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Hydrogen sulfide (H2S), mainly produced from L-cysteine (Cys), renders bacteria highly resistant to oxidative stress. This mitigation of oxidative stress was suggested to be an important survival mechanism to achieve antimicrobial resistance (AMR) in many pathogenic bacteria. CyuR (known as DecR or YbaO) is a recently characterized Cys-dependent transcription regulator, responsible for the activation of thecyuAPoperon and generation of hydrogen sulfide from Cys. Despite its potential importance, the regulatory network of CyuR remains poorly understood. In this study, we investigated the roles of the CyuR regulon in a Cys-dependent AMR mechanism inE. colistrains. We found: 1) Cys metabolism has a significant role in AMR and its effect is conserved in manyE. colistrains, including clinical isolates; 2) CyuR negatively controls the expression ofmdlABencoding a transporter that exports antibiotics such as cefazolin and vancomycin; 3) CyuR binds to a DNA sequence motif ‘GAAwAAATTGTxGxxATTTsyCC’ in the absence of Cys, confirmed by anin vitrobinding assay; and 4) CyuR may regulate 25 additional genes as suggested byin silicomotif scanning and transcriptome sequencing. Collectively, our findings expanded the understanding of the biological roles of CyuR relevant to antibiotic resistance associated with Cys.

Although strain tolerance to high product concentrations is a barrier to the economically viable biomanufacturing of industrial chemicals, chemical tolerance mechanisms are often unknown. To reveal tolerance mechanisms, an automated platform was utilized to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals (1,2-propanediol, 2,3-butanediol, glutarate, adipate, putrescine, hexamethylenediamine, butanol, isobutyrate, coumarate, octanoate, hexanoate), reaching tolerance at concentrations 60%–400% higher than initial toxic levels. Sequencing genomes of 223 isolates from 89 populations, reverse engineering, and cross-compound tolerance profiling were employed to uncover tolerance mechanisms. We show that: 1) cells are tolerized via frequent mutation of membrane transporters or cell wall-associated proteins (e.g., ProV, KgtP, SapB, NagA, NagC, MreB), transcription and translation machineries (e.g., RpoA, RpoB, RpoC, RpsA, RpsG, NusA, Rho), stress signaling proteins (e.g., RelA, SspA, SpoT, YobF), and for certain chemicals, regulators and enzymes in metabolism (e.g., MetJ, NadR, GudD, PurT); 2) osmotic stress plays a significant role in tolerance when chemical concentrations exceed a general threshold and mutated genes frequently overlap with those enabling chemical tolerance in membrane transporters and cell wall-associated proteins; 3) tolerization to a specific chemical generally improves tolerance to structurally similar compounds whereas a tradeoff can occur on dissimilar chemicals, and 4) using pre-tolerized starting isolates can hugely enhance the subsequent production of chemicals when a production pathway is inserted in many, but not all, evolved tolerized host strains, underpinning the need for evolving multiple parallel populations. Taken as a whole, this study provides a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 223 chemical tolerant isolates.

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. In this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (

Streptococcus pyogenes can cause a wide variety of acute infections throughout the body of its human host. The underlying transcriptional regulatory network (TRN) is responsible for altering the physiological state of the bacterium to adapt to each host environment. Consequently, an in-depth understanding the comprehensive dynamics of its TRN could inform new therapeutic strategies. Here, we compiled 116 existing high-quality RNA-seq data sets of S. pyogenes serotype M1, and estimated the TRN structure in a top-down fashion by performing independent component analysis (ICA). The algorithm computed 42 independently modulated sets of genes (iModulons). Four iModulons contained nga-ifs-slo virulence-related operon, which allowed us to identify carbon sources that control its expression. In particular, dextrin utilization upregulated nga-ifs-slo operon by activation of two-component regulatory system CovRS-related iModulons, and changed bacterial hemolytic activity compared to glucose or maltose utilization. Finally, we show that the iModulon-based TRN structure can be used to simplify interpretation of noisy bacterial transcriptome at the infection site.

