Publications by authors named "Seon-Won Kim"

85 Publications

Metabolic Engineering of for Production of α-Santalene, a Precursor of Sandalwood Oil.

J Agric Food Chem 2021 Nov 28;69(44):13135-13142. Epub 2021 Oct 28.

School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215006, People's Republic of China.

α-Santalene belongs to a class of natural compounds with many physiological functions and medical applications. Advances in metabolic engineering enable non-native hosts (e.g., ) to produce α-santalene, the precursor of sandalwood oil. However, imbalances in enzymatic activity often result in a metabolic burden on hosts and repress the synthetic capacity of the desired product. In this work, we manipulated ribosome binding sites (RBSs) to optimize an α-santalene synthetic operon in , and the best engineered NA-IS strain could produce α-santalene at a titer of 412 mg·L. Concerning the observation of the inverse correlation between indole synthesis and α-santalene production, this study speculated that indole-associated amino acid metabolism would be competitive to the synthesis of α-santalene rather than indole toxicity itself. The deletion of could lead to a 1.5-fold increase in α-santalene production to a titer of 599 mg·L in NA-IS. Our results suggested that the optimization of RBS sets of the synthetic module and attenuation of the competitive pathway are promising approaches for improving the production of terpenoids including α-santalene.
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http://dx.doi.org/10.1021/acs.jafc.1c05486DOI Listing
November 2021

Metagenomic analysis of gut microbiome reveals a dynamic change in Alistipes onderdonkii in the preclinical model of pancreatic cancer, suppressing its proliferation.

Appl Microbiol Biotechnol 2021 Nov 14;105(21-22):8343-8358. Epub 2021 Oct 14.

Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, 05505, Republic of Korea.

Pancreatic cancer is a lethal cancer with aggressive and invasive characteristics. By the time it is diagnosed, patients already have tumors extended to other organs and show extremely low survival rates. The gut microbiome is known to be associated with many diseases and its imbalance affects the pathogenesis of pancreatic cancer. In this study, we established an orthotopic, patient-derived xenograft model to identify how the gut microbiome is linked to pancreatic ductal adenocarcinoma (PDAC). Using the 16S rDNA metagenomic sequencing, we revealed that the levels of Alistipes onderdonkii and Roseburia hominis decreased in the gut microbiome of the PDAC model. To explore the crosstalk between the two bacteria and PDAC cells, we collected the supernatant of the bacteria or cancer cell culture medium and treated it in a cross manner. While the cancer cell medium did not affect bacterial growth, we observed that the A. onderdonkii medium suppressed the growth of the pancreatic primary cancer cells. Using the bromodeoxyuridine/7-amino-actinomycin D (BrdU/7-AAD) staining assay, we confirmed that the A. onderdonkii medium inhibited the proliferation of the pancreatic primary cancer cells. Furthermore, RNA-seq analysis revealed that the A. onderdonkii medium induced unique transcriptomic alterations in the PDAC cells, compared to the normal pancreatic cells. Altogether, our data suggest that the reduction in the A. onderdonkii in the gut microbiome provides a proliferation advantage to the pancreatic cancer cells. KEY POINTS: • Metagenome analysis of pancreatic cancer model reveals A. onderdonkii downregulation. • A. onderdonkii culture supernatant suppressed the proliferation of pancreatic cancer cells. • RNA seq data reveals typical gene expression changes induced by A. onderdonkii.
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http://dx.doi.org/10.1007/s00253-021-11617-zDOI Listing
November 2021

The promoter sequences of lettuce cis-prenyltransferase and its binding protein specify gene expression in laticifers.

Planta 2021 Jan 28;253(2):51. Epub 2021 Jan 28.

Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada.

