Prospects of microbial cell factories developed through systems metabolic engineering.

While academic-level studies on metabolic engineering of microorganisms for production of chemicals and fuels are ever growing, a significantly lower number of such production processes have reached commercial-scale. In this work, we review the challenges associated with moving from laboratory-scale demonstration of microbial chemical or fuel production to actual commercialization, focusing on key requirements on the production organism that need to be considered during the metabolic engineering process. Metabolic engineering strategies should take into account techno-economic factors such as the choice of feedstock, the product yield, productivity and titre, and the cost effectiveness of midstream and downstream processes.

Single Walled Carbon Nanotube-Based Electrical Biosensor for the Label-Free Detection of Pathogenic Bacteria.

We herein describe the development of a single-walled carbon nanotube (SWNT)-based electrical biosensor consisting of a two-terminal resistor, and report its use for the specific, label-free detection of pathogenic bacteria via changes in conductance. The ability of this biosensor to recognize different pathogenic bacteria was analyzed, and conditions were optimized with different probe concentrations. Using this system, the reference strains and clinical isolates of Staphylococcus aureus and Escherichia coli were successfully detected; in both cases, the sensor showed a detection limit of 10 CFU.

Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota.

Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses.

Unlabelled: Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence of C.

Systems Metabolic Engineering of Escherichia coli.

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E.

Stable and enhanced gene expression in Clostridium acetobutylicum using synthetic untranslated regions with a stem-loop.

Gene overexpression is one of the most basic strategies in metabolic engineering, but the factors determining gene expression levels have been poorly studied in Clostridium species. In this study, we found that a short single-stranded 5' untranslated region (UTR) sequence led to decreased gene expression in Clostridium acetobutylicum. Using an in vitro enzyme assay and reverse transcription-quantitative PCR, we found that addition of a small stem-loop at the 5' end of mRNA increased mRNA levels and thereby protein expression levels up to 4.

Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites.

Covering: 2012 to 2016Metabolic engineering using systems biology tools is increasingly applied to overproduce secondary metabolites for their potential industrial production. In this Highlight, recent relevant metabolic engineering studies are analyzed with emphasis on host selection and engineering approaches for the optimal production of various prokaryotic secondary metabolites: native versus heterologous hosts (e.g.

Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.

Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA.

Production of succinic acid by metabolically engineered microorganisms.

Succinic acid (SA) has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. For the economical bio-based production of SA, extensive research works have been performed on developing microbial strains by metabolic engineering as well as fermentation and downstream processes. Here we review metabolic engineering strategies applied for bio-based production of SA using representative microorganisms, including Saccharomyces cerevisiae, Pichia kudriavzevii, Escherichia coli, Mannheimia succiniciproducens, Basfia succiniciproducens, Actinobacillus succinogenes, and Corynebacterium glutamicum.

Hemoglobin-mimetic oxygen adsorbent prepared via self-assembly of cysteinyl bolaamphiphiles.

In this study, a novel cysteinyl bolaamphiphilic molecule was synthesized and its self-assembled planar suprastructure was applied as a biomimetic matrix to create a hemoglobin-mimetic oxygen adsorbent that exploits the ability of cysteine thiols to bind hemin. Self-assembly of the cysteinyl bolaamphiphilic molecule exposed cysteine thiols on its surface in the presence of β-mercaptoethanol, known to reduce disulfide bonds, without which, helically coiled structures were generated. The self-assembled planar structure was used as a soft matrix to create a hemoglobin-mimetic oxygen adsorbent.

Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol.

Biocompatible chemistry is gaining increasing attention because of its potential within biotechnology for expanding the repertoire of biological transformations carried out by enzymes. Here we demonstrate how biocompatible chemistry can be used for synthesizing valuable compounds as well as for linking metabolic pathways to achieve redox balance and rescued growth. By comprehensive rerouting of metabolism, activation of respiration, and finally metal ion catalysis, we successfully managed to convert the homolactic bacterium Lactococcus lactis into a homo-diacetyl producer with high titer (95mM or 8.

One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli.

Poly(lactate-co-glycolate) (PLGA) is a widely used biodegradable and biocompatible synthetic polymer. Here we report one-step fermentative production of PLGA in engineered Escherichia coli harboring an evolved polyhydroxyalkanoate (PHA) synthase that polymerizes D-lactyl-CoA and glycolyl-CoA into PLGA. Introduction of the Dahms pathway enables production of glycolate from xylose.

Enzymatic formation of carbohydrate rings catalyzed by single-walled carbon nanotubes.

Macrocyclic carbohydrate rings were formed via enzymatic reactions around single-walled carbon nanotubes (SWNTs) as a catalyst. Cyclodextrin glucanotransferase, starch substrate and SWNTs were reacted in buffer solution to yield cyclodextrin (CD) rings wrapped around individual SWNTs. Atomic force microscopy showed the resulting complexes to be rings of 12-50 nm in diameter, which were highly soluble and dispersed in aqueous solution.

