High Yield Fermentation of L-serine in Recombinant Escherichia coli via Co-localization of SerB and EamA through Protein Scaffold

Kim-Ngan T. Tran,쿠마라벨 아쇽 쿠마,정재훈,홍순호 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.2

L-serine is a non-essential amino acid which has a wide range of applications and plays an important role as a building block for growing cells. L-serine microbial development is considered a difficult activity due to Lserine's central role in cellular metabolism with 2 main degradation pathways. A novel strategy is needed to overcome the L-serine degradation pathway and low Lserine tolerance of Escherichia coli for efficient L-serine production. A synthetic protein scaffold between SerB and EamA was introduced in this study to physically combine the two enzymes. Through this strategy, the L-serine production is more efficient than in competing pathways. By the introduction of a synthetic protein scaffold without metabolic pathway engineering or addition of glycine, 1.8 g/L of L-serine was produced at pH7 and 37°C. By fermentation, 9.4 g/L of serine was produced at a yield of 0.34 mol/mol glucose. These results suggest that the carbon flux was successfully directed to the L-serine secretion pathway without knocking out a competing pathway or adding expensive glycine.

Impact of the Synthetic Scaffold Strategy on the Metabolic Pathway Engineering

Kim-Ngan T. Tran,쿠마라벨 아쇽 쿠마,홍순호 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.3

For the development of the efficient bio-refinery process or biochemical producer, metabolic engineering has become an attractive choice recently. However, engineered metabolic pathways often suffer from flux imbalances due to a lack of corresponding regulatory mechanisms associated with natural metabolism. The interaction among different enzymes within a metabolic pathway plays an important role in regulating the efficiency of metabolic processes. Consequently, the creation of protein scaffolds has helped with the spatial co-localization of proteins in metabolic engineering. Research on protein scaffolds indicated scaffold systems may enhance metabolic productivity further. In this review, the specificity, selectivity, and regulatory mechanisms of protein-protein interactions are discussed in the context of the important effects that they exert on various biological processes.

Forskolin rescues hypoxia-induced cognitive dysfunction in zebrafish: a potential role of HBP/O-GlcNAc cycling

Quynh T N Nguyen,Jiwon Park,Dong Yeol Kim,Sang-Min Kim,Hyun Jae Sung,Thuy-Duong Thi Tran,Ngan An Bui,Inn-Oc Han 한국당과학회 2022 한국당과학회 학술대회 Vol.2022 No.07

Repeated sublethal hypoxia exposure is reported to accelerate brain inflammation, and to affect the initiation and progression of cognitive dysfunction. Forskolin, an adenylate cyclase activator, is well-known as an inducer of the cAMP/PKA/CREB signaling. It has been shown that forskolin protects against various neuronal complications and induces long term memory; however, the underlying mechanism remains unclear. In this study, we showed that intraperitoneal administration of 200ng/g forskolin for 5 days on zebrafish notably recovered hypoxia-induced social interaction impairment and learning memory deficit. Forskolin suppressed hypoxia-induced neuroinflammation indicated by the decrease of NF-kB signaling and GFAP expression. On mouse neuroblastoma cell line, forskolin notably rescued hypoxia-induced cell death and dysfunction. The hypoxia model either in vivo or in vitro exerted a downregulation of PKA/CREB signaling and its downstream, c-Fos and BDNF expression, which was restored by forskolin. As previous studies proposed that hypoxia-induced hypo-O-GlcNAcylation could be an important causal factor for cognitive defect, we further investigated whether the HBP/O-GlcNAcylation cycling was changed by forskolin. Intriguingly, we observed that following forskolin treatment, OGT protein level (enzyme controls the addition of GlcNAc group to target proteins) induced considerably, which was accompanied by the upregulated O-GlcNAcylation level. Under forskolin treatment, O-GlcNAcylation flux on hypoxia-exposed zebrafish was reserved in line with the recovery of other inflammation and learning/memory markers. Next, we studied further about potential regulation of O-GlcNAcylation on the neuroprotective effect of forskolin. Zebrafish were treated with 500ng/g 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GFAT1 (enzyme that catalyzes the reaction from fructose-6-P to glucosamine-6-P), to downregulate the O-GlcNAcylation of proteins. It was observed that DON treatment led to learning/memory impairment, which was rescued by forskolin. Moreover, forskolin conserved hypo-O-GlcNAcylation and neuroinflammation following DON administration. Further experiments are being conducted to examine how HBP/O-GlcNAc flux is involved in the beneficial effect of forskolin on cognitive function.

Anti-inflammatory Triterpenoid Saponins from the Stem Bark of <i>Kalopanax pictus</i>

Quang, Tran H.,Ngan, Nguyen T. T.,Minh, Chau V.,Kiem, Phan V.,Nhiem, Nguyen X.,Tai, Bui H.,Thao, Nguyen P.,Tung, Nguyen H.,Song, Seok B.,Kim, Young H. American Chemical Society and American Society of 2011 Journal of natural products Vol.74 No.9