Main Conclusion: Promoters of lettuce cis-prenyltransferase 3 (LsCPT3) and CPT-binding protein 2 (LsCBP2) specify gene expression in laticifers, as supported by in situ β-glucuronidase stains and microsection analysis. Lettuce (Lactuca sativa) has articulated laticifers alongside vascular bundles. In the cytoplasm of laticifers, natural rubber (cis-1,4-polyisoprene) is synthesized by cis-prenyltransferase (LsCPT3) and CPT-binding protein (LsCBP2), both of which form an enzyme complex. Here we determined the gene structures of LsCPT3 and LsCBP2 and characterized their promoter activities using β-glucuronidase (GUS) reporter assays in stable transgenic lines of lettuce. LsCPT3 has a single 7.4-kb intron while LsCBP2 has seven introns including a 940-bp intron in the 5'-untranslated region (UTR). Serially truncated LsCPT3 promoters (2.3 kb, 1.6 kb, and 1.1 kb) and the LsCBP2 promoter with (1.7 kb) or without (0.8 kb) the 940-bp introns were fused to GUS to examine their promoter activities. In situ GUS stains of the transgenic plants revealed that the 1.1-kb LsCPT3 and 0.8-kb LsCBP2 promoter without the 5'-UTR intron are sufficient to express GUS exclusively in laticifers. Fluorometric assays showed that the LsCBP2 promoter was several-fold stronger than the CaMV35S promoter and was ~ 400 times stronger than the LsCPT3 promoter in latex. Histochemical analyses confirmed that both promoters express GUS exclusively in laticifers, recognized by characteristic fused multicellular structures. We concluded that both the LsCPT3 and LsCBP2 promoters specify gene expression in laticifers, and the LsCBP2 promoter displays stronger expression than the CaMV35S promoter in laticifers. For the LsCPT3 promoter, it appears that unknown cis-elements outside of the currently examined LsCPT3 promoter are required to enhance LsCPT3 expression.
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http://dx.doi.org/10.1007/s00425-021-03566-8DOI Listing
January 2021

Association of pericardial adipose tissue with left ventricular structure and function: a region-specific effect?

Cardiovasc Diabetol 2021 01 25;20(1):26. Epub 2021 Jan 25.

Division of Cardiology, Korea University Ansan Hospital, 123, Jeokgeum-ro, Danwon-gu, Gyeonggi-do, 15355, Ansan, South Korea.

Background: The independent role of pericardial adipose tissue (PAT) as an ectopic fat associated with cardiovascular disease (CVD) remains controversial. This study aimed to determine whether PAT is associated with left ventricular (LV) structure and function independent of other markers of general obesity.

Methods: We studied 2471 participants (50.9 % women) without known CVD from the Korean Genome Epidemiology Study, who underwent 2D-echocardiography with tissue Doppler imaging (TDI) and computed tomography measurement for PAT.

Results: Study participants with more PAT were more likely to be men and had higher cardiometabolic indices, including blood pressure, glucose, and cholesterol levels (all P < 0.001). Greater pericardial fat levels across quartiles of PAT were associated with increased LV mass index and left atrial volume index (all P < 0.001) and decreased systolic (P = 0.015) and early diastolic (P < 0.001) TDI velocities, except for LV ejection fraction. These associations remained after a multivariable-adjusted model for traditional CV risk factors and persisted even after additional adjustment for general adiposity measures, such as waist circumference and body mass index. PAT was also the only obesity index independently associated with systolic TDI velocity (P < 0.001).

Conclusions: PAT was associated with subclinical LV structural and functional deterioration, and these associations were independent of and stronger than with general and abdominal obesity measures.
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http://dx.doi.org/10.1186/s12933-021-01219-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836147PMC
January 2021

Regulatory molecule cAMP changes cell fitness of the engineered Escherichia coli for terpenoids production.

Metab Eng 2021 05 24;65:178-184. Epub 2020 Nov 24.

Division of Applied Life Science (BK21 Four), PMBBRC, Gyeongsang National University, Jinju, Republic of Korea. Electronic address:

Terpenoids are a class of natural compounds with many important functions and applications. They are synthesized from a long synthetic pathway of isoprenyl unit coupling with the myriads of terpene synthases. Owing to the catalytic divergence of terpenoids synthesis, microbial production of terpenoids is compromised to the complexity of pathway engineering and suffers from the metabolic engineering burden. In this work, the adaptive Escherichia coli HP variant exhibited a general cell fitness in terpenoid synthesis. Especially, it could yield taxadiene of 193.2 mg/L in a test tube culture, which is a five-fold increase over the production in the wild type E. coli DH5α. Mutational analyses indicated that IS10 insertion in adenylate cyclase CyaA (CyaA) resulted in lowering intracellular cyclic AMP (cAMP), which could regulate its receptor protein CRP to rewire cell metabolism and contributed to the improved cell fitness. Our results suggested a way to manipulate cell fitness for terpenoids production and other products.
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http://dx.doi.org/10.1016/j.ymben.2020.11.009DOI Listing
May 2021

Extension of cell membrane boosting squalene production in the engineered Escherichia coli.

Biotechnol Bioeng 2020 11 30;117(11):3499-3507. Epub 2020 Jul 30.

School of Biology and Basic Medical Sciences, Soochow University, Suzhou, China.