Rare-cold-inducible 2 (RCI2) genes are involved in plant response to abiotic stresses. In this study, we report the functional role of a Camelina RCI2, CsRCI2D, in plant salt stress response. The localization of CsRCI2D was observed in the plasma membrane and intracellular membranes by confocal analysis in tobacco leaf and western blot analysis in Camelina. The full length CsRCI2D cDNA clone was not able to complement the salt sensitivity of △spmp3 lacking the PMP3 gene. However, a C-terminal tail deleted CsRCI2D cDNA was able to restore the level of salt tolerance to that of WT. CsRCI2D-overexpressing Camelina showed better germination rate and seedling growth. CsRCI2D overexpression decreased Na+ accumulation in both roots and shoots but increased K+ accumulation in the shoots under salt stress. Furthermore, CsRCI2D-overexpressing Camelina displayed lower H2O2 and malondialdehyde (MDA) upon salt stress. Under normal growth condition, CsRCI2D-overexpressing Camelina showed higher transcript levels of all antioxidant genes (CsCuSODs, CsMnSOD1, CsFeSODs, CsCATs, CsAPX1, and CsGR), whereas salt stress significantly induced all antioxidant genes in WT. These results indicate that salt-upregulated CsRCI2D plays a positive role in Camelina seed germination and seedling growth under salt stress by maintenance of ion homeostasis and modulating the expression of antioxidant-related genes.

Although plant rare cold inducible 2 (RCI2), a homolog of yeast PMP3, has been considered to play important roles in responses to abiotic stress in plants, the functional role of RCI2 in salt-stress tolerance remains largely unknown for crop plants. In this study, we determined the function of CsRCI2H from Camelina under salt stress. Subcellular localization analysis of the yellow fluorescent protein (YFP)-CsRCI2H fusion protein revealed that CsRCI2H localizes to the plasma membrane in tobacco leaves. Expression of CsRCI2H was greatly increased in the presence of 150 mM NaCl. CsRCI2H compensated for the salt sensitivity of the yeast mutant ∆pmp3 lacking the PMP3 gene. CsRCI2H-overexpressing transgenic Camelina showed improved seed germination, root growth and fresh weight under high salt stress. CsRCI2H-overexpressing transgenic Camelina displayed lower accumulation of Na+ in the roots and shoots but higher K+ and Ca2+ accumulation in the shoots than wild type plants under salt stress. Furthermore, CsRCI2H-overexpressing Camelina displayed higher stomatal conductance and lower malondialdehyde under salt stress. Transcript levels of ion-homeostasis related genes, such as CsSOS1, CsSOS3, and CsHKT1, were significantly increased in the CsRCI2H-overexpressing transgenic plants upon salt treatment. These results suggest that Na+ and K+ homeostasis can be controlled by the Na+ and K+ transport systems throughout the CsRCI2H-overexpressing transgenic Camelina plant under salt-stress conditions. Collectively, these results indicate that CsRCI2H contributes to salt tolerance in Camelina during seed germination and seedling growth by reducing Na+ toxicity in the plant cells via regulation of ion homeostasis.

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bioprocesses.

Bacterial gene expression is orchestrated by numerous transcription factors (TFs). Elucidating how gene expression is regulated is fundamental to understanding bacterial physiology and engineering it for practical use. In this study, a machine-learning approach was applied to uncover the genome-scale transcriptional regulatory network (TRN) in Pseudomonas putida KT2440, an important organism for bioproduction. We performed independent component analysis of a compendium of 321 high-quality gene expression profiles, which were previously published or newly generated in this study. We identified 84 groups of independently modulated genes (iModulons) that explain 75.7% of the total variance in the compendium. With these iModulons, we (i) expand our understanding of the regulatory functions of 39 iModulon associated TFs (e.g., HexR, Zur) by systematic comparison with 1993 previously reported TF-gene interactions; (ii) outline transcriptional changes after the transition from the exponential growth to stationary phases; (iii) capture group of genes required for utilizing diverse carbon sources and increased stationary response with slower growth rates; (iv) unveil multiple evolutionary strategies of transcriptome reallocation to achieve fast growth rates; and (v) define an osmotic stimulon, which includes the Type VI secretion system, as coordination of multiple iModulon activity changes. Taken together, this study provides the first quantitative genome-scale TRN for P. putida KT2440 and a basis for a comprehensive understanding of its complex transcriptome changes in a variety of physiological states.

