Regulatory properties of transcription factors with diverse mechanistic function.

Transcription factors (TFs) regulate the process of transcription through the modulation of different kinetic steps. Although models can often describe the observed transcriptional output of a measured gene, predicting a TFs role on a given promoter requires an understanding of how the TF alters each step of the transcription process. In this work, we use a simple model of transcription to assess the role of promoter identity, and the degree to which TFs alter binding of RNAP (stabilization) and initiation of transcription (acceleration) on three primary characteristics: the range of steady-state regulation, cell-to-cell variability in expression, and the dynamic response time of a regulated gene. We find that steady state regulation and the response time of a gene behave uniquely for TFs that regulate incoherently, i.e that speed up one step but slow the other. We also find that incoherent TFs have dynamic implications, with one type of incoherent mode configuring the promoter to respond more slowly at intermediate TF concentrations. We also demonstrate that the noise of gene expression for these TFs is sensitive to promoter strength, with a distinct non-monotonic profile that is apparent under stronger promoters. Taken together, our work uncovers the coupling between promoters and TF regulatory modes with implications for understanding natural promoters and engineering synthetic gene circuits with desired expression properties.

Tunable transcription factor library for robust quantification of regulatory properties in Escherichia coli.

Predicting the quantitative regulatory function of transcription factors (TFs) based on factors such as binding sequence, binding location, and promoter type is not possible. The interconnected nature of gene networks and the difficulty in tuning individual TF concentrations make the isolated study of TF function challenging. Here, we present a library of Escherichia coli strains designed to allow for precise control of the concentration of individual TFs enabling the study of the role of TF concentration on physiology and regulation. We demonstrate the usefulness of this resource by measuring the regulatory function of the zinc-responsive TF, ZntR, and the paralogous TF pair, GalR/GalS. For ZntR, we find that zinc alters ZntR regulatory function in a way that enables activation of the regulated gene to be robust with respect to ZntR concentration. For GalR and GalS, we are able to demonstrate that these paralogous TFs have fundamentally distinct regulatory roles beyond differences in binding affinity.

Quantifying the regulatory role of individual transcription factors in Escherichia coli.

Gene regulation often results from the action of multiple transcription factors (TFs) acting at a promoter, obscuring the individual regulatory effect of each TF on RNA polymerase (RNAP). Here we measure the fundamental regulatory interactions of TFs in E. coli by designing synthetic target genes that isolate individual TFs' regulatory effects. Using a thermodynamic model, each TF's regulatory interactions are decoupled from TF occupancy and interpreted as acting through (de)stabilization of RNAP and (de)acceleration of transcription initiation. We find that the contribution of each mechanism depends on TF identity and binding location; regulation immediately downstream of the promoter is insensitive to TF identity, but the same TFs regulate by distinct mechanisms upstream of the promoter. These two mechanisms are uncoupled and can act coherently, to reinforce the observed regulatory role (activation/repression), or incoherently, wherein the TF regulates two distinct steps with opposing effects.

Inherent regulatory asymmetry emanating from network architecture in a prevalent autoregulatory motif.

Predicting gene expression from DNA sequence remains a major goal in the field of gene regulation. A challenge to this goal is the connectivity of the network, whose role in altering gene expression remains unclear. Here, we study a common autoregulatory network motif, the negative single-input module, to explore the regulatory properties inherited from the motif. Using stochastic simulations and a synthetic biology approach in E. coli, we find that the TF gene and its target genes have inherent asymmetry in regulation, even when their promoters are identical; the TF gene being more repressed than its targets. The magnitude of asymmetry depends on network features such as network size and TF-binding affinities. Intriguingly, asymmetry disappears when the growth rate is too fast or too slow and is most significant for typical growth conditions. These results highlight the importance of accounting for network architecture in quantitative models of gene expression.

Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries.

Complete hydrolysis of cellulose has been a key characteristic of biomass technology because of the limitation of industrial production hosts to use cellodextrin, the partial hydrolysis product of cellulose. Cellobiose, a ß-1,4-linked glucose dimer, is a major cellodextrin of the enzymatic hydrolysis (via endoglucanase and exoglucanase) of cellulose. Conversion of cellobiose to glucose is executed by ß-glucosidase. The complete extracellular hydrolysis of celluloses has several critical barriers in biomass technology. An alternative bioengineering strategy to make the bioprocessing less challenging is to engineer microbes with the abilities to hydrolyze and assimilate the cellulosic-hydrolysate cellodextrin. Microorganisms engineered to metabolize cellobiose rather than the monomeric glucose can provide several advantages for lignocellulose-based biorefineries. This review describes the recent advances and challenges in engineering efficient intracellular cellobiose metabolism in industrial hosts. This review also describes the limitations of and future prospectives in engineering intracellular cellobiose metabolism.

Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli.

Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (ß-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

A simple and effective method for construction of Escherichia coli strains proficient for genome engineering.

Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.

Feasibilities of consolidated bioprocessing microbes: from pretreatment to biofuel production.

Lignocelluloses are rich sugar treasures, which can be converted to useful commodities such as biofuel with the help of efficient combination of enzymes and microbes. Although several bioprocessing approaches have been proposed, biofuel production from lignocelluloses is limited because of economically infeasible technologies for pretreatment, saccharification and fermentation. Use of consolidated bioprocessing (CBP) microbes is the most promising method for the cost-effective production of biofuels. However, lignocelluloses are obtained from highly diverse environment and hence are heterogeneous in nature. Therefore, it is necessary to develop and integrate tailor-designed pretreatment processes and efficient microbes that can thrive on many different kinds of biomass. In this review, the progress towards the construction of consolidated bioprocessing microbes, which can efficiently convert heterogeneous lignocellulosic biomass to bioenergy, has been discussed; in addition, the potential and constraints of current bioprocessing technologies for cellulosic biofuel production have been discussed.

Complete genome sequence of Brucella abortus A13334, a new strain isolated from the fetal gastric fluid of dairy cattle.

Brucella abortus is a major pathogen that infects livestock and humans. A new strain of B. abortus (A13334) was isolated from the fetal gastric fluid of a dairy cow, with the aim of using it to compare genetic properties, analyze virulence factor, and survey the epidemiological relationship to other Brucella species. Here, we report the complete and annotated genome sequence of B. abortus A13334.

Complete genome sequence of Brucella canis strain HSK A52141, isolated from the blood of an infected dog.

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.

Heterologous expression of plant cell wall degrading enzymes for effective production of cellulosic biofuels.

A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies.