pharmaceuticals Review Fermentation Strategies for Production of Pharmaceutical Terpenoids in Engineered Yeast Erdem Carsanba 1,2, Manuela Pintado 2 and Carla Oliveira 2,* 1 Amyris BioProducts Portugal, Unipessoal, Lda. Rua Diogo Botelho 1327, 4169-005 Porto, Portugal; [email protected] 2 CBQF—Centro de Biotecnologia e Química Fina—Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal; [email protected] * Correspondence: [email protected] Abstract: Terpenoids, also known as isoprenoids, are a broad and diverse class of plant natural prod- ucts with significant industrial and pharmaceutical importance. Many of these natural products have antitumor, anti-inflammatory, antibacterial, antiviral, and antimalarial effects, support transdermal absorption, prevent and treat cardiovascular diseases, and have hypoglycemic activities. Produc- tion of these compounds are generally carried out through extraction from their natural sources or chemical synthesis. However, these processes are generally unsustainable, produce low yield, and result in wasting of substantial resources, most of them limited. Microbial production of terpenoids provides a sustainable and environment-friendly alternative. In recent years, the yeast Saccharomyces cerevisiae has become a suitable cell factory for industrial terpenoid biosynthesis due to developments in omics studies (genomics, transcriptomics, metabolomics, proteomics), and mathematical modeling. Besides that, fermentation development has a significant importance on achieving high titer, yield, and productivity (TYP) of these compounds. Up to now, there have been many studies and reviews Citation: Carsanba, E.; Pintado, M.; reporting metabolic strategies for terpene biosynthesis. However, fermentation strategies have not Oliveira, C. Fermentation Strategies been yet comprehensively discussed in the literature. This review summarizes recent studies of for Production of Pharmaceutical recombinant production of pharmaceutically important terpenoids by engineered yeast, S. cerevisiae, Terpenoids in Engineered Yeast. with special focus on fermentation strategies to increase TYP in order to meet industrial demands to Pharmaceuticals 2021, 14, 295. https:// feed the pharmaceutical market. Factors affecting recombinant terpenoids production are reviewed doi.org/10.3390/ph14040295 (strain design and fermentation parameters) and types of fermentation process (batch, fed-batch, and continuous) are discussed. Academic Editor: Marialuigia Fantacuzzi Keywords: terpenoids; S. cerevisiae; pharmaceutics; fermentation; fed-batch Received: 9 March 2021 Accepted: 24 March 2021 Published: 26 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral Terpenoids, also known as isoprenoids or terpenes, constitute one of the largest chem- with regard to jurisdictional claims in ically and structurally diverse group of natural products with over 80,000 members in published maps and institutional affil- a widespread family (currently stated in the Dictionary of Natural Products database, iations. http://dnp.chemnetbase.com, accessed on 26 March 2021) [1–4]. Although the majority of terpenoids are predominantly present in plants, other organisms such as bacteria, fungi, insects, and animals contain them as well [5]. Their essential biological functions can be counted as the regulation of cell growth and defense in plants (e.g., tocopherol, gibberellin), and intracellular electron transport, glycoproteins biosynthesis and cell membranes forma- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. tion in animals (e.g., cholesterol) [6]. Owing to their structural variety, there exist diverse This article is an open access article terpenoid activities, such as mediating symbiotic or antagonistic interactions between distributed under the terms and organisms to electron transfer, protein prenylation, or contribution to membrane fluidity, conditions of the Creative Commons which make these compounds highly useful to be applied in various industries: food, Attribution (CC BY) license (https:// cosmetics, fine chemistry, pharmacy, agriculture, and biofuel [5]. creativecommons.org/licenses/by/ Terpenoids are formed from several isoprene (2-methylbuta-1,3-diene; IUPAC 1997) 4.0/). (CH2=C(CH3)–CH=CH2) structural units (or building blocks) synthesized from meval- Pharmaceuticals 2021, 14, 295. https://doi.org/10.3390/ph14040295 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 31 Pharmaceuticals 2021, 14, 295 2 of 29 Terpenoids are formed from several isoprene (2-methylbuta-1,3-diene; IUPAC 1997) (CH2=C(CH3)−CH=CH2) structural units (or building blocks) synthesized from mevalo- onatenate (MVA)(MVA) oror 2-C-methyl-d-erythritol2-C-methyl-d-erythritol 