Hongweon Lee

Korea Research Institute of Bioscience and Biotechnology KRIBB | KRIBB · Biotechnology Process Engineering Center

α, ω-Dicarboxylic acids (DCAs) are multipurpose chemicals widely used in polymers, perfumes, plasticizers, lubricants, and adhesives. The biotransformation of DCAs from alkanes and fatty acids by microorganisms has attracted recent interest, since synthesis via chemical oxidation causes problems in terms of the environment and safety. We isolated a...

Supporting Information

The physiological responses of microorganisms to specific nutrient limitation can be regulated at the transcriptional levels. In this study, in order to develop the Pichia pastoris-derived promoter inducible by nutrient-limited condition, we constructed cDNA libraries using RT-PCR of total RNA from P. pastoris in steady-states of phosphate-limited...

A highly productive fed-batch fermentation process was developed for the production of L-ornithine by using a new stabilized strain, Brevibacterium ketoglutamicum BK52. Fed-batch cultures with a continuous feeding of the complex medium were conducted on various operating conditions. The optimal concentration of phosphate in the complex medium was 2...

L-Threonine fermentation process was constructed on batch and fed-batch culture by using Escherichia coli MT201. The production type of L-threonine was observed as growth-associated production in batch culture. In fed-batch culture studying optimal concentration of yeast extract in feeding media, when 600 g/l of glucose and 60 g/l of yeast extract...

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon...

Volatile compounds can be produced by fermentation from genetically engineered microorganisms. Escherichia coli strains are mainly used for isoprene production owing to their higher titers; however, this has thus far been confined to only strains BL21, BL21 (DE3), Rosetta, and BW25113. Here, we tested four groups of E. coli strains for improved iso...

Yarrowia lipolytica is a nonconventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knock-out mutant strains from polyploid cells requires significa...

Sebacic acid (SA) is an aliphatic ten-carbon dicarboxylic acid (1,10-decanedioic acid) with a variety of industrial applications, including the production of plasticizers, lubricants, cosmetics, and plastics. Currently, SA has been produced exclusively from alkaline pyrolysis of castor oil. Herein, we present an environmentally friendly green route...

Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutrop...

Heart failure (HF) is a coronary disease that affects people worldwide and has a high mortality rate. N-terminal pro-brain natriuretic peptide (NT-proBNP) has been proven to be a useful and accurate biomarker for diagnosing systolic HF. Here, we report a strategy for the high-level production of recombinant (r)NT-proBNP in Escherichia coli. An Fh8...

Sustainable manufacture of dicarboxylic acids (DCAs), which are used as raw materials for multiple commercial products, has been an area of considerable research interest in recent years. Traditional chemical-based manufacture of DCAs suffers from limitations such as harsh operational conditions and generation of hazardous by-products. Microbiologi...

Background As a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast Candida tropicalis has been used as a host for DCA production, additional strains are...

Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available...

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current s...

The study describes the development of a vaccine using microcrystalline cellulose (Avicel PH-101) as a delivery carrier of recombinant protein-based antigen against erysipelas. Recombinant SpaA, surface protective protein, from a gram-positive pathogen Erysipelothrix rhusiopathiae was fused to a cellulose-binding domain (CBD) from Trichoderma harzi...

Biotransformation of fatty acid methyl esters to dicarboxylic acids has attracted much attention in recent years; however, reports of sebacic acid production using such biotransformation remain few. The toxicity of decanoic acid is the main challenge for this process. Decane induction has been reported to be essential to activate the enzymes involv...

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing ace...

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon ov...

Polyhydroxyalkonate (PHA) is a type of polymer that has the potential to replace petro-based plastics. To make PHA production more economically feasible, there is a need to find a new carbon source and engineer microbes to produce a commercially valuable polymer. Coffee waste is an inexpensive raw material that contains fatty acids. It can act as a...

A sulfur-oxidizing chemolithoautotrophic bacterium, Sulfurovum lithotrophicum 42BKTT, isolated from hydrothermal sediments in Okinawa, Japan, has been used industrially for CO2 bio-mitigation owing to its ability to convert CO2 into C5H8NO4− at a high rate of specific mitigation (0.42 g CO2/cell/h). The genome of S. lithotrophicum 42BKTT comprised...

Controlling residual glucose concentration is important for improving productivity in L-threonine fermentation. In this study, we developed a procedure to automatically control the feeding quantity of glucose solution as a function of ammonia-water consumption rate. The feeding ratio (Rc/n) of glucose and ammonia water was pre-determined via a stoi...

Metabolite production through a multistep metabolic pathway can often be increased by efficient substrate channeling created by spatial sequestration of the metabolic reactions. Here, Tya, a structural component in the Ty1 retrotransposon element that forms virus-like particles (VLPs) in Saccharomyces cerevisiae, was used to spatially organize enzy...

Lignocellulose is now a promising raw material for the biofuel production. However, lignin complex and crystalline cellulose require pretreatment steps for break-down of the crystalline structure of cellulose for the generation of fermentable sugars. However, several fermentation inhibitors are generated with sugar compounds majorly furfural. Mitig...

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing ace...

Objectives: To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris. Results: Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameter...

Recently, the bio-production of α,ω-dicarboxylic acids (DCAs) has gained significant attention, which potentially leads to the replacement of the conventional petroleum-based products. In this regard, the lipid accumulating yeast Candida tropicalis, has been recognized as a promising microbial host for DCA biosynthesis: it possess the unique ω-oxid...

Enolase (ENO) is one of the surface-exposed proteins of Streptococcus iniae, which previously had been identified as a plasminogen-binding protein. In this study, ENO was evaluated to induce cross-protective immunity against S. iniae and S. parauberis which are major pathogens causing streptococcosis in fish. Immunoblot analysis shows that S. iniae...

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory el...

Furfural is a toxic by-product formulated from pretreatment processes of lignocellulosic biomass. In order to utilize the lignocellulosic biomass on isobutanol production, inhibitory effect of the furfural on isobutanol production was investigated and combinatorial application of two oxidoreductases, FucO and YqhD, was suggested as an alternative s...

Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRN...

Streptococcus iniae is associated with morbidity in commercial fish species, especially in olive flounders (Paralichthys olivaceus), and was recently identified as an emerging human pathogen. Here, we report the complete 2.09-Mb genome sequence of S. iniae strain YSFST01-82, isolated from an olive flounder with streptococcosis disease in Jeju, Sout...

Clitocybin A is a novel anti-wrinkle cosmetic agent produced by the strain from a Korean native mushroom Clitocybe aurantiaca. In this study, fermentation, extraction, and purification conditions for a large scale production of clitocybin A were optimized, and its cytotoxicity and inhibition activity on the expression of matrix metalloproteinase-1...

For the isolation of a ε-caprolactam-degrading microbe from wastewaters of a factory producing caprolactam, we applied a chemostat-enrichment technique with a selective medium containing caprolactam as sole source of carbon and nitrogen. This allowed for the isolation of a novel caprolactam-degrading microbe, identified as Acinetobacter calcoacetic...

Corynebacterium glutamicum is one of the well-studied industrial strain that is used for the production of nucleotides and amino acids. Recently, it has also been studied as a possible producer of organic acids such as succinic acid, based on its ability to produce organic acids under an oxygen deprivation condition. In this study, we conducted the...

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dime...

The removal of Gal80 protein by gene disruption turned into efficient GAL promoter‐driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P GAL10 ) in Δgal80 mutant that does not require galactose as an ind...

Overexpression of Bcl-2, a typical anti-apoptotic protein, is one of the most effective means to maintain mitochondria integrity in recombinant CHO (rCHO) cell culture treated with sodium butyrate (NaBu). NaBu is known as a typical specific productivity-enhancing factor and also a well-known apoptosis inducer. Bcl-2 is distributed to and functions...

The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. By using lipase B from C. antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL) (10) ) in Δgal80 mutant that does not require galactose as an i...

Haemophilus parasuis causes contagious porcine Glässer's disease leading to severe losses in the swine industry. In this study, we established an efficient Escherichia colibased system for the expression of H. parasuis major outer-membrane protein (MOMP) that has been known as a good vaccine candidate against Glässer's disease. Use of an E. coli-de...

The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the sele...

Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S(3)N(10) pept...

We explored the physiological and metabolic effects of different carbon sources (glucose, fructose, and glucose/fructose mixture) in phosphoglucose isomerase (pgi) knockout Escherichia coli mutant producing shikimic acid (SA). It was observed that the pgi(-) mutant grown on glucose exhibited significantly lower cell growth compared with the pgi(+)...

Constitutively active Ras (CA-Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA-Ras overexpres...

Haemophilus parasuis causes contagious porcine Glässer's disease, which is occurring worldwide and leads to severe losses in the pig industry. To identify novel antigen candidates against this disease, 22 surface-exposed or secreted proteins were selected from the annotated H. parasuis genome by reverse vaccinology strategy. Expression of these pro...

We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For th...

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed the galactose-fr...

Word document contains details on the calculation of biomass composition, carbon balance and ATP maintenance requirement.

Excel file contains metabolic reaction, metabolite and gene lists.

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a c...

An efficient yeast gene expression system with GAL10 promoter that does not require galactose as an inducer was developed using Δgal80 mutant strain of Saccharomyces cerevisiae. We constructed several combinations of gal mutations (Δgal1, Δgal80, Δmig1, Δmig2, and Δgal6) of S. cerevisiae and tested for their effect on efficiency of recombinant prot...

Efficient expression and purification of bioactive recombinant human interleukin-6 (hIL6) was successfully achieved in Escherichia coli (E. coli) by fusion of the maltose-binding protein (MBP) with hIL6 and the insertion of oligopeptide linkers. MBP/hIL6 was over-expressed in the soluble form at a concentration of approximately 2.5g/L. For hIL6 rec...

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na+)-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the P...

Effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate, and glycerol) on shikimic acid (SA) biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for overproducing SA. At the outset, constraints-based flux analysis using the genome-scale in silico model of E. coli...

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 m...

The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P TEF1 ) in P. pastoris was investigated with comparison to the glyceralde...

L-threonine production was investigated in a minimal salt medium using L-threonine-overproducing Escherichia coli MT201, derived from E. coli K-12. It was observed that dry cell weight reached 12.5 g/l with 15.9 g/lL-threonine. To increase dry cell weight and L-threonine production, the fermentation process was optimized. When biotin was added as g...

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the f...