Sensitive Methods to Detect Single-Stranded Nucleic Acids of Food Pathogens Based on Cell-Free Protein Synthesis and Retroreflection Signal Detection.

Cell-free protein synthesis (CFPS) has recently gained considerable attention as a new platform for developing methods to detect various molecules, ranging from small chemicals to biological macromolecules. Retroreflection has been used as an alternative signal to develop analytical methods because it can be detected by using a simple instrument comprising a white light source and a camera. Here, we report a novel reporter protein that couples the capability of CFPS and the simplicity of retroreflection signal detection. The design of the reporter was based on two pairs of protein-peptide interactions, SpyCatcher003-SpyTag003 and MDM2-PMI(N8A). MDM2-MDM2-SpyCatcher003 was decided as the reporter protein, and the two peptides, SpyTag003 and PMI(N8A), were immobilized on the surfaces of retroreflective Janus particles and microfluidic chips, respectively. The developed retroreflection signal detection system was combined with a previously reported CFPS reaction that can transduce the presence of a single-stranded nucleic acid into protein synthesis. The resulting methods were applied to detect 16S rRNAs of several foodborne pathogens. Concentration-dependent relationships were observed over a range of 10° fM to 102 pM, with the limits of detection being single-digit femtomolar concentrations. Considering the designability of the CFPS system for other targets, the retroreflection signal detection method will enable the development of novel methods to detect various molecules.

Assay methods based on proximity-enhanced reactions for detecting non-nucleic acid molecules.

Accurate and reliable detection of biological molecules such as nucleic acids, proteins, and small molecules is essential for the diagnosis and treatment of diseases. While simple homogeneous assays have been developed and are widely used for detecting nucleic acids, non-nucleic acid molecules such as proteins and small molecules are usually analyzed using methods that require time-consuming procedures and highly trained personnel. Recently, methods using proximity-enhanced reactions (PERs) have been developed for detecting non-nucleic acids. These reactions can be conducted in a homogeneous liquid phase via a single-step procedure. Herein, we review three assays based on PERs for the detection of non-nucleic acid molecules: proximity ligation assay, proximity extension assay, and proximity proteolysis assay.

Engineering an aldehyde dehydrogenase toward its substrates, 3-hydroxypropanal and NAD+, for enhancing the production of 3-hydroxypropionic acid.

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD+ regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD+, were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).