A metabolite profiling study of the antibiotic producing bacterium Streptomyces coelicolor A3(2) has been performed. The aim of this study was to monitor intracellular metabolite pool changes occurring as strains of S. coelicolor react to nutrient depletion with metabolic re-modeling, so-called metabolic switching, and transition from growth to secondary metabolite production phase. Two different culture media were applied, providing depletion of the key nutrients phosphate and L-glutamate, respectively, as the triggers for metabolic switching. Targeted GC-MS and LC-MS methods were employed to quantify important primary metabolite groups like amino acids, organic acids, sugar phosphates and other phosphorylated metabolites, and nucleotides in time-course samples withdrawn from fully-controlled batch fermentations. A general decline, starting already in the early growth phase, was observed for nucleotide pools and phosphorylated metabolite pools for both the phosphate and glutamate limited cultures. The change in amino acid and organic acid pools were more scattered, especially in the phosphate limited situation while a general decrease in amino acid and non-amino organic acid pools was observed in the L-glutamate limited situation. A phoP deletion mutant showed basically the same metabolite pool changes as the wild-type strain M145 when cultivated on phosphate limited medium. This implies that the inactivation of the phoP gene has only little effect on the detected metabolite levels in the cell. The energy charge was found to be relatively constant during growth, transition and secondary metabolite production phase. The results of this study and the employed targeted metabolite profiling methodology are directly relevant for the evaluation of precursor metabolite and energy supply for both natural and heterologous production of secondary metabolites in S. coelicolor.

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).

Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42 %, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates.

Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast Yarrowia lipolytica for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD ⁺ –dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism Y. lipolytica for competitive industrial-scale production of itaconic acid.

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.