A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor.

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-ß-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-ß-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Identification of novel biomarkers of inflammation in Atlantic salmon (Salmo salar L.) by a plasma proteomic approach.

Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding proper biomarkers of stress, inflammation and infection is necessary for surveillance and documentation of fish health. In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected fish and non-infected controls. The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2, LCP1) and soluble toll-like receptor 5 (sTLR5) were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity immunoglobulin Fc gamma receptor I (FcγR1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like protein. However, various fibrinogen components, CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers were further verified by Western blot analysis of plasma and real time PCR of spleen and liver, including CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 h after infection, and a CATH2 increase was observed from 72 h in plasma of infected fish. Real time PCR of selected genes confirmed increased transcription of CATH1 and CATH2. In addition, serum amyloid A mRNA significantly increased in liver and spleen after bacterial infection. However, transcription of L-plastin was not consistently induced in liver and spleen. The results of the present study reveal novel and promising biomarkers of the acute phase response and inflammation in Atlantic salmon.

Enzyme-Constrained Models and Omics Analysis of Streptomyces coelicolor Reveal Metabolic Changes that Enhance Heterologous Production.

Many biosynthetic gene clusters (BGCs) require heterologous expression to realize their genetic potential, including silent and metagenomic BGCs. Although the engineered Streptomyces coelicolor M1152 is a widely used host for heterologous expression of BGCs, a systemic understanding of how its genetic modifications affect the metabolism is lacking and limiting further development. We performed a comparative analysis of M1152 and its ancestor M145, connecting information from proteomics, transcriptomics, and cultivation data into a comprehensive picture of the metabolic differences between these strains. Instrumental to this comparison was the application of an improved consensus genome-scale metabolic model (GEM) of S. coelicolor. Although many metabolic patterns are retained in M1152, we find that this strain suffers from oxidative stress, possibly caused by increased oxidative metabolism. Furthermore, precursor availability is likely not limiting polyketide production, implying that other strategies could be beneficial for further development of S. coelicolor for heterologous production of novel compounds.

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium.

The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall of Streptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down of tagO expression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that the Streptomyces cell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.

Production of poly-ß-1,6-N-acetylglucosamine by MatAB is required for hyphal aggregation and hydrophilic surface adhesion by Streptomyces.

Streptomycetes are multicellular filamentous microorganisms, and major producers of industrial enzymes and bioactive compounds such as antibiotics and anticancer drugs. The mycelial lifestyle plays an important role in the productivity during industrial fermentations. The hyphae of liquid-grown streptomycetes can self-aggregate into pellets, which hampers their industrial exploitation. Here we show that the Mat complex, which is required for pellet formation, catalyzes the synthesis of extracellular poly-ß-1,6-N-acetylglucosamine (PNAG) in the model organisms Streptomyces coelicolor and Streptomyces lividans. Extracellular accumulation of PNAG allows Streptomyces to attach to hydrophilic surfaces, while attachment to hydrophobic surfaces requires a cellulase-degradable extracellular polymer (EPS) produced by CslA. Over-expression of matAB was sufficient to restore pellet formation to cslA null mutants of S. lividans. The two EPS systems together increase the robustness of mycelial pellets. These new insights allow better control of liquid-culture morphology of streptomycetes, which may be harnessed to improve growth and industrial exploitation of these highly versatile natural product and enzyme producers.

Microencapsulation extends mycelial viability of Streptomyces lividans 66 and increases enzyme production.

BACKGROUND: Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics. RESULTS: Here, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth. CONCLUSIONS: Our data demonstrate the utility of micro-encapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of streptomycetes.

SParticle, an algorithm for the analysis of filamentous microorganisms in submerged cultures.

Streptomycetes are filamentous bacteria that produce a plethora of bioactive natural products and industrial enzymes. Their mycelial lifestyle typically results in high heterogeneity in bioreactors, with morphologies ranging from fragments and open mycelial mats to dense pellets. There is a strong correlation between morphology and production in submerged cultures, with small and open mycelia favouring enzyme production, while most antibiotics are produced mainly in pellets. Here we describe SParticle, a Streptomyces Particle analysis method that combines whole slide imaging with automated image analysis to characterize the morphology of submerged grown Streptomyces cultures. SParticle allows the analysis of over a thousand particles per hour, offering a high throughput method for the imaging and statistical analysis of mycelial morphologies. The software is available as a plugin for the open source software ImageJ and allows users to create custom filters for other microbes. Therefore, SParticle is a widely applicable tool for the analysis of filamentous microorganisms in submerged cultures.

Morphology-driven downscaling of Streptomyces lividans to micro-cultivation.

