Functional characterization of cinnamate 4-hydroxylase from Helianthus annuus Linn using a fusion protein method

Wang, Ziwen; Jian, Xiangyun; Zhao, Yucheng; Li, Shan; Sui, Ziwei; Li, Li; Kong, Ling‐Yi; Luo, Jun

doi:10.1016/j.gene.2020.144950

Cited by 7 publications

(7 citation statements)

References 40 publications

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“…Whole‐cell bioconversion experiments demonstrated that the optimal strain, E. coli B01 ( At PAL2 from Arabidopsis thaliana ), could produce 1.7 g/L of CA from 3.3 g/L of l ‐phenylalanine (Table S7 and Figure S1). For module II, a functional CPR encoding‐gene from Arabidopsis thaliana ( At ATR2) was selected due to its excellent solubility when expressed in E. coli (Wang et al, 2020). Six C4H enzymes were identified from both Brenda and UniProt (https://www.uniprot.org/) databases, and different co‐expressed and fusion proteins were constructed using the linker peptide “GSTSSGSG” (Wang et al, 2020) and RBS.…”

Section: Resultsmentioning

confidence: 99%

“…For the functional expression of C4H genes in E. coli BL21, constructs fusing the C‐terminus of C4H with the N‐terminus of ATR2 were synthesized, utilizing the linker sequences “GSTSSGSG” (Y. Li et al, 2018; Wang et al, 2020). The methodology exemplified by the creation of the expression vector pET28a‐ At C4H ∆3–23 ‐ At ATR2 ∆2–72 proceeded as follows: (i) Membrane localization sequences in the N‐terminal regions of 2‐23 for At C4H and 2‐135 for At ATR2 were removed.…”

Section: Methodsmentioning

confidence: 99%

“…Six C4H enzymes were identified from both Brenda and UniProt (https://www. uniprot.org/) databases, and different co-expressed and fusion proteins were constructed using the linker peptide "GSTSSGSG" (Wang et al, 2020) and RBS. This resulted in the generation of strains E. coli B09-B14 and R01-R05, using the same method as Module I (Figure 1b).…”

Section: Design and Reconstruction Of P-coumaric Acid (P-ca) Biosynth...mentioning

confidence: 99%

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Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

Qiu,

Wang,

Zuo

et al. 2024

Biotech & Bioengineering

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P‐coumaric acid (p‐CA), a pant metabolite with antioxidant and anti‐inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p‐CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l‐phenylalanine‐producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p‐CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N‐terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3‐fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9‐folds, 1.3‐folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p‐CA titer, respectively. These optimizations significantly enhance p‐CA production, in a 5‐L fermenter using fed‐batch fermentation, the p‐CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Design and Reconstruction Of P-coumaric Acid (P-ca) Biosynth...mentioning

confidence: 99%

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Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

Qiu,

Wang,

Zuo

et al. 2024

Biotech & Bioengineering

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“…For example, OsPAL genes induce immunity against M. oryzae, Rhizoctonia solani, and Xanthomonas oryzae pv oryzae (Xoo; Duan et al, 2014;Tonnessen et al, 2015). C4H encodes p-coumaric acid hydroxylation from cinnamic acid, which drives lignification and biosynthesis of other essential defense metabolites (Li et al, 2020a,b;Wang et al, 2020). The soybean C4H1 gene has been linked to defensive lignification against Phytophthora sojae.…”

Section: Discussionmentioning

confidence: 99%

Hrip1 mediates rice cell wall fortification and phytoalexins elicitation to confer immunity against Magnaporthe oryzae

Ninkuu

Yan²,

Zhang³

et al. 2022

Front. Plant Sci.

