidating clear patterns is speculative, it is believed that some improvements or optimizations of this study could offer promising knowledge which can serve as useful reference in futur

Transcriptome and translatome profiles of Streptomyces species in different growth phases.

Streptomyces are efficient producers of various bioactive compounds, which are mostly synthesized by their secondary metabolite biosynthetic gene clusters (smBGCs). The smBGCs are tightly controlled by complex regulatory systems at transcriptional and translational levels to effectively utilize precursors that are supplied by primary metabolism. Thus, dynamic changes in gene expression in response to cellular status at both the transcriptional and translational levels should be elucidated to directly reflect protein levels, rapid downstream responses, and cellular energy costs. In this study, RNA-Seq and ribosome profiling were performed for five industrially important Streptomyces species at different growth phases, for the deep sequencing of total mRNA, and only those mRNA fragments that are protected by translating ribosomes, respectively. Herein, 12.0 to 763.8 million raw reads were sufficiently obtained with high quality of more than 80% for the Phred score Q30 and high reproducibility. These data provide a comprehensive understanding of the transcriptional and translational landscape across the Streptomyces species and contribute to facilitating the rational engineering of secondary metabolite production

Optimized submerged batch fermentation for metabolic switching in Streptomyces yanglinensis 3–10 providing platform for reveromycin A and B biosynthesis, engineering, and production

The cultivation system requires that the approach providing biomass for all types of metabolic analysis is of excellent quality and reliability. This study was conducted to enhance the efficiency and yield of antifungal substance (AFS) production in Streptomyces yanglinensis 3–10 by optimizing operation conditions of aeration, agitation, carbon source, and incubation time in a fermenter. Dissolved oxygen (DO) and pH were found to play significant roles in AFS production. The optimum pH for the production of AFS in S. yanglinensis 3–10 was found to be 6.5. As the AFS synthesis is generally thought to be an aerobic process, DO plays a significant role. The synthesis of bioactive compounds can vary depending on how DO affects growth rate. This study validates that the high growth rate and antifungal activity required a minimum DO concentration of approximately 20% saturation. The DO supply in a fermenter can be raised once agitation and aeration have been adjusted. Consequently, DO can stimulate the development of bacteria and enzyme production. A large shearing effect could result from the extreme agitation, harming the cell and deactivating its products. The highest inhibition zone diameter (IZD) was obtained with 3% starch, making starch a more efficient carbon source than glucose. Temperature is another important factor affecting AFS production. The needed fermentation time would increase and AFS production would be reduced by the too-low operating temperature. Furthermore, large-scale fermenters are challenging to manage at temperatures that are far below from room temperature. According to this research, 28°C is the ideal temperature for the fermentation of S. yanglinensis 3–10. The current study deals with the optimization of submerged batch fermentation involving the modification of operation conditions to effectively enhance the efficiency and yield of AFS production in S. yanglinensis 3–10

Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)

-Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2)

Preliminary investigation of growth and antimicrobial production by streptomyces polyantibioticus : from shake flask to stirred tank bioreactor

Resistance to antibiotics by microbial pathogens continues to be a major global health problem. Treatment of bacterial infections is becoming increasingly complex and expensive. Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is affected by antibiotic resistance. In South Africa, the Western Province is the worst affected, with an increasing incidence of both multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis. Both resistant forms of TB increase the length of treatment to almost 24 months and cost by as much as 1400 times that of regular anti-tubercular chemotherapy. A potential solution to this problem is the discovery of new drugs, which can be obtained from natural sources. Actinomycetes are good sources of these drugs, with over 45% of current medicines derived from these bacteria. The actinobacterium Streptomyces polyantibioticus SPRT (SPRT) was locally isolated and first described by Le Roes (2006). It has been shown to produce bioactive molecules active against a range of bacteria, including compounds (drugs) that have anti-tubercular properties. One of the anti-tubercular molecules was identified as 2,5-diphenyloxazole (DPO). DPO is currently used as a component of scintillation fluid for its luminescent properties and is synthesised chemically in industry. SPRT is the only reported biological source of DPO, it is however not yet produced commercially via a biological route. The present study was performed to inform future process development of DPO production from SPRT. An investigation into the growth and production of antimicrobial compounds from submerged cultures of SPRT in shake flasks, and scale-up of the process into a laboratory stirred tank bioreactor (STR) was done in the present study. The work focused on obtaining growth kinetics and suitable operating conditions for cultivation. Characterisation of the growth profile of SPRT and determination of the kinetic growth parameters was carried out. Additionally, the antimicrobial production phases, and factors influencing their production was investigated. It was determined that the most reliable method of measuring biomass concentration was by dry cell gravimetric measurement of whole shake flasks following vacuum filtration, as it best suited the non-homogenous filamentous nature of SPRT

Produção de Biomassa probiótica e enriquecida com selênio de Saccharomyces boulardii utilizando melaço de cana-de-açucar

