Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov., isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Central Andes in Chile.

Two novel Micromonospora strains, STR1-7T and STR1S-6T, were isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Atacama Desert, Chile. Chemotaxonomic, cultural and phenotypic features confirmed that the isolates belonged to the genus Micromonospora. They grew from 20 to 37 °C, from pH7 to 8 and in the presence of up to 3 %, w/v NaCl. The isolates formed distinct branches in Micromonospora gene trees based on 16S rRNA gene sequences and on a multi-locus sequence analysis of conserved house-keeping genes. A phylogenomic tree generated from the draft genomes of the isolates and their closest phylogenetic neighbours showed that isolate STR1-7T is most closely related to Micromonospora orduensis S2509T, and isolate STR1S-6 T forms a distinct branch that is most closely related to 12 validly named Micromonospora species, including Micromonospora saelicesensis the earliest proposed member of the group. The isolates were separated from one another and from their closest phylogenomic neighbours using a combination of chemotaxonomic, genomic and phenotypic features, and by low average nucleotide index and digital DNA-DNA hybridization values. Consequently, it is proposed that isolates STR1-7T and STR1S-6T be recognized as representing new species in the genus Micromonospora, namely as Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov.; the type strains are STR1-7T (=CECT 9665T=LMG 30768T) and STR1S-6T (=CECT 9666T=LMG 30770T), respectively. Genome mining showed that the isolates have the capacity to produce novel specialized metabolites, notably antibiotics and compounds that promote plant growth, as well as a broad-range of stress-related genes that provide an insight into how they cope with harsh abiotic conditions that prevail in high-altitude Atacama Desert soils.

Mutactimycin AP, a New Mutactimycin Isolated from an Actinobacteria from the Atacama Desert.

Bacteria belonging to the phylum Actinobacteria are a very good source of antibiotics, and indeed dominate the current clinical antibiotic space. This paper reports Mutactimycin AP, a new compound belonging to an anthracycline-type family of antibiotics, isolated from a Saccharothrix sp. This actinobacterial strain was isolated from the rhizosphere of lupine plants growing in the extreme hyper-arid Atacama Desert. Structural characterization was carried out using electrospray ionization-mass spectrometry (ESI-MS) and NMR spectroscopy in combination with molecular modelling. The compound was tested against the ESKAPE pathogens, where it showed activity against MRSA and five strains associated with bovine mastitis, where it showed activity against Enterococcus pseudoavium and Staphylycoccus Aureus subsp. Aureus.

Cutting the Gordian knot: early and complete amino acid sequence confirmation of class II lasso peptides by HCD fragmentation.

Lasso peptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Their proteolytic and thermal stability alongside their growing potential as therapeutics has increased attention to these antimicrobial peptides. With the advent of genome mining, the discovery of RiPPs allows for the accurate prediction of putatively encoded structures, however, MSn experiments only provide partial sequence confirmation, therefore 2D NMR experiments are necessary for characterisation. Multiple MS/MS techniques were applied to two structurally characterized lasso peptides, huascopeptin and leepeptin, and one uncharacterized lasso peptide, citrulassin C, which was not isolable in sufficient quantity for NMR analysis. We have shown that MS2 can be used to elucidate the full amino acid sequences previously predicted with genome mining for this compound class. HCD was able to open the macrocycles and fragment the newly opened linear peptides, confirming the complete amino acid sequences of the characterised lasso peptides. In addition, to determine if this technique could be applied at the earliest stages of the isolation process, we targeted a lasso peptide found by genome mining, citrulassin C, and were able to fully elucidate the amino acid sequence using only MS2 from a semi-crude extract of Streptomyces huasconensis HST28T.

Downsizing Class II Lasso Peptides: Genome Mining-Guided Isolation of Huascopeptin Containing the First Gly1-Asp7 Macrocycle.

A new lasso peptide, huascopeptin, was isolated following genome-mined discovery of a new biosynthetic gene cluster in extremotolerant Streptomyces huasconensis HST28T from Salar de Huasco, Atacama Desert, Chile. Compound 1 is a 13-residue class II lasso peptide containing a novel Gly1-Asp7 macrolactam ring, a three-residue loop, and a three-residue tail, making it the smallest lasso peptide isolated to date. The lasso structure was confirmed using NOE restraint-based molecular dynamics simulations.

