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Exploiting transcriptomic data for metabolic engineering: toward a systematic strain design
Kim, Minsuk,Park, Beom Gi,Kim, Joonwon,Kim, Jin Young,Kim, Byung-Gee Elsevier 2018 Current opinion in biotechnology Vol.54 No.-
<P>Transcriptomics is now recognized as a primary tool for metabolic engineering as it can be used for identifying new strain designs by diagnosing current states of microbial cells. This review summarizes current application of transcriptomic data for strain design. Along with a few successful examples, limitations of conventionally used differentially expressed gene-based strain design approaches have been discussed, which have been major reasons why transcriptomic data are considerably underutilized. Recently, integrative network-based approaches interpreting transcriptomic data in the context of biological networks were invented to provide complimentary solutions for metabolic engineering by overcoming the limitations of conventional approaches. Here, we highlight recent pioneering studies in which integrative network-based methods have been used for providing novel strain designs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Transcriptomic data can facilitate strain design but yet underutilized. </LI> <LI> Differentially expressed genes have been primary targets for metabolic engineering. </LI> <LI> Integrative network-based analysis of transcriptomic data could provide novel strain design. </LI> <LI> More computational tools are needed to fully exploit transcriptomic data for strain design. </LI> </UL> </P>
Kim, Joonwon,Yoo, Hee-Wang,Kim, Minsuk,Kim, Eun-Jung,Sung, Changmin,Lee, Pyung-Gang,Park, Beom Gi,Kim, Byung-Gee Elsevier 2018 Metabolic engineering Vol.47 No.-
<P><B>Abstract</B></P> <P>ω-Hydroxy palmitic acid (ω-HPA) is a valuable compound for an ingredient of artificially synthesized ceramides and an additive for lubricants and adhesives. Production of such a fatty acid derivative is limited by chemical catalysis, but plausible by biocatalysis. However, its low productivity issue, including formations of unsaturated fatty acid (UFA) byproducts in host cells, remains as a hurdle toward industrial biological processes. In this study, to achieve selective and high-level production of ω-HPA from glucose in <I>Escherichia coli</I>, FadR, a native transcriptional regulator of fatty acid metabolism, and its regulon were engineered. First, FadR was co-expressed with a thioesterase with a specificity toward palmitic acid production to enhance palmitic acid production yield, but a considerable quantity of UFAs was also produced. In order to avoid the UFA production caused by <I>fadR</I> overexpression, FadR regulon was rewired by i) mutating FadR consensus binding sites of <I>fabA</I> or <I>fabB</I>, ii) integrating <I>fabZ</I> into <I>fabI</I> operon, and iii) enhancing the strength of <I>fabI</I> promoter. This approach led to dramatic increases in both proportion (48.3–83.0%) and titer (377.8 mg/L to 675.8 mg/L) of palmitic acid, mainly due to the decrease in UFA synthesis. Introducing a fatty acid ω-hydroxylase, CYP153A35, into the engineered strain resulted in a highly selective production of ω-HPA (83.5 mg/L) accounting for 87.5% of total ω-hydroxy fatty acids. Furthermore, strategies, such as i) enhancement in CYP153A35 activity, ii) expression of a fatty acid transporter, iii) supplementation of triton X-100, and iv) separation of the ω-HPA synthetic pathway into two strains for a co-culture system, were applied and resulted in 401.0 mg/L of ω-HPA production. For such selective productions of palmitic acid and ω-HPA, the rewiring of FadR regulation in <I>E. coli</I> is a promising strategy to develop an industrial process with economical downstream processing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> FadR regulon was rewired by mutating FadR binding sites of promoters. </LI> <LI> Rewiring FadR regulation decreased the degree of unsaturation from 33.4% to 5.9% in free fatty acid profile. </LI> <LI> Co-culture system enhanced productivity by relieving the possible metabolic burden. </LI> <LI> In total, 401.0 mg/L of ω-hydroxy palmitic acid was produced in high percentages. </LI> </UL> </P>
Kim, Minsuk,Sang Yi, Jeong,Kim, Joonwon,Kim, Ji-Nu,Kim, Min Woo,Kim, Byung-Gee Wiley 2014 Biotechnology journal Vol.9 No.9
<P>Streptomycetes are industrially and pharmaceutically important bacteria that produce a variety of secondary metabolites including antibiotics. Streptomycetes have a complex metabolic network responsible for the production of secondary metabolites and the utilization of organic residues present in soil. In this study, we reconstructed a high-quality metabolic model for Streptomyces coelicolor A3(2), designated iMK1208, in order to understand and engineer the metabolism of this model species. In comparison to iIB711, the previous metabolic model for S. coelicolor, the predictive power of iMK1208 was enhanced by the recent insights that enabled the incorporation of an updated biomass equation, stoichiometric matrix, and energetic parameters. iMK1208 was validated by comparing predictions with the experimental data for growth capability in various growth media. Furthermore, we applied a strain-design algorithm, flux scanning based on enforced objective flux (FSEOF), to iMK1208 for actinorhodin overproduction. FSEOF results identified not only previously known gene overexpression targets such as actII-ORF4 and acetyl-CoA carboxylase, but also novel targets such as branched-chain α-keto acid dehydrogenase (BCDH). We constructed and evaluated the BCDH overexpression mutant, which showed a 52-fold increase in actinorhodin production, validating the prediction power of iMK1208. Hence iMK1208 was shown to be a useful and valuable framework for studying the biotechnologically important Streptomyces species using the principles of systems biology and metabolic engineering.</P>
마이크로/나노 사이즈의 표면 개질을 통한 풀 비등 임계열유속 향상을 위한 실험적 연구
김선태(Seontae Kim),김형모(Hyungmo Kim),안호선(Ho Seon Ahn),조항진(Hangjin Jo),김준원(Joonwon Kim),김무환(Moo Hwan Kim) 대한기계학회 2008 대한기계학회 춘추학술대회 Vol.2008 No.5
It is well known that modifying the geometry or characteristics of heater surface is one of the effective ways in enhancing CHF. Also many researchers have been struggling to develop fine and fancy heater surface by various methods so far. Lately, there were lots of research about enormous CHF enhancement with nanofluids, which is well dispersed fluid with nano-sized particles, and it was proved that this phenomenon definitely depends on deposition of nanoparticle over heater surface. We have observed micro/nano multi-scale geometry and high wettability characteristic on naturally developed nanoparticle deposited heater surface from boiling experiment with nanofluid. On the base of this observation, we made micro/nano multi-scale surface with good wettability by MEMs technique, and conducted CHF experiments with them.