Actinobacteria are prolific producers of secondary metabolites and industrially relevant enzymes. Growth of these mycelial micro-organisms in small culture volumes is challenging due to their complex morphology. Since morphology and production are typically linked, scaling down culture volumes requires better control over morphogenesis. In larger scale platforms, ranging from shake flasks to bioreactors, the hydrodynamics play an important role in shaping the morphology and determining product formation. Here, we report on the effects of agitation on the mycelial morphology of Streptomyces lividans grown in microtitre plates. Our work shows that at the appropriate agitation rates cultures can be scaled down to volumes as small as 100 µl while maintaining the same morphology as seen in larger scale platforms. Using image analysis and principal component analysis we compared the morphologies of the cultures; when agitated at 1400-1600 rpm the mycelial morphology in micro-cultures was similar to that obtained in shake flasks, while product formation was also maintained. Our study shows that the morphology of actinobacteria in micro-cultures can be controlled in a similar manner as in larger scale cultures by carefully controlling the mixing rate. This could facilitate high-throughput screening and upscaling.

Aggregation of germlings is a major contributing factor towards mycelial heterogeneity of Streptomyces.

Streptomycetes are filamentous bacteria that produce numerous valuable compounds, including the majority of clinically used antibiotics. At an industrial scale, most of these compounds are produced in bioreactors. Growth of streptomycetes under these conditions is characterized by the formation of complex mycelial particles, whose sizes follow a bimodal distribution. Given the correlation between specific productivity and morphology, this size heterogeneity poses a potential drawback in industry. Recent work indicates that mycelial morphology is controlled by a number of genes that encode proteins required for the synthesis of cell surface-associated glycans. Using a quantifiable system based on fluorescent markers, we here show that these glycans mediate aggregation between germlings and young mycelia, yielding mycelial particles that originate from many different individuals. We also demonstrate that at later time points aggregation between distinct particles is no longer detectable. Notably, the absence of the corresponding glycan synthases yields mycelia that are homogeneous in size, identifying mycelial aggregation as a driving factor towards size heterogeneity. Given that aggregation is widespread within streptomycetes and can also occur between different Streptomyces strains, our work paves the way to improve Streptomyces as a cell factory for the production of known metabolites, but possibly also to discover new ones.

A novel locus for mycelial aggregation forms a gateway to improved Streptomyces cell factories.

BACKGROUND: Streptomycetes produce a plethora of natural products including antibiotics and anticancer drugs, as well as many industrial enzymes. Their mycelial life style is a major bottleneck for industrial exploitation and over decades strain improvement programs have selected production strains with better growth properties. Uncovering the nature of the underlying mutations should allow the ready transfer of desirable traits to other production hosts. RESULTS: Here we report that the mat gene cluster, which was identified through reverse engineering of a non-pelleting mutant selected in a chemostat, is key to pellet formation of Streptomyces lividans. Deletion of matA or matB, which encode putative polysaccharide synthases, effects mycelial metamorphosis, with very small and open mycelia. Growth rate and productivity of the matAB null mutant were increased by over 60% as compared to the wild-type strain. CONCLUSION: Here, we present a way to counteract pellet formation by streptomycetes, which is one of the major bottlenecks in their industrial application. The mat locus is an ideal target for rational strain design approaches aimed at improving streptomycetes as industrial production hosts.

Morphogenesis of Streptomyces in submerged cultures.

Members of the genus Streptomyces are mycelial bacteria that undergo a complex multicellular life cycle and propagate via sporulation. Streptomycetes are important industrial microorganisms, as they produce a plethora of medically relevant natural products, including the majority of clinically important antibiotics, as well as a wide range of enzymes with industrial application. While development of Streptomyces in surface-grown cultures is well studied, relatively little is known of the parameters that determine morphogenesis in submerged cultures. Here, growth is characterized by the formation of mycelial networks and pellets. From the perspective of industrial fermentations, such mycelial growth is unattractive, as it is associated with slow growth, heterogeneous cultures, and high viscosity. Here, we review the current insights into the genetic and environmental factors that determine mycelial growth and morphology in liquid-grown cultures. The genetic factors include cell-matrix proteins and extracellular polymers, morphoproteins with specific roles in liquid-culture morphogenesis, with the SsgA-like proteins as well-studied examples, and programmed cell death. Environmental factors refer in particular to those dictated by process engineering, such as growth media and reactor set-up. These insights are then integrated to provide perspectives as to how this knowledge can be applied to improve streptomycetes for industrial applications.

Functional characterization of a Penicillium chrysogenum mutanase gene induced upon co-cultivation with Bacillus subtilis.

BACKGROUND: Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis. RESULTS: Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative α-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active α-1,3 endoglucanase. CONCLUSION: Time-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding α-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides.