Dae-Hee Lee

Korea Research Institute of Bioscience and Biotechnology KRIBB | KRIBB · Synthetic Biology Research Center

Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by buildi...

Methanotrophs are promising and sustainable cell factory platforms owing to their ability to convert the most potent greenhouse gas, methane to valuable bioproducts. Genetic engineering toolkits for methanotrophs are extremely limited. Here, we present a phenol-inducible promoter for the high-level expression of exogenous genes in methanotrophs. Th...

A genetically encoded biosensor for microbiome engineering is a promising emerging approach for the diagnosis, treatment, and prevention of disease. Engineered bacteria equipped with the genetically encoded biosensor can sense, record, and respond to in vivo disease signals. Recent advances in the design–build–test–learn (DBTL) cycle of genetically...

The past decade has seen a spurt in the number of studies on human microbiome engineering, featuring the microbiota‐mediated modulation of various disease states. Such efforts are ascribed largely to the fact that the microbiome profoundly impacts host physiology, metabolism, and immunity. In this chapter, we provide a brief overview of how gut mic...

Background The gut microbiota is associated with diverse age-related disorders. Several rejuvenation methods, such as probiotic administration and faecal microbiota transplantation, have been applied to alter the gut microbiome and promote healthy ageing. Nevertheless, prolongation of the health span of aged mice by remodelling the gut microbiome r...

The microbial conversion of glycerol into value-added commodity products has emerged as an attractive means to meet the demands of biosustainability. However, glycerol is a non-preferential carbon source for productive fermentation because of its low energy density. We employed evolutionary and metabolic engineering in tandem to construct an Escher...

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. t...

As the bioconversion of methane becomes increasingly important for bio-industrial and environmental applications, methanotrophs have received much attention for their ability to convert methane under ambient conditions. This includes the extensive reporting of methanotroph engineering for the conversion of methane to biochemicals. To further increa...

Genetic circuits have been developed for quantitative measurement of enzyme activity, metabolic engineering of strain development, and dynamic regulation of microbial cells. A genetic circuit consists of several bio-elements, including enzymes and regulatory cassettes, that can generate the desired output signal, which is then used as a precise cri...

Bacteria initiate complicated signaling cascades from the detection of intracellular metabolites or exogenous substances by hundreds of transcription factors, which have been widely investigated as genetically-encoded biosensors for molecular recognition. However, the limited number of transcription factors and their broad substrate specificity res...

Lysozyme is widely used as a model protein in studies of structure–function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme scr...

Acetate has attracted great attention as a carbon source to develop economically feasible bioprocesses for sustainable bioproducts. Acetate is a less-preferred carbon source and a well-known growth inhibitor of Escherichia coli. In this study, we carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, and...

Microbes reprogrammed using advanced genetic circuits are envisaged as emerging living diagnostics for a wide range of diseases and play key roles in regulating gut microbiota to treat disease-associated symptoms in a non-invasive manner. Here, we developed a designer probiotic Escherichia coli that senses and responds to nitrate, a biomarker of gu...

Methanotrophs with soluble methane monooxygenase (sMMO) show high potential for various ecological and biotechnological applications. Here, we developed a high throughput method to identify sMMO-producing microbes by integrating droplet microfluidics and a genetic circuit-based biosensor system. sMMO-producers and sensor cells were encapsulated in...

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ54)-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compare...

Bioconversion of C1 chemicals such as methane and methanol into higher carbon-chain chemicals has been widely studied. Methanol oxidation catalyzed by methanol dehydrogenase (Mdh) is one of the key steps in methanol utilization in bacterial methylotrophy. In bacteria, few NAD+-dependent Mdhs have been reported that convert methanol to formaldehyde....

Methylomonas koyamae LM6 is a potential methanotrophic bacterium of interest for methane bioconversion. Here, we report the complete genome sequence of M. koyamae LM6, which contains 4,337 predicted open reading frames on one chromosome (4,894,002 bp) and one plasmid (186,658 bp), with genes involved in methane oxidation.

Anaerobic digestion (AD) of secondary sludge is a rate-limiting step due to the bacterial cell wall. In this study, experiments were performed to characterize secondary sludges from three wastewater treatment plants (WWTPs), and to investigate the feasibility of using bacteriophage lysozymes to speed up AD by accelerating the degradation of bacteri...

(−)-α-Bisabolol, a naturally occurring sesquiterpene alcohol, has been used in pharmaceuticals and cosmetics owing to its beneficial effects on inflammation and skin healing. Previously, we reported the high production of (−)-α-bisabolol by fed-batch fermentation using engineered Escherichia coli (E. coli) expressing the exogenous mevalonate (MVA)...

The microbial assimilation of one-carbon (C1) gases is a topic of interest, given that products developed using this pathway have the potential to act as promising substrates for the synthesis of valuable chemicals via enzymatic oxidation or C–C bonding. Despite extensive studies on C1 gas assimilation pathways, their key enzymes have yet to be sub...

Human enteropathogenic bacteria has been reported to be transmitted by fresh vegitables. Shigella , which infects primates, is reportedly transmitted by fresh vegetables; however, its molecular interactions with plants have not been elucidated. Here, we show that four Shigella strains, S. boydii ( S. b ), S. sonnei , S. flexneri 2a ( S. f 2a), and...

Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease‐deficient Cas9 gene and a d...

Biocatalytic cyclization is highly desirable for efficient synthesis of biologically derived chemical substances, such as the commodity chemicals ε-caprolactam and δ-valerolactam. To identify biocatalysts in lactam biosynthesis, we develop a caprolactam-detecting genetic enzyme screening system (CL-GESS). The Alcaligenes faecalis regulatory protein...