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YgfI, YiaU, YneJ, YcaN, YbhD. We show that: 1) YbdO regulation has an effect on bacterial growth at low pH with citrate supplementation. YbdO is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); 2) YgfI activates the dhaKLM operon that encodes the phosphotransferase system involved in glycerol and dihydroxyacetone utilization; 3) YiaU regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase; 4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen sources; and 5) YbhD and YcaN regulate adjacent y-genes on the genome and YbeF is involved in flagella gene regulation. We have thus established the functions for four LTFs and identified the target genes for three LTFs.

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YgfI, YiaU, YneJ, YcaN, YbhD. We show that: 1) YbdO regulation has an effect on bacterial growth at low pH with citrate supplementation. YbdO is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); 2) YgfI activates the dhaKLM operon that encodes the phosphotransferase system involved in glycerol and dihydroxyacetone utilization; 3) YiaU regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase; 4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen sources; and 5) YbhD and YcaN regulate adjacent y-genes on the genome and YbeF is involved in flagella gene regulation. We have thus established the functions for four LTFs and identified the target genes for three LTFs.IMPORTANCEThe reconstruction of the transcriptional regulatory network (TRN) is important for gram-negative bacteria such as E. coli. LysR-type TFs are abundant in Enterobacteria, but many LTF functions still remain unknown. Here we report putative functions of uncharacterized TFs based on multi-omics data related to L-threonine, L-glutamate, and putrescine utilization. Amino acids (AAs) and polyamines are important sources of nitrogen for many microorganisms, but the increase in one amino acid or putrescine concentration in a minimal medium also induces stress. Although polyamine metabolism has been studied, the TRN that controls the putrescine (Ptr) and AA utilization at minimal medium conditions are still poorly understood. The function of previously uncharacterized transcriptional regulators YbdO, YgfI, and YneJ (PtrR) were identified in Escherichia coli. PtrR is important for Ptr and L-glutamate utilization, while YgfI transcriptional regulation was found to be important for growth on L-threonine and glycerol as a carbon source.

Although cellular secretion is important in industrial biotechnology, its assessment is difficult due to the lack of efficient analytical methods. This study describes a synthetic cellular communication-based microfluidic platform for screening strains with the improved secretion of 3-hydroxypropionic acid (3-HP), an industry-relevant platform chemical. 3-HP-secreting cells were compartmentalized in droplets, with receiving cells equipped with a genetic circuit that converts the 3-HP secretion level into an easily detectable signal. This platform was applied to identify Escherichia coli genes that enhance the secretion of 3-HP. As a result, two genes (setA, encoding a sugar exporter, and yjcO, encoding a Sel1 repeat-containing protein) found by this platform enhance the secretion of 3-HP and its production. Given the increasing design capability for chemical-detecting cells, this platform has considerable potential in identifying efflux pumps for not only 3-HP but also many important chemicals.

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively., Rodionova et al. find that the putative transporter gene, ydhC and its regulator ydhB are involved in purine transportation and that the expression of the ydhC gene is activated by the YdhB in E. coli. The authors suggest renaming the regulator PunR and the transporter PunC, respectively.

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., This research was supported by the C1 Gas Refinery Program (NRF-2018M3D3A1A01055754 and NRF-2015M3D3A1A01064882), the Global Research Laboratory Program (NRF-2016K1A1A2912829), and the Bio & Medical Technology Development Program (NRF-2018M3A9H3020459) through the National Research Foundation of Korea (NRF of Korea), funded by the Ministry of Science and ICT (MSIT). S.W.S. is partially supported by the Creative-Pioneering Researchers Program through Seoul National University (SNU).