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Magnaporthe oryzae is a potent fungus that adversely affects rice yield. Combinatorial techniques of prevention, toxic chemicals, and fungicide are used to remedy rice blast infection. We reported the role of Hrip1 in cell death elicitation and expression of systematic acquired resistance that could potentially stifle M. oryzae infection. In this study, transcriptome and metabolomic techniques were used to investigate the mechanism by which Hrip1 reprogramed the transcriptome of rice seedlings to confer immunity against M. oryzae. Our results showed that Hrip1 induces cell wall thickening and phytoalexin elicitation to confer immunity against M. oryzae infection. Hrip1 activates key lignin biosynthetic genes and myeloblastosis transcription factors that act as molecular switches for lignin production. Lignin content was increased by 68.46% and more after 48 h onwards in Hrip1-treated seedlings compared to the control treatment. Further analysis of cell wall morphology using the transmission electron microscopy technique revealed over 100% cell wall robustness. Hrip1 also induced the expression of 24 diterpene synthases. These include class I and II terpene synthases, cytochrome P450 subfamilies (OsCYP76M and OsCYP71Z), and momilactones synthases. The relationship between the expression of these genes and metabolic elicitation was analyzed using ultra-performance liquid chromatography–tandem mass spectrometry. Enhanced amounts of momilactones A and B, oryzalactone, and phytocassane A and G were detected in the Hrip1-treated leaves. We also identified seven benzoxazinoid genes (BX1-BX7) that could improve rice immunity. Our findings show that Hrip1 confers dual immunity by leveraging lignin and phytoalexins for physical and chemical resistance. This study provides novel insights into the mechanisms underlying Hrip1-treated plant immunity.

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“…Anthocyanins belong to secondary metabolites, which stability is affected by the self-chemical structure and environmental factors. Structural genes mainly encode crucial enzymes in the anthocyanin biosynthesis pathway, such as phenylalanine ammonialyase (PAL) [ 3 ], cinnamic acid hydroxylase [ 4 ], chalcone synthase (CHS) [ 5 ], chalcone isomerase (CHI) [ 6 ], diflavonol-4-reductase (DFR) [ 7 ], anthocyanin synthase (ANS) [ 8 ]. Additionally, regulatory genes mainly encode transcription factors to regulate the spatio-temporal expression of structural genes in the anthocyanin biosynthesis pathway.…”

Section: Introductionmentioning

confidence: 99%

MYB Transcription Factor OsC1PLSr Involves the Regulation of Purple Leaf Sheath in Rice

Zou

Wang

Sun

et al. 2023

IJMS

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Although several regulators associated with purple traits in rice have been identified, the genetic basis of the purple sheath remains unclear. In the present study, F2-1 and F2-2 populations were constructed using purple sheath (H93S) and green sheath (R1173 and YHSM), respectively. In order to identify QTL loci in purple sheaths, BSA analyses were performed on the two F2 populations. A crucial QTL for purple sheath was identified, tentatively named qPLSr6, and was located in the 4.61 Mb to 6.03 Mb region of chromosome 6. Combined with expression pattern analysis of candidate genes, LOC_Os06g10350 (OsC1PLSr) was suggested as a candidate gene. The homozygous mutant KO-1 and KO-2 created through CRISPR/Cas9 editing, lost their purple leaf sheath. The RT-PCR revealed that OsC1PLSr, anthocyanin synthase (ANS), diflavonol-4-reductase (DFR), flavanone-3-hydroxylase (F3H), and flavanone-3′-hydroxylase (F3′H) expression levels were dramatically down-regulated in the mutants. The yeast report system indicated that the 145–272 aa region at the C-terminal of OsC1PLSr is a positive transcriptional activation domain. The results indicated that OsC1PLSr synthesized anthocyanins by regulating the expression of ANS, DFR, F3H, and F3′H. This study provides new insights into the genetic basis of the purple sheath.

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Functional characterization of cinnamate 4-hydroxylase from Helianthus annuus Linn using a fusion protein method

Cited by 7 publications

References 40 publications

Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

Hrip1 mediates rice cell wall fortification and phytoalexins elicitation to confer immunity against Magnaporthe oryzae

MYB Transcription Factor OsC1PLSr Involves the Regulation of Purple Leaf Sheath in Rice

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