Development of a Plasmid-Free Biosynthetic Pathway for Enhanced Muconic Acid Production in Pseudomonas chlororaphis HT66

ABSTRACT Two seven-gene phenazine biosynthetic loci were cloned fromPseudomonas aeruginosa PAO1. The operons, designatedphzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes,phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone inEscherichia coli or in enzymes, pyocyanin-nonproducingP. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene,phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosaconsisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.

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Bioprocess development for muconic acid production from aromatic compounds and lignin

Green Chemistry ◽

10.1039/c8gc02519c ◽

2018 ◽

Vol 20 (21) ◽

pp. 5007-5019 ◽

Cited By ~ 41

Author(s):

Davinia Salvachúa ◽

Christopher W. Johnson ◽

Christine A. Singer ◽

Holly Rohrer ◽

Darren J. Peterson ◽

...

Keyword(s):

Aromatic Compounds ◽

Acid Production ◽

Process Development ◽

Bioprocess Development ◽

Muconic Acid ◽

Bioreactor Process

This work shows parallel strain and bioreactor process development to improve muconic acid production from aromatic compounds and lignin.

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Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

10.1101/2020.04.13.039354 ◽

2020 ◽

Author(s):

Chaofeng Li ◽

Xiaofeng Lin ◽

Xing Ling ◽

Shuo Li ◽

Hao Fang

Keyword(s):

Acid Production ◽

Biosynthetic Pathway ◽

Microbial Consortium ◽

Consolidated Bioprocessing ◽

Glucaric Acid ◽

Work Distribution ◽

Fed Batch Fermentation ◽

Excellent Work ◽

Simultaneous Saccharification ◽

Rut C30

AbstractThe biomanufacturing of D-glucaric acid has been attracted increasing interest and the industrial yeast Saccharomyces cerevisiae is regarded as an excellent host for D-glucaric acid production. Here we constructed the biosynthetic pathway of D-glucaric acid in S. cerevisiae INVSc1 whose opi1 was knocked out and obtained two engineered strains, LGA-1 and LGA-C, producing record breaking titers of D-glucaric acid, 9.53 ± 0.46 g/L and 11.21 ± 0.63 g/L D-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in the mode of fed-batch fermentation, respectively. Due to the genetic stability and the outperformance in subsequent applications, however, LGA-1 was a preferable strain. As one of the top chemicals from biomass, there have been no reports on D-glucaric acid production from lignocellulose, which is the most abundant renewable on earth. Therefore, the biorefinery processes of lignocellulose for D-glucaric acid production including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated in this work and CBP by an artificial microbial consortium composed of Trichoderma reesei Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high D-glucaric acid titers and yields after 7 d fermentation, 0.54 ± 0.12 g/L D-glucaric acid from 15 g/L Avicel, and 0.45 ± 0.06 g/L D-glucaric acid from 15 g/L steam exploded corn stover (SECS), respectively. In attempts to design the microbial consortium for more efficient CBP the team consisted of the two members, T. reesei Rut-C30 and S. cerevisiae LGA-1, was found to be the best with excellent work distribution and collaboration. This desirable and promising approach for direction production of D-glucaric acid from lignocellulose deserves extensive and in-depth research.

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Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos

Molecules ◽

10.3390/molecules27010143 ◽

2021 ◽

Vol 27 (1) ◽

pp. 143

Author(s):

Wenjun Zhu ◽

Ying Liu ◽

Jing Tang ◽

Heping Liu ◽

Naliang Jing ◽

...

Keyword(s):

Acid Production ◽

Chemical Structure ◽

Biosynthetic Pathway ◽

Target Genes ◽

High Efficiency ◽

Antioxidant Activities ◽

Acid Synthesis ◽

Active Components ◽

Pachymic Acid ◽

Liquid Chromatography Tandem Mass

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

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Improvement of cis,cis-Muconic Acid Production in Saccharomyces cerevisiae through Biosensor-Aided Genome Engineering

ACS Synthetic Biology ◽

10.1021/acssynbio.9b00477 ◽

2020 ◽

Vol 9 (3) ◽

pp. 634-646 ◽

Cited By ~ 7

Author(s):

Guokun Wang ◽

Süleyman Øzmerih ◽

Rogério Guerreiro ◽

Ana C. Meireles ◽

Ana Carolas ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Acid Production ◽

Genome Engineering ◽

Muconic Acid

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Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440

Metabolic Engineering ◽

10.1016/j.ymben.2020.01.001 ◽

2020 ◽

Vol 59 ◽

pp. 64-75 ◽

Cited By ~ 16

Author(s):

Gayle J. Bentley ◽

Niju Narayanan ◽

Ramesh K. Jha ◽

Davinia Salvachúa ◽

Joshua R. Elmore ◽

...

Keyword(s):

Glucose Metabolism ◽

Pseudomonas Putida ◽

Acid Production ◽

Pseudomonas Putida Kt2440 ◽

Muconic Acid

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Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas chlororaphis Strain UFB2

International Journal of Molecular Sciences ◽

10.3390/ijms20010061 ◽

2018 ◽

Vol 20 (1) ◽

pp. 61 ◽

Cited By ~ 3

Author(s):

Boitumelo Setlhare ◽

Ajit Kumar ◽

Mduduzi Mokoena ◽

Ademola Olaniran

Keyword(s):

Specific Activity ◽

Protein A ◽

Total Yield ◽

Gel Filtration ◽

Cell Extract ◽

Dimensional Structure ◽

Pseudomonas Chlororaphis ◽

Muconic Acid ◽

Crude Cell Extract ◽

Catechol 1,2 Dioxygenase

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver–Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.

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