4-phosphate4-phosphate (MEP)(MEP) (which(which is is also also known known as as the the non-mevalonate)non-mevalonate) pathway pathway [ 2[2,3].,3]. Through Through the the condensation condensation reactions reactions of of these these units, units, sev- sev- eraleral large large and and more more complex complex compounds compounds are are produced. produced. According According to to the the number number of of C5 C5 isopreneisoprene units, units, terpenoids terpenoids are are mainly mainly classified classified in in hemiterpenoid hemiterpenoid (C5), (C5), monoterpenoid monoterpenoid (C10),(C10), sesquiterpenoid sesquiterpenoid (C15), (C15), diterpenoid diterpenoid (C20), (C20), triterpenoid triterpenoid (C30), (C30), tetraterpenoid tetraterpenoid (C40), (C40), andand polyterpenoid polyterpenoid (C (C >˃40) 40) [2,3,6]. [2,3,6]. Terpenoids Terpenoids are are mostly mostly formed formed through through continuous continuous ad- additiondition of of head head to to tail tail manner of building blocks,blocks, whichwhich areareisoprene isoprene diphosphate diphosphate (IPP) (IPP) andand dimethylallyl dimethylallyl diphosphate diphosphate (DMAPP). (DMAPP). For For instance, instance, the the precursor precursor of of monoterpenoids, monoterpenoids, geranylgeranyl diphosphate diphosphate (GPP), (GPP), is is produced produced by by head head to to tail tail condensation condensation of of these these two two build- build- inging blocks. blocks. Later,Later, additionaddition of of IPP IPP leads leads to to th thee formation formation of offarnesyl farnesyl diphosphate diphosphate (FPP) (FPP) and andgeranylgeranyl geranylgeranyl diphosphate diphosphate (GGPP), (GGPP), which which are are precursors ofof sesquiterpenoidsesquiterpenoid andand diterpenoid.diterpenoid. OnOn the other hand hand,, head head to to head head condensation condensation ofof two two molecules molecules of FPP of FPP and andGGPP, GGPP, respectively, respectively, forms forms squalene squalene and and phytoene, phytoene, the the precursors precursors of oftriterpenoid triterpenoid and and tet- tetraterpenoidraterpenoid [6,7] [6,7 ](Figure (Figure 11).). Furthermore,Furthermore, thesethese precursorsprecursors are are exposed exposed to to oxidation, oxidation, by by cytochromecytochrome P450 P450 oxygenases, oxygenases, and and glycosylation, glycosylation, by by glycosyltransferases glycosyltransferases to to form form various various terpenoids. In addition, prenyltransferases concatenates isoprene-derived precursors to terpenoids. In addition, prenyltransferases concatenates isoprene-derived precursors to fatty acid-derived precursor to synthase complex terpenoids or meroterpenoids, which fatty acid-derived precursor to synthase complex terpenoids or meroterpenoids, which consist of the medically important compounds, cannabinoids [2]. consist of the medically important compounds, cannabinoids [2]. FigureFigure 1. 1.The The condensation condensation reactions reactions of of terpenoid terpenoid biosynthesis: biosynthesis: ( A(A)) Head Head to to tail tail condensation condensationand and (B()B Tail) Tail to to tail tail condensation condensation (Adapted (Adapted from from [7 ]).[7]). FromFrom early early ancient ancient times times upup toto now,now, terpenoidsterpenoids have been playing playing important important roles roles in inmany many medical medical treatments treatments as as pharmaceuticals pharmaceuticals with with diverse diverse biomedical biomedical activities, activities, such such as as antimalarial, anticancer, anti-inflammatory, antibacterial, antiviral, hypoglycemic, pre- venting and treating cardiovascular diseases, and promoting the transdermal absorption. Pharmaceuticals 2021, 14, 295 3 of 29 Besides that, they can have insecticidal, immunomodulatory, antioxidant, antiaging, and neuroprotective effects [2,3,8,9]. They are usually produced from natural resources through extraction or by chemical synthesis, but the obtained yields are generally low due to inherently low level of target compound and the necessity of complex extraction and chemical synthesis methods, which are commercially infeasible [10]. In most cases, the natural production method, extracting terpenoids from original sources (e.g., taxol from yew tree and artemisinin from the plant Artemisia annua) generally fails in terms of quality and supply management due to seasonal and geographical alterations [5]. In addition, plant engineering for terpenoid production is difficult and complex due to tissue specific expression and loss of volatile products by evaporation, and productivity and yields are very low [11]. Due to these limitations, microbial production of terpenoids has received