Serratia marcescens에서 글리옥실산이 Prodigiosin 생합성에 미치는 연구

최병범,방선권 한국식품영양학회 1997 韓國食品營養學會誌 Vol.10 No.4

최소 배지에 여러 아미노산과 대사 산물을 첨가하여 혐기성 조건하에서 배양시킨 Serratia marcescens ATCC 25419 세포 추출물에서 prodigiosin의 생합성을 조사한 결과 아미노산과 대사 산물들은 prodigiosin 생합성을 대조군보다 50∼80% 정도 감소시켰다. 글리옥실산은 1∼3mM에서 prodigiosin의 생합성을 대조군보다 20∼40% 정도 증가시켰고, 특히 5mM의 농도에서는 최대 122% 증가시켰으나, 20∼30mM에서 prodigiosin의 생합성을 50∼90% 정도 감소시켰다. 한편, 혐기성과 호기성 조건의 피루브산과 α-케토부티르산은 호기성 조건의 글리옥실산과 함께 조사한 모든 농도(0.5∼30mM)에서 prodigiosin이 생성되지 않았으며 또한, 혐기성 조건하에서 높은 농도(20mM 이상)의 글리옥실산은 S. marcescens의 성장을 현저하게 억제시켰다. 이러한 결과들로부터 글리옥실산은 피루브산과 α-케토부티르산과는 다르게 낮은 농도(1∼5mM)에서는 S. marcescens의 1, 2차 대사물질로 작용하여 prodigiosin 생합성을 증가시키지만, 높은 농도(20mM 이상)에서는 S. marcescens의 성장을 억제시켜 2차 대사물질인 prodigiosin의 생합성도 억제된다고 사료된다. The effects of amino acids and metabolites in growth media on the biosynthesis of prodigiosin from Serratia marcescens ATCC 25419 were examined. The prodigiosin synthesis was decreased approximately by 50 to 80% by several amino acids and metabolites tested. The prodigiosin synthesis was increased approximately by 20 to 40% by a low concentration of glyoxylate(1 to 3mM) and outstandingly increased by 122% at 5mM concentration under anaerobic condition. However, the prodigiosin synthesis was decreased approximately by 50 to 90% at a high concentration(20 to 30mM) under anaerobic condition. The prodigiosin was not synthesized by pyruvate and α-ketobutyrate under aerobic and anaerobic condition, with addition to glyoxylate under aerobic condition, among the range from 0.5 to 30mM, while the cell growth under anaerobic condition was decreased distinctly by high concentration(20mM above) of glyoxylate. These data suggest that the growth and prodigiosin of S. marcescens is positively regulated by a low concentration of glyoxylate (1∼5mM), but repressed by a high concentration of glyoxylate (20mM above) unlike pyruvate and α-ketobutyrate.

Crystal structure and functional analysis of isocitrate lyases from Magnaporthe oryzae and Fusarium graminearum

Park, Y.,Cho, Y.,Lee, Y.H.,Lee, Y.W.,Rhee, S. Academic Press 2016 Journal of structural biology Vol.194 No.3

<P>The glyoxylate cycle bypasses a CO2-generating step in the tricarboxylic acid (TCA) cycle and efficiently assimilates C-2 compounds into intermediates that can be used in later steps of the TCA cycle. It plays an essential role in pathogen survival during host infection such that the enzymes involved in this cycle have been suggested as potential drug targets against human pathogens. Isocitrate lyase (ICL) catalyzes the first-step reaction of the glyoxylate cycle, using isocitrate from the TCA cycle as the substrate to produce succinate and glyoxylate. In this study we report the crystal structure of Magnaporthe oryzae ICL in both the ligand-free form and as a complex with Mg2+, glyoxylate, and glycerol, as well as the structure of the Fusarium graminearum ICL complexed with Mn2+ and malonate. We also describe the ligand-induced conformational changes in the catalytic loop and C-terminal region, both of which are essential for catalysis. Using various mutant ICLs in an activity assay, we gained insight into the function of residues within the active site. These structural and functional analyses provide detailed information with regard to fungal ICLs. (C) 2016 Elsevier Inc. All rights reserved.</P>

