Jean Franco Castro

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

ABSTRACT Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.

Acidithiobacillus ferrooxidans's comprehensive model driven analysis of the electron transfer metabolism and synthetic strain design for biomining applications

Acidithiobacillus ferrooxidans is a gram-negative chemolithoautotrophic γ-proteobacterium. It typically grows at an external pH of 2 using the oxidation of ferrous ions by oxygen, producing ferric ions and water, while fixing carbon dioxide from the environment. A. ferrooxidans is of great interest for biomining and environmental applications, as it can process mineral ores and alleviate the negative environmental consequences derived from the mining processes. In this study, the first genome-scale metabolic reconstruction of A. ferrooxidans ATCC 23270 was generated (iMC507). A total of 587 metabolic and transport/exchange reactions, 507 genes and 573 metabolites organized in over 42 subsystems were incorporated into the model. Based on a new genetic algorithm approach, that integrates flux balance analysis, chemiosmotic theory, and physiological data, the proton translocation stoichiometry for a number of enzymes and maintenance parameters under aerobic chemolithoautotrophic conditions using three different electron donors were estimated. Furthermore, a detailed electron transfer and carbon flux distributions during chemolithoautotrophic growth using ferrous ion, tetrathionate and thiosulfate were determined and reported. Finally, 134 growth-coupled designs were calculated that enables Extracellular Polysaccharide production. iMC507 serves as a knowledgebase for summarizing and categorizing the information currently available for A. ferrooxidans and enables the understanding and engineering of Acidithiobacillus and similar species from a comprehensive model-driven perspective for biomining applications.

Streptomyces aridus sp. nov., isolated from a high altitude Atacama Desert soil and emended description of Streptomyces noboritoensis Isono et al. 1957

A polyphasic study was undertaken to determine the taxonomic status of a Streptomyces strain which had been isolated from a high altitude Atacama Desert soil and shown to have bioactive properties. The strain, isolate H9T, was found to have chemotaxonomic, cultural and morphological properties that place it in the genus Streptomyces. 16S rRNA gene sequence analyses showed that the isolate forms a distinct branch at the periphery of a well-delineated subclade in the Streptomyces 16S rRNA gene tree together with the type strains of Streptomyces crystallinus, Streptomyces melanogenes and Streptomyces noboritoensis. Multi-locus sequence analysis (MLSA) based on five house-keeping gene alleles showed that isolate H9T is closely related to the latter two type strains and to Streptomyces polyantibioticus NRRL B-24448T. The isolate was distinguished readily from the type strains of S. melanogenes, S. noboritoensis and S. polyantibioticus using a combination of phenotypic properties. Consequently, the isolate is considered to represent a new species of Streptomyces for which the name Streptomyces aridus sp. nov. is proposed; the type strain is H9T (=NCIMB 14965T=NRRL B65268T). In addition, the MLSA and phenotypic data show that the S. melanogenes and S. noboritoensis type strains belong to a single species, it is proposed that S. melanogenes be recognised as a heterotypic synonym of S. noboritoensis for which an emended description is given.

Analysis of metabolic networks of Streptomyces leeuwenhoekii C34 by means of a genome scale model: Prediction of modifications that enhance the production of specialized metabolites

The first genome scale model (GSM) for Streptomyces leeuwenhoekii C34 was developed to study the biosynthesis pathways of specialized metabolites and to find metabolic engineering targets for enhancing their production. The model, iVR1007, consists of 1,722 reactions, 1,463 metabolites, and 1,007 genes, it includes the biosynthesis pathways of chaxamycins, chaxalactins, desferrioxamines, ectoine, and other specialized metabolites. iVR1007 was validated using experimental information of growth on 166 different sources of carbon, nitrogen and phosphorous, showing an 83.7% accuracy. The model was used to predict metabolic engineering targets for enhancing the biosynthesis of chaxamycins and chaxalactins. Gene knockouts, such as sle03600 (L‐homoserine O‐acetyltransferase), and sle39090 (trehalose‐phosphate synthase), that enhance the production of the specialized metabolites by increasing the pool of precursors were identified. Using the algorithm of flux scanning based on enforced objective flux (FSEOF) implemented in python, 35 and 25 over‐expression targets for increasing the production of chaxamycin A and chaxalactin A, respectively, that were not directly associated with their biosynthesis routes were identified. Nineteen over‐expression targets that were common to the two specialized metabolites studied, like the over‐expression of the acetyl carboxylase complex (sle47660 (accA) and any of the following genes: sle44630 (accA_1) or sle39830 (accA_2) or sle27560 (bccA) or sle59710) were identified. The predicted knockouts and over‐expression targets will be used to perform metabolic engineering of S. leeuwenhoekii C34 and obtain overproducer strains.