Although enzymes are efficient catalysts capable of converting various substrates into desired products with high specificity under mild conditions, their effectiveness as catalysts is substantially reduced when substrates are poorly water-soluble. In this study, to expedite the enzymatic conversion of a hydrophobic substrate, we use a bicontinuous interfacially jammed emulsion gel (bijel) which provides large interfacial area between two immiscible liquids: oil and water. Using lipase-catalyzed hydrolysis of tributyrin as a model reaction in a batch mode, we show that bijels can be used as media to enable enzymatic reaction. The bijel system gives a four-fold increase in the initial reaction rate in comparison to a stirred biphasic medium. Our results demonstrate that bijels are powerful biphasic reaction media to accelerate enzymatic reactions with various hydrophobic reagents. This work also demonstrates that bijels can potentially be used as reaction media to enable continuous reactive separations.

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs; 34 of them were found to be DNA-binding protein. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. These TFs were found to have various roles in regulating primary cellular processes in E. coli. Taken together, this study: (1) significantly expands the number of confirmed TFs, close to the estimated total of about 280 TFs; (2) predicts the putative functions of the newly discovered TFs, and (3) confirms the functions of representative TFs through mutant phenotypes.

Microbial consortia have been considered potential platforms for bioprocessing applications. However, the complexity in process control owing to the use of multiple strains necessitates the use of an efficient population control strategy. Herein, we report circuit-guided synthetic acclimation as a strategy to improve biochemical production by a microbial consortium. We designed a consortium comprising alginate-utilizing Vibrio sp. dhg and 3-hydroxypropionic acid (3-HP)-producing Escherichia coli strains for the direct conversion of alginate to 3-HP. We introduced a genetic circuit, named “Population guider”, in the E. coli strain, which degrades ampicillin only when 3-HP is produced. In the presence of ampicillin as a selection pressure, the consortium was successfully acclimated for increased 3-HP production by 4.3-fold compared to that by a simple co-culturing consortium during a 48-h fermentation. We believe this concept is a useful strategy for the development of robust consortium-based bioprocesses., This research was supported by the C1 Gas Refinery Program [NRF-2018M3D3A1A01055754] and National Research Foundation of Korea grant [NRF-2019R1A2C2084631].

Carbon monoxide (CO) is a promising carbon source for producing value-added biochemicals via microbial fermentation. However, its microbial conversion has been challenging because of difficulties in genetic engineering of CO-utilizing microorganisms and, more importantly, maintaining CO consumption which is negatively affected by the toxicity of CO and accumulated byproducts. To overcome these issues, we devised mutualistic microbial consortia, co-culturing Eubacterium limosum and genetically engineered Escherichia coli for the production of 3-hydroxypropionic acid (3-HP) and itaconic acid (ITA). During the co-culture, E. limosum assimilated CO and produced acetate, a toxic by-product, while E. coli utilized acetate as a sole carbon source. We found that this mutualistic interaction dramatically stabilized and improved CO consumption of E. limosum compared to monoculture. Consequently, the improved CO consumption allowed successful production of 3-HP and ITA from CO. This study is the first demonstration of value-added biochemical production from CO using a microbial consortium. Moreover, it suggests that synthetic mutualistic microbial consortium can serve as a powerful platform for the valorization of CO.

Although polyamines, such as putrescine (Ptr), induce envelope stress for bacteria, they are important as nitrogen and carbon sources. Ptr utilization in Escherichia coli involves protein glutamylation, and glutamate stands at a crossroads between catabolism and anabolism. This communication reports that the transcription factor YneJ, here renamed PtrR, is involved in the regulation of a small regulatory RNA gene, fnrS, and an operon, yneIHGF, encoding succinate-semialdehyde dehydrogenase, Sad (YneI), glutaminase, GlsB (YneH), and several other genes. The yneI promoter is activated during putrescine utilization under nitrogen/carbon starvation conditions, and we show that PtrR is important for the putrescine stress response. It is also a repressor of fnrS gene expression, involved in the cascade regulation of mRNA synthesis for the marA and sodB genes, involved in antibiotic responses. PtrR transcriptional regulation of fnrS leads to a regulatory cascade induced by this small RNA that affects mRNA levels of ompF and the multidrug resistance regulator, MarA. We propose that PtrR functions as a dual activator/repressor, and that its regulation is important for the responses to different stress conditions involving L-glutamine/L-glutamate and putrescine utilization.IMPORTANCEPutrescine is an important source of nitrogen for many organisms, but it also induces stress. Although its metabolism has been studied extensively, the regulatory mechanisms that control the stress response are still poorly understood. This study reveals that the HTH-type transcriptional regulator, YneJ in Escherichia coli, here re-named PtrR, is important for the putrescine stress response, in part because it plays a role in outer membrane porin regulation as a sensor in a regulatory cascade. Direct PtrR transcriptional regulation of the fnrS, yneI (sad), gltS and ptrR genes is documented and rationalized, and nine PtrR binding sites were identified using ChIP-Exo. A ptrR mutant exhibited altered resistance to a tetracycline group of antibiotics under microaerophilic conditions, suggesting that PtrR indirectly controls expression of porin genes such as ompF.

