The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating -OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J 2 K 2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the -OH of the tyrosyl moiety to yield the tethered -ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile -ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity.

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations- electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system's capabilities through CRISPR-based engineering of two test cases- 1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

34Bacteria often possess alternative sigma factors that initiate the transcription of 35 specific genes under environmental stresses, the largest and most diverse group being 36 the extracytoplasmic function (ECF) sigma factors. The regulation of ECF activity is 37 crucial for ensuring the distinct transcription of stress responsive genes only occurs 38 under the appropriate conditions. While most ECFs are comprised of only the core σ 2 39 and σ 4 regions, a unique form of ECF sigma factor regulation also contains a C-terminal 40 extension bearing homology to the NTF2 superfamily of protein domains. While 41 previous work has shown that this NTF2 domain can affect transcriptional activity in vivo 42 in ECF41 and ECF42, its role in the newly classified ECF56 subgroup is unknown. In 43 this work, we show that truncation of the C-terminus of the ECF56 sigma factor 44 SVEN_4562 of Streptomyces venezuelae upregulates its activity in a hybrid assay. 45 Through transcriptomics in S. venezuelae, we found that this truncated ECF56 sigma 46 factor has a highly conserved promoter sequence in vivo. Bioinformatic assays 47 ECF56 family of sigma factors is highly conserved and performs important functions yet 53 to be understood in Actinobacteria. 54 55 Importance 56 Most ECF sigma-factors rely on anti-sigma factor regulation; in contrast, the 57 unique classes of ECF sigma-factors that contain a C-terminal extension are thought to 58 respond directly to an environmental signal. Here we show that the cis-acting regulatory 59 element of the ECF56 regulon is likely highly conserved in many Actinobacteria, with 60 exact nucleotide level conservation over ~2 billion years of evolution. The high 61 conservation of this genetic architecture, as well as a conserved gene content within the 62 regulon, strongly point to a specialized and important role in Actinobacteria biology. 63 64