Orientadora : Profa. Dra. Luciana Porto de Souza VandenbergheCo-orientadora : Profa. Dra. Cristine RodriguesCo-orientador : Prof. Dr. Carlos Ricardo SoccolDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 12/03/2013Inclui referênciasÁrea de concentração: Agroindústria e biocombustíveisResumo: Os probióticos são micro-organismos vivos que produzem efeitos benéficos à saúde do indivíduo, quando administrados em quantidades adequadas. A maioria são bactérias, sendo a Saccharomyces boulardii uma das leveduras que apresentam propriedades probióticas. O micromineral selênio possui efeitos nutricionais e é um elemento traço essencial necessário para o adequado funcionamento do sistema antioxidante do organismo. Uma das formas mais usadas de suplementação nutricional de selênio é a levedura rica em selênio, fonte rica do selênio orgânico selenometionina, a melhor fonte para os organismos. Todos os produtos existentes no mercado de levedura rica em selênio são produzidos com a levedura S. cerevisiae e os estudos sobre a produção de probióticos são voltados para bactérias, não sendo encontrados estudos sobre a produção biotecnológica da levedura probiótica S. boulardii. Dessa forma, o objetivo deste trabalho foi desenvolver um bioprocesso para a produção de biomassa probiótica S. boulardii e também desta levedura enriquecida com selênio (Se) orgânico, através de fermentação submersa utilizando o subproduto agroindustrial melaço de cana-de-açúcar. Duas cepas de S. boulardii, SbM e Sb4308, passaram pelos estudos de otimização do meio de cultivo. A cepa Sb4308 foi selecionada por apresentar maior produção de biomassa e foi utilizada nos estudos de bioacúmulo de Se. Os crescimentos das cepas a 30?C e 37?C foram comparados, sendo selecionada 30?C como temperatura de cultivo. Iniciou-se a otimização dos componentes do meio de cultivo com um screening de 11 componentes em um Planejamento tipo Plackett & Burman, no qual 5 foram significativos, e seguiram para um planejamento fatorial incompleto 2 (5-2), sendo selecionadas 3 variáveis significativas. A terceira etapa da otimização foi realizada com o suporte de um planejamento do tipo DCCR 23. Neste planejamento tipo DCCR foi encontrada a região ótima das concentrações das variáveis para a cepa SbM, sendo glicose 40 g/L, melaço de cana 50 g/L e extrato de levedura 12 g/L, cuja produção de biomassa foi de 9,66 g/L. Seguiu-se os estudos para a cepa Sb4308 com aumento da concentração da fonte de carbono, pelos quais foram encontradas as condições selecionadas: melaço de cana 150 g/L e extrato de levedura 12 g/L, obtendo-se uma produção de 11,03 g/L de biomassa seca. Após a definição do meio de cultivo e da cepa, foram realizados os estudos das cinéticas de produção do inóculo e da biomassa; os tempos de cultivos determinados foram 20 h e 24 h, respectivamente. Prosseguiu-se com o estudo da fermentação em batelada em biorreator BioFlo? 110, obtendo-se uma produção de 11,37 g/L de biomassa seca, com o cultivo a 30?C, 30% OD, agitação mínima de 300 rpm e pH inicial de 5,5. Posteriormente foram realizados estudos do processo em batelada alimentada em frascos de Erlemeyer e as melhores condições encontradas foram: alimentação no tempo de 4 h com melaço de cana a 100 g/L. Nesta etapa, a produção de biomassa seca foi de 13,18 g/L em 10 h de cultivo, com uma produtividade de 1,32 g/L.h. Obteve-se assim, um aumento de produtividade de 1624% em relação às condições iniciais (0,08 g/L.h em meio YM líquido). Iniciaram-se os estudos de bioacúmulo de Se e os valores ótimos encontrados foram: adição de 100 ppm de Se no cultivo no tempo de 10 h. Foi testada uma adaptação prévia de Sb4308 a concentrações crescentes de Se no meio, para uma comparação entre a cepa selvagem e a cepa adaptada. A cepa selvagem teve um maior crescimento e melhor bioacúmulo. A determinação de Se inorgânico total livre foi realizada através de espectrofotometria da reação colorimétrica com Azure B e a determinação de Se total bioacumulado foi realizada através de espectrofotometria de absorção atômica. Um estudo da cinética de crescimento e bioacúmulo de selênio pela levedura Sb4308 em fermentação em batelada alimentada foi realizado em biorreator BioFlo? 110, obtendo-se uma produção de 12,73 g/L de biomassa seca e 3.053,00 ?g de Se/g de biomassa. Foram iniciados estudos de especiação e quantificação de Se orgânico bioacumulado através de cromatografia líquida acoplada à espectrometria de massa. Palavras-chave: Levedura, Saccharomyces boulardii, probiótico, selênio orgânico, fermentação submersa.Abstract: Probiotics are live microorganisms that produce beneficial effects on the health of the individual, when administered in adequate amounts. Most are bacterias, being Saccharomyces boulardii one of yeasts that have probiotic properties. Selenium has nutritional effects and is an essential trace element necessary for the proper functioning of the body's antioxidant system. One of the most widely used forms of nutritional supplementation of selenium is the yeast rich in selenium, a rich source of the organic selenium selenomethionine, the best source for the organisms. All products on the market of yeast rich in selenium are produced with the yeast S. cerevisiae and studies about the production of probiotics are facing bacterias, did not find studies about the biotechnological production of the probiotic yeast S. boulardii. Thus, the aim of this study was to develop a bioprocess for the production of probiotic biomass Saccharomyces boulardii and also this yeast enriched with organic selenium (Se) through submerged fermentation using the agroindustrial byproduct sugarcane molasses. Two strains of S. boulardii, SbM and Sb4308, passed by the optimization studies of the medium. Sb4308 was selected due to its higher biomass production and has been used in studies of selenium bioaccumulation. The growths at 30?C and 37?C were compared, determining that the cultivation temperature was 30?C. The optimization of the components of the culture medium began with a screening of 11 components in Plackett & Burman design, in which 5 components were significant. Then an incomplete factorial design 2(5-2) was performed, being selected three significant variables. The third step of optimization was performed with the support of a DCCR 23 design. In DCCR was found the optimal region of concentrations of the variables for strain SbM, being glucose 40 g/L, sugarcane molasses 50 g/L and yeast extract 12 g/L, whose biomass was 9.661g/L. Followed studies for Sb4308 strain with increased concentration of carbon source, whereby selected conditions were found: sugarcane molasses 150 g/L and yeast extract 12 g/L, yielding of 11.03 g/L dry biomass. After defining the culture medium and the strain, kinetic studies of the inoculum and biomass production were conducted, and the times were determined for 20 h and 24 h, respectively. Proceeded with the study of the fermentation batch in the bioreactor Bioflo?110, the dry biomass obtained was 11.37 g/L, by cultivating at 30?C, DO 30%, minimum agitation of 300 rpm and pH initial 5.5. Subsequent studies about feed fermentation were conducted in flasks Erlemeyers, being the fed in time of 4 h of sugarcane molasses 100 g/L the best conditions found. At this stage the dry biomass was 13.18 g/L in 10 h of cultive, with a productivity of 1.32 g/L.h. There was thus obtained a productivity increase of 1624% in relation of the initial conditions (0.08 g/L.h in liquid YM medium). Followed then for selenium bioaccumulation studies and the optimum values found were: addition of 100 ppm of Se in cultivation at the time of 10 h. A prior adaptation was tested to Sb4308 in increasing concentrations of Se for a comparison between the wild strain and the adapted strain. The wild strain had higher growth and better bioaccumulation. The determination of free total Se was performed by spectrophotometry of color reaction with Azure B and determination of Se bioaccumulated was performed by atomic absorption spectrophotometry. The study of the kinetics of growth and bioaccumulation of selenium by yeast Sb4308 in the fed-batch fermentation was performed in a bioreactor Bioflo?110, that gave a yield of 12.73 g/L dry biomass and 3053.00 ?g of Se/g biomass. Speciation studies and quantification of organic selenium in biomass were initiated through liquid chromatography coupled to mass spectrometry. Keywords: yeast, Saccharomyces boulardii, probiotic, organic selenium, submerged fermentation

