Metabolic engineering of Saccharomyces cerevisiae for chelerythrine biosynthesis.
Saccharomyces cerevisiae
Chelerythrine
Metabolic engineering
Microbial cell factory
Natural product biosynthesis
Synthetic biology
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
21 Jun 2024
21 Jun 2024
Historique:
received:
01
11
2023
accepted:
03
06
2024
medline:
21
6
2024
pubmed:
21
6
2024
entrez:
20
6
2024
Statut:
epublish
Résumé
Chelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming. The first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain. This is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.
Sections du résumé
BACKGROUND
BACKGROUND
Chelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming.
RESULTS
RESULTS
The first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain.
CONCLUSIONS
CONCLUSIONS
This is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.
Identifiants
pubmed: 38902758
doi: 10.1186/s12934-024-02448-4
pii: 10.1186/s12934-024-02448-4
doi:
Substances chimiques
chelerythrine
E3B045W6X0
Benzophenanthridines
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
183Subventions
Organisme : National Natural Science Foundation of China
ID : 21977067
Organisme : National Key Research and Development Program of China
ID : 2020YFA0907700
Informations de copyright
© 2024. The Author(s).
Références
Li YR, Smolke CD. Engineering biosynthesis of the anticancer alkaloid noscapine in yeast. Nat Commun. 2016;7:12137.
pubmed: 27378283
pmcid: 4935968
Ehrenworth AM, Peralta-Yahya P. Accelerating the semisynthesis of alkaloid-based drugs through metabolic engineering. Nat Chem Biol. 2017;13:249–58.
pubmed: 28199297
Casciaro B, Mangiardi L, Cappiello F, Romeo I, Loffredo MR, Iazzetti A, et al. Naturally-occurring alkaloids of plant origin as potential antimicrobials against antibiotic-resistant infections. Molecules. 2020;25:3619.
pubmed: 32784887
pmcid: 7466045
He N, Wang PQ, Wang PY, Ma CY, Kang WY. Antibacterial mechanism of chelerythrine isolated from root of Toddalia Asiatica (Linn) Lam. BMC Complement Altern Med. 2018;18:261.
pubmed: 30257662
pmcid: 6158911
Fan L, Fan Y, Liu L, Tao WW, Shan X, Dong Y, et al. Chelerythrine attenuates the inflammation of lipopolysaccharide-induced acute lung inflammation through NF-κB signaling pathway mediated by Nrf2. Front Pharmacol. 2018;9:1047.
pubmed: 30319404
pmcid: 6169195
Kosina P, Walterova D, Ulrichova J, Lichnovsky V, Stiborova M, Rydlova H, et al. Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment. Food Chem Toxicol. 2004;42:85–91.
pubmed: 14630132
Bampidis V, Azimonti G, Bastos MD, Christensen H, Dusemund B, Durjava M, et al. Safety and efficacy of a feed additive consisting of Macleaya cordata (Willd.) R. Br. Extract and leaves (Sangrovit (R) extra) for all poultry species (excluding laying and breeding birds) (Phytobiotics Futterzusatzstoffe GmbH). EFSA J. 2023;21:e08052.
pubmed: 37304353
pmcid: 10251260
Castanon JIR. History of the use of antibiotic as growth promoters in European poultry feeds. Poult Sci. 2007;86:2466–71.
pubmed: 17954599
Wei QH, Cui DZ, Liu XF, Chai YY, Zhao N, Wang JY, et al. In vitro antifungal activity and possible mechanisms of action of chelerythrine. Pestic Biochem Physiol. 2020;164:140–8.
pubmed: 32284120
Lv P, Huang KL, Xie LG, Xu XH. Palladium-catalyzed tandem reaction to construct benzo[c]phenanthridine: application to the total synthesis of benzo[c]phenanthridine alkaloids. Org Biomol Chem. 2011;9:3133–5.
pubmed: 21423927
Suchomelová J, Bochořáková H, Paulová H, Musil P, Táborská E. HPLC quantification of seven quaternary benzo[c]phenanthridine alkaloids in six species of the family Papaveraceae. J Pharm Biomed Anal. 2007;44:283–7.
pubmed: 17367981
Georgianna DR, Mayfield SP. Exploiting diversity and synthetic biology for the production of algal biofuels. Nature. 2012;488:329–35.
pubmed: 22895338
Trenchard IJ, Smolke CD. Engineering strategies for the fermentative production of plant alkaloids in yeast. Metab Eng. 2015;30:96–104.
pubmed: 25981946
pmcid: 4519383
Leonard E, Runguphan W, O’Connor S, Prather KJ. Opportunities in metabolic engineering to facilitate scalable alkaloid production. Nat Chem Biol. 2009;5:292–300.
pubmed: 19377455
DeLoache WC, Russ ZN, Narcross L, Gonzales AM, Martin VJJ, Dueber JE. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat Chem Biol 2105;11:465–71.