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.
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http://dx.doi.org/10.1002/bit.27511DOI Listing
November 2020

Crystal structure of geranylgeranyl pyrophosphate synthase (crtE) from Nonlabens dokdonensis DSW-6.

Biochem Biophys Res Commun 2019 10 16;518(3):479-485. Epub 2019 Aug 16.

School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu, 41566, Republic of Korea; KNU Institute for Microorganisms, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 41566, Republic of Korea. Electronic address:

Isoprenoids comprise a diverse group of natural products with a broad range of metabolic functions. Isoprenoids are synthesized from prenyl pyrophosphates by prenyltransferases that catalyze the isoprenoid chain-elongation process to different chain lengths. We hereby present the crystal structure of geranylgeranyl pyrophosphate synthase from the marine flavobacterium Nonlabens dokdonensis DSW-6 (NdGGPPS). NdGGPPS forms a hexamer composed of homodimeric trimer, and the monomeric structure is composed of 15 α-helices (α1-α15). In this structure, we observed the binding of one pyrophosphate molecule and two glycerol molecules that mimicked substrate binding to the enzyme. The substrate binding site of NdGGPPS contains large hydrophobic residues such as Phe, His and Tyr, and structural and amino acids sequence analyses thereof suggest that the protein belongs to the short-chain prenyltransferase family.
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http://dx.doi.org/10.1016/j.bbrc.2019.08.071DOI Listing
October 2019

Challenges and tackles in metabolic engineering for microbial production of carotenoids.

Microb Cell Fact 2019 Mar 18;18(1):55. Epub 2019 Mar 18.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, Republic of Korea.

Naturally occurring carotenoids have been isolated and used as colorants, antioxidants, nutrients, etc. in many fields. There is an ever-growing demand for carotenoids production. To comfort this, microbial production of carotenoids is an attractive alternative to current extraction from natural sources. This review summarizes the biosynthetic pathway of carotenoids and progresses in metabolic engineering of various microorganisms for carotenoid production. The advances in synthetic pathway and systems biology lead to many versatile engineering tools available to manipulate microorganisms. In this context, challenges and possible directions are also discussed to provide an insight of microbial engineering for improved production of carotenoids in the future.
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http://dx.doi.org/10.1186/s12934-019-1105-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421696PMC
March 2019

Microbial Platform for Terpenoid Production: and Yeast.

Front Microbiol 2018 12;9:2460. Epub 2018 Oct 12.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju, South Korea.

Terpenoids, also called isoprenoids, are a large and highly diverse family of natural products with important medical and industrial properties. However, a limited production of terpenoids from natural resources constrains their use of either bulk commodity products or high valuable products. Microbial production of terpenoids from and yeasts provides a promising alternative owing to available genetic tools in pathway engineering and genome editing, and a comprehensive understanding of their metabolisms. This review summarizes recent progresses in engineering of industrial model strains, and yeasts, for terpenoids production. With advances of synthetic biology and systems biology, both strains are expected to present the great potential as a platform of terpenoid synthesis.
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http://dx.doi.org/10.3389/fmicb.2018.02460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194902PMC
October 2018

Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production.

Appl Microbiol Biotechnol 2019 Jan 20;103(1):211-223. Epub 2018 Oct 20.

School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8 mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.
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http://dx.doi.org/10.1007/s00253-018-9449-8DOI Listing
January 2019

A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli.

ACS Synth Biol 2018 10 10;7(10):2379-2390. Epub 2018 Oct 10.

Synthetic Biology and Bioengineering Research Center , Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141 , Republic of Korea.

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 isoprene as its less-preferred effector molecule; thus, we amplified the reporter gene expression using a T7 RNA polymerase-mediated transcriptional cascade and iteratively tuned the promoter transcribing tbuT to improve the sensitivity for detecting isoprene. When the engineered E. coli cells expressed heterologous genes for isoprene biosynthesis, the intracellular isoprene was expelled and the tbuT transcription factor was subsequently activated, leading to gfp expression. The chromosomal isoprene biosensor showed a linear correlation between GFP fluorescence and intracellular isoprene concentration. Using this chromosomal isoprene biosensor, we successfully identified the highest isoprene producer among four different E. coli strains producing different amounts of isoprene. The isoprene biosensor presented here can enable high-throughput screening of isoprene synthases and metabolic pathways for efficient and sustainable production of bioisoprene in engineered microbes.
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http://dx.doi.org/10.1021/acssynbio.8b00164DOI Listing
October 2018

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

J Biosci Bioeng 2019 Jan 30;127(1):121-127. Epub 2018 Jul 30.

