Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora for glucosylation of selected flavones chrysin and luteolin. UDP-glucose pool was enhanced in E. coli cell cytosol by introducing heterologous UDP-glucose biosynthestic genes i.e., gluco...
Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora for glucosylation of selected flavones chrysin and luteolin. UDP-glucose pool was enhanced in E. coli cell cytosol by introducing heterologous UDP-glucose biosynthestic genes i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector. The C-glucosylated products were analyzed by high performance liquid chromatography-photo diode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16
µM) and 11% (9 mg/L; 22 µM) conversion of chrysin to chrysin 6-C-β-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilator diffusion protein enhanced the production of chrysin 6-C-β-D-glucoside by approximately 1.4–fold, thus producing 10 mg/L (12%, 24 µM) and 14 mg/L (17%, 34
µM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of
additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab scale fermentor at 3-L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 µM) conversion of chrysin to chrysin 6-C-β-D-glucoside at 48h upon supplementation of 200 µM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 µM) in 50 mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble, and more stable to high temperature, different pH range, and β-glucosidase enzyme, than O-glucosylated derivative of chrysin.
Resveratrol, a major stilbene phytoalexin is valuable polyphenol having beneficial effects to human health. It has potent antioxidant and antitumor effects and promotes longevity. In this study, resveratrol and its ortho-hydroxylated derivative piceatannol were biosynthesized by modular pathway engineering in E. coli. The biosynthetic pathway was divided into three different modules. Module I includes polyketide biosynthetic genes; module II genes include acetyl-CoA and malonyl-CoA pool-enhancing genes from three different organisms, and module III genes are regiospecific 3′- hydroxylating enzymes. Biosynthetic genes include 4-coumarate:CoA ligase from Petroselinum crispum (Pc4CL2) and a codon optimized stilbene synthase from Vitis vinifera (VvSTS) (module I) accompanied by acetyl-CoA and malonyl-CoA building blocks supplying enzyme complexes (module II) from Nocardia farcinica (nfa9890, nfa9940, nfa9950, nfa3550; module IIa), E. coli (accCADB; module IIb), and Streptomyces venezuelae (matBC; module IIc). HpaBC complex (module III) from
E. coli was overexpressed for the hydroxylation.
E. coli BL21(DE3) with module I produced 8.6 mg/L of resveratrol from
exogenously fed 1 mM p-coumaric acid. Combination of module I and acetyl-CoA supplementing module IIa genes from N. farcinica produced 2.5-fold higher (60 mg/L) titer of resveratrol than the module IIb genes from E. coli. The exogenous supplementation of sodium acetate further enhanced production to 64 mg/L. Furthermore, module I with module IIc harboring matBC from S. venezulae produced
73 mg/L of resveratrol, which was elevated to 151 mg/L upon supplementing disodium malonate exogenously. This increment is 17.5-fold higher than module I harboring E. coli BL21(DE3). The combination of module I and two different module II genes yielded 137 mg/L resveratrol when supplemented with both sodium acetate and disodium malonate.
The high resveratrol-producing combination module was further modified with incorporation of hpaBC for the ortho-hydroxylation of resveratrol to produce piceatannol. The engineered strain harboring modules I, IIc and III produced 124 mg/L of piceatannol, the highest titer after 72 h in disodium malonate-supplemented strain, which is 2-fold higher than in non-supplemented strain. The remaining resveratrol was about 30 mg/L. Furthermore, caffeic acid (85.5 mg/L) was also produced in the same strain. Resveratrol and piceatannol were biosynthesized along with caffeic acid by three different modules overexpressing acetate and malonate assimilation pathway genes from three different sources. The production titer of both resveratrol and piceatannol may be enhanced by blocking acetyl-CoA and malonyl-CoA utilizing genes in host strain.
Moreover, we attempted to produce diverse derivatives of diadzein by biotransformation. O-methyltransferase from S. avermetilis was used for methylation.
YjiC, a substrate promiscious and regio-flexible glycosyltransferase, from Bacillus was used for glycosylation. In order to tune the glycosylation, the glycosyltransferase was expressed under theophylline inducible system, where the methylation and glycosylation steps were controlled for production of methylated, glycosylated and both methylated/glycosylated derivatives. Hence, with this approach, we could generate formonentin and ononin. Particularly, the tuning by controlled expression provided a platform for generating the onionin in higher titer. The cytotoxic effect and wound healing assay were tested for all the diadzein derivatives. The deriavative compounds inhibited proliferation of each cancer cell line with different sensitivity to growth inhibition. Notably, methylated product and double modified glycosylated/methylated showed comparatively better growth inhibition than control product. Methylated derivative also reduced the migration capacity of AGS gastric cancer cells.
Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora for glucosylation of selected flavones chrysin and luteolin. UDP-glucose pool was enhanced in E. coli cell cytosol by introducing heterologous UDP-glucose biosynthestic genes i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector. The C-glucosylated products were analyzed by high performance liquid chromatography-photo diode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16
µM) and 11% (9 mg/L; 22 µM) conversion of chrysin to chrysin 6-C-β-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilator diffusion protein enhanced the production of chrysin 6-C-β-D-glucoside by approximately 1.4–fold, thus producing 10 mg/L (12%, 24 µM) and 14 mg/L (17%, 34
µM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of
additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab scale fermentor at 3-L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 µM) conversion of chrysin to chrysin 6-C-β-D-glucoside at 48h upon supplementation of 200 µM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 µM) in 50 mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble, and more stable to high temperature, different pH range, and β-glucosidase enzyme, than O-glucosylated derivative of chrysin.
Resveratrol, a major stilbene phytoalexin is valuable polyphenol having beneficial effects to human health. It has potent antioxidant and antitumor effects and promotes longevity. In this study, resveratrol and its ortho-hydroxylated derivative piceatannol were biosynthesized by modular pathway engineering in E. coli. The biosynthetic pathway was divided into three different modules. Module I includes polyketide biosynthetic genes; module II genes include acetyl-CoA and malonyl-CoA pool-enhancing genes from three different organisms, and module III genes are regiospecific 3′- hydroxylating enzymes. Biosynthetic genes include 4-coumarate:CoA ligase from Petroselinum crispum (Pc4CL2) and a codon optimized stilbene synthase from Vitis vinifera (VvSTS) (module I) accompanied by acetyl-CoA and malonyl-CoA building blocks supplying enzyme complexes (module II) from Nocardia farcinica (nfa9890, nfa9940, nfa9950, nfa3550; module IIa), E. coli (accCADB; module IIb), and Streptomyces venezuelae (matBC; module IIc). HpaBC complex (module III) from
E. coli was overexpressed for the hydroxylation.
E. coli BL21(DE3) with module I produced 8.6 mg/L of resveratrol from
exogenously fed 1 mM p-coumaric acid. Combination of module I and acetyl-CoA supplementing module IIa genes from N. farcinica produced 2.5-fold higher (60 mg/L) titer of resveratrol than the module IIb genes from E. coli. The exogenous supplementation of sodium acetate further enhanced production to 64 mg/L. Furthermore, module I with module IIc harboring matBC from S. venezulae produced
73 mg/L of resveratrol, which was elevated to 151 mg/L upon supplementing disodium malonate exogenously. This increment is 17.5-fold higher than module I harboring E. coli BL21(DE3). The combination of module I and two different module II genes yielded 137 mg/L resveratrol when supplemented with both sodium acetate and disodium malonate.
The high resveratrol-producing combination module was further modified with incorporation of hpaBC for the ortho-hydroxylation of resveratrol to produce piceatannol. The engineered strain harboring modules I, IIc and III produced 124 mg/L of piceatannol, the highest titer after 72 h in disodium malonate-supplemented strain, which is 2-fold higher than in non-supplemented strain. The remaining resveratrol was about 30 mg/L. Furthermore, caffeic acid (85.5 mg/L) was also produced in the same strain. Resveratrol and piceatannol were biosynthesized along with caffeic acid by three different modules overexpressing acetate and malonate assimilation pathway genes from three different sources. The production titer of both resveratrol and piceatannol may be enhanced by blocking acetyl-CoA and malonyl-CoA utilizing genes in host strain.
Moreover, we attempted to produce diverse derivatives of diadzein by biotransformation. O-methyltransferase from S. avermetilis was used for methylation.
YjiC, a substrate promiscious and regio-flexible glycosyltransferase, from Bacillus was used for glycosylation. In order to tune the glycosylation, the glycosyltransferase was expressed under theophylline inducible system, where the methylation and glycosylation steps were controlled for production of methylated, glycosylated and both methylated/glycosylated derivatives. Hence, with this approach, we could generate formonentin and ononin. Particularly, the tuning by controlled expression provided a platform for generating the onionin in higher titer. The cytotoxic effect and wound healing assay were tested for all the diadzein derivatives. The deriavative compounds inhibited proliferation of each cancer cell line with different sensitivity to growth inhibition. Notably, methylated product and double modified glycosylated/methylated showed comparatively better growth inhibition than control product. Methylated derivative also reduced the migration capacity of AGS gastric cancer cells.
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