Pyne ME, Kevvai K, Grewal PS, Narcross L, Choi B, Bourgeois L, et al. A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids. Nat Commun. 2020;11:3337.
pubmed: 32620756
pmcid: 7335070
Hawkins KM, Smolke CD. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol. 2008;4:564–73.
pubmed: 18690217
pmcid: 2830865
Liu XB, Liu YS, Huang P, Ma YS, Qing ZX, Tang Q, et al. The genome of medicinal plant Macleaya cordata provides new insights into benzylisoquinoline alkaloids metabolism. Mol Plant. 2017;10:975–89.
pubmed: 28552780
Naito Y, Hino K, Bono H, Ui-Tei K. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 2015;31:1120–3.
pubmed: 25414360
Westfall PJ, Pitera DJ, Lenihan JR, Eng D, Woolard FX, Regentin R, et al. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci USA. 2012;109:E111–8.
pubmed: 22247290
pmcid: 3271868
Liu TF, Gou YW, Zhang B, Gao R, Dong C, Qi MM, et al. Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast. Biotechnol Bioeng. 2022;119:1314–26.
pubmed: 35060115
Fossati E, Ekins A, Narcross L, Zhu Y, Falgueyret JP, Beaudoin GAW, et al. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat Commun. 2014;5:3283.
pubmed: 24513861
Xool-Tamayo J, Tamayo-Ordoñez Y, Monforte-González M, Muñoz-Sánchez JA, Vázquez-Flota F. Alkaloid Biosynthesis in the early stages of the germination of Argemone mexicana L. (Papaveraceae). Plants. 2021;10:2226.
pubmed: 34686035
pmcid: 8538276
Dittrich H, Kutchan TM. Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. Proc Natl Acad Sci USA. 1991;88:9969–73.
pubmed: 1946465
pmcid: 52848
Bird DA, Facchini PJ. Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant. Planta. 2001;213:888–97.
pubmed: 11722125
Takeshita N, Fujiwara H, Mimura H, Fitchen JH, Yamada Y, Sato F. Molecular cloning and characterization of S-adenosyl-L-methionine: scoulerine-9-O-methyltransferase from cultured cells of Coptis Japonica. Plant Cell Physiol. 1995;36:29–36.
pubmed: 7719631
Valentic TR, Payne JT, Smolke CD. Structure-guided engineering of a scoulerine 9-O-methyltransferase enables the biosynthesis of tetrahydropalmatrubine and tetrahydropalmatine in yeast. ACS Catal. 2020;10:4497–509.
Díaz Chávez ML, Rolf M, Gesell A, Kutchan TM. Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana. Arch Biochem Biopyhs. 2011;507:186–93.
Hannemann F, Bichet A, Ewen KM, Bernhardt R. Cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta. 2007;1770:330–4.
pubmed: 16978787
Beaudoin GAW, Facchini PJ. Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis. Biochem Biophys Res Commun. 2013;431:597–603.
pubmed: 23313486
Liu QL, Liu Y, Li G, Savolainen O, Chen Y, Nielsen J. De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories. Nat Commun. 2021;12:6085.
pubmed: 34667183
pmcid: 8526750
Wei YF, Ang EL, Zhao HM. Recent developments in the application of P450 based biocatalysts. Curr Opin Chem Biol. 2018;43:1–7.
pubmed: 29100098
Kim JE, Jang IS, Son SH, Ko YJ, Cho BK, Kim SC, et al. Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway. Metab Eng. 2019;56:50–9.
pubmed: 31445083
Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, et al. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc Natl Acad Sci USA. 2008;105:18525–30.
pubmed: 19011102
pmcid: 2587584
Schuck S, Prinz WA, Thorn KS, Voss C, Walter P. Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol. 2009;187:525–36.
pubmed: 19948500
pmcid: 2779237
Urban P, Mignotte C, Kazmaier M, Delorme F, Pompon D. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J Biol Chem. 1997;272:19176–86.
pubmed: 9235908
Michener JK, Nielsen J, Smolke CD. Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases. Proc Natl Acad Sci USA. 2012;109:19504–9.
pubmed: 23129650
pmcid: 3511110
Zhang TT, Bu PL, Zeng J, Vancura A. Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression. J Biol Chem. 2017;292:16942–54.
pubmed: 28830930
pmcid: 5641876
Hu BD, Yu HB, Zhou JW, Li JH, Chen J, Du GC, et al. Whole-cell P450 biocatalysis using engineered Escherichia coli with fine-tuned heme biosynthesis. Adv Sci. 2022;10:e2205580.
Oh EJ, Ha SJ, Kim SR, Lee WH, Galazka JM, Gate JHD, et al. Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccharomyces cerevisiae. Metab Eng. 2013;15:226–34.
pubmed: 23103205
Yu T, Zhou YJJ, Huang MT, Liu QL, Pereira R, David F, et al. Reprogramming yeast metabolism from alcoholic fermentation to lipogenesis. Cell. 2018;174:1549–58.
pubmed: 30100189
Frick O, Wittmann C. Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13 C flux analysis. Microb Cell Fact. 2005;4:30.
pubmed: 16269086
pmcid: 1291395
Shitan N, Kato K, Shoji T. Alkaloid transporters in plants. Plant Biotechnol. 2014;31:453–63.
Young ND, Debelle F, Oldroyd GED, Geurts R, Cannon SB, Udvardi MK, et al. The medicago genome provides insight into the evolution of rhizobial symbioses. Nature. 2011;480:520–4.
pubmed: 22089132
pmcid: 3272368
Yu F, De Luca V. ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus. Proc Natl Acad Sci USA. 2013;110:15830–5.
pubmed: 24019465
pmcid: 3785729
Shitan N, Dalmas F, Dan K, Kato N, Ueda K, Sato F, et al. Characterization of Coptis Japonica CjABCB2, an ATP-binding cassette protein involved in alkaloid transport. Phytochemistry. 2013;91:109–16.
pubmed: 22410351
Cravens A, Payne J, Smolke CD. Synthetic biology strategies for microbial biosynthesis of plant natural product. Nat Commun. 2019;10:2142.
pubmed: 31086174
pmcid: 6513858
Renault H, Bassard JE, Hamberger B, Werck-Reichhart D. Cytochrome P450-mediated metabolic engineering: current progress and future challenges. Curr Opin Plant Biol. 2014;19:27–34.
pubmed: 24709279
Chen RB, Gao JQ, Yu W, Chen XH, Zhai XX, Chen Y, et al. Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast. Nat Chem Biol. 2022;18:520–9.
pubmed: 35484257