Biotechnology Process Engineering Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 30 Yeongudanji-ro, Cheongwon-gu, Cheongju, Chungcheongbuk-do 28116, Republic of Korea; Department of Bioprocess Engineering, University of Science and Technology (UST) of Korea, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea. Electronic address:

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 systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.
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http://dx.doi.org/10.1016/j.jbiosc.2018.07.007DOI Listing
January 2019

Metabolic engineering of for production of mixed isoprenoid alcohols and their derivatives.

Biotechnol Biofuels 2018 24;11:210. Epub 2018 Jul 24.

1Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju, 52828 Republic of Korea.

Background: Current petroleum-derived fuels such as gasoline (C-C) and diesel (C-C) are complex mixtures of hydrocarbons with different chain lengths and chemical structures. Isoprenoids are hydrocarbon-based compounds with different carbon chain lengths and diverse chemical structures, similar to petroleum. Thus, isoprenoid alcohols such as isopentenol (C), geraniol (C), and farnesol (C) have been considered to be ideal biofuel candidates. NudB, a native phosphatase of , is reported to dephosphorylate isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into isopentenol. However, no attention has been paid to its promiscuous activity toward longer chain length (C-C) prenyl diphosphates.

Results: In this study, the promiscuous activity of NudB toward geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) was applied for the production of isoprenoid alcohol mixtures, including isopentenol, geraniol, and farnesol, and their derivatives. was engineered to produce a mixture of C and C alcohols by overexpressing NudB (dihydroneopterin triphosphate diphosphohydrolase) and IspA (FPP synthase) along with a heterologous MVA pathway, which resulted in a total of up to 1652 mg/L mixture of C and C alcohols and their derivatives. The production was further increased to 2027 mg/L by overexpression of another endogenous phosphatase, AphA, in addition to NudB. Production of DMAPP- and FPP-derived alcohols and their derivatives was significantly increased with an increase in the gene dosage of , encoding IPP isomerase (IDI), indicating a potential modulation of the composition of the alcohols mixture according to the expression level of IDI. When IspA was replaced with its mutant IspA*, generating GPP in the production strain, a total of 1418 mg/L of the isoprenoid mixture was obtained containing C alcohols as a main component.

Conclusions: The promiscuous activity of NudB was newly identified and successfully used for production of isoprenoid-based alcohol mixtures, which are suitable as next-generation biofuels or commodity chemicals. This is the first successful report on high-titer production of an isoprenoid alcohol-based mixture. The engineering approaches can provide a valuable platform for production of other isoprenoid mixtures via a proportional modulation of IPP, DMAPP, GPP, and FPP syntheses.
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http://dx.doi.org/10.1186/s13068-018-1210-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058358PMC
July 2018

Ty1-fused protein-body formation for spatial organization of metabolic pathways in Saccharomyces cerevisiae.

Biotechnol Bioeng 2018 03 27;115(3):694-704. Epub 2017 Nov 27.

Biotechnology Process Engineering Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea.

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 enzymes involved in a metabolic pathway into a multi-enzyme protein body in yeast. As a proof of principle, Tya fusion to three key enzymes involved in biosynthesis of the isoprenoids farnesene and farnesol was tested to assess its potential to improve productivity. The Tya-fusion protein resulted in three and fourfold increases in farnesene and farnesol production, respectively, as compared with that observed in a non-fused control. Specifically, two-phase partitioning fed-batch fermentations of S. cerevisiae ATCC200589 overexpressing Tya-fused enzymes (tHmg1, IspA, and α-farnesene synthase) yielded 930 ± 40 mg/L of farnesene after 7 days. Additionally, we observed that the Tya-fusion proteins tended to partition into particulate fractions upon 100,000g ultracentrifugation, suggesting the formation of large aggregates of protein bodies, with their particulate structure also observed by transmission electron microscopy. The dramatic increase in the biosynthetic productivity of metabolites via use of a Tya-fusion protein suggested that this approach might be useful for the creation of multi-enzyme complexes to improve metabolic engineering in yeast.
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http://dx.doi.org/10.1002/bit.26493DOI Listing
March 2018

Metabolic engineering and synthetic biology approaches driving isoprenoid production in Escherichia coli.

Bioresour Technol 2017 Oct 29;241:430-438. Epub 2017 May 29.