Atacama Database: a platform of the microbiome of the Atacama Desert.

The Atacama Desert is one of the oldest and driest places on Earth. In the last decade, microbial richness and diversity has been acknowledged as an important biological resource of this region. Owing to the value of the microbial diversity apparent in potential biotechnology applications and conservation purposes, it is necessary to catalogue these microbial communities to promote research activities and help to preserve the wide range of ecological niches of the Atacama region. A prototype Atacama Database has been designed and it provides a description of the rich microbial diversity of the Atacama Desert, and helps to visualise available literature resources. Data has been collected, curated, and organised into several categories to generate a single record for each organism in the database that covers classification, isolation metadata, morphology, physiology, genome and metabolism information. The current version of Atacama Database contains 2302 microorganisms and includes cultured and uncultured organisms retrieved from different environments within the desert between 1984 and 2016. These organisms are distributed in bacterial, archaeal or eukaryotic domains, along with those that are unclassified taxonomically. The initial prototype of the Atacama Database includes a basic search and taxonomic and advanced search tools to allow identification and comparison of microbial populations, and space distribution within this biome. A geolocation search was implemented to visualise the microbial diversity of the ecological niches defined by sectors and extract general information of the sampling sites. This effort will aid understanding of the microbial ecology of the desert, microbial population dynamics, seasonal behaviour, impact of climate change over time, and reveal further biotechnological applications of these microorganisms. The Atacama Database is freely available at: https://www.atacamadb.cl.

Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin.

Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

Micromonospora acroterricola sp. nov., a novel actinobacterium isolated from a high altitude Atacama Desert soil.

A Micromonospora strain, designated 5R2A7T, isolated from a high altitude Atacama Desert soil was examined by using a polyphasic approach. Strain 5R2A7T was found to have morphological, chemotaxonomic and cultural characteristics typical of members of the genus Micromonospora. The cell wall contains meso- and hydroxy-diaminopimelic acid, the major whole-cell sugars are glucose, ribose and xylose, the predominant menaquinones MK-10(H4), MK-10(H6), MK-10(H8) and MK-9(H6), the major polar lipids diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown glycolipid, and the predominant cellular fatty acids iso-C16 : 0, iso-C15 : 0 and 10-methyl C17 : 0. The digital genomic DNA G+C content is 72.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain 5R2A7T was closely related to Micromonospora coriariae DSM 44875T (99.8 %) and Micromonospora cremea CR30T (99.7 %), and was separated readily from the latter, its closest phylogenetic neighbour, based on gyrB and multilocus sequence data, by low average nucleotide identity (92.59 %) and in silico DNA-DNA relatedness (51.7 %) values calculated from draft genome assemblies and by a range of chemotaxonomic and phenotypic properties. Consequently, strain 5R2A7T is considered to represent a novel species of Micromonospora for which the name Micromonospora acroterricola sp. nov. is proposed. The type strain is 5R2A7T (=LMG 30755T=CECT 9656T).

New genus-specific primers for PCR identification of Rubrobacter strains.

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes.

Streptomyces altiplanensis sp. nov., an alkalitolerant species isolated from Chilean Altiplano soil, and emended description of Streptomyces chryseus (Krasil'nikov et al. 1965) Pridham 1970.

