Coelimycin Synthesis Activatory Proteins Are Key Regulators of Specialized Metabolism and Precursor Flux in Streptomyces coelicolor A3(2).

Many microbial specialized metabolites are industrially relevant agents but also serve as signaling molecules in intra-species and even inter-kingdom interactions. In the antibiotic-producing Streptomyces, members of the SARP (Streptomyces antibiotic regulatory proteins) family of regulators are often encoded within biosynthetic gene clusters and serve as their direct activators. Coelimycin is the earliest, colored specialized metabolite synthesized in the life cycle of the model organism Streptomyces coelicolor A3(2). Deletion of its two SARP activators cpkO and cpkN abolished coelimycin synthesis and resulted in dramatic changes in the production of the later, stationary-phase antibiotics. The underlying mechanisms of these phenotypes were deregulation of precursor flux and quorum sensing, as shown by label-free, bottom-up shotgun proteomics. Detailed profiling of promoter activities demonstrated that CpkO is the upper-level cluster activator that induces CpkN, while CpkN activates type II thioesterase ScoT, necessary for coelimycin synthesis. What is more, we show that cpkN is regulated by quorum sensing gamma-butyrolactone receptor ScbR.

A GntR-Like Transcription Factor HypR Regulates Expression of Genes Associated With L-Hydroxyproline Utilization in Streptomyces coelicolor A3(2).

Bacteria from the genus Streptomyces have been long exploited as the most prolific producers of antibiotics, other secondary metabolites and enzymes. They are important members of soil microbial communities that can adapt to changing conditions thank to the fine regulation of gene expression in response to environmental signals. Streptomyces coelicolor A3(2) is a model organism for molecular studies with the most deeply recognized interactions within the complex metabolic and regulatory network. However, details about molecular signals recognized by specialized regulatory proteins as well as their direct targets are often missing. We describe here a zinc-binding protein HypR (SCO6294) which belongs to FadR subfamily of GntR-like regulators. The DNA sequence 5'-TACAATGTCAC-3' recognized by the HypR protein in its own promoter region was identified by DNase I footprinting. Binding of six DNA fragments containing similar sequences located in other promoter regions were confirmed by the electrophoretic mobility shift assay (EMSA). The sequences of 7 in vitro-determined binding sites were assembled to generate a logo of the HypR binding motif, 5'-CTNTGC(A/C)ATGTCAC-3'. Comparison of luciferase reporter genes expression under the control of cloned promoter regions in S. coelicolor A3(2) wild type and deletion mutant strains revealed, that the HypR protein acts as a repressor of its target genes. Genes belonging to the regulon of HypR code for enzymes putatively involved in collagen degradation and utilization of L-hydroxyproline (L-Hyp) as concluded from predicted structure and conserved domains. Their transcription is induced in the wild type strain by the addition of L-Hyp to the culture medium. Moreover, knockout of one of the genes from the predicted L-Hyp utilization operon abolished the ability of the strain to grow on L-Hyp as a sole source of carbon. To our knowledge, this work is the first indication of the existence of the pathway of L-hydroxyproline catabolism in Streptomycetes.

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains.

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in an S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, 'genetically unaltered' hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.

FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae.

Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can mediate RNA-guided DNA cleavage at targeted genomic loci in the popular model and industrial yeast Saccharomyces cerevisiae. FnCpf1 very efficiently and precisely promoted repair DNA recombination with efficiencies up to 100%. Furthermore, FnCpf1 was shown to introduce point mutations with high fidelity. While editing multiple loci with Cas9 is hampered by the need for multiple or complex expression constructs, processing itself a customized CRISPR array FnCpf1 was able to edit four genes simultaneously in yeast with a 100% efficiency. A remarkable observation was the unexpected, strong preference of FnCpf1 to cleave DNA at target sites harbouring 5'-TTTV-3' PAM sequences, a motif reported to be favoured by Cpf1 homologs of Acidaminococcus and Lachnospiraceae. The present study supplies several experimentally tested guidelines for crRNA design, as well as plasmids for FnCpf1 expression and easy construction of crRNA expression cassettes in S. cerevisiae. FnCpf1 proves to be a powerful addition to S. cerevisiae CRISPR toolbox.