Division of Applied Life Science (BK21 Plus), PMBBRC, Institute of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea. Electronic address:

Isoprenoids comprise the largest family of natural organic compounds with many useful applications in the pharmaceutical, nutraceutical, and industrial fields. Rapid developments in metabolic engineering and synthetic biology have facilitated the engineering of isoprenoid biosynthetic pathways in Escherichia coli to induce high levels of production of many different isoprenoids. In this review, the stem pathways for synthesizing isoprene units as well as the branch pathways deriving diverse isoprenoids from the isoprene units have been summarized. The review also highlights the metabolic engineering efforts made for the biosynthesis of hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, carotenoids, retinoids, and coenzyme Q in E. coli. Perspectives and future directions for the synthesis of novel isoprenoids, decoration of isoprenoids using cytochrome P450 enzymes, and secretion or storage of isoprenoids in E. coli have also been included.
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http://dx.doi.org/10.1016/j.biortech.2017.05.168DOI Listing
October 2017

Reassessing Escherichia coli as a cell factory for biofuel production.

Curr Opin Biotechnol 2017 06 11;45:92-103. Epub 2017 Mar 11.

Division of Applied Life Science (BK21 Plus), PMBBRC, Institute of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea. Electronic address:

Via metabolic engineering, industrial microorganisms have the potential to convert renewable substrates into a wide range of biofuels that can address energy security and environmental challenges associated with current fossil fuels. The user-friendly bacterium, Escherichia coli, remains one of the most frequently used hosts for demonstrating production of biofuel candidates including alcohol-, fatty acid- and terpenoid-based biofuels. In this review, we summarize the metabolic pathways for synthesis of these biofuels and assess enabling technologies that assist in regulating biofuel synthesis pathways and rapidly assembling novel E. coli strains. These advances maintain E. coli's position as a prominent host for developing cell factories for biofuel production.
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http://dx.doi.org/10.1016/j.copbio.2017.02.010DOI Listing
June 2017

Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH.

J Biotechnol 2017 Apr 7;248:1-8. Epub 2017 Mar 7.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 52828, South Korea. Electronic address:

The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway.
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http://dx.doi.org/10.1016/j.jbiotec.2017.03.005DOI Listing
April 2017

Erratum to: Fermentative production and direct extraction of (-)-α-bisabolol in metabolically engineered Escherichia coli.

Microb Cell Fact 2017 Jan 31;16(1):19. Epub 2017 Jan 31.

Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.

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http://dx.doi.org/10.1186/s12934-017-0635-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282890PMC
January 2017

Isoprene production by Escherichia coli through the exogenous mevalonate pathway with reduced formation of fermentation byproducts.

Microb Cell Fact 2016 Dec 23;15(1):214. Epub 2016 Dec 23.

Division of Applied Life Science (BK21 Plus), PMBBRC, Institute of Agricultural and Life Science, Gyeongsang National University, Jinju, 52828, South Korea.

Background: Isoprene, a volatile C5 hydrocarbon, is an important platform chemical used in the manufacturing of synthetic rubber for tires and various other applications, such as elastomers and adhesives.

Results: In this study, Escherichia coli MG1655 harboring Populus trichocarpa isoprene synthase (PtispS) and the exogenous mevalonate (MVA) pathway produced 80 mg/L isoprene. Codon optimization and optimal expression of the ispS gene via adjustment of the RBS strength and inducer concentration increased isoprene production to 199 and 337 mg/L, respectively. To augment expression of MVA pathway genes, the MVA pathway was cloned on a high-copy plasmid (pBR322 origin) with a strong promoter (P), which resulted in an additional increase in isoprene production up to 956 mg/L. To reduce the formation of byproducts derived from acetyl-CoA (an initial substrate of the MVA pathway), nine relevant genes were deleted to generate the E. coli AceCo strain (E. coli MG1655 ΔackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA). The AceCo strain harboring the ispS gene and MVA pathway showed enhanced isoprene production of 1832 mg/L in flask culture with reduced accumulation of byproducts.

Conclusions: We achieved a 23-fold increase in isoprene production by codon optimization of PtispS, augmentation of the MVA pathway, and deletion of genes involved in byproduct formation.
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http://dx.doi.org/10.1186/s12934-016-0612-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180398PMC
December 2016

Fermentative production and direct extraction of (-)-α-bisabolol in metabolically engineered Escherichia coli.

Microb Cell Fact 2016 Nov 8;15(1):185. Epub 2016 Nov 8.

Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.

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 tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (-)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock.

