173 research outputs found
Serial adaptive laboratory evolution enhances mixed carbon metabolic capacity of Escherichia coli
No full textMicrobes have inherent capacities for utilizing various carbon sources, however they often exhibit sub-par fitness due to low metabolic efficiency. To test whether a bacterial strain can optimally utilize multiple carbon sources, Escherichia coli was serially evolved in L -lactate and glycerol. This yielded two end -point strains that evolved first in L -lactate then in glycerol, and vice versa. The end -point strains displayed a universal growth advantage on single and a mixture of adaptive carbon sources, enabled by a concerted action of carbon source-specialists and generalist mutants. The combination of just four variants of glpK , ppsA , ydcI , and rph-pyrE , accounted for more than 80% of end -point strain fitness. In addition, machine learning analysis revealed a coordinated activity of transcriptional regulators imparting condition-specific regulation of gene expression. The effectiveness of the serial adaptive laboratory evolution (ALE) scheme in bioproduction applications was assessed under single and mixed-carbon culture conditions, in which serial ALE strain exhibited superior productivity of acetoin compared to ancestral strains. Together, systems-level analysis elucidated the molecular basis of serial evolution, which hold potential utility in bioproduction applications.
Sequence and function relationship of Escherichia coli flavin mononucleotide binding fluorescent protein
No full textFlavin mononucleotide (FMN)-binding fluorescent proteins can provide in vivo reporter system, without oxygen. Here, we present the functional landscape of individual amino acid sequence of Escherichia coli FMN-binding fluorescent protein (EcFbFP). We used random mutagenesis to generate the mutant libraries, which were screened by function loss or retained. The function and sequence relationship of the mutants were analyzed in single high-throughput sequencing, resulting in 329 tolerant mutations and 259 sensitive mutations that show retained fluorescence or loss of fluorescence respectively. In addition, the enrichment of tolerant or sensitive mutations in each amino acid residues were weighed to find functionally important residues n EcFbFP. The mutation enrichment analysis show that the positions critical to the function of EcFbFP lies among the FMN binding pocket, turns and loops of the protein where dynamic conformational changes occur and the Glu56-Lys97 salt bridge which is critical to structural stability of EcFbFP. Collectively, the mutational scanning results provide a functional landscape of each amino acid of EcFbFP
Transcriptional regulatory networks of the human gut symbiont Bacteroides thetaiotaomicron are uncovered using machine learning.
No full textBacteroides thetaiotaomicron VPI-5482 is a prominent human gut symbiont of increasing importance to human health and therapeutic applications. Despite its significance, the transcriptional regulatory network (TRN) governing its survival and resilience in vivo remains poorly understood. Here, we present BtModulome, a comprehensive transcriptional regulatory framework derived from independent component analysis of 461 RNA-seq datasets spanning diverse niche-specific conditions and genetic backgrounds. This analysis revealed the BtModulome consisting of 110 independently modulated gene sets (iModulons), explaining 72.9% of the variance in the RNA-seq dataset. We validated strong associations with 39 known regulators and identified 311 novel regulator-regulon relationships, accounting for 22.4% expansion of the known TRN of B. thetaiotaomicron. In addition, we functionally characterized 11 ECF-σs, including SigW-1, which orchestrates arylsulfatase expression critical for host colonization, and SigH-1, which mediates (p)ppGpp-dependent stringent response. Integration of iModulon activities with multi-omics datasets provided mechanistic insights into stress responses and carbon utilization both in vitro and in vivo. This comprehensive TRN framework establishes a foundation for understanding adaptive mechanisms in gut commensals and demonstrates the utility of module-centric analysis for biological discovery in non-model organisms
CRISPRi screening reveals E. coli's anaerobic-like respiratory adaptations to gentamicin: membrane depolarization by CpxR
No full textBacterial genes serve diverse cellular functions and many affect fitness in response to environmental challenges. We employed CRISPR interference screening to investigate the fitness effect of each gene in Escherichia coli exposed to gentamicin, aiming to understand the cellular defense mechanisms. Our findings revealed that ribosomal proteins, ribosome-associated proteins, toxin-antitoxin systems, and outer membrane proteins strongly influence the fitness of E. coli in gentamicin. Notably, gentamicin-induced fitness changes resembled those under anaerobic conditions, where resistance to gentamicin was observed. Specifically, genes related to the biosynthesis of cofactors and electron carriers, crucial for the respiratory system, showed reduced essentiality under both gentamicin and anaerobic conditions, suggesting a disruption in membrane potential leading to limited gentamicin uptake. Transcriptomic and genome-wide binding analyses identified the two-component system CpxR as a key regulator of respiratory systems in response to gentamicin. Our study provides insights into cellular defense mechanisms, offering potential strategies for combating antibiotic resistance. IMPORTANCE Bacteria can adapt to a variety of stressful environments, including antibiotic exposure. The mechanisms underlying antibiotic resistance remain an active area of investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) interference enables specific silencing of gene expression, allowing researchers to assess the fitness effects of gene knockdowns under given conditions. Using genome-wide CRISPR interference screening on Escherichia coli exposed to gentamicin, we identified anaerobic-like fitness effects of genes involved in respiration and the maintenance of membrane potential-key processes that facilitate gentamicin entrance into the cell. Transcriptomic analysis and immunoprecipitation assays further indicated that the two-component system CpxR modulates respiratory adaptations in response to gentamicin challenge. These findings shed light on the development of antibiotic resistance in bacteria and may offer new insight into strategies for treating gentamicin-resistant pathogens.