Metabolic Engineering of Escherichia coli for the Production of 3-Hydroxypropionic Acid and Malonic Acid through β-Alanine Route.

Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the β-alanine (BA) route. First, various combinations of downstream enzymes were screened and BA pyruvate transaminase (encoded by pa0132) from P. aeruginosa was selected to generate malonic semialdehyde (MSA) from BA.

Editorial: Biotechnology Journal brings more than biotechnology.

Biotechnology Journal always brings the state-of-the-art biotechnologies to our readers. Different from other topical issues, this issue of Biotechnology Journal is complied with a series of exiting reviews and research articles from spontaneous submissions, again, addressing society's actual problems and needs. The progress is a real testimony how biotechnology contributes to achievements in healthcare, better utilization of resources, and a bio-based economy.

CRISPy-web: An online resource to design sgRNAs for CRISPR applications.

CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology, allowing the targeted engineering of a wide range of genomes. The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections. It now sees widespread application in genome engineering workflows, especially using the endonuclease Cas9.

Microfluidic high-throughput selection of microalgal strains with superior photosynthetic productivity using competitive phototaxis.

Microalgae possess great potential as a source of sustainable energy, but the intrinsic inefficiency of photosynthesis is a major challenge to realize this potential. Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report a microfluidic screening using competitive phototaxis of the model alga, Chlamydomonas reinhardtii, for rapid isolation of strains with improved photosynthetic efficiencies.

Precisely Determining Ultralow level UO2(2+) in Natural Water with Plasmonic Nanowire Interstice Sensor.

Uranium is an essential raw material in nuclear energy generation; however, its use raises concerns about the possibility of severe damage to human health and the natural environment. In this work, we report an ultrasensitive uranyl ion (UO2(2+)) detection method in natural water that uses a plasmonic nanowire interstice (PNI) sensor combined with a DNAzyme-cleaved reaction. UO2(2+) induces the cleavage of DNAzymes into enzyme strands and released strands, which include Raman-active molecules.

One hundred years of clostridial butanol fermentation.

Butanol has been widely used as an important industrial solvent and feedstock for chemical production. Also, its superior fuel properties compared with ethanol make butanol a good substitute for gasoline. Butanol can be efficiently produced by the genus Clostridium through the acetone-butanol-ethanol (ABE) fermentation, one of the oldest industrial fermentation processes.

Electro-triggering and electrochemical monitoring of dopamine exocytosis from a single cell by using ultrathin electrodes based on Au nanowires.

A sophisticated set of an Au nanowire (NW) stimulator-Au NW detector system is developed for electrical cell stimulation and electrochemical analysis of subsequent exocytosis with very high spatial resolution. Dopamine release from a rat pheochromocytoma cell is more stimulated by a more negative voltage pulse. This system could help to improve the therapeutic efficacy of electrotherapies by providing valuable information on their healing mechanism.

Flux-sum analysis identifies metabolite targets for strain improvement.

Background: Rational design of microbial strains for enhanced cellular physiology through in silico analysis has been reported in many metabolic engineering studies. Such in silico techniques typically involve the analysis of a metabolic model describing the metabolic and physiological states under various perturbed conditions, thereby identifying genetic targets to be manipulated for strain improvement. More often than not, the activation/inhibition of multiple reactions is necessary to produce a predicted change for improvement of cellular properties or states.

Optical Biosensors for the Detection of Pathogenic Microorganisms.

Pathogenic microorganisms are causative agents of various infectious diseases that are becoming increasingly serious worldwide. For the successful treatment of pathogenic infection, the rapid and accurate detection of multiple pathogenic microorganisms is of great importance in all areas related to health and safety. Among various sensor systems, optical biosensors allow easy-to-use, rapid, portable, multiplexed, and cost-effective diagnosis.

Label-Free and Real-Time Detection of Avian Influenza Using Nanowire Field Effect Transistors.

Real-time and label-free detection of antibodies from avian influenza (anti-AI) in an aqueous solution is demonstrated with the use of a nanowire field effect transistor. A real-time measurement system is constructed without leakage paths through the solution medium. The current through the nanowire changes significantly after an injection of an anti-AI solution onto the device, which was previously functionalized by the antigen of AI as a probe of anti-AI.

Systems strategies for developing industrial microbial strains.

Industrial strain development requires system-wide engineering and optimization of cellular metabolism while considering industrially relevant fermentation and recovery processes. It can be conceptualized as several strategies, which may be implemented in an iterative fashion and in different orders. The key challenges have been the time-, cost- and labor-intensive processes of strain development owing to the difficulties in understanding complex interactions among the metabolic, gene regulatory and signaling networks at the cell level, which are collectively represented as overall system performance under industrial fermentation conditions.