SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and controller various physiological processes. Its availability also believed to be bottleneck in the biological production numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, SAM-dependent transcriptional regulator, core component. corepressor which suppresses MetJ promoter with increasing cellular concentration (SAM-OFF sensor). The application...

Arsenite [As(III)] is a toxic substance widely present on Earth, and the development of low-cost simple microbial-based As(III) sensors has been attracting attention. Recently, we discovered that protein LuxR, which contains multiple cysteine residues with high affinity for As(III), forms an insoluble structure upon binding to exhibits OFF-switching properties as quorum sensing transcriptional activator. Based this property, LuxR sensor operates new principle distinct from conventional...

The fusion of different proteins can result in the linkage-dependent emergence molecular switches. We inserted chloramphenicol acetyltransferase (CAT) from Escherichia coli into a loop d-fucose-responsive mutant transcription factor AraC, using linker libraries with various lengths. found that on-switches tend to emerge when two are fused linkers long enough fill gap distance residues be connected, while fusing short or zero results frequent off-switches. Both types switches rapidly evolved...

Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it challenging to evolve without sacrificing maximum output level. This problem also comes from absence truly tunable negative selection methods. paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated previously generated leaky LuxR, quorum-sensing transcription activator Vibrio fischeri,...

LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to signaling molecules acyl-homoserine lactones (acyl-HSLs). The function homologs is remarkably plastic, naturally occurring acyl-HSLs structurally diverse. To investigate molecular basis functional plasticity Vibrio fischeri LuxR, we directed evolution toward three different specificities laboratory. We found an orthogonal pair mutants specific...

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due lack systems that meets three requirements: low cost, safety, tight control, inducer molecules. Previously, we developed high-spec gene induction system controlled by safe cheap choline. However, requires relatively high concentration (~100 mM) choline fully induce under...

Abstract Binding-induced folding 1–4 (BIF) is a promising mechanism that can be used to rapidly convert binders into sensors/regulators without allosteric design. Here we showed regulatory proteins AraC acquire BIF compromising their inherent mechanisms, with high frequency upon mutations. This opened an opportunity compare the evolutionary capacity of and non-allosteric modes specific sensory protein. We found evolved novel function far more in mode than mode. newly acquired...

The cellular activities of gymnosperms monoterpene synthases are largely compromised due to their requirement for manganese, which is deficient in microbial cells. Through site-saturation mutagenesis the residue adjacent metal-binding glutamate, we found that pinene synthase highly mutable at this position yet drastically alter metal binding preference, thereby quickly improving performance heterologous hosts.

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in variety of biological functions. SAM also required for the biosynthesis valuable methylated compounds, but its supply bottleneck these biosynthetic pathways. To overcome this to reconfigure homeostasis, high-throughput sensing system changes intracellular availability required. We constructed plasmid can detect factors alter using minimal components. It does so by placing...

Abstract The fusion of different proteins can result in the linkage-dependent emergence molecular switches. In cases where allosteric regulation is designed between input and output modules proteins, it hard to predict whether on-switching or off-switching will occur. However, binding-induced folding, a non-allosteric switch mechanism, has potential quickly establish mutually regulatory relationship two fused way occur would be predictable. We inserted chloramphenicol acetyltransferase (CAT)...

Visualization and quantification of intracellular molecules mammalian cells are crucial steps in clinical diagnosis, drug development, basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium have limited reproducibility recovery efficiency. Here we present a microfluidic processor that can perform four-step exchange medium, simply by introducing suspension fluid reagents into...

In this report, the anther collected data observed through summer season of five months from May to September in thirty one observatory points Aomori prefecture 1949, defined number (in %) blowing “1/2(NE+SE)+ENE+ESE+E” wind as prevailing easterlies, and classified into three groups by x s easterlies these shown fig. 2, too, described charasteristics topographycal region belongs each group table 2.

Nanometre- to micrometre-sized solid dust particles play a vital role in star and planet formations. Despite of their importance, however, our understanding physical chemical properties is still provisional. We have conducted condensation experiment the vapour generated from starting material having nearly cosmic proportions elements. A laser flash heating subsequent cooling has produced diverse type nanoparticles simultaneously. Here we introduce four types as potential space: amorphous...

Abstract In Gegenwart des Dipeptids Z‐Leu‐His und anionischer Micellen aus Cetyltrimethylammoniumbromid wird bevorzugt das L‐Enantiomer Phenylalanats (Ia) hydrolysiert.