Journal: Nature Communications
Article Title: De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts
doi: 10.1038/s41467-022-30826-2
Figure Lengend Snippet: a Illustration of the modularized platform for producing and exporting rubusoside. Module A (terpene synthesis module) incorporates modifications designed to divert carbon flux to diterpene metabolic and brought ent-kaurene biosynthesis. Engineering yeast into the efficient platform to produce rubusoside by introducing Module B (P450s module) and Module C (rubusoside synthesis module). Module D (UDP-glucose synthesis module) provides glycoside ligands for producing rubusoside. Module E (rubusoside exporter module) is a possible exportation system of rubusoside. ERG10 acetyl-CoA C-acetyltransferase, ERG13 hydroxymethylglutaryl-CoA synthase, HMG1 hydroxymethylglutaryl-CoA reductase, tHMG1 truncated hydroxymethylglutaryl-CoA reductase, ERG12 mevalonate kinase, ERG8 phosphomevalonate kinase, IDI1 isopentenyl diphosphate delta-isomerase, ERG20 bifunctional (2E,6E)-farnesyl diphosphate, BST1 farnesyltranstransferase, KS kaurene synthase, KO ent-kaurene oxidase, KAH kaurenoic acid 13α-hydroxylase, UGT74G1 UDP-glycosyltransferase 74G1, UGT85C2 UDP-glycosyltransferase 85C2. FPS F112A mutant farnesyl pyrophosphate synthase. Glc-6-P glucose-6-phosphate, Acetyl-CoA acetyl coenzyme A, IPP isopentenyl diphosphate, GPP Geranyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl pyrophosphate, DMAPP dimethylallyl diphosphate, EKA ent-kaurenoic acid, 13-SMG (5ξ,8α,9ξ,10α,13α)-13-(β-D-Glucopyranosyloxy) kaur-16-en-18-säure, 19-SMG 1-O-[(5ξ,8α,9ξ,10α,13α)-13-Hydroxy-18-oxokaur-16-én-18-yl]-β-D-glucopyranose. All the heterologous genes were controlled by GAL promoters. b Increased ent-kaurene biosynthesis by eliminating the rate-limiting steps in MVA pathway (overexpressed tHMG1 and IDI1 , SGN02) and avoiding competition for FPP with the monoterpene synthesis pathway (introduced FPS F112A , SGN03). All the heterologous genes were controlled by GAL promoters. c HPLC spectra of ent-kaurenoic acid (EKA), steviol, rubusoside, and their standards. RT retention time. d LC-MS analysis results of EKA, steviol, and rubusoside in negative ion mode. Source data are provided as a Source Data file. e The rubusoside titer difference in the intracellular and extracellular of the SGN06 strain. b , e Data are presented as mean values ± SD from three independent biological replicates ( n = 3), the circles represent individual data points. Significance ( p -value) was evaluated by two-sided t -test.
Article Snippet: The codon-optimized genes KS (NCBI Accession Number: Q9UVY5 ), FPS F112A (NCBI Accession Number: P08836.2 ), KO (NCBI Accession Number: AAQ63464.1 ), KAH (NCBI Accession Number: NP_197872.1 ) , CPR1 (NCBI Accession Number: ABB88839.2 ), UGT74G1 (NCBI Accession Number: Q6VAA6.1 ), UGT85C2 (NCBI Accession Number: Q6VAB0.1 ), UGT91D2 (NCBI Accession Number: B3VI56.1 ), UGT76G1 (NCBI Accession Number: AGL95113.1 ), and EUGT11 (NCBI Accession Number: XP_015629141.1 ) were synthesized by GenScript.
Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy
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![a Illustration of the modularized platform for producing and exporting rubusoside. Module A (terpene synthesis module) incorporates modifications designed to divert carbon flux to diterpene metabolic and brought ent-kaurene biosynthesis. Engineering yeast into the efficient platform to produce rubusoside by introducing Module B (P450s module) and Module C (rubusoside synthesis module). Module D (UDP-glucose synthesis module) provides glycoside ligands for producing rubusoside. Module E (rubusoside exporter module) is a possible exportation system of rubusoside. ERG10 acetyl-CoA C-acetyltransferase, ERG13 hydroxymethylglutaryl-CoA synthase, HMG1 hydroxymethylglutaryl-CoA reductase, tHMG1 truncated hydroxymethylglutaryl-CoA reductase, ERG12 mevalonate kinase, ERG8 phosphomevalonate kinase, IDI1 isopentenyl diphosphate delta-isomerase, ERG20 bifunctional (2E,6E)-farnesyl diphosphate, BST1 farnesyltranstransferase, KS kaurene synthase, KO ent-kaurene oxidase, KAH kaurenoic acid 13α-hydroxylase, UGT74G1 UDP-glycosyltransferase 74G1, UGT85C2 UDP-glycosyltransferase 85C2. FPS <t>F112A</t> mutant farnesyl pyrophosphate synthase. Glc-6-P glucose-6-phosphate, Acetyl-CoA acetyl coenzyme A, IPP isopentenyl diphosphate, GPP Geranyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl pyrophosphate, DMAPP dimethylallyl diphosphate, EKA ent-kaurenoic acid, 13-SMG (5ξ,8α,9ξ,10α,13α)-13-(β-D-Glucopyranosyloxy) kaur-16-en-18-säure, 19-SMG 1-O-[(5ξ,8α,9ξ,10α,13α)-13-Hydroxy-18-oxokaur-16-én-18-yl]-β-D-glucopyranose. All the heterologous genes were controlled by GAL promoters. b Increased ent-kaurene biosynthesis by eliminating the rate-limiting steps in MVA pathway (overexpressed tHMG1 and IDI1 , SGN02) and avoiding competition for FPP with the monoterpene synthesis pathway (introduced FPS F112A , SGN03). All the heterologous genes were controlled by GAL promoters. c HPLC spectra of ent-kaurenoic acid (EKA), steviol, rubusoside, and their standards. RT retention time. d LC-MS analysis results of EKA, steviol, and rubusoside in negative ion mode. Source data are provided as a Source Data file. e The rubusoside titer difference in the intracellular and extracellular of the SGN06 strain. b , e Data are presented as mean values ± SD from three independent biological replicates ( n = 3), the circles represent individual data points. Significance ( p -value) was evaluated by two-sided t -test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0076/pmc09160076/pmc09160076__41467_2022_30826_Fig1_HTML.jpg)