Results: Here, we created an Escherichia coli strain producing (-)-α-bisabolol at high titer and developed an in situ extraction method of (-)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (-)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (-)-α-bisabolol during fermentation and showed high yield recovery (>98%).

Conclusions: The engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (-)-α-bisabolol at large scale.
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http://dx.doi.org/10.1186/s12934-016-0588-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101696PMC
November 2016

CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production.

Metab Eng 2016 11 26;38:228-240. Epub 2016 Aug 26.

Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34113, Republic of Korea. Electronic address:

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) is emerging as a promising tool for transcriptional modulation. In this study, we developed a regulatable CRISPRi system for fine-tuning biosynthetic pathways and thus directing carbon flux toward target product synthesis. By exploiting engineered Escherichia coli harboring a biosynthetic mevalonate (MVA) pathway and plant-derived terpenoid synthases, the CRISPRi system successfully modulated the expression of all the MVA pathway genes in the context of operon and blocked the transcription of the acetoacetyl-CoA thiolase enzyme that catalyzes the first step in the MVA pathway. This CRISPRi-guided balancing of expression of MVA pathway genes led to enhanced production of (-)-α-bisabolol (C15) and lycopene (C40) and alleviation of cell growth inhibition that may be caused by expression of multiple enzymes or production of toxic intermediate metabolites in the MVA pathway. Coupling CRISPRi to cell growth by regulating an endogenous essential gene (ispA) increased isoprene (C5) production. The regulatable CRISPRi system proved to be a robust platform for systematic modulation of biosynthetic and endogenous gene expression, and can be used to tune biosynthetic metabolic pathways. Its application can enable the development of microbial 'smart cell' factories that can produce other industrially valuable products in the future.
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http://dx.doi.org/10.1016/j.ymben.2016.08.006DOI Listing
November 2016

Farnesol production in Escherichia coli through the construction of a farnesol biosynthesis pathway - application of PgpB and YbjG phosphatases.

Biotechnol J 2016 Oct 29;11(10):1291-1297. Epub 2016 Aug 29.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju, Republic of Korea.

Farnesol is a sesquiterpenoid alcohol that has important industrial and medical potential. It is usually synthesized from farnesyl diphosphate (FPP) by farnesol synthase in plants. FPP accumulation can cause up-regulation of phosphatases capable of FPP hydrolysis, resulting in farnesol production in Escherichia coli. We found that PgpB and YbjG, two integral membrane phosphatases, can hydrolyze FPP into farnesol. Overexpression of FPP synthase (IspA) and PgpB, along with a heterologous mevalonate pathway, enabled recombinant E. coli to produce 526.1 mg/L of farnesol. This result indicates that the phosphatases PgpB and YbjG can be used to construct a novel farnesol synthesis pathway for mass production in E. coli.
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http://dx.doi.org/10.1002/biot.201600250DOI Listing
October 2016

Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production.

Microb Cell Fact 2016 Jan 19;15:14. Epub 2016 Jan 19.

Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea.

Background: Protoilludene is a valuable sesquiterpene and serves as a precursor for several medicinal compounds and antimicrobial chemicals. It can be synthesized by heterologous expression of protoilludene synthase in Escherichia coli with overexpression of mevalonate (MVA) or methylerythritol-phosphate (MEP) pathway, and farnesyl diphosphate (FPP) synthase. Here, we present E. coli as a cell factory for protoilludene production.

Results: Protoilludene was successfully produced in E. coli by overexpression of a hybrid exogenous MVA pathway, endogenous FPP synthase (IspA), and protoilludene synthase (OMP7) of Omphalotus olearius. For improving protoilludene production, the MVA pathway was engineered to increase synthesis of building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by sequential order permutation of the lower MVA portion (MvL), the alteration of promoters and copy numbers for the upper MVA portion (MvU), and the coordination of both portions, resulting in an efficient entire MVA pathway. To reduce the accumulation of mevalonate observed in the culture broth due to lower efficiency of the MvL than the MvU, the MvL was further engineered by homolog substitution with the corresponding genes from Staphylococcus aureus. Finally, the highest protoilludene production of 1199 mg/L was obtained from recombinant E. coli harboring the optimized hybrid MVA pathway in a test tube culture.

Conclusions: This is the first report of microbial synthesis of protoilludene by using an engineered E. coli strain. The protoilludene production was increased by approx. Thousandfold from an initial titer of 1.14 mg/L. The strategies of both the sequential order permutation and homolog substitution could provide a new perspective of engineering MVA pathway, and be applied to optimization of other metabolic pathways.
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http://dx.doi.org/10.1186/s12934-016-0409-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719686PMC
January 2016

Dynamic interplay of multidrug transporters with TolC for isoprenol tolerance in Escherichia coli.