Isoprene is a valuable precursor for synthetic rubber and a signature product of terpenoid pathways. Here, we developed an isoprene biosensor by employing a TbuT transcriptional regulator of Ralstonia pickettii to express a fluorescent reporter gene in response to intracellular isoprene in engineered Escherichia coli. The TbuT regulator recognizes...

ω-Hydroxyundec-9-enoic acid (ω-HUA) was reported as a valuable medium-chain fatty acid with industrial potentials. For bioconversion of ricinoleic acid to ω-HUA, in this study, an alcohol dehydrogenase (Adh) from Micrococcus luteus, a Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440 and an esterase (Pfe1) from Pseudomonas fluores...

CelR is a transcriptional regulator that controls the expression of cellulases catalyzing cellulose hydrolysis. However, the structural mechanism of its regulation has remained unclear. Here, we report the first structure of CelR, in this case with cellobiose bound. CelR consists of a DNA binding domain (DBD) and a regulatory domain (RD), and homod...

Background Isoprene is a five-carbon chemical that is an important starting material for the synthesis of rubber, elastomers, and medicines. Although many plants produce huge amounts of isoprene, it is very difficult to obtain isoprene directly from plants because of its high volatility and increasing environmental regulations. Over the last decade...

Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the...

Background Multiplex control of metabolic pathway genes is essential for maximizing product titers and conversion yields of fuels, chemicals, and pharmaceuticals in metabolic engineering. To achieve this goal, artificial transcriptional regulators, such as clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi), ha...

Successful utilization of cellulose as renewable biomass depends on the development of economically feasible technologies, which can aid in enzymatic hydrolysis. In this study, we developed a whole-cell biosensor for detecting cellulolytic activity that relies on the recognition of cellobiose using the transcriptional factor CelR from Thermobifida...

Inefficient transport of xylose into Saccharomyces cerevisiae is a major hurdle for production of xylitol, a natural sweetener with five carbons. To facilitate the xylose transport and hence increase xylose conversion to xylitol, the araE gene encoding an arabinose:H⁺ symporter (AraE) from Bacillus subtilis and the XYL1 gene from Scheffersomyces st...

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane prote...

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is an emerging technology for artificial gene regulation. Type II CRISPR-Cas endonuclease Cas9 is the most widely used protein for gene regulation with CRISPRi. Here, we present type V-A CRISPR-Cas endonuclease Cpf1-based CRISPRi. We constructed an l-rhamnose-inducible...

Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the format...

Determination of molecular mass of GusA and CBD fused GusA. (A) SDS-PAGE analysis of purified enzymes from each purification steps. Lane 1, crude extract of GusA; lane 2, IMAC column product (purified GusA enzyme) Lane 3, crude extract of GusA-CBD; Lane 4, IMAC column product (GusA-CBD); Lane 5, crude extract of CBD-GusA; Lane 6, IMAC column produc...

The specific activity of GusA, GusA-CBD, CBD-GusA. (TIF)

Schematic representation of enzyme catalysts with self-assembled CBDs. The GusA (yellow)-CBD (grey) complex with flexible linker was present in three states depending on the environmental conditions. (TIF)

Primers used in this study. (TIF)

Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with t...

Effect of insert size and composition. A. Composition and sizes of various clones in this study. cat represents chloramphenicol-resistant gene activated by homologous recombination. B. Images of ChroV-JM109(DE3) colonies expressing various target genes: 0.7, 2kb insert expressed green fluorescence, while 4kb insert produced yellow pigmented colonie...

Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions o...

Background(−)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (−)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tre...

Escherichia coli was metabolically engineered for the production of d-ribose, a functional five-carbon sugar, from xylose. For the accumulation of d-ribose, two genes of transketolase catalyzing the conversion of d-ribose-5-phosphate to sedoheptulose-7-phosphate in pentose phosphate pathway were disrupted to create a transketolase-deficient E. coli...

Methods: for simple and efficient regulation of metabolic pathway genes are essential for maximizing product titers and conversion yields, and for minimizing the metabolic burden caused by heterologous expression of multiple genes often in the operon context. Clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi)...

The development of synthetic biological devices has increased rapidly in recent years and the practical benefits of such biological devices are becoming increasingly clear. Here, we further improved the design of a previously reported high-throughput genetic enzyme screening system by investigating device-compatible biological components and phenol...

Bacterial persisters are a small fraction of quiescent cells that survive lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the...

Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. To accomplish successful overexpression of membrane proteins in a workhorse organism such as E. coli, defined and rational optimization strategies based on an understanding of the genetic background of the t...

PacBio's long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of second-generation, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In...

Fluorescence resonance energy transfer (FRET)-based sensors transduce ligand recognition into a change in the fluorophore spectrum, as ligand binding alters the distance between and orientation of two fluorescent proteins. Here, we report a dramatic increase in the signal intensity of FRET-based sugar sensors with bacterial periplasmic binding prot...

Background Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.ResultsThe gene encoding a novel AP...

Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from...

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosom...

Sugar transport is very critical in developing an efficient and rapid conversion process of a mixture of sugars by engineered microorganisms. By using expressed sequence tag data generated for the fructophilic yeast Candida magnoliae JH110, we identified two fructose-specific transporters, CmFSY1 and CmFFZ1, which show high homology with known fruc...

Spacing between the −10 and −35 promoter elements. The distribution of the number of base pairs separating the −10 promoter element from the −35 promoter element for each unique transcription start site. (PDF)

AT content in the regions surrounding promoters. The AT fraction is shown for each promoter motif determined. The plot is shown ± 300 bp with respect to the 3′ end of the −10 promoter element. (PDF)