Development of web-based supporting tools for generic diagnostic reservoir operation
No full textResearch in the field of reservoir operation has been conducted for decades, ending with numerous numerical simulation and optimization models. However, few models are adopted in real-world applications due to enormous complexity and high computational burden requited by these models. Analytical optimization studies and data-based models have been receiving attention as a complement to numerical models, and the cloud-computing technology is increasingly adopted in building decision support systems to make academic advances accessible to practitioners. In this thesis, a web-based support system for reservoir operation, named diagnostic generic reservoir operation tools (DROT), is developed and deployed for public access. DROT implements generic rules and algorithms derived from analytical studies and general data mining techniques, and provides diagnostic information for a particular reservoir operation problem. In addition, auxiliary functionalities including historical operation data retrieval from public databases and interactive data visualization are also provided within DROT. The operation tools implemented in the current version of DROT are based on several existing publications, and can be used for multiple purposes, such as determining hedge release for water supply reservoir, improving use of forecast information in reservoir operation, and selecting most relevant variables in modeling water resources systems. Besides, the algorithm for operating multi-reservoirs system is also available within DROT. The demonstration of DROT for practical operation cases is provided using synthetic data or case studies from existing publications.Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-08-01The student, Donghui Li, accepted the attached license on 2020-07-15 at 11:02.The student, Donghui Li, submitted this Thesis for approval on 2020-07-15 at 11:15.This Thesis was approved for publication on 2020-07-20 at 10:29.DSpace SAF Submission Ingestion Package generated from Vireo submission #15632 on 2020-10-02 at 15:50:52Made available in DSpace on 2020-10-07T22:49:59Z (GMT). No. of bitstreams: 2
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고효율 바이오케미컬 생산을 위한 최소유전체 대장균 개발
No full text학위논문(박사) - 한국과학기술원 : 생명과학과, 2021.2,[v, 179 p. :]Synthetic biology aims to design and construct biological system with novel and better functionalities. One fascinating approach to accomplish the goal is a streamlined genome, often referred as minimal genome, with fewer number of non-essential genes. A number of Escherichia coli strains harboring minimal genomes have been constructed by sequential genome reduction mostly without growth retardation in rich media. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. However, when genome-reduced strains are grown in minimal medium, their growth rate is often reduced. The decreased growth rate has been attributed to our limited understanding of some bacterial genome processes, such as synthetic lethality and interactions between interconnected cellular components, making it difficult to be applied in practical use. In this study, I utilized systems biology approach to re-optimize growth performance of a genome-reduced E. coli and developed an efficient chassis for efficient bio-production using a novel synthetic biology tool. First, I deployed adaptive laboratory evolution (ALE) to allow re-optimization of the perturbations during genome reduction. The evolved strain exhibited a comparable growth rate to wild-type E. coli. Next, multi-omic analysis of the evolved strain was performed to elucidate the cause of growth retardation and design principles for downstream applications. The analysis revealed transcriptome- and translatome-wide changes that remodel metabolism and growth. Lastly, I developed a novel synthetic biology tool and implemented it into the evolved strain to improve bio-production. The engineered strain showed superior capability of producing various biochemicals and proteins than other E. coli strains. Taken together, the comprehensive analysis of a genome-reduced strain provides valuable insight for cellular design and the engineered genome-reduced strain is a promising chassis for biochemical production.한국과학기술원 :생명과학과
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No full textI hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners
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