Sung, Changmin,Jung, Eunok,Choi, Kwon-Young,Bae, Jin-hyung,Kim, Minsuk,Kim, Joonwon,Kim, Eun-Jung,Kim, Pyoung Il,Kim, Byung-Gee Springer-Verlag 2015 Applied microbiology and biotechnology Vol.99 No.16
<P>Hydroxylated fatty acids (HFAs) are used as important precursors for bulk and fine chemicals in the chemical industry. Here, to overproduce long-chain (C16-C18) fatty acids and hydroxy fatty acid, their biosynthetic pathways including thioesterase (Lreu_0335) from Lactobacillus reuteri DSM20016, beta-hydroxyacyl-ACP dehydratase (fabZ) from Escherichia coli, and a P450 system (i.e., CYP153A from Marinobacter aquaeolei VT8 and camA/camB from Pseudomonas putida ATCC17453) were overexpressed. Acyl-CoA synthase (fadD) involved in fatty acid degradation by beta-oxidation was also deleted in E. coli BW25113. The engineered E. coli FFA4 strain without the P450 system could produce 503.0 mg/l of palmitic (C-16) and 508.4 mg/l of stearic (C-18) acids, of which the amounts are ca. 1.6- and 2.3-fold higher than those of the wild type. On the other hand, the E. coli HFA4 strain including the P450 system for omega-hydroxylation could produce 211.7 mg/l of omega-hydroxy palmitic acid, which was 42.1 +/- 0.1 % of the generated palmitic acid, indicating that the hydroxylation reaction was the rate-determining step for the HFA production. For the maximum production of omega-hydroxy palmitic acid, NADH, i.e., an essential cofactor for P450 reaction, was overproduced by the integration of NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii into E. coli chromosome and the deletion of alcohol dehydrogenase (ADH). Finally, the NADH-level-optimized E. coli strain produced 610 mg/l of omega-hydroxy palmitic acid (omega-HPA), which was almost a threefold increase in its yield compared to the same strain without NADH overproduction.</P>
A superhydrophobic dual-scale engineered lotus leaf
Kim, Donghyun,Kim, Joonwon,Park, Hyun C,Lee, Kun-H,Hwang, Woonbong IOP 2008 JOURNAL OF MICROMECHANICS AND MICROENGINEERING - Vol.18 No.1
<P>A surface was created with the same superhydrophobic property as the lotus leaf (lotus effect) by dipping of sandblasted porous alumina into polytetrafluoroethylene (PTFE, Teflon®: DuPont™) solution. The fabricated engineered lotus leaf had PTFE dual-scale structures. This fabrication process has several advantages, including low fabrication cost, simplicity and easy coverage of a large area. The sandblasted porous alumina template was fabricated by sandblasting of an aluminum sheet and anodization in oxalic acid. To obtain PTFE dual-scale structures, PTFE replication based on the dipping method was used, with a 0.3 w% PTFE solution. To remove the aluminum and alumina layers, wet etching by chromic and phosphoric acid mixed solution and liquid HgCl<SUB>2</SUB> solution was used. The fabricated surface has a superhydrophobic property whose apparent contact angle of the PTFE dual-scale structures was approximately 165° and sliding angle is less than 2°.</P>
Kim, Ji Eun,Lim, Joonwon,Lee, Gil Yong,Choi, Sun Hee,Maiti, Uday Narayan,Lee, Won Jun,Lee, Ho Jin,Kim, Sang Ouk American Chemical Society 2016 ACS APPLIED MATERIALS & INTERFACES Vol.8 No.3
<P>Electrochemical oxygen redox reactions are the crucial elements for energy conversion and storage including fuel cells and metal air batteries. Despite tremendous research efforts, developing high-efficient, low-cost, and durable bifunctional oxygen catalysts remains a major challenge. We report a new class of hybrid material consisting of subnanometer thick amorphous cobalt hydroxide anchored on NCNT as a durable ORR/OER bifunctional catalyst. Although amorphous cobalt species-based catalysts are known as good OER catalysts, hybridizing with NCNT successfully enhanced ORR activity by promoting a 4e reduction pathway. Abundant charge carriers in amorphous cobalt hydroxide are found to trigger the superior OER activity with high current density and low Tafel slope as low as 36 mV/decade. A remarkably high OER turnover frequency (TOF) of 2.3 s(-1) at an overpotential of 300 mV was obtained, one of the highest values reported so far. Moreover, the catalytic activity was maintained over 120 h of cycling. The unique subnanometer scale morphology of amorphous hydroxide cobalt species along with intimate cobalt species-NCNT interaction minimizes the deactivation of catalyst during prolonged repeated cycles.</P>