scholarly journals Overexpression of PtDXS Enhances Stress Resistance in Poplars

2019 ◽  
Vol 20 (7) ◽  
pp. 1669 ◽  
Author(s):  
Hui Wei ◽  
Ali Movahedi ◽  
Chen Xu ◽  
Weibo Sun ◽  
Amir Almasi Zadeh Yaghuti ◽  
...  
Keyword(s):  
Populus Trichocarpa ◽  
Terpenoid Biosynthesis ◽  
Functional Protein ◽  
Methylerythritol Phosphate Pathway ◽  
E Coli ◽  
Methylerythritol Phosphate ◽  
Transgenic Lines ◽  
Key Enzyme ◽  
Young Leaves ◽  
Rate Limiting

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the rate-limiting enzyme in the plastidial methylerythritol phosphate pathway (MEP). In this study, PtDXS (XM_024607716.1) was isolated from Populus trichocarpa. A bioinformatics analysis revealed that PtDXS had high homology with the DXSs of other plant species. PtDXS expression differed among plant tissues and was highest in young leaves and lowest in roots. The recombinant protein was produced in Escherichia coli BL21 (DE3), purified, and its activity evaluated. The purified protein was capable of catalyzing the formation of 1-deoxy-d-xylulose-5-phosphate (DXP) from glyceraldehyde-3-phosphate and pyruvate. A functional color assay in E. coli harboring pAC-BETA indicated that PtDXS encodes a functional protein involved in the biosynthesis of isoprenoid precursors. The treatment of P. trichocarpa seedlings with 200 μM abscisic acid (ABA), 200 mM NaCl, 10% polyethylene glycol6000, and 2 mM H2O2 resulted in increased expression of PtDXS. The ABA and gibberellic acid contents of the transgenic lines (Poplar Nanlin 895) were higher than wild types, suggesting that DXS is important in terpenoid biosynthesis. Overexpression of PtDXS enhanced resistance to S. populiperda infection. Furthermore, the transgenic lines showed decreased feeding by Micromelalopha troglodyta, supporting the notion that PtDXS is a key enzyme in terpenoid biosynthesis.

scholarly journals Inhibitory Activity of Proanthocyanidins Against Escherichia coli 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase

2021 ◽  
Vol 16 (11) ◽  
pp. 1934578X2110564
Author(s):  
Xian Hui ◽  
Bo-Rong Zhu ◽  
Long-Long Wu ◽  
Wen-Yun Gao ◽  
Yi-Ming Li ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Moderate Activity ◽  
Terpenoid Biosynthesis ◽  
Inhibitory Effects ◽  
Methylerythritol Phosphate Pathway ◽  
Methylerythritol Phosphate ◽  
Structure Activity ◽  
Initial Characterization ◽  
Key Enzyme ◽  
Relationship Of

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is a key enzyme in the methylerythritol phosphate pathway for terpenoid biosynthesis. Furthermore, it is an ideal target for the screening of novel antibiotics because it is present in causative organisms, but absent from humans. To identify more lipophilic DXR inhibitors from natural resources, we tested the DXR inhibitory activity of five proanthocyanidins in this study. The results indicated that all these compounds specifically restrained the activity of DXR, with procyanid B2 exhibiting a relatively low effect against DXR (IC50 ∼ 305 μM) and procyanid C1 displaying moderate activity (IC50 75.1 μM). The other three compounds cinnamtannin A2, cinnamtannin B1, and cinnamtannin D1 (IC50 ∼ 89.3, 105.0, and 97.8 μM, respectively) showed DXR inhibitory effects that were slightly weaker than that of procyanid C1. In addition, based on the initial characterization, the structure–activity relationship of this series of compounds against DXR is discussed.

scholarly journals Modulating the Precursor and Terpene Synthase Supply for the Whole-Cell Biocatalytic Production of the Sesquiterpene (+)-Zizaene in a Pathway Engineered E. coli

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 478 ◽  
Author(s):  
Francisco Aguilar ◽  
Thomas Scheper ◽  
Sascha Beutel
Keyword(s):  
Essential Oil ◽  
Metabolic Flux ◽  
Mevalonate Pathway ◽  
Terpene Synthase ◽  
Methylerythritol Phosphate Pathway ◽  
Main Compound ◽  
E Coli ◽  
Methylerythritol Phosphate ◽  
Cosmetic Industry ◽  
Multiple Copies