<P>Five new compounds, 16,23,29-trihydroxy-3-oxo-olean-12-en-28-oic acid (<B>1</B>), 4,23,29-trihydroxy-3,4-<I>seco</I>-olean-12-en-3-oate-28-oic acid (<B>2</B>), 3β,6β,23-trihydroxyolean-12-en-28-oic acid 28-<I>O</I>-β-<SMALL>d</SMALL>-glucopyranoside (<B>3</B>), 3-<I>O</I>-[2,3-di<I>-O</I>-acetyl-α-<SMALL>l</SMALL>-arabinopyranosyl]hederagenin 28-<I>O</I>-α-<SMALL>l</SMALL>-rhamnopyranosyl-(1→4)-β-<SMALL>d</SMALL>-glucopyranosyl-(1→6)-β-<SMALL>d</SMALL>-glucopyranoside (<B>4</B>), and 3-<I>O</I>-[3,4-di-<I>O</I>-acetyl-α-<SMALL>l</SMALL>-arabinopyranosyl]hederagenin 28-<I>O</I>-α-<SMALL>l</SMALL>-rhamnopyranosyl-(1→4)-β-<SMALL>d</SMALL>-glucopyranosyl-(1→6)-β-<SMALL>d</SMALL>-glucopyranoside (<B>5</B>), as well as 10 known compounds (<B>6</B>–<B>15</B>), were isolated from the stem bark of <I>Kalopanax pictus</I>. Compounds <B>1</B>–<B>5</B> and <B>7</B>–<B>14</B> inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC<SUB>50</SUB> values ranging from 0.6<B></B>to 16.4 μM. Furthermore, the transcriptional inhibitory function of these compounds was confirmed on the basis of decreases in COX-2 and iNOS gene expression in HepG2 cells. The structure–activity relationship of the compounds with respect to anti-inflammatory activity is also discussed.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jnprdf/2011/jnprdf.2011.74.issue-9/np200382s/production/images/medium/np-2011-00382s_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/np200382s'>ACS Electronic Supporting Info</A></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/np200382s'>ACS Electronic Supporting Info</A></P>

REM sleep deprivation impairs learning and memory by decreasing brain O-GlcNAc cycling in mouse

Sang-Min Kim,Jiwon Park,Hyun Jae Sung,Ngan An Bui,Quynh T N Nguyen,Thuy-Duong Thi Tran,Dong Yeol Kim,Inn-Oc Han 한국당과학회 2022 한국당과학회 학술대회 Vol.2022 No.07

Rapid eye movement (REM) sleep is associated with learning and memory (L/M) functions. Here, we demonstrate that REM sleep deprivation (REMSD)-induced impairment of contextual fear memory and spatial memory in mouse is linked to a downregulation in hexosamine biosynthetic pathway (HBP)/O-GlcNAc flux in mouse brain. In mice exposed to REMSD, O-GlcNAcylation proteins and O-GlcNAc transferase (OGT) were decreased while O-GlcNAcase was increased compared to control mouse brain. Foot shock fear conditioning (FC) induced activation of protein kinase A (PKA) and cAMP response element binding protein (CREB), which were significantly inhibited in brains of the REMSD group. Intriguingly, increasing O-GlcNAc cycling with glucosamine (GlcN) or OGA inhibitor, Thiamet G, restored defects in L/M functions and FC-induced PKA/CREB activation induced by REMSD. On behavior test, GlcN restored the contextual fear memory and spatial memory impairment induced by REMSD. Furthermore, Thiamet G restored the REMSD-induced decrease in dendritic spine density and learning and memory impairment. Suppression of O-GlcNAcylation by the glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitor, 6‐diazo‐5‐oxo‐l‐norleucine (DON), or OGT inhibitor, OSMI-1, impaired memory function and inhibited FC-induced PKA/CREB activation. To our knowledge, this is the first study to provide comprehensive evidence of dynamic O-GlcNAcylation changes during the L/M process in mice and defects in this pathway in the brain of REM sleep-deprived mice. Our collective results highlight HBP/O-GlcNAc cycling as a novel molecular link between sleep and cognitive function.

Glucosamine relieves high fat diet-induced fatty liver through autophagy

Jiwon Park,Quynh T N Nguyen,Dong Yeol Kim,Sang-Min Kim,Thuy-Duong Thi Tran,Hyun Jae Sung,Ngan An Bui,Inn-Oc Han 한국당과학회 2022 한국당과학회 학술대회 Vol.2022 No.07

Glucosamine (GlcN) is the amino sugar that increases the protein O-GlcNAcylation level by going into the hexosamine biosynthesis pathway (HBP pathway). Previous studies have reported that GlcN regulates autophagy in various organs including the brain, cartilage, and retina. However, its regulating mechanism and role in the liver are still unclear. Here, we observed glucosamine induces autophagy in human hepatocytes (HepG2). Moreover, LC3, one of the most important factors of autophagy, was upregulated by GlcN at the transcriptional and protein levels. We hypothesized that the HBP pathway is involved in GlcN-induced autophagy. Therefore, examined with inhibition of two enzymes, O-GlcNacase and O-GlcNAc transferase, which have an opposite role in the HBP pathway. As a result, the autophagy process was regulated by O-GlcNAcylation levels. To provide the role of GlcN in the liver autophagy process, we conducted the GlcN effect on the fatty liver, known autophagy disrupted disease. Palmitic acid (PA) induced autophagy inhibition and fat accumulation was relieved by GlcN in HepG2 cells. In addition, high-fat diet-induced fatty liver was restored by GlcN through autophagy in mice. Our results indicate that GlcN increased autophagy flux and through this, lipid accumulation can be restored both in vivo and in vitro.

Improved Itaconic Acid Production by Employing Protein Scaffold Between GltA, AcnA, and CadA in Recombinant Escherichia coli

Jae Hoon JEONG,Ashokkumar KUMARAVEL,Kim-Ngan T TRAN,Soon Ho HONG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.4

Enhanced Production of L-serine in Escherichia coli Through Synthetic Protein Scaffold of SerB, SerC, and EamA

Ashokkumar KUMARAVEL,Jae Hoon JEONG,Kim-Ngan T. TRAN,Soon Ho HONG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.4