Sci Rep 2015 Nov 13;5:16505. Epub 2015 Nov 13.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea.

Engineering of efflux pumps is a promising way to improve host's tolerance to biofuels such as medium-chain alcohols (CmOHs); however, this strategy is restricted by poor understanding of the efflux pumps engaged in extrusion of solvents. In this study, several Escherichia coli mutants of multidrug transporters were evaluated for isoprenol tolerance. Susceptible phenotypes were observed in the mutants with individual deletion of six transporters, AcrD, EmrAB, MacAB, MdtBC, MdtJI and YdiM, whereas inactivation of AcrAB transporter resulted in an improved tolerance to isoprenol and other CmOHs. AcrAB is the major transporter forming tripartite transperiplasmic complex with outer membrane channel TolC for direct extrusion of toxic molecules in E. coli. The AcrAB inactivation enables to enhance TolC availability for the multidrug transporters associated with extrusion of CmOHs and increase the tolerance to CmOHs including isoprenol. It is assumed that outer membrane channel TolC plays an important role in extrusion of isoprenol and other CmOHs.
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http://dx.doi.org/10.1038/srep16505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643228PMC
November 2015

Functionalized Polyacrylonitrile Nanofibrous Membranes for Covalent Immobilization of Glucose Oxidase.

J Biomed Nanotechnol 2015 Jan;11(1):143-9

Nanofibrous membrane (NFM) with uniform morphology and large surface area was prepared from 10% solution of polyacrylonitrile (PAN) in N,N-dimethylformamide by electrospinning technique. NFM was chemically modified for use as a support for the immobilization of glucose oxidase. Chemical modification of NFM was carried out by two different methods. In the first method, the cyano groups of PAN were modified to amino groups by a two-step process, while in the second method the carboxylic groups were generated first and then further reacted with hexamethylene diamine to create a reactive spacer arm for the immobilization of enzyme. Scanning electron microscopy studies showed that the surface morphology of NFM was not changed by chemical modification and its mechanical strength was improved. The immobilized glucose oxidase (GOx) retained 54 and 60% of its original activity up to 25 cycles with the PAN NFMs modified by the first and the second method, respectively. The GOx-immobilized NFM from the second method showed promising performance with higher enzyme immobilization, activity retention, and favorable kinetic parameters.
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http://dx.doi.org/10.1166/jbn.2015.2028DOI Listing
January 2015

Determination of single nucleotide variants in Escherichia coli DH5α by using short-read sequencing.

FEMS Microbiol Lett 2015 Jun 30;362(11). Epub 2015 Apr 30.

Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea Intelligent Synthetic Biology Center, Daejeon 305-701, Republic of Korea

Escherichia coli DH5α is a common laboratory strain that provides an important platform for routine use in cloning and synthetic biology applications. Many synthetic circuits have been constructed and successfully expressed in E. coli DH5α; however, its genome sequence has not been determined yet. Here, we determined E. coli DH5α genome sequence and identified genetic mutations that affect its phenotypic functions by using short-read sequencing. The sequencing results clearly described the genotypes of E. coli DH5α, which aid in further studies using the strain. Additionally, we observed 105 single nucleotide variants (SNVs), 83% of which were detected in protein-coding regions compared to the parental strain E. coli DH1. Interestingly, 23% of the protein-coding regions have mutations in their amino acid residues, whose biological functions were categorized into two-component systems, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis. These results underscore the advantages of E. coli DH5α, which tolerates the components of transformation buffer and expresses foreign plasmids efficiently. Moreover, these SNVs were also observed in the commercially available strain. These data provide the genetic information of E. coli DH5α for its future application in metabolic engineering and synthetic biology.
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http://dx.doi.org/10.1093/femsle/fnv073DOI Listing
June 2015

Selective retinol production by modulating the composition of retinoids from metabolically engineered E. coli.

Biotechnol Bioeng 2015 Aug 12;112(8):1604-12. Epub 2015 May 12.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea.