The vetiver essential oil from Chrysopogon zizanioides contains fragrant sesquiterpenes used widely in the formulation of nearly 20% of men’s cosmetics. The growing demand and issues in the supply have raised interest in the microbial production of the sesquiterpene khusimol, the main compound of the vetiver essential oil due to its woody smell. In this study, we engineered the biosynthetic pathway for the production of (+)-zizaene, the immediate precursor of khusimol. A systematic approach of metabolic engineering in Escherichia coli was applied to modulate the critical bottlenecks of the metabolic flux towards (+)-zizaene. Initially, production of (+)-zizaene was possible with the endogenous methylerythritol phosphate pathway and the codon-optimized zizaene synthase (ZS). Raising the precursor E,E-farnesyl diphosphate supply through the mevalonate pathway improved the (+)-zizaene titers 2.7-fold, although a limitation of the ZS supply was observed. To increase the ZS supply, distinct promoters were tested for the expression of the ZS gene, which augmented 7.2-fold in the (+)-zizaene titers. Final metabolic enhancement for the ZS supply by using a multi-plasmid strain harboring multiple copies of the ZS gene improved the (+)-zizaene titers 1.3-fold. The optimization of the fermentation conditions increased the (+)-zizaene titers 2.2-fold, achieving the highest (+)-zizaene titer of 25.09 mg L−1. This study provides an alternative strategy to enhance the terpene synthase supply for the engineering of isoprenoids. Moreover, it demonstrates the development of a novel microbial platform for the sustainable production of fragrant molecules for the cosmetic industry.

scholarly journals 1-Deoxy-d-Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 inRhodobacter capsulatus

2001 ◽  
Vol 183 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Frederick M. Hahn ◽  
Lisa M. Eubanks ◽  
Charles A. Testa ◽  
Brian S. J. Blagg ◽  
Jonathan A. Baker ◽  
...  
Keyword(s):  
Rhodobacter Capsulatus ◽  
Open Reading Frames ◽  
Reading Frame ◽  
Methylerythritol Phosphate Pathway ◽  
E Coli ◽  
Methylerythritol Phosphate ◽  
Steady State Kinetic ◽  
Photosynthetic Machinery ◽  
State Kinetic ◽  
Phosphate Synthase

ABSTRACT In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and d-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-d-xylulose 5-phosphate synthase. InRhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-d-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-d-xylulose 5-phosphate synthase A and B, containing a His6 tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-d-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-d-xylulose 5-phosphate synthase A and B: Km pyruvate = 0.61 and 3.0 mM, Km d-glyceraldehyde 3-phosphate = 150 and 120 μM, andV max = 1.9 and 1.4 μmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-d-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.

scholarly journals Complete Genome Sequence of Psychrobacter alimentarius PAMC 27889, a Psychrophile Isolated from an Antarctic Rock Sample

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Jaejin Lee ◽  
Miye Kwon ◽  
Jae Young Yang ◽  
Jusun Woo ◽  
Hong Kum Lee ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Rock Sample ◽  
Psychrophilic Bacterium ◽  
Terpenoid Biosynthesis ◽  
Gram Negative ◽  
Methylerythritol Phosphate Pathway ◽  
Methylerythritol Phosphate ◽  
Benzoate Degradation

Psychrobacter alimentarius PAMC 27889, a Gram-negative, psychrophilic bacterium, was isolated from an Antarctic rock sample. Here, we report the complete genome of P. alimentarius PAMC 27889, which has the nonmevalonate methylerythritol phosphate pathway of terpenoid biosynthesis and a complete gene cluster for benzoate degradation.

scholarly journals Metabolome and proteome analyses reveal transcriptional misregulation in glycolysis of engineered E. coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chun-Ying Wang ◽  
Martin Lempp ◽  
Niklas Farke ◽  
Stefano Donati ◽  
Timo Glatter ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Metabolic Pathways ◽  
Glycerol Production ◽  
Inducible Promoters ◽  
E Coli ◽  
Inhibition Of Glycolysis ◽  
Underlying Mechanisms ◽  
Rate Limiting ◽  
Engineered Bacteria

AbstractSynthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.

scholarly journals High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

2013 ◽  
Vol 97 (9) ◽  
pp. 3893-3900 ◽  
Author(s):  
Odile Francesca Restaino ◽  
Ujjwal Bhaskar ◽  
Priscilla Paul ◽  
Lingyun Li ◽  
Mario De Rosa ◽  
...  
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Coli Strain ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Bioengineered Heparin ◽  
Key Enzyme
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