Triterpenoids, one of the most diverse classes of natural products, have been used for centuries as active ingredients in essential oils and Chinese medicines and are of interest for many industrial applications ranging from low-calorie sweeteners to cosmetic ingredients and vaccine adjuvants. However, not only can the extraction from plant material be cumbersome due to low concentrations of the specific triterpenoid, but concerns are also increasing regarding the sustainability of wild plant harvest while meeting market demands. The alternative is to produce triterpenoids with engineered microbes. Here, we present a generally applicable strategy for triterpenoid production in the yeast Saccharomyces cerevisiae based on a modified oxidosqualene cyclase Erg7. The modification reduces the flux into the sterol pathway while increasing the precursor supply for triterpenoid production. The minimally engineered strain was exploited for the exemplary production of the lupane triterpenoids betulin, betulin aldehyde, and betulinic acid at a total titer above 6 g/L, the highest reported so far. To further highlight the chassis concept, squalene, oleanane- and dammarane-type triterpenoids were synthesized to titers at a similar gram scale. We propose the developed baker’s yeast as a host for the thousands of triterpenoid synthesis pathways from plants, reducing the pressure on the natural resources.

Chemie Ingenieur TechnikVolume 90, Issue 9 p. 1255-1256 VortragFree Access Metabolic engineering of Saccharomyces cerevisiae for cyclic triterpenoid production B. E. Ebert, Corresponding Author B. E. Ebert birgitta.ebert@rwth-aachen.de RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanyCorrespondence: B. E. Ebert (birgitta.ebert@rwth-aachen.de), RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorK. Walter, K. Walter RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorJ. Maury, J. Maury DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorC. Lang, C. Lang Organobalance GmbH, Gustav-Meyer-Allee 25, 13355 Berlin, GermanySearch for more papers by this authorJ. Förster, J. Förster DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorL. M. Blank, L. M. Blank RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorE. Czarnotta, E. Czarnotta RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorC. Knuf, C. Knuf DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorS. A. Jacobsen, S. A. Jacobsen DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorH. Guo, H. Guo RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorA. Lewandowski, A. Lewandowski Organobalance GmbH, Gustav-Meyer-Allee 25, 13355 Berlin, GermanySearch for more papers by this authorT. Polakowski, T. Polakowski Organobalance GmbH, Gustav-Meyer-Allee 25, 13355 Berlin, GermanySearch for more papers by this author B. E. Ebert, Corresponding Author B. E. Ebert birgitta.ebert@rwth-aachen.de RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanyCorrespondence: B. E. Ebert (birgitta.ebert@rwth-aachen.de), RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorK. Walter, K. Walter RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorJ. Maury, J. Maury DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorC. Lang, C. Lang Organobalance GmbH, Gustav-Meyer-Allee 25, 13355 Berlin, GermanySearch for more papers by this authorJ. Förster, J. Förster DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorL. M. Blank, L. M. Blank RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorE. Czarnotta, E. Czarnotta RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorC. Knuf, C. Knuf DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorS. A. Jacobsen, S. A. Jacobsen DTU Denmark, Novo Nordisk Foundation Center for Biosustainability, Kemitorvet 220, 2800 Lyngby, DenmarkSearch for more papers by this authorH. Guo, H. Guo RWTH Aachen University, iAMB – Institute of Applied Microbiology, Worringer Weg 1, 52056 Aachen, GermanySearch for more papers by this authorA. Lewandowski, A. Lewandowski Organobalance GmbH, Gustav-Meyer-Allee 25, 13355 Berlin, GermanySearch for more papers by this authorT. Polakowski, T. Polakowski Organobalance GmbH, Gustav-Meyer-Allee 25, 13355 Berlin, GermanySearch for more papers by this author First published: 24 August 2018 https://doi.org/10.1002/cite.201855271AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume90, Issue9Special Issue: ProcessNet-Jahrestagung und 33. DECHEMA-Jahrestagung der Biotechnologen 2018September 2018Pages 1255-1256 RelatedInformation

Chemie Ingenieur TechnikVolume 86, Issue 9 p. 1405-1405 VortragFree Access Metabolic Engineering von Saccharomyces cerevisiae für die Produktion zyklischer Triterpenoide K. Walter, Corresponding Author K. Walter kerstin.walter@rwth-aachen.de RWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, GermanyRWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, Germany===Search for more papers by this authorDr. B. E. Ebert, Dr. B. E. Ebert RWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, GermanySearch for more papers by this authorProf. C. Lang, Prof. C. Lang Organobalance GmbH, Gustav-Meyer-Allee 25, D-13355 Berlin, GermanySearch for more papers by this authorProf. L. M. Blank, Prof. L. M. Blank RWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, GermanySearch for more papers by this author K. Walter, Corresponding Author K. Walter kerstin.walter@rwth-aachen.de RWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, GermanyRWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, Germany===Search for more papers by this authorDr. B. E. Ebert, Dr. B. E. Ebert RWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, GermanySearch for more papers by this authorProf. C. Lang, Prof. C. Lang Organobalance GmbH, Gustav-Meyer-Allee 25, D-13355 Berlin, GermanySearch for more papers by this authorProf. L. M. Blank, Prof. L. M. Blank RWTH Aachen, iAMB – Institute für Angewandte Mikrobiologie, Worringer Weg 1, D-52074 Aachen, GermanySearch for more papers by this author First published: 28 August 2014 https://doi.org/10.1002/cite.201450328AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 K. Muffler, Process Biochem. 2011, 46 (1), 1– 15. 2 F. B. Mullauer, Anti-Cancer Drugs 2010, 21 (3), 215– 227. Volume86, Issue9Special Issue: ProcessNet-Jahrestagung 2014 und 31. DECHEMA-Jahrestagung der BiotechnologenSeptember, 2014Pages 1405-1405 ReferencesRelatedInformation

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.

The sterol-acyl transferase encoded by the gene ARE2 was transcriptionally deregulated in the yeast Saccharomyces cerevisiae to understand its role in sterol storage and sterol enrichment. Our results show that sterols can indeed be enriched in yeast by enhancing the capacity of the cells to esterify sterols, ARE2 overexpression had no impact on the accumulation of the early sterols such as lanosterol, but influenced the later intermediates and the end product ergosterol. Thus an enhanced conversion of free sterols to their esterified counterparts may provide a tool to increase the overall sterol content of the yeast cell. We have previously shown that the overexpression of a truncated version of the key enzyme of the early sterol pathway, HMG-CoA reductase (HMG1), leads to an increase in the early sterols such as lanosterol and zymosterol. The simultaneous deregulation of both genes in one strain produces a cumulative effect in that both early and late sterols are enhanced. Karmellae-like structures can be detected when Are2p is overexpressed. Are2p therefore constitutes a new member of the karmellae-inducing protein family.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate-limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1–552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene.

Birgitta E, Ebert, iAMB Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany birgitta.ebert@rwth-aachen.de Eik Czarnotta, Kerstin Walter, Lars M. Blank, iAMB Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany Christoph Knuf, Jérôme Maury, Simo A. Jacobsen, Jochen Förster, Novo Nordisk Foundation Center for Biosustainability, DTU Denmark, Denmark Anna Lewandowski, Thomas Polakowski, Christine Lang, Organobalance GmbH, Berlin, Germany