Overproduction of recombinant <i>E. coli</i> malate synthase enhances <i>Chlamydomonas reinhardtii</i> biomass by upregulating heterotrophic metabolism

Paik, Sang-Min,Kim, Joonwon,Jin, EonSeon,Jeon, Noo Li Elsevier 2019 Bioresource technology Vol.272 No.-

<P><B>Abstract</B></P> <P>High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic <I>Chlamydomonas reinhardtii</I> was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (<I>MDH4</I>) and acetyl-CoA synthetase (<I>ACS3</I>), isocitrate lyase (<I>ICL1</I>) and malate synthase (<I>MAS1</I>), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic F<SUB>v</SUB>/F<SUB>m</SUB> was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Chloroplastic transgenic <I>C. reinhardtii</I> was developed that stably expressed malate synthase. </LI> <LI> This transgenic strain showed a more 19% increase in dry cell weight than wild-type strain. </LI> <LI> Transcripts of <I>MDH4</I>, <I>ACS3</I>, <I>ICL1</I>, and <I>MAS1</I> were increased, and F<SUB>v</SUB>/F<SUB>m</SUB> was decreased. </LI> <LI> Upregulation of heterotrophic metabolism might be involved in biomass increase. </LI> <LI> This could serve as a valuable strain for treating wastewater containing acetate and glyoxylate. </LI> </UL> </P>

Site-specific Disruption of Glyoxylate Bypass and Its Effect in Lysine-producing Corynebacterium lactofermentum Strain

( Youn Hee Kim ),( Heung Shick Lee ) 한국미생물생명공학회 1996 Journal of microbiology and biotechnology Vol.6 No.5

2LO-4 Revisiting glyoxylate cycle for production of valuable chemicals

노명현,임현규,정규열 한국공업화학회 2017 한국공업화학회 연구논문 초록집 Vol.2017 No.1

The glyoxylate cycle is a bypass of the TCA cycle without carbon loss and enables microorganisms to utilize simple organic compounds as their sole carbon source. Despite these characteristics, engineering strategies based on the cycle have been limited a few; the inactivation of a transcriptional regulator (iclR) which represses the expression of aceBAK in the cycle. Here, we have devised a novel strategy to precisely regulate the glyoxylate cycle rather than the simple amplification. The activity of isocitrate lyase (aceA) was taken under control for the overall regulation of the glyoxylate cycle considering its thermodynamic properties. The strategy has been successfully applied to the production of 5-aminolevulinic acid and itaconic acid in Escherichia coli. This is the first attempt to precisely control the glyoxylate cycle, and the results suggest that elaborate tuning of the cycle can be a powerful strategy to facilitate efficient production of many other TCA derivatives.

TCA cycle‐independent acetate metabolism via the glyoxylate cycle in <i>Saccharomyces cerevisiae</i>

Lee, Yong Joo,Jang, Jin Won,Kim, Kyung Jin,Maeng, Pil Jae John Wiley Sons, Ltd. 2011 Yeast Vol.28 No.2

