Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthaseby Codruta Ignea, Fotini A. Trikka, Alexandros K. Nikolaidis, Panagiota Georgantea, Efstathia Ioannou, Sofia Loupassaki, Panagiotis Kefalas, Angelos K. Kanellis, Vassilios Roussis, Antonios M. Makris, Sotirios C. Kampranis

Metabolic Engineering


Applied Microbiology and Biotechnology / Biotechnology / Bioengineering


Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase

Codruta Ignea a, Fotini A. Trikka b, Alexandros K. Nikolaidis b, Panagiota Georgantea c,

Efstathia Ioannou c, Sofia Loupassaki a, Panagiotis Kefalas a, Angelos K. Kanellis d,

Vassilios Roussis c, Antonios M. Makris b, Sotirios C. Kampranis a,e,nQ1 a Mediterranean Agronomic Institute of Chania, P.O. Box 85, Chania 73100, Greece b Institute of Applied Biosciences – Centre for Research and Technology Hellas (INAB-CERTH), P.O. Box 60361, Thermi, 57001 Thessaloniki, Greece c Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou,

Athens 15771, Greece d Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece e Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion 71003, Greece a r t i c l e i n f o

Article history:

Received 22 January 2014

Received in revised form 1 August 2014

Accepted 20 October 2014




Protein engineering

Geranylgeranyl diphosphate

Terpene synthase a b s t r a c t

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any

GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms. & 2014 International Metabolic Engineering Society. Published by Elsevier Inc. 1. Introduction

Terpenes are a large class of natural compounds whose numerous members have attracted industrial interest as flavors and fragrances, pharmaceuticals, or biofuels. According to the number of carbon atoms in their skeleton, basic terpene structures are classified into different groups, such as monoterpenes (C10), sesquiterpenes (C15), or diterpenes (C20). These are formed by the action of terpene synthases on prenyl diphosphate substrates of the same size, i.e., geranyl diphosphate (GPP) for monoterpenes, farnesyl diphosphates (FPP) for sesquiterpenes, or geranylgeranyl diphosphate (GGPP) for diterpenes (Fig. 1). The prenyl diphosphate substrates themselves are formed by the sequential addition of the 5-carbon building block isopentenyl diphosphate (IPP) initially on dimethylallyl diphosphate (DMAPP), and, subsequently, on the resulting GPP and FPP, by specific prenyl diphosphate synthases of varying specificity (e.g. FPP synthase in mammals, GGPP and FPP synthases in plants, or Erg20p and Bts1p in Saccharomyces cerevisiae). Further modification of the various terpene scaffolds produced by this modular biosynthetic process gives rise to a vast diversity of natural structures with more than 50,000 members (Dewick, 2009; McGarvey and Croteau, 1995).

Several members of the 20-carbon diterpene group have found industrial application. For example, paclitaxel (or taxol; from the pacific yew Taxus brevifolia) prevents microtubule de-polymerization and is widely used as a chemotherapeutic agent (Goldspiel, 1997; Schiff et al., 1979). Total paclitaxel synthesis is feasible, but not 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91

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Metabolic Engineering http://dx.doi.org/10.1016/j.ymben.2014.10.008 1096-7176/& 2014 International Metabolic Engineering Society. Published by Elsevier Inc.

Abbreviations: geranyl diphosphate, GPP; dimethylallyl diphosphate, DMAPP; isopentenyl diphosphate, IPP; farnesyl diphosphate, FPP; geranylgeranyl diphosphate, GGPP; nerolidol, NOH; farnesol, FOH; geraniol, GOH; linalool, LOH; geranylgeraniol, GGOH; geranyllinalool, GLOH; 8-hydroxycopalyl diphosphate, 8OH-CPP; Salvia sclarea labdenediol diphosphate synthase, SsLPP; Salvia sclarea sclareol synthase, SsSCLS; Cistus creticus geranylgeranyl diphosphate synthase,

CcGGPPS; Cistus creticus 8-hydroxycopalyl diphosphate synthase, CcCLS; Salvia pomifera copalyl diphosphate synthase, SpCDS; Salvia fruticosa copalyl diphosphate synthase, SfCDS; Nicotiana tabacum abienol synthase, NtABS; abietadiene synthase,

PaLAS; X. dendrorhous phytoene desaturase, crtI; X. dendrorhous phytoene synthase/lycopene cyclase, crtYB; X. dendrorhous GGPP synthase, crtE; dry cell weight, DCW n Corresponding author at: Department of BiochemistryQ3 , School of Medicine,

University of Crete, P.O. Box 2208, Heraklion 71003, Greece.

E-mail address: s.kampranis@med.uoc.gr (S.C. Kampranis).

Please cite this article as: Ignea, C., et al., Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase. Metab. Eng. (2014), http://dx.doi.org/10.1016/j.ymben.2014.10.008i

Metabolic Engineering ∎ (∎∎∎∎) ∎∎∎–∎∎∎ commercially viable, and medicinal paclitaxel can be produced either by semi-synthesis from 10-deacetyl baccatin or by Taxus cell cultures (Bringi et al., 1993; Gibson, 1999). Another diterpene-derived highvalue compound is ambroxan (Fig. 2), an important base molecule in perfumery which serves as a substitute for ambergris, a substance excreted from the injured intestines of whales (Ohloff, 1982). Currently, ambroxan is synthesized from sclareol, a diterpene diol obtained from Salvia sclarea plants (Barton et al., 1994). Abienol (Fig. 2), a related labdane-type diterpene alcohol, is also used as a precursor for ambroxan synthesis (Barrero et al., 1993).