Evolutionary engineering of microbes for the production of metabolites requires efficient screening methods to test vast mutant libraries. Genetically encoded biosensors are regarded as promising screening devices owing to their wide range of detectable ligands and great applicability to high-throughput screening and selection. Here, we reviewed the current progress in design and optimization of biosensors for high-throughput screening of chemicals. First, we summarized genetic parts of biosensors and strategies for their discovery and development. Next, we explained the properties of biosensors that are relevant to high-throughput screening. Finally, we described various methods for tuning biosensors to fulfill requirements of an efficient screening.

Genetic circuits are composed of input, logic, and output parts. Construction of complex circuits for practical applications requires numerous tunable genetic parts. However, the limited diversity and complicated tuning methods used for the input parts hinders the scalability of genetic circuits. Therefore, a new type of input part is required that responds to diverse signals and enables easy tuning. Here, we developed RNA-protein hybrid input parts that combine a riboswitch and orthogonal transcriptional repressors. The hybrid inputs successfully regulated the transcription of an output in response to the input signal detected by the riboswitch and resulted in signal inversion because of the expression of transcriptional repressors. Dose-response parameters including fold-change and half-maximal effective concentration were easily modulated and amplified simply by changing the promoter strength. Furthermore, the hybrid input detected both exogenous and endogenous signals, indicating potential applications in metabolite sensing. This hybrid input part could be highly extensible considering the rich variety of components.

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP from glycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has been revealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield and productivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the two enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a synthetic selection device using a 3-HP-responsive transcription factor and developed a selection approach for screening 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding site library of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichment of strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a 2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach will provide the simple and efficient tool to engineer the pathway enzymes in metabolic engineering.

One of the great advantages of microbial fermentation is the capacity to convert various carbon compounds into value-added chemicals. In this regard, there have been many efforts to engineer microorganisms to facilitate utilization of abundant carbon sources. Recently, the potential of acetate as a feedstock has been discovered; efforts have been made to produce various biochemicals from acetate based on understanding of its metabolism. In this review, we discuss the potential sources of acetate and summarized the recent progress to improve acetate utilization with microorganisms. Furthermore, we also describe representative studies that engineered microorganisms for the production of biochemicals from acetate.

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of E. coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

Summary The production of coenzyme B12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B12 using a previously engineered coenzyme B12-producing E. coli strain. To select a high producer, the coenzyme B12-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B12-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B12 validated the coenzyme B12-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B12 production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B12-producing strains., Graphical Abstract, Highlights • The auxotrophic selection strategy was applied to coenzyme B12 production • Coenzyme B12-independent methionine synthase was deleted for auxotroph system • The auxotrophic strategy could significantly enhance the coenzyme B12 production • Optimization of the auxotroph system further enhanced the coenzyme B12 production, Biological Sciences; Bioengineering; Metabolic Engineering