A system-wide stable isotope labeling approach for connecting natural products to their biosynthetic gene clusters

Although the first bacterial genome sequence was published almost 20 years ago, there is still no generalizable method for automatically assigning natural products to their cognate biosynthetic gene clusters (BGCs). This thesis describes the development of a mass spectrometry-based parallel stable isotope labeling (SIL) platform, termed IsoAnalyst, which automatically associates metabolite stable isotope labeling patterns with BGC structure prediction in order to connect natural products to their cognate BGCs. The parallel SIL experiments were optimized for small scale and a custom tool written in Python was developed for the untargeted detection and interpretation of SIL labeling patterns. This approach was validated in the industrial production strains Saccharopolyspora erythraea and Amycolatopsis mediterranei demonstrating that the compounds erythromycin A and rifamycin SV respectively, could be associated with the proper BGCs based on the distribution of isotopomer labeling patterns. The method was further validated by connecting known biosynthetic intermediates of these compounds to their associated BGCs and the identification of various siderophores through a combination of SIL labeling patterns and MS/MS fragmentation data. Extension to environmental organisms using a sequenced Micromonospora sp. from our Actinobacterial isolate library led to the discovery of lobosamide D, a new member of the lobosamide family of natural products, and an update to the lobosamide BGC to include relevant tailoring enzymes. This discovery illustrates the power of the IsoAnalyst platform for identifying new compounds, linking molecules to BGCs, and generating new knowledge about biosynthesis