A polyphasic approach was used for evaluating the taxonomic status of strain HST21T isolated from Salar de Huasco in the Atacama Desert. The results of 16S rRNA gene and multilocus sequence phylogenetic analyses assigned strain HST21T to the genus Streptomyceswith Streptomyces albidochromogenes DSM 41800Tand Streptomyces flavidovirens DSM 40150T as its nearest neighbours. Digital DNA-DNA hydridization (dDDH) and average nucleotide identity (ANI) values between the genome sequences of strain HST21T and S. albidochromogenes DSM 41800T (35.6 and 88.2 %) and S. flavidovirens DSM 40105T (47.2 and 88.8 %) were below the thresholds of 70  and 95-96 % for prokaryotic conspecific assignation. Phenotypic, chemotaxonomic and genetic results distinguished strain HST21T from its closest neighbours. Strain HST21T is characterized by the presence of ll-diaminopimelic acid in its peptidoglycan layer; glucose and ribose as whole cell sugars; diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, glycophospholipids, unknown lipids and phospholipids as polar lipids; and anteiso-C15 : 0 (21.6 %) and anteiso-C17 : 0 (20.5 %) as major fatty acids (>15 %). Based on these results, strain HST21T merits recognition as a novel species, for which the name Streptomyces altiplanensis sp. nov. is proposed. The type strain is HST21T=DSM 107267T=CECT 9647T. While analysing the phylogenies of strain HST21T, Streptomyces chryseus DSM 40420T and Streptomyces helvaticus DSM 40431T were found to have 100 % 16S rRNA gene sequence similarity with digital DNA-DNA hydridization (dDDH) and average nucleotide identity (ANI) values of 95.3 and 99.4 %, respectively. Therefore, S. helvaticus is considered as a later heterotypic synonym of S. chryseus and, consequently, we emend the description of S. chryseus.

Streptomyces huasconensis sp. nov., an haloalkalitolerant actinobacterium isolated from a high altitude saline wetland at the Chilean Altiplano.

Streptomyces strain HST28T isolated from the Salar de Huasco, an athalassohaline and poly-extreme high altitude saline wetland located in northern Chile, was the subject of a polyphasic taxonomic study. Strain HST28T showed morphological and chemotaxonomic features in line with its classification in the genus Streptomyces. Optimal growth of strain HST28T was obtained at 28 °C, pH 8-9 and up to 10 % (w/v) NaCl. Single (16S rRNA) and multi-locus gene sequence analyses showed that strain HST28T had a distinct phylogenetic position from its closest relatives, the type strains of Steptomyces aureus and Streptomyces kanamyceticus. Digital DNA-DNA hybridization (23.3 and 31.0 %) and average nucleotide identity (79.3 and 85.6 %) values between strain HST28T and its corresponding relatives mentioned above were below the threshold of 70 and 96 %, respectively, defined for assigning a prokaryotic strains to the same species. Strain HST28T was characterised by the presence of ll-diaminopimelic acid in its peptidoglycan layer; galactose, glucose, ribose and traces of arabinose and mannose as whole-cell sugars; phosphatidylmethylethanolamine, phosphatidylinositol, aminolipid, glycophospholipid and an unidentified lipid as polar lipids; and the predominating menaquinones MK-9(H6), MK-9(H8) and MK-9(H4) (>20 %) as well as anteiso-C15 : 0 and anteiso-C17 : 0 as major fatty acids (>15 %). Based on the phenotypic and genetic results, strain HST28T (DSM 107268T=CECT 9648T) merits recognition as a new species named Streptomyces huasconensis sp. nov.

Use of genome-scale models to get new insights into the marine actinomycete genus Salinispora.

BACKGROUND: There is little published regarding metabolism of Salinispora species. In continuation with efforts performed towards this goal, this study is focused on new insights into the metabolism of the three-identified species of Salinispora using constraints-based modeling. At present, only one manually curated genome-scale metabolic model (GSM) for Salinispora tropica strain CNB-440T has been built despite the role of Salinispora strains in drug discovery. RESULTS: Here, we updated, and expanded the scope of the model of Salinispora tropica CNB-440T, and GSMs were constructed for two sequenced type strains covering the three-identified species. We also constructed a Salinispora core model that contains the genes shared by 93 sequenced strains and a few non-conserved genes associated with essential reactions. The models predicted no auxotrophies for essential amino acids, which was corroborated experimentally using a defined minimal medium (DMM). Experimental observations suggest possible sulfur accumulation. The Core metabolic content shows that the biosynthesis of specialised metabolites is the less conserved subsystem. Sets of reactions were analyzed to explore the differences between the reconstructions. Unique reactions associated to each GSM were mainly due to genome sequence data except for the ST-CNB440 reconstruction. In this case, additional reactions were added from experimental evidence. This reveals that by reaction content the ST-CNB440 model is different from the other species models. The differences identified in reaction content between models gave rise to different functional predictions of essential nutrient usage by each species in DMM. Furthermore, models were used to evaluate in silico single gene knockouts under DMM and complex medium. Cluster analysis of these results shows that ST-CNB440, and SP-CNR114 models are more similar when considering predicted essential genes. CONCLUSIONS: Models were built for each of the three currently identified Salinispora species, and a core model representing the conserved metabolic capabilities of Salinispora was constructed. Models will allow in silico metabolism studies of Salinispora strains, and help researchers to guide and increase the production of specialised metabolites. Also, models can be used as templates to build GSMs models of closely related organisms with high biotechnology potential.

Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the Antarctic bacteria Pseudoalteromonas sp. 2-3.

α-Amylase is an endo-acting enzyme which catalyzes random hydrolysis of starch. These enzymes are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. The use of active enzymes at low temperatures has a high potential because these enzymes would avoid the demand for heating during the process thereby reducing costs. In this work, the gene of α-amylase from Pseudoalteromonas sp. 2-3 (Antarctic bacteria) has been sequenced and expressed in Escherichia coli BL21(DE3). The ORF of the α-amylase gene cloned into pET22b(+) is 1824 bp long and codes for a protein of 607 amino acid residues including a His6-tag. The mature protein has a calculated molecular mass of 68.8 kDa. Recombinant α-amylase was purified with Ni-NTA affinity chromatography. The purified enzyme is active on potato starch with a Km of 6.94 mg/ml and Vmax of 0.27 mg/ml*min. The pH optimum is 8.0 and the optimal temperature is 20 °C. This enzyme was strongly activated by Ca2+; results consistent with other α-amylases. To the best of our knowledge, this enzyme has the lowest temperature optimum so far reported for α-amylases.

Discovery of Half-life of Circulating Hepatitis B Surface Antigen in Patients With Chronic Hepatitis B Infection Using Heavy Water Labeling.

In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. The mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which reflects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients.

Treatment of hepatitis C with direct-acting antivirals significantly reduces liver-related hospitalizations in patients with cirrhosis.

BACKGROUND: The availability of direct-acting antivirals (DAA) for the treatment of hepatitis C (HCV) has resulted in the ability to safely and effectively treat patients with cirrhosis and end-stage liver disease. However, information is limited with regard to the impact of DAA treatment on inpatient health-related resource utilization in patients with advanced HCV-related cirrhosis. We aimed to ascertain the impact of DAA treatment on the frequency of liver-related hospitalizations and associated costs in patients with cirrhosis. PATIENTS AND METHODS: Retrospective cohort analysis carried out at a single US reference center that compared patients with HCV cirrhosis according to treatment status: the untreated group (January 2011 to December 2013) and the DAA-treated group (January 2014 to March 2017). The primary outcome was the difference in the incidence rate of liver-related hospitalizations. Secondary outcomes included differences in the incidence of hepatocellular carcinoma, liver transplant, and all-cause mortality. We calculated the projected savings per-patient treated per-year on the basis of calculated hospitalization rate stratified by Child-Turquotte-Pugh (CTP) score. RESULTS: Baseline characteristics were similar between the untreated (n=182) and DAA-treated (n=196) cohorts. Mean follow-up time in the untreated and treated cohort was 20.4 and 17.7 months, respectively. The incidence rates of liver-related hospitalizations were 29.1/100 and 10.4/100 person-years of follow-up (P≤0.0001) in the untreated and treated cohorts, respectively. This was accounted for by a decreased incidence of hospitalizations in patients with CTP-A (75.8%) and CTP-B (64.5%), but not CTP-C. CONCLUSION: Successful DAA treatment reduces hospitalization rate and resource utilization costs in patients with CTP-A and CTP-B, but not in those with CTP-C.

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.

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.

Introducing the Atacama Desert.

This brief introduction is intended to orientate the reader with respect to the principal environmental and historical features of the Atacama Desert, the oldest and continuously driest non-polar temperate desert on Earth. Exploration of its microbiology is relatively recent but both fundamental and applied research activities have grown dramatically in recent years reflecting the substantial interest in its microbial diversity, ecology, biogeochemistry, natural product potential and Mars-analogue properties of this unique and invigorating environment.

Relevance of Local Flexibility Near the Active Site for Enzymatic Catalysis: Biochemical Characterization and Engineering of Cellulase Cel5A From Bacillus agaradherans.

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations.

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.