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

Deciphering the Molecular Underpinnings of the Cryptic Cellobiose Metabolism in Escherichia coli : An omics guided approach to modularize CBP microbes

Department Of Biomedical EngineeringConsolidated bioprocessing (CBP) is an efficient process that combines saccharification and fermentation of lignocellulosic biomass into a single microbial host. The choice of ideal microbes for CBP is challenging as it demands efficient functioning of several complex traits including expression of sets of saccharifying enzymes, metabolism of wide range of substrates, tolerance to various inhibitors, and high yield of desired products. One approach to develop an ideal host for CBP is to mimic the native cellulolytic microbes. In accordance with this, most native cellulolytic microbes metabolize cellulose in the form of cellobiose to obtain energetic benefits for growth on cellulose and to avoid feed-back inhibition of cellulase by glucose or cellobiose. In this study, we constructed cellobiose-metabolizing Escherichia coli (named as strain OSS, Original Synthetic Strain or ESS, Evolved Synthetic Strain which differ in their ability to ferment cellobiose) by exploiting its native cryptic chb and asc operons with an aim for using it as a platform host for Consolidated bioprocessing (CBP) or Simultaneous Saccharification and Fermentation (SSF) process. In addition to paving way for efficient consolidated bioprocessing, in depth analysis of strain ESS revealed several interesting molecular mysteries and regulations related to the cryptic operons of E. coli. Noteworthy is the significance of the ascB gene of asc operon which was previously considered less significant for growth on cellobiose. Here, we show through a combination of conventional genetics, adaptive evolution and targeted genome engineering that the ascB gene could serve as one of the most efficient ??-glucosidases or even a stand-alone ??-glucosidase for cellobiose metabolism in E. coli. In addition, we show that this gene ascB is being controlled by another putative promoter within the operon apart from the cryptic promoter of the asc operon thus opening new directions on the evolution and regulation of these operons. A combination of recombinant DNA technology and high-throughput screening process revealed that a combination of these cryptic operons could help in extending the substrate range of E. coli to metabolize several glucosidases including cellobiose, salicin, arbutin, gentiobiose, raffinose, and amygdalin. It is not just sufficient to enhance the cellobiose metabolic rate in order to make a strain more proficient for consolidated bioprocessing; it is also necessary that cellobiose is metabolized as efficiently as glucose. For instance, glucose is metabolized in a respiro-fermentative mode resulting in the secretion of large amount of acetate due to overflow metabolism whereas cellobiose is metabolized in a respirative manner secreting acetate only during the stationary phase. Such differences urge the need to understand and rewire the central carbon metabolism for an efficient cellobiose metabolism. Here we show that rewiring the flux through the TCA cycle could help in enhancing the cellobiose metabolism in E. coli. We demonstrate here that the transcription factor, YebK helps in enhancing the cellobiose metabolic activity through modulations in the central carbon metabolism. YebK recognizes the two major central carbon intermediates, 2-keto-3-deoxy-phosphogluconate (KDPG) and alpha-ketoglutarate (AKG), and co-ordinates the regulations of the TCA cycle of E. coli. Such regulation is particularly dominant during the down-shift of cells from rich nutrient source to a minimal nutrient source. Finally, we also demonstrate the application of the strain ESS, engineered with efficient cellobiose metabolism for co-metabolism of multiple carbon sources or in consolidated bioprocessing for growth with cellulose as a sole carbon source. Thus, the strain ESS would serve as a potential host for consolidated bio-processing or in simultaneous saccharification and fermentation process. The strain ESS could also serve as a molecular bag to decipher challenging queries related to the evolution of the cryptic genes of E. coli. Finally, through this study we also show that the putative transcription factor binding sites or transcription start sites (TSS) reported within the coding/intergenic regions identified through ChIP-sequencing or deep RNA sequencing technologies could serve as a potential target for metabolic engineering and strain optimization.ope

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.open8

Quantifying the regulatory role of individual transcription factors in Escherichia coli [preprint]

Transcription factors (TFs) modulate gene expression by binding to regulatory DNA sequences surrounding target genes. To isolate the fundamental regulatory interactions of E. coli TFs, we measure regulation of TFs acting on synthetic target genes that are designed to isolate the individual TF regulatory effect. This data is interpreted through a thermodynamic model that decouples the role of TF copy number and TF binding affinity from the interactions of the TF on RNA polymerase through two distinct mechanisms: (de)stabilization of the polymerase and (de)acceleration of transcription initiation. We find the contribution of each mechanism towards the observed regulation depends on TF identity and binding location; for the set of TFs studied here, regulation immediately downstream of the promoter is not sensitive to TF identity, however these same TFs regulate through distinct mechanisms at an upstream binding site. Furthermore, depending on binding location, these two mechanisms of regulation can act coherently, to reinforce the observed regulatory role (activation or repression), or incoherently, where the TF regulates two distinct steps with opposing effect

Rewiring carbon catabolite repression for microbial cell factory

Carbon catabolite repression (CCR) is a key regulatory system found in most microorganisms that ensures preferential utilization of energy-efficient carbon sources. CCR helps microorganisms obtain a proper balance between their metabolic capacity and the maximum sugar uptake capability. It also constrains the deregulated utilization of a preferred cognate substrate, enabling microorganisms to survive and dominate in natural environments. On the other side of the same coin lies the tenacious bottleneck in microbial production of bioproducts that employs a combination of carbon sources in varied proportion, such as lignocellulose-derived sugar mixtures. Preferential sugar uptake combined with the transcriptional and/or enzymatic exclusion of less preferred sugars turns out one of the major barriers in increasing the yield and productivity of fermentation process. Accumulation of the unused substrate also complicates the downstream processes used to extract the desired product. To overcome this difficulty and to develop tailor-made strains for specific metabolic engineering goals, quantitative and systemic understanding of the molecular interaction map behind CCR is a prerequisite. Here we comparatively review the universal and strain-specific features of CCR circuitry and discuss the recent efforts in developing synthetic cell factories devoid of CCR particularly for lignocellulose-based biorefinery.close11

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, (beta-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 metabolismopen1

Metabolomics methods for the synthetic biology of secondary metabolism

Many microbial secondary metabolites are of high biotechnological value for medicine, agriculture, and the food industry. Bacterial genome mining has revealed numerous novel secondary metabolite biosynthetic gene clusters, which encode the potential to synthesize a large diversity of compounds that have never been observed before. The stimulation or “awakening” of this cryptic microbial secondary metabolism has naturally attracted the attention of synthetic microbiologists, who exploit recent advances in DNA sequencing and synthesis to achieve unprecedented control over metabolic pathways. One of the indispensable tools in the synthetic biology toolbox is metabolomics, the global quantification of small biomolecules. This review illustrates the pivotal role of metabolomics for the synthetic microbiology of secondary metabolism, including its crucial role in novel compound discovery in microbes, the examination of side products of engineered metabolic pathways, as well as the identification of major bottlenecks for the overproduction of compounds of interest, especially in combination with metabolic modeling. We conclude by highlighting remaining challenges and recent technological advances that will drive metabolomics towards fulfilling its potential as a cornerstone technology of synthetic microbiology

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 l 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.open7

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.open2