Sailesh Malla

Production of 7-O-methyl aromadendrin, a medicinally valuable flavonoid, in Escherichia coli.

ABSTRACT 7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate–coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.

Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli.

This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum seropedicae SmR1 responds to naringenin, while the repressor QdoR from Bacillus subtilis is inactivated by quercetin and kaempferol. Both biosensors showed over a 7-fold increase of the fluorescent signal after addition of their specific effectors, and a linear correlation between the fluorescence intensity and externally added flavonoid concentration. The QdoR-biosensor was successfully applied for detection of kaempferol production in vivo at the single cell level by fluorescence-activated cell sorting. Furthermore, the amount of kaempferol produced highly correlated with the specific fluorescence of E. coli cells containing a flavonol synthase from Arabidopsis thaliana (fls1). We expect the designed biosensors to be applied for isolation of genes involved in flavonoid biosynthetic pathways.

Regiospecific modifications of naringenin for astragalin production in Escherichia coli.

We report the production of astragalin (AST) from regiospecific modifications of naringenin (NRN) in Escherichia coli BL21(DE3). The exogenously supplied NRN was converted into dihydrokaempferol (DHK) and then kaempferol (KMF) in the presence of flavanone‐3‐hydroxylase (f3h) and flavonone synthase (fls1) from Arabidopsis thaliana, respectively. KMF was further modified to produce AST by 3‐O‐glucosylation utilizing the endogeneous UDP‐glucose in presence of UGT78K1 from Glycine max. To increase the intracellular UDP‐glucose concentration by channeling the carbon flux toward UDP‐glucose at the branch point of glucose‐6‐phosphate (G6P), the chromosomal glucose phosphate isomerase (pgi) and D‐glucose‐6‐phosphate dehydrogenase (zwf) were knocked‐out in E. coli BL21(DE3). The two enzymes directly involved in the synthesis of UDP‐glucose from G6P, phosphoglucomutase (nfa44530) from Nocardia farcinia and glucose‐1‐phosphate uridylyltransferase (galU) from E. coli K12 were overexpressed, which successfully diverted the carbon flow from glycolysis to the synthesis of UDP‐glucose. Furthermore, to prevent the dissociation of UDP‐glucose into UDP and glucose, the UDP‐glucose hydrolase (ushA) was deleted. The E. coli ΔpgiΔzwfΔushA mutant harboring the UDP‐glucose biosynthetic pathway and the aforementioned genes for the regiospecific glucosylation produced 109.3 mg/L (244 µM) of AST representing 48.8% conversion from 500 µM of NRN in 60 h without any supplementation of extracellular UDP‐glucose. Biotechnol. Bioeng. 2013; 110:2525–2535.

Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli

Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimers disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols.

Fungal cytochrome P450 monooxygenases of Fusarium oxysporum for the synthesis of ω-hydroxy fatty acids in engineered Saccharomyces cerevisiae

BackgroundOmega hydroxy fatty acids (ω-OHFAs) are multifunctional compounds that act as the basis for the production of various industrial products with broad commercial and pharmaceutical implications. However, the terminal oxygenation of saturated or unsaturated fatty acids for the synthesis of ω-OHFAs is intricate to accomplish through chemocatalysis, due to the selectivity and controlled reactivity in C-H oxygenation reactions. Cytochrome P450, the ubiquitous enzyme is capable of catalyzing the selective terminal omega hydroxylation naturally in biological kingdom.ResultsTo gain a deep insight on the biochemical role of fungal P450s towards the production of omega hydroxy fatty acids, two cytochrome P450 monooxygenases from Fusarium oxysporum (FoCYP), FoCYP539A7 and FoCYP655C2; were identified, cloned, and heterologously expressed in Saccharomyces cerevisiae. For the efficient production of ω-OHFAs, the S. cerevisiae was engineered to disrupt the acyl-CoA oxidase enzyme and the β-oxidation pathway inactivated (ΔPox1) S. cerevisiae mutant was generated. To elucidate the significance of the interaction of redox mechanism, FoCYPs were reconstituted with the heterologous and homologous reductase systems - S. cerevisiae CPR (ScCPR) and F. oxysporum CPR (FoCPR). To further improve the yield, the effect of pH was analyzed and the homologous FoCYP-FoCPR system efficiently hydroxylated caprylic acid, capric acid and lauric acid into their respective ω-hydroxy fatty acids with 56%, 79% and 67% conversion. Furthermore, based on computational simulations, we identified the key residues (Asn106 of FoCYP539A7 and Arg235 of FoCYP655C2) responsible for the recognition of fatty acids and demonstrated the structural insights of the active site of FoCYPs.ConclusionFungal CYP monooxygenases, FoCYP539A7 and FoCYP655C2 with its homologous redox partner, FoCPR constitutes a promising catalyst due to its high regio- and stereo-selectivity in the hydroxylation of fatty acids and in the substantial production of industrially valuable ω-hydroxy fatty acids.

A sustainable route to produce the scytonemin precursor using Escherichia coli

Scytonemin is an indolic–phenolic natural product with potent pharmaceutical activities and possible application as a sunscreen. However, the productivity of the existing synthesis systems restrains its applications in medicine and cosmetics. In this paper, we report the generation of the monomer moiety of scytonemin from tryptophan and tyrosine in Escherichia coli. We heterologously expressed the biosynthetic pathway from Nostoc punctiforme and discovered that only three enzymes from N. punctiforme are required for the in vivo production of the monomer moiety of scytonemin in E. coli. We also found that the constructed recombinant E. coli strains are capable of producing novel alkaloids as shunt products. The recombinant E. coli strain expressing the putative scytonemin biosynthetic gene cluster produced 4.2 mg L−1 (2.46 μg mg−1 dry cell weight) of the monomer moiety of scytonemin without supplementation of extracellular substrates whereas upon supplementation with 1 mM of the substrates to the E. coli strain harboring scyABC genes, 8.9 mg L−1 (4.56 μg mg−1 dry cell weight) of the monomer moiety of scytonemin was produced in 5 days. Combining this cell factory with the previously described chemical dimerization process will contribute to a sustainable production of semi-synthetic scytonemin.

deFUME: Dynamic exploration of functional metagenomic sequencing data

BackgroundFunctional metagenomic selections represent a powerful technique that is widely applied for identification of novel genes from complex metagenomic sources. However, whereas hundreds to thousands of clones can be easily generated and sequenced over a few days of experiments, analyzing the data is time consuming and constitutes a major bottleneck for experimental researchers in the field.FindingsHere we present the deFUME web server, an easy-to-use web-based interface for processing, annotation and visualization of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The web-server integrates multiple analysis steps into one single workflow: read assembly, open reading frame prediction, and annotation with BLAST, InterPro and GO classifiers. Analysis results are visualized in an online dynamic web-interface.ConclusionThe deFUME webserver provides a fast track from raw sequence to a comprehensive visual data overview that facilitates effortless inspection of gene function, clustering and distribution. The webserver is available at cbs.dtu.dk/services/deFUME/and the source code is distributed at github.com/EvdH0/deFUME.