Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli

ABSTRACT Oxidation of malate to oxaloacetate in Escherichia colican be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37 ) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16 ), encoded by the genemqo (previously called yojH). Expression of themqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repressmqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genessfcA and b2463 (coding for EC 1.1.1.38 and EC1.1.1.40 , respectively) and of the phosphoenolpyruvate synthase (EC2.7.9.2 ) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of themdh single mutant to that of the double mutant containingmdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.

Download Full-text

Respiratory chain components are required for peptidoglycan recognition protein-induced thiol depletion and killing in Bacillus subtilis and Escherichia coli

Scientific Reports ◽

10.1038/s41598-020-79811-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chun-Kai Yang ◽

Des R. Kashyap ◽

Dominik A. Kowalczyk ◽

David Z. Rudner ◽

Xindan Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Shikimate Pathway ◽

High Survival ◽

E Coli ◽

Peptidoglycan Recognition Protein ◽

Transposon Insertion ◽

Transport Chain ◽

Recognition Protein ◽

Ndh Genes

AbstractMammalian peptidoglycan recognition proteins (PGRPs or PGLYRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. Tn-seq screening of Bacillus subtilis transposon insertion library revealed that mutants in the shikimate pathway of chorismate synthesis had high survival following PGLYRP4 treatment. Deletion mutants for these genes had decreased amounts of menaquinone (MK), increased resistance to killing, and attenuated depletion of thiols following PGLYRP4 treatment. These effects were reversed by MK or reproduced by inhibiting MK synthesis. Deletion of cytochrome aa3-600 or NADH dehydrogenase (NDH) genes also increased B. subtilis resistance to PGLYRP4-induced killing and attenuated thiol depletion. PGLYRP4 treatment also inhibited B. subtilis respiration. Similarly in Escherichia coli, deletion of ubiquinone (UQ) synthesis, formate dehydrogenases (FDH), NDH-1, or cytochrome bd-I genes attenuated PGLYRP4-induced thiol depletion. PGLYRP4-induced low level of cytoplasmic membrane depolarization in B. subtilis and E. coli was likely not responsible for thiol depletion. Thus, our results show that the respiratory electron transport chain components, cytochrome aa3-600, MK, and NDH in B. subtilis, and cytochrome bd-I, UQ, FDH-O, and NDH-1 in E. coli, are required for both PGLYRP4-induced killing and thiol depletion and indicate conservation of the PGLYRP4-induced thiol depletion and killing mechanisms in Gram-positive and Gram-negative bacteria.

Download Full-text

Synthesis of three major auxins from glucose in EngineeredEscherichia coli

10.1101/347930 ◽

2018 ◽

Author(s):

Daoyi Guo ◽

Lihua Zhang ◽

Sijia Kong ◽

Zhijie Liu ◽

Xu Chu ◽

...

Keyword(s):

Plant Growth ◽

Phenylacetic Acid ◽

De Novo ◽

Shikimate Pathway ◽

Aromatic Amino Acids ◽

Plant Growth And Development ◽

E Coli ◽

Naturally Occurring ◽

Hydroxyphenylacetic Acid ◽

Indole 3 Acetic Acid

ABSTRACTIndole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In addition, phenylacetic acid (PAA) and 4-hydroxyphenylacetic acid (4HPA) can also play a role as auxin in some plants. In recent years, several microbes have been metabolically engineered to produce IAA from L-tryptophan. In this study, we showed that aminotransferasearo8and decarboxylasekdcfromSaccharomyces cerevisiae, and aldehyde dehydrogenasealdHfromEscherichia colihave broad substrate ranges and can catalyze the conversion of three kinds of aromatic amino acids (L-tryptophan, L-tyrosine or L-phenylalanine) to the corresponding IAA, 4HPA and PAA. Subsequently, three de novo biosynthetic pathways for the production of IAA, PAA and 4HPA from glucose were constructed inE. colithrough strengthening the shikimate pathway. This study described here shows the way for the development of agricultural microorganism for biosynthesis of plant auxin and promoting plant growth in the future.

Download Full-text

Corynebacterium glutamicum Contains 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate Synthases That Display Novel Biochemical Features

Applied and Environmental Microbiology ◽

10.1128/aem.00262-08 ◽

2008 ◽

Vol 74 (17) ◽

pp. 5497-5503 ◽

Cited By ~ 21

Author(s):

Ya-Jun Liu ◽

Pan-Pan Li ◽

Ke-Xin Zhao ◽

Bao-Jun Wang ◽

Cheng-Ying Jiang ◽

...

Keyword(s):

Amino Acids ◽

Corynebacterium Glutamicum ◽

Feedback Inhibition ◽

Shikimate Pathway ◽

Aromatic Amino Acids ◽

Cloning And Expression ◽

Dahp Synthase ◽

E Coli ◽

Phenotypic Alteration ◽

In The Wild

ABSTRACT 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.