Geodermatophilus chilensis sp. nov., from soil of the Yungay core-region of the Atacama Desert, Chile

A polyphasic study was undertaken to establish the taxonomic status of three representative Geodermatophilus strains isolated from an extreme hyper-arid Atacama Desert soil. The strains, isolates B12T, B20 and B25, were found to have chemotaxonomic and morphological properties characteristic of the genus Geodermatophilus. The isolates shared a broad range of chemotaxonomic, cultural and physiological features, formed a well-supported branch in the Geodermatophilus 16S rRNA gene tree in which they were most closely associated with the type strain of Geodermatophilus obscurus. They were distinguished from the latter by BOX-PCR fingerprint patterns and by chemotaxonomic and other phenotypic properties. Average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between the whole genome sequences of isolate B12T and G. obscurus DSM 43160T were 89.28%, 87.27% and 37.4%, respectively, metrics consistent with its classification as a separate species. On the basis of these data, it is proposed that the isolates be assigned to the genus Geodermatophilus as Geodermatophilus chilensis sp. nov. with isolate B12T (CECT 9483T=NCIMB 15089T) as the type strain. Analysis of the whole genome sequence of G. chilensis B12T with 5341 open reading frames and a genome size of 5.5Mb highlighted genes and gene clusters that encode for properties relevant to its adaptation to extreme environmental conditions prevalent in extreme hyper-arid Atacama Desert soils.

Blastococcus atacamensis sp. nov., a novel strain adapted to life in the Yungay core region of the Atacama Desert

A polyphasic study was undertaken to establish the taxonomic status of a Blastococcus strain isolated from an extreme hyper-arid Atacama Desert soil. The isolate, strain P6T, was found to have chemotaxonomic and morphological properties consistent with its classification in the genus Blastococcus. It was shown to form a well-supported branch in the Blastococcus 16S rRNA gene tree together with the type strains of Blastococcus capsensis and Blastococcus saxobsidens and was distinguished from the latter, its close phylogenetic neighbour, by a broad range of phenotypic properties. The draft genome sequence of isolate P6T showed 84.6 % average nucleotide identity, 83.0 % average amino acid identity and a digital DNA-DNA hybridisation value of 27.8 % in comparison with the genome sequence of B. saxobsidens DSM 44509T, values consistent with its assignment to a separate species. Based on these data it is proposed that isolate P6T (NCIMB 15090T=NRRL B-65468T) be assigned to the genus Blastococcus as Blastococcus atacamensis sp. nov. Analysis of the whole genome sequence of B. atacamensis P6T, with 3778 open reading frames and a genome size of 3.9 Mb showed the presence of genes and gene clusters that encode for properties that reflect its adaptation to the extreme environmental conditions that prevail in Atacama Desert soils.

The ‘gifted’ actinomycete Streptomyces leeuwenhoekii

Streptomyces leeuwenhoekii strains C34T, C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34T.

Metabolic modelling and flux analysis of microorganisms from the Atacama Desert used in biotechnological processes

Metabolic modelling is a useful tool that enables the rational design of metabolic engineering experiments and the study of the unique capabilities of biotechnologically important microorganisms. The extreme abiotic conditions of the Atacama Desert have selected microbial diversity with exceptional characteristics that can be applied in the mining industry for bioleaching processes and for production of specialised metabolites with antimicrobial, antifungal, antiviral, antitumoral, among other activities. In this review we summarise the scientific data available of the use of metabolic modelling and flux analysis to improve the performance of Atacama Desert microorganisms in biotechnological applications.