Characterization and Application of Embden-Meyerhof-Parnas Pathway-disrupted Escherichia coli for Production of 3-hydroxypropionic Acid

Kyung Hyun CHO,Young Shin RYU,Yuchan KIM,Sung Kuk LEE 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

In nature, Escherichia coli mainly relies on Embden-Meyerhof-Parnas pathway (EMPP) for utilizing glucose. On the other hand, strains such as Pseudomonas catabolize glucose via Entner-Doudoroff pathway (EDP) and pentose phosphate pathway (PPP). Different natures of microorganisms are often attributed to distinct structure of glycolysis critical in feeding metabolic currencies such as precursor, redox and energy for anabolism. A flux rewiring from EMPP to EDP or PPP in E. coli can supply energy and reducing power required for cell growth and production of valuable chemicals such as 3-hydroxypropionic acid (3-HP). However, the EMPP-disrupted E. coli did not grow on glucose as a sole carbon source. To recover its growth rate, we applied adaptive laboratory evolution (ALE). Then, by applying genotypic- and phenotypic-analysis, the mutations that contributed growth recovery of the evolved strain was characterized. Basis of the knowledge obtained from ALE, we further engineered the evolved strain in order to enhance production performance of 3-HP which demands high amount of NADPH. The evolved strain produced 2.7 g/L of 3-HP while unevolved strain produced 0.7 g/L of 3-HP.

Biosynthesis of L-ergothioneine by Regulating Sulfur Assimilation in Metabolically Engineered Corynebacterium glutamicum

Minhye KIM,Jun Won OH,Hyun Jin JEONG,Young Jin KO,Sung Ok HAN 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

L-Ergothioneine (EGT), which is known as a sulfur-containing histidine compound, has been regarded as a strong antioxidant in the cosmetic and food industries by using as an antiaging material and food preservative. It is commonly extracted from edible mushrooms, but it still has limited productivity. Here, we developed metabolically engineered Corynebacterium glutamicum producing EGT without any precursor addition. We first reinforced sulfur assimilation by exchanging a native promoter and enhanced the cysteine synthetic pathway. In this strain, EGT production was 1.48-fold more increased compared to that of control. To confirm the effects of enhanced L-histidine production, the pentose phosphate pathway was reinforced by introducing a synthetic promoter. This strain produced a 1.89-fold increased EGT compared to the strain in which only the sulfur assimilatory pathway was enhanced. Finally, the strain, which was consolidated all sulfur assimilatory, histidine, and cysteine pathways, was developed, and it synthesized a 2.95-fold increase of EGT. In this study, we suggested major-related pathways for EGT synthesis, and we first synthesized EGT by accumulating precursors in C. glutamicum.

Function of the pentose phosphate pathway and its key enzyme, transketolase, in the regulation of the meiotic cell cycle in oocytes

Kim, Yunna,Kim, Eun-Young,Seo, You-Mi,Yoon, Tae Ki,Lee, Woo-Sik,Lee, Kyung-Ah The Korean Society for Reproductive Medicine 2012 Clinical and Experimental Reproductive Medicine Vol.39 No.2

Objective: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). Methods: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). Results: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. Conclusion: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.

The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy

( Eunae Sandra Cho ),( Yong Hoon Cha ),( Hyun Sil Kim ),( Nam Hee Kim ),( Jong In Yook ) 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.1

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeu-tic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphoryla-tion required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progres-sion as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress.

The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy

Cho, Eunae Sandra,Cha, Yong Hoon,Kim, Hyun Sil,Kim, Nam Hee,Yook, Jong In The Korean Society of Applied Pharmacology 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.1

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeutic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphorylation required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progression as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress.

Heteroexpression and Functional Characterization of Glucose 6-Phosphate Dehydrogenase from Industrial Aspergillus oryzae

( Hongwei Guo ),( Jinyao Han ),( Jingjing Wu ),( Hongwen Chen ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.4

The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was 50°C. This enzyme also had a half-life of 33.3 min at 40°C. Kinetics assay showed that AoG6PDH was strictly dependent on NADP+ (K_m = 6.3 μM, k_cat = 1000.0 s^-1, k_cat/K_m =158.7 s^-1·μM^-1) as cofactor. The K_m and k_cat/K_m values of glucose-6-phosphate were 109.7 s^-1·μM^-1 and 9.1 s^-1·μM^-1 respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where NADP⁺ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.

6-aminonicotinamide ameliorates pulmonary fibrosis by suppressing the TGF-β1-activated Smad signaling pathway

( Miae Kim ),( Jin Woo Song ),( Kwanghon Choi ),( Su Jin Moon ),( Hyun Ju Yoo ) 대한결핵 및 호흡기학회 2019 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.127 No.-

Background: Pentose phosphate (PP) pathway is one of the major metabolic pathways associated with glucose metabolism. Glucose 6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of PP pathway and its function is inhibited by 6-aminonicotinamide (6-AN). This study aims to investigate the role of the PP pathway in idiopathic pulmonary fibrosis (IPF). Methods: The metabolites of PP pathway were analyzed with Agilent 7890/5975 GC/MSD system and HP-5 MS column in the human lung tissues (IPF =31 and control=20). The roles of G6PD were evaluated using fibrotic markers in fibroblasts or epithelial cells treated with transforming growth factor-β1 (TGF-β1). The antifibrotic role of 6-AN was assessed in a bleomycin-induced lung fibrosis mice model. The levels of proteins in cell lysates or tissues were measured by western blot assays or RT-PCR. Results: The levels of PP pathway metabolites in IPF lung tissues were significantly elevated than those in control lung tissues. Moreover, protein expression of G6PD was increased in fibroblasts by TGF-β1 stimulation. 6-AN and G6PD specific siRNA reduced TGF-β1 induced mRNA or protein expression levels of collagen type-I and -SMA in fibroblasts. In addition, 6-AN decreased the TGF-β1 induced mRNA and protein expression levels of epithelial mesenchymal transition factor in Beas-2b cells. In bleomycin treated mice, 6-AN also decreased levels of hydroxyproline in the lung compared to that of control mice. Conclusions: Our findings indicate that inhibition of G6PD may have anti-fibrotic effects on pulmonary fibrosis, suggesting that G6PD is implicated as a potential therapeutic target in IPF.

Effects of Increased NADPH Concentration by Metabolic Engineering of the Pentose Phosphate Pathway on Antibiotic Production and Sporulation in Streptomyces lividans TK24

( Xue-mei Jin ),( Yong-keun Chang ),( Jae Hag Lee ),( Soon-kwang Hong ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.10

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.

PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration

Kang, Hojin,Jo, Areum,Kim, Hyein,Khang, Rin,Lee, Ji-Yeong,Kim, Hanna,Park, Chi-Hu,Choi, Jeong-Yun,Lee, Yunjong,Shin, Joo-Ho Academic Press 2018 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H<SUB>2</SUB>O<SUB>2</SUB>, and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PARIS reprograms glucose metabolism via TKT suppression. </LI> <LI> Increased oxidative stress by TKT reduction induces HIF-1α. </LI> <LI> TKT overexpression and HIF-1α knock-down partially rescues PARIS toxicity. </LI> </UL> </P>

Activation of Pentose Phosphate Pathway in Escherichia coli for the Co-production of Hydrogen and Ethanol from Glucose

Balaji SUNDARA SEKAR,Eunhee SEOL,Sunghoon PARK 한국생물공학회 2016 한국생물공학회 학술대회 Vol.2016 No.10