Download Full-text

Engineering the shikimate pathway for biosynthesis of molecules with pharmaceutical activities in E. coli

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2016.01.016 ◽

2016 ◽

Vol 42 ◽

pp. 1-6 ◽

Cited By ~ 41

Author(s):

Ming Jiang ◽

Haoran Zhang

Keyword(s):

Shikimate Pathway ◽

E Coli

Download Full-text

The serC-aro A operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways

Biochemical Journal ◽

10.1042/bj2340049 ◽

1986 ◽

Vol 234 (1) ◽

pp. 49-57 ◽

Cited By ~ 36

Author(s):

K Duncan ◽

J R Coggins

Keyword(s):

Amino Acid ◽

Biosynthetic Pathway ◽

Shikimate Pathway ◽

Biosynthetic Pathways ◽

The Novel ◽

Base Pairs ◽

Reading Frame ◽

E Coli ◽

Phosphoserine Aminotransferase ◽

Aroa Gene

Sub-cloning experiments aimed at precisely locating the E. coli aroA gene, which encodes the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase, showed that in certain constructions, which remain capable of complementing an auxotrophic aroA mutation, expression of aroA is reduced. DNA sequence analysis revealed that a sequence approx. 1200 base pairs (bp) upstream of aroA is necessary for its expression. An open reading frame was identified in this region which encodes a protein of 362 amino acids with a calculated Mr of 39,834 and which ends 70 bp before the start of the aroA coding sequence. This gene has been identified as serC, the structural gene for 3-phosphoserine aminotransferase, an enzyme of the serine biosynthetic pathway. Both genes are expressed as a polycistronic message which is transcribed from a promotor located 58 bp upstream of serC. Evidence is presented which confirms that the aroA and serC genes constitute an operon which has the novel feature of encoding enzymes from two different amino acid biosynthetic pathways.

Download Full-text

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.01148-12 ◽

2012 ◽

Vol 78 (17) ◽

pp. 6203-6216 ◽

Cited By ~ 99

Author(s):

Daisuke Koma ◽

Hayato Yamanaka ◽

Kunihiko Moriyoshi ◽

Takashi Ohmoto ◽

Kiyofumi Sakai

Keyword(s):

Escherichia Coli ◽

Aromatic Compounds ◽

Phenylacetic Acid ◽

Shikimate Pathway ◽

Phenyllactic Acid ◽

Content Type ◽

E Coli ◽

Dehydrogenase Gene ◽

Hydroxyphenylacetic Acid ◽

By Products

ABSTRACTEscherichia coliwas metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) fromCupriavidus necatorwas introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) fromAzospirillum brasilenseand the phenylacetaldehyde dehydrogenase gene (feaB) fromE. coliwere introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereasipdCand the alcohol dehydrogenase gene (adhC) fromLactobacillus breviswere introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases inE. coliencoded by theyqhD,yjgB, andyahKgenes. Cointroduction and cooverexpression of each gene withipdCin the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of theyahKgene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion offeaB,pheA, and/ortyrAgenes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

Download Full-text

Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli

Applied Biological Chemistry ◽

10.1186/s13765-021-00643-0 ◽

2021 ◽

Vol 64 (1) ◽

Author(s):

Shin-Won Lee ◽

Han Kim ◽

Joong-Hoon Ahn

Keyword(s):

Escherichia Coli ◽

Caffeic Acid ◽

Shikimate Pathway ◽

Acid Synthesis ◽

Biological Properties ◽

E Coli ◽

Pathway Gene ◽

Coa Ligase ◽

Prunus Yedoensis ◽

Tyrosine Ammonia Lyase

AbstractHydroxycinnamic acids (HCs) are natural compounds that form conjugates with diverse compounds in nature. Ethyl caffeate (EC) is a conjugate of caffeic acid (an HC) and ethanol. It has been found in several plants, including Prunus yedoensis, Polygonum amplexicaule, and Ligularia fischeri. Although it exhibits anticancer, anti-inflammatory, and antifibrotic activities, its biosynthetic pathway in plants still remains unknown. This study aimed to design an EC synthesis pathway and clone genes relevant to the same. Genes involved in the caffeic acid synthesis pathway (tyrosine ammonia-lyase (TAL) and p-coumaric acid hydroxylase (HpaBC)) were introduced into Escherichia coli along with 4-coumaroyl CoA ligase (4CL) and acyltransferases (AtCAT) cloned from Arabidopsis thaliana. In presence of ethanol, E. coli harboring the above genes successfully synthesized EC. Providing more tyrosine through the overexpression of shikimate-pathway gene-module construct and using E. coli mutant enhanced EC yield; approximately 116.7 mg/L EC could be synthesized in the process. Synthesis of four more alkyl caffeates was confirmed in this study; these might potentially possess novel biological properties, which would require further investigation.

Download Full-text