Enhanced Production of Soluble Recombinant Proteins With an In Situ-Removable Fusion Partner in a Cell-Free Synthesis System.

High-yield production of soluble protein is a common concern in diverse fields of biotechnology. In this study, a strategy of using an engineered nucleotide sequence of ubiquitin for enhancing the production of soluble proteins in a cell-free synthesis system is presented. When examined for a series of proteins that otherwise were poorly expressed, N-terminal fusion with ubiquitin significantly increased both the expression levels and solubility of the translational products. The effect of ubiquitin fusion was also markedly augmented by engineering the nucleotide sequence of ubiquitin, leading to several fold enhancements in soluble production of target proteins. Recombinant proteins were produced with their native amino acid sequences through in situ removal of ubiquitin during cell-free synthesis reactions in the presence of a deubiquitinase. The presented strategy could be employed as a facile route to prepare soluble proteins required for various applications.

Cell-free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery.

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017.

Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

Production of 3-hydroxypropionic acid from glycerol by recombinant Pseudomonas denitrificans.

3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12 -dependent glycerol dehydratase and an NAD(P)(+) -dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol.

Cell-free platforms for flexible expression and screening of enzymes.

As was witnessed from PCR technology, in vitro applications of biosynthetic machinery can expand the horizon of biotechnology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocess. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables instant creation of protein molecules from diverse sources of genetic information. Enzyme discovery and engineering is the field of particular interest among the possible applications of cell-free protein synthesis since many of the intrinsic limitations associated with traditional cell-based expression screening of enzymes can be effectively addressed. Cell-free synthesis not only offers excellent throughput in the generation of enzymes, it allows facile integration of expression and analysis of enzymes, greatly accelerating the process of enzyme discovery. This review article is thus intended to survey recent progress in cell-free protein synthesis technology focused on its applications in enzyme expression and screening.

Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains.

Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reductase (MCR). The recombinant E. coli overexpressing mcr under the T5 promoter showed MCR activity of 0.015 U mg⁻¹ protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA carboxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding α-ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway.