Mining of Laticifer Specific Promoter in Lettuce

Moonhyuk KWON,Elysabeth K BARNES,Connor HODGINS,Edward YEUNG,Yang QU,Seon-Won KIM,Dae-Kyun RO 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Lettuce (Lactuca sativa) is widely used as a model plant due to advantages such as early flowering, easy transfection, tissue culture, and regeneration. Lettuce laticifers forming a pipe-like structure along the vasculature, and the cytoplasmic content of laticifers is latex. In lettuce latex, terpenoids (natural rubber and sesquiterpene lactone) is synthesized and accumulated as major metabolites. For the heterologous terpenoids production, the lettuce laticifer is suitable tissue because of the enriched terpenoids precursors. However, the lettuce laticifer-specific promoter has not been characterized yet. Here, we identified the promoter of cis-prenyltransferase isoform 3 (LsCPT3) and CPT-binding protein isoform 2 (LsCBP2), the essential genes in natural rubber biosynthesis. From β-glucuronidase (GUS) staining and histochemical sectioning of GUS-stained stem, we proved that the LsCPT3 and LsCBP2 promoter enabled to drive GUS expressions exclusively in laticifers. The LsCBP2 promoter was about 400 folds stronger than the LsCPT3 promoter in laticifers. The LsCBP2 promoter is useful for the latex cell engineering in lettuce.

Characterization of Burkholderia glumae BGR1 4-Hydroxy-3-methylbut-2-enyl Diphosphate Reductase (HDR), the Terminal Enzyme in 2-C-Methyl-Derythritol 4-Phosphate (MEP) Pathway

Kwon, Moonhyuk,Shin, Bok-Kyu,Lee, Jaekyoung,Han, Jaehong,Kim, Soo-Un The Korean Society for Applied Biological Chemistr 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.1

4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the ultimate enzyme in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway converting (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Burkholderia glumae, a Gram-negative rice-pathogenic bacterium, harbors 2 hdr genes and lacks isopentenyl diphosphate isomerase (idi). Both HDR enzymes could complement E. coli hdr deletion mutant (DYTL1). Both of the recombinant HDR proteins, BgHDR1 and BgHDR2, catalyzed reduction of HMBPP into IPP and DMAPP at a ratio of 2:1, in contrast to 5:1 ratio of other bacterial HDRs so far characterized. The $k_{cat}$ and $K_m$ values of BgHDR1 and BgHDR2 were 187.0 $min^{-1}$ and 6.0 ${\mu}M$ and 66.6 $min^{-1}$ and 21.2 ${\mu}M$, respectively. Physiological significance of the kinetic properties was discussed.

A spatial downscaling of soil moisture from rainfall, temperature, and AMSR2 using a Gaussian-mixture nonstationary hidden Markov model

Kwon, Moonhyuk,Kwon, Hyun-Han,Han, Dawei Elsevier 2018 Journal of hydrology Vol.564 No.-

<P><B>Abstract</B></P> <P>A multivariate stochastic soil moisture (SM) estimation approach based on a Gaussian-mixture nonstationary hidden Markov model (GM-NHMM) is introduced in this study to spatially disaggregate the AMSR2 SM data for multiple locations in the Yongdam dam watershed in South Korea. Rainfall and air temperature are considered as additional predictors in the proposed modeling framework. In GM-NHMM, a six-state model is constructed with three predictors representing an unobserved state associated with SM. It is clearly seen that the rainfall predictor plays a substantial role in achieving the overall predictability. Using weather variables (i.e., rainfall and temperature) can be effective in picking up some of the predictability of local SM that is not captured by the AMSR2 data. On the other hand, larger scale dynamic features identified from the AMSR2 data seem to facilitate the identification of regional spatial patterns of SM. The efficiency of the proposed model is compared with that of an ordinary regression model (OLR) using the same predictors. The mean correlation coefficient of the proposed model is about 0.78, which is significantly greater than that of the OLR at about 0.49. The proposed GM-NHMM method not only provides a better representation of the observed SM than the OLR model but also preserves the spatial coherence across all stations reasonably well.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A nonstationary HMM model is employed to spatially downscale the soil moisture data. </LI> <LI> Rainfall predictor plays a substantial role in achieving the overall predictability. </LI> <LI> Climate data are effective in picking up the predictability of local soil moisture. </LI> <LI> The proposed model preserves the spatial coherence across stations reasonably well. </LI> </UL> </P>

Characterization of a Piperonal Synthase in Pipper nigrum

Moonhyuk KWON,Zhehao JIN,Juraithip WUNGSINTAWEEKUL,Dae-Kyun RO,Seon-Won KIM,Soo-un KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Piperonal with a characteristic cherry-like aroma is a simple aromatic aldehyde compound, which has been widely used in flavor and fragrance industries. The aldehyde is also a crucial aroma-imparting molecule of black pepper (Piper nigrum). The bioinformatic analysis of P. nigrum transcriptome data predicted a hydratase-lyase with conserved cysteine proteinases in excess of 68% identity. The hydratase-lyase catalyzed specific side-chain cleavage of 3,4-methylenedioxycinnamic acid (3,4-MDCA) to produce 3,4-methylenedioxybenzaldehyde (piperonal) in vitro and in the gene-harboring yeast. The enzyme was named 3,4-MDCA hydratase-lyase (PnMCHL) or piperonal synthase. However, purported substrates such as cinnamic, coumaric, caffeic, ferulic, and piperic acids did not generate corresponding cleavage products in transgenic yeast. The optimal pH for in-vitro PnMHL was 7.0, and it had Km value for 317.2 μM and kcat for 2.7 s<SUP>-1</SUP>. The enzyme was highly expressed in the leaves, followed by fruits.

Engineering and Enhancing Carotenoids Accumulation in Methylotroph, Methylobacterium organophilum DSM-760

Hawaibam Birla SINGH,Moonhyuk KWON,Seon-Won KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Carotenoids, a group of widely distributed natural products, are extensively utilized in food, pharmacological, and nutraceutical industries. Recently microbial bio-carotenoids production is gaining much interest because of its fast, safe, and varying ability to produce different types of carotenoids using various carbon sources. In this study, Methylobacterium organophilum DSM-760, a pink pigmented, fast-growing facultative methylotroph, was selected for carotenoid production using methanol as a sole carbon source. The native carotenoid accumulation was significantly increased with the MEP pathway augmentation via overexpression of the bottleneck genes (1-Deoxy-D-xylulose 5-phosphate synthase (dsx) and 2-C-methylerythritol 4-phosphate reductoisomerase (dxr)) under the strong promoter PmxaF. Furthermore, the overexpression of methylotrophic carotenogenic genes, along with deletion of carotenoid hydroxylating genes like crtC and crtD, increased the native carotenoids accumulation up to 10 folds. Our results highlight that M. organophilum DSM-760 is a potential microbial platform for producing valuable carotenoids using inexpensive and viable carbon feedstock such as methanol. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A5A8029490).

Functional identification of a <i>Lippia dulcis</i> bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif

Hurd, Matthew C.,Kwon, Moonhyuk,Ro, Dae-Kyun Elsevier 2017 Biochemical and biophysical research communication Vol.490 No.3

<P><B>Abstract</B></P> <P> <I>Lippia dulcis</I> (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of <I>L</I>. <I>dulcis</I> leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (<I>LdTPS3</I> and <I>LdTPS9</I>) were isolated from <I>L</I>. <I>dulcis</I> leaf cDNA. To elucidate their catalytic functions, <I>E. coli</I>-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). <I>In vitro</I> enzyme assays showed that <I>LdTPS3</I> encodes bornyl diphosphate synthase (thus named <I>LdBPPS</I>) while <I>LdTPS9</I> encodes linalool synthase. Interestingly, the <I>N</I>-terminus of LdBPPS possesses two arginine-rich (RRX<SUB>8</SUB>W) motifs, and enzyme assays showed that the presence of both RRX<SUB>8</SUB>W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX<SUB>8</SUB>W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and <I>L. dulcis</I>. The discovery of LdBPPS opens up the possibility of engineering <I>L</I>. <I>dulcis</I> to remove the undesirable product, camphor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Lippia dulcis</I> (Aztec herb) produces intensely sweet sesquiterpenoid, hernandulcin. </LI> <LI> A toxic terpene, camphor, is synthesized in <I>L. dulcis</I> alongside hernandulcin. </LI> <LI> Bornyl diphosphate synthase (BPPS) was first identified from <I>L. dulcis.</I> </LI> <LI> A repeat of arginine-rich motif inhibits <I>L. dulcis</I> BPPS activity. </LI> <LI> Sage and L. <I>dulcis</I> BPPS have independently evolved for the same catalytic activity. </LI> </UL> </P>

Increased Retinoids Production in Recombinant E. coli through Addition of Glyoxylic Acid

Ji-Bin PARK,Seong-Hee JEONG,Moonhyuk KWON,Seon-Won KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Retinoids are synthesized chemically and used as cosmetic agents and effective pharmaceuticals for skin diseases. Chemical processes are expensive, and purification steps are complex, so microbial production systems have emerged as an alternative. However, production using the engineered E. coli MG1655 strain was insufficient due to by-products such as pyruvate and acetyl-CoA. The genes involved in the by-product metabolism were deleted in MG1655 resulting in the mutant called AceCo. Although higher lycopene was produced in the AceCo strain, retinoids production was decreased, and we presumed that reduction was by the glyoxylate cycle. After adding 10 g/L of the glyoxylic acid to the culture media, the retinoids production increased up to 80% after 72 hours. The AceCo strain will be widely used for the production of another terpenoid.

Enhancement of Carotenoid Production and Industrial Applicability by Reconstruction of the Mevalonate Pathway in Escherichia coli

Min-Kyoung KANG,Minh Phuong NGUYEN,Moonhyuk KWON,Seon-Won KIM 한국생물공학회 2023 한국생물공학회 학술대회 Vol.2023 No.10

Reconstitution of the Mevalonate Pathway for the Establishment of Industrial Isoprenoid Producing Escherichia coli Cell Factories

Min-Kyoung KANG,Minh Phuong NGUYEN,Moonhyuk KWON,Seon-Won KIM 한국생물공학회 2023 한국생물공학회 학술대회 Vol.2023 No.4

Strategies to Mitigate Enteric Methane Emissions from Ruminant Animals

( Tenzin Tseten ),( Rey Anthony Sanjorjo ),( Moonhyuk Kwon ),( Seon-won Kim ) 한국미생물 · 생명공학회 2022 Journal of microbiology and biotechnology Vol.32 No.3

Human activities account for approximately two-thirds of global methane emissions, wherein the livestock sector is the single massive methane emitter. Methane is a potent greenhouse gas of over 21 times the warming effect of carbon dioxide. In the rumen, methanogens produce methane as a by-product of anaerobic fermentation. Methane released from ruminants is considered as a loss of feed energy that could otherwise be used for productivity. Economic progress and growing population will inflate meat and milk product demands, causing elevated methane emissions from this sector. In this review, diverse approaches from feed manipulation to the supplementation of organic and inorganic feed additives and direct-fed microbial in mitigating enteric methane emissions from ruminant livestock are summarized. These approaches directly or indirectly alter the rumen microbial structure thereby reducing rumen methanogenesis. Though many inorganic feed additives have remarkably reduced methane emissions from ruminants, their usage as feed additives remains unappealing because of health and safety concerns. Hence, feed additives sourced from biological materials such as direct-fed microbials have emerged as a promising technique in mitigating enteric methane emissions.