Synthetic biosensor accelerates evolution by rewiring carbon metabolism toward a specific metabolite.

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.

Signal amplification and optimization of riboswitch-based hybrid inputs by modular and titratable toehold switches.

BACKGROUND: Synthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive sensor modules for use in synthetic biology applications. However, tuning the fold-change of riboswitch circuits is challenging because a deep understanding of the riboswitch mechanism and screening of mutant libraries is generally required. Therefore, novel molecular parts and strategies for straightforward tuning of the fold-change of riboswitch circuits are needed. RESULTS: In this study, we devised a toehold switch-based modulator approach that combines a hybrid input construct consisting of a riboswitch and transcriptional repressor and de-novo-designed riboregulators named toehold switches. First, the introduction of a pair of toehold switches and triggers as a downstream signal-processing module to the hybrid input for coenzyme B12 resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on balancing the expression levels of the RNA components greatly improved the fold-change from 260- to 887-fold depending on the promoter and host strain. Further characterizations confirmed low leakiness and high orthogonality of five toehold switch pairs, indicating the broad applicability of this strategy to riboswitch tuning. CONCLUSIONS: The toehold switch-based modulator substantially improved the fold-change compared to the previous sensors with only the hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.

Molecular parts and genetic circuits for metabolic engineering of microorganisms.

Microbial conversion of biomass into value-added biochemicals is a highly sustainable process compared to petroleum-based production. In this regard, microorganisms have been engineered via simple overexpression or deletion of metabolic genes to facilitate the production. However, the producer microorganisms require complex regulatory circuits to maximize productivity and performance. To address this issue, diverse genetic circuits have been developed that allow cells to minimize their metabolic burden, overcome metabolic imbalances and respond to a dynamically changing environment. In this review, we briefly explain the basic strategy for constructing genetic circuits by assembling molecular parts such as input, operation and output modules. Next, we describe recent applications of the circuits in the metabolic engineering of microorganisms to improve biochemical production. Beyond those achievements, genetic circuits will facilitate more innovative approaches to future strain development through mining and engineering new genetic elements and improving the complexity of genetic circuit design.

Optimization of hexanoic acid production in recombinant Escherichia coli by precise flux rebalancing.

The aim of this study is to demonstrate that rebalancing of metabolic fluxes at acetyl-CoA branch node can substantially improve the titer and productivity of hexanoic acid in recombinant Escherichia coli strains. First, a hexanoic acid-producing E. coli strain was constructed by expressing genes encoding ß-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Megasphaera sp. MH in a butyric acid producer strain. Next, metabolic flux was optimized at the acetyl-CoA branch node by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Four synthetic 5'-untranslated regions were designed for atoB using UTR Designer to modulate the expression level of the gene. Notably, the productivity of the optimized strain (14.7 mg/L/h) was the highest among recombinant E. coli strains in literature when using a similar inoculum size for fermentation. These results show that fine-tuning the expression level of atoB is critical for production of hexanoic acid.

On-chip analysis, indexing and screening for chemical producing bacteria in a microfluidic static droplet array.

Economic production of chemicals from microbes necessitates development of high-producing strains and an efficient screening technology is crucial to maximize the effect of the most popular strain improvement method, the combinatorial approach. However, high-throughput screening has been limited for assessment of diverse intracellular metabolites at the single-cell level. Herein, we established a screening platform that couples a microfluidic static droplet array (SDA) and an artificial riboswitch to analyse intracellular metabolite concentration from single microbial cells. Using this system, we entrapped single Escherichia coli cells in SDA to measure intracellular l-tryptophan concentrations. It was validated that intracellular l-tryptophan concentration can be evaluated by the fluorescence from the riboswitch. Moreover, high-producing strains were successfully screened from a mutagenized library, exhibiting up to 145% productivity compared to its parental strain. This platform will be widely applicable to strain improvement for diverse metabolites by developing new artificial riboswitches.

Improving the electrical properties of lanthanum silicate films on ge metal oxide semiconductor capacitors by adopting interfacial barrier and capping layers.

The electrical properties of La-silicate films grown by atomic layer deposition (ALD) on Ge substrates with different film configurations, such as various Si concentrations, Al2O3 interfacial passivation layers, and SiO2 capping layers, were examined. La-silicate thin films were deposited using alternating injections of the La[N{Si(CH3)3}2]3 precursor with O3 as the La and O precursors, respectively, at a substrate temperature of 310 °C. The Si concentration in the La-silicate films was further controlled by adding ALD cycles of SiO2. For comparison, La2O3 films were also grown using [La((i)PrCp)3] and O3 as the La precursor and oxygen source, respectively, at the identical substrate temperature. The capacitance-voltage (C-V) hysteresis decreased with an increasing Si concentration in the La-silicate films, although the films showed a slight increase in the capacitance equivalent oxide thickness. The adoption of Al2O3 at the interface as a passivation layer resulted in lower C-V hysteresis and a low leakage current density. The C-V hysteresis voltages of the La-silicate films with Al2O3 passivation and SiO2 capping layers was significantly decreased to ∼0.1 V, whereas the single layer La-silicate film showed a hysteresis voltage as large as ∼1.0 V.