Retinoids can be produced from E. coli when introduced with the β-carotene biosynthesis pathway and the BCMO gene. E. coli has no inherent metabolic pathways related to retinoids, therefore only retinal should be produced from the cleavage of β-carotene by BCMO. However, retinol and retinyl acetate were also produced in significant amounts, by the non-specific activity of inherent promiscuous enzymes or the antibiotic resistance marker of the retinal-producing plasmids. Retinol was produced by the ybbO gene of E. coli which encodes oxidoreductase and retinyl acetate was produced by the chloramphenicol resistance gene, called cat gene which encodes chloramphenicol acetyltransferase, present within the pS-NA plasmid that also contains the mevalonate pathway. The composition of retinoids could be modulated by manipulating the relevant genes. The composition of retinol, a commercially important retinoid, was significantly increased by the overexpression of ybbO gene and the removal of cat gene in the recombinant E. coli, which suggests the possibility of selective retinoid production in the future.
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http://dx.doi.org/10.1002/bit.25577DOI Listing
August 2015

Geranyl diphosphate synthase: an important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli.

Enzyme Microb Technol 2015 Jan 25;68:50-5. Epub 2014 Oct 25.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea. Electronic address:

The expression level of geranyl diphosphate synthase (GPPS) was suspected to play a key role for geraniol production in recombinant Escherichia coli harboring an entire mevalonate pathway operon and a geraniol synthesis operon. The expression of GPPS was optimized by using ribosomal binding sites (RBSs) designed to have different translation initiation rates (TIRs). The RBS strength in TIR window of 500 arbitrary unit (au)-1400 au for GPPS appears to be suitable for balancing the geraniol biosynthesis pathway in this study. With the TIR of 500 au, the highest production titer of geraniol was obtained at a level of 1119mg/L, which represented a 6-fold increase in comparison with the previous titer of 183mg/L. The TIRs of GPPS locating out of range of the optimal window (500-1400 au) caused significant decreases of cell growth and geraniol production. It was suspected to result from metabolic imbalance and plasmid instability in geraniol production by inappropriate expression level of GPP synthase. Our results collectively indicated GPPS as an important regulation point in balancing a recombinant geraniol synthesis pathway. The GPPS-based regulation approach could be applicable for optimizing microbial production of other monoterpenes.
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http://dx.doi.org/10.1016/j.enzmictec.2014.10.005DOI Listing
January 2015

Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose.

Appl Microbiol Biotechnol 2015 Apr 11;99(7):3041-55. Epub 2014 Oct 11.

Division of Applied Life Sciences (BK21), SSAC and PMBBRC, Gyeongsang National University, Jinju, 660-701, Republic of Korea.

The modular auxiliary activity (AA) family of proteins is believed to cause amorphogenesis in addition to oxidative cleavage of crystalline cellulose although the supporting evidence is limited. HcAA10-2 is a modular AA10 family protein (58 kDa) composed of a AA10 module and a family two carbohydrate binding module (CBM2), joined by a long stretch of 222 amino acids of unknown function. The protein was expressed in Escherichia coli and purified to homogeneity. Scanning electron microscopy and X-ray diffraction analysis of Avicel treated with HcAA10-2 provided evidence for the disruption of the cellulose microfibrils ("amorphogenesis") and reduction of the crystallinity index, resulting in a twofold increase of cellulase adsorption on the polysaccharide surface. HcAA10-2 exhibited weak endoglucanase-like activity toward soluble cellulose and cello-oligosaccharides with an optimum at pH 6.5 and 45 °C. HcAA10-2 catalyzed oxidative cleavage of crystalline cellulose released native and oxidized cello-oligosaccharides in the presence of copper and an electron donor such as ascorbic acid. Multiple sequence alignment indicated that His1, His109, and Phe197 in the AA10 module formed the conserved copper-binding site. The reducing sugar released from Avicel by the endoglucanase Cel5 and Celluclast accompanying HcAA10-2 was increased by four- and sixfold, respectively. Moreover, HcAA10-2 and Celluclast acted synergistically on pretreated wheat straw biomass resulting in a threefold increase in reducing sugar than Celluclast alone. Taken together, these results suggest that HcAA10-2 is a novel multifunctional modular AA10 protein possessing amorphogenesis, weak endoglucanase, and oxidative cleavage activities useful for efficient degradation of crystalline cellulose.
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http://dx.doi.org/10.1007/s00253-014-6116-6DOI Listing
April 2015

Synthetic biology and metabolic engineering for marine carotenoids: new opportunities and future prospects.

Mar Drugs 2014 Sep 17;12(9):4810-32. Epub 2014 Sep 17.

Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 660-701, Korea.

Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.
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http://dx.doi.org/10.3390/md12094810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178492PMC
September 2014
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