<P><B>Abstract</B></P><P>In <I>Saccharomyces cerevisiae</I>, the accepted theory is that due to TCA cycle dysfunction, the <I>Δcit1</I> mutant lacking the mitochondrial enzyme citrate synthase (Cit1) cannot grow on acetate, regardless of the presence of the peroxisomal isoenzyme (Cit2). In this study, we re‐evaluated the roles of Cit1 and Cit2 in acetate utilization and examined the pathway of acetate metabolism by analysing mutants defective in TCA or glyoxylate cycle enzymes. Although <I>Δcit1</I> cells showed significantly reduced growth on rich acetate medium (YPA), they exhibited growth similar to <I>Δcit2</I> and the wild‐type cells on minimal acetate medium (YNBA). Impaired acetate utilization by <I>Δcit1Δcit2</I> cells on YNBA was restored by ectopic expression of either Cit2 or its cytoplasmically localized variants. Deletion of any of <B>the genes</B> for the <B>enzymes</B> solely involved in the TCA cycle (<I>IDH1, KGD1</I> and <I>LSC1</I>), except for <I>SDH1</I>, caused little defect in acetate utilization on YNBA but resulted in significant growth impairment on YPA. In contrast, cells lacking any of the genes involved in the glyoxylate cycle (<I>ACO1, FUM1, MLS1, ICL1</I> and <I>MDH2</I>) did not grow on either YNBA or YPA. Deletion of <I>SFC1</I> encoding the succinate–fumarate carrier also caused similar growth defects on YNBA. Our results suggest that in <I>S. cerevisiae</I> the glyoxylate cycle functions as a competent metabolic pathway for acetate utilization on YNBA, while both the <B>TCA and</B> glyoxylate cycles are essential for growth on YPA. Copyright © 2010 John Wiley & Sons, Ltd.</P>

Precise tuning of glyoxylate cycle for efficient conversion of acetate into value-added chemicals

노명현,임현규,조민지,우성화,강채원,임대균,오민규,정규열 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.0

Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid and tyrosine from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium. Thereafter, acetate uptake pathway and checmical production pathway were amplified. To further enhance the production, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. As the results, production of itaconic acid and tyrosine could be significantly enhanced and these efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.

Role of Glyoxylate Shunt in Oxidative Stress Response

Ahn, Sungeun,Jung, Jaejoon,Jang, In-Ae,Madsen, Eugene L.,Park, Woojun American Society for Biochemistry and Molecular Bi 2016 The Journal of biological chemistry Vol.291 No.22

<P>The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.</P>

Effects of Isocitrate Lyase Inhibitors on Spore Germination and Appressorium Development in Magnaporthe grisea

Kim Seung-Young,Park Jin-Soo,Oh Ki-Bong The Korean Society for Microbiology and Biotechnol 2006 Journal of microbiology and biotechnology Vol.16 No.7

The glyoxylate cycle can conserve carbons and adequately supply tricarboxylic acid (TCA) cycle intermediates for biosynthesis when microorganisms grow on $C_{2}$ carbon sources. It has been reported that isocitrate lyase (ICL1), a key enzyme of the glyoxylate cycle, is highly induced when Magnaporthe grisea, the causal agent of rice blast, infects its host. Therefore, the glyoxylate cycle is considered as a new target for antifungal agents. A 1.6-kb DNA fragment encoding the ICL1 from M. grisea KJ201 was amplified by PCR, cloned into a vector providing His-tag at the N-terminus, expressed in Escherichia coli, and purified using Ni-NTA affinity chromatography. The molecular mass of the purified ICL1 was approximately 60 kDa, as determined by SDS-PAGE. The ICL1 inhibitory effects of TCA cycle intermediates and their analogs were investigated. Among them, 3-nitropropionate was found to be the strongest inhibitor with an $IC_{50}$ value of $11.0{\mu}g/ml$. 3-Nitropropionate inhibited the appressorium development in M. grisea at the ${\mu}M$ level, whereas conidia germination remained unaffected. This compound also inhibited the mycelial growth of the fungus on minimal medium containing acetate as a $C_{2}$ carbon source. These results suggest that ICL1 plays a crucial role in appressorium formation of M. grisea and is a new target for the control of phytopathogenic fungal infection.

Precise control of glyoxylate cycle for production of tyrosine from acetate in Escherichia coli.

조민지,노명현,임현규,강채원,임대균,오민규,정규열 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

Acetate is one of promising feedstocks owing to its cheap and great abundance. Considering that tyrosine production is shifting to microbial production method, its production from acetate can be attempted to improve the economic feasibility. We engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux redistribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was controlled by varied expression of isocitrate lyase. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield. This is the first demonstration of tyrosine production from acetate and our strategies would be widely applicable to the production of various chemicals from acetate.