Rare cold-inducible 2 (RCI2) proteins are small hydrophobic proteins that are known to be localized in cellular membranes. The function of RCI2 proteins has been reported to be associated with low-temperature, salt, and drought stress tolerances as a membrane potential regulator; however, the specific functions are still unknown. The PIP2 (plasma membrane intrinsic protein 2) aquaporins are proteins that transport water and small solutes into the cell. The expression and activity of PIP2 proteins, like RCI2, are also related to salt- and drought-stress tolerance. In this study, we identified novel protein interactions between RCI2 and PIP2; 1, including protein accumulation changes in the bioenergy crop Camelina sativa L. under various NaCl stress conditions. Accumulation of both CsRCI2E and CsRCI2F proteins increased with NaCl stress; however, to differing levels depending on the NaCl stress intensity. A co-immunoprecipitation test revealed interaction between CsRCI2E-CsPIP2 and CsRCI2F-CsPIP2. Moreover, co-expression of the four CsRCI2 proteins with CsPIP2; 1 in Xenopus laevis oocytes reduced water transport activity. Furthermore, the abundance of CsPIP2; 1 protein was decreased under CsRCI2E and CsRCI2F co-expression. These results suggest that NaCl-induced expression of CsRCI2E and CsRCI2F contributes to the regulation of CsPIP2; 1.

Bio-based chemical production has enormous potential as an alternative to petroleum-based chemical production. To establish the economic feasibility of bio-based production, the metabolism of microbial cell factories should be engineered to maximize yield, titer, and productivity. Methods for regulating gene expression are critical for metabolic engineering because enzymes, the final products of gene expression, catalyze most of the reactions required to synthesize chemical products. Recent advances in the development of synthetic regulatory tools have enabled more complex but precise control of gene expression in either static or dynamic mode. These molecular tools have been successfully applied in the metabolic engineering of microorganisms to produce diverse chemicals. Here, we review the synthetic regulatory tools along with the design strategies and applications in the context of metabolic engineering.

Summary: The production of coenzyme B12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B12 using a previously engineered coenzyme B12-producing E. coli strain. To select a high producer, the coenzyme B12-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B12-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B12 validated the coenzyme B12-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B12 production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B12-producing strains. : Biological Sciences; Bioengineering; Metabolic Engineering Subject Areas: Biological Sciences, Bioengineering, Metabolic Engineering

Acetate, which is an abundant carbon source, is a potential feedstock for microbial processes that produce diverse value-added chemicals. In this study, we produced 3-hydroxypropionic acid (3-HP) from acetate with engineered Escherichia coli. For the efficient conversion of acetate to 3-HP, we initially introduced heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus. Then, the acetate assimilating pathway and glyoxylate shunt pathway were activated by overexpressing acs (encoding acetyl-CoA synthetase) and deleting iclR (encoding the glyoxylate shunt pathway repressor). Because a key precursor malonyl-CoA is also consumed for fatty acid synthesis, we decreased carbon flux to fatty acid synthesis by adding cerulenin. Subsequently, we found that inhibiting fatty acid synthesis dramatically improved 3-HP production (3.00 g/L of 3-HP from 8.98 g/L of acetate). The results indicated that acetate can be used as a promising carbon source for microbial processes and that 3-HP can be produced from acetate with a high yield (44.6% of the theoretical maximum yield).

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.

Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.

Microbial production of 5-aminolevulinic acid (ALA) has received much attention because of its potential in clinical applications. Overexpression along with the deciphering of regulation of the related enzymes and an analogue transporter yielded remarkable achievements in ALA production. Nonetheless, there is significant room for carbon flux optimization to enhance ALA production. The aim of this study was precise carbon flux optimization for high ALA production in Escherichia coli expressing the ALA biosynthetic pathway. Initially, genes hemA and hemL were overexpressed with strong promoters and synthetic 5'-untranslated regions (5'-UTRs). Then, the tricarboxylic acid (TCA) cycle was blocked to force carbon flux toward the ALA production pathway by deletion of sucA. Although the resulting strain showed a severe metabolic imbalance and low ALA production, further precise tuning of carbon flux to the glyoxylate cycle by varying the transcriptional strength of aceA led to substantially improved cell growth and ALA production. Thus, this precise tuning of the glyoxylate cycle in a quantitative manner should also enable efficient production of other value-added products derived from the TCA cycle.