processes Review The Potential Production of the Bioactive Compound Pinene Using Whey Permeate Derrick Risner, Maria L. Marco, Sara A. Pace and Edward S. Spang * Department of Food Science and Technology, University of California, Davis, CA 95616, USA; [email protected] (D.R.); [email protected] (M.L.M.); [email protected] (S.A.P.) * Correspondence: [email protected]; Tel.: +1-530-752-1255 Received: 26 December 2019; Accepted: 19 February 2020; Published: 26 February 2020 Abstract: Pinene is a secondary plant metabolite that has functional properties as a flavor additive as well as potential cognitive health benefits. Although pinene is present in low concentrations in several plants, it is possible to engineer microorganisms to produce pinene. However, feedstock cost is currently limiting the industrial scale-up of microbial pinene production. One potential solution is to leverage waste streams such as whey permeate as an alternative to expensive feedstocks. Whey permeate is a sterile-filtered dairy effluent that contains 4.5% weight/weight lactose, and it must be processed or disposed of due its high biochemical oxygen demand, often at significant cost to the producer. Approximately 180 million m3 of whey is produced annually in the U.S., and only half of this quantity receives additional processing for the recovery of lactose. Given that organisms such as recombinant Escherichia coli grow on untreated whey permeate, there is an opportunity for dairy producers to microbially produce pinene and reduce the biological oxygen demand of whey permeate via microbial lactose consumption. The process would convert a waste stream into a valuable coproduct. This review examines the current approaches for microbial pinene production, and the suitability of whey permeate as a medium for microbial pinene production. Keywords: terpene; pinene; Escherichia coli; whey; whey permeate; biosynthesis; microbial 1. Introduction Terpenoids/isoprenoids are multifunctional organic compounds that contribute to an array of applications and are currently used as solvents, fragrances, natural pesticides, lubricants, flavoring agents, and in nutraceutical/medical applications [1–4]. Terpenoids/isoprenoids are hydrocarbons comprised of five carbon isoprene units and are classified by their number of carbon atoms, namely hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30) tetraterpenoids (C40) and polyterpenoids (C45+). Terpenoid precursors are produced in most organisms via the mevalonate (MVA) (Figure1) and /or the 2-C-methyl-D-erythrithol 4-phosphate/ 1-deoxy-D-xylose 5 phosphate (MEP/DOXP) pathway (Figure2) and are synthesized via terpenoid synthases [5]. Pinene is synthesized from isopentenyl pyrophosphate produced from the MVA or MEP/DOXP pathway via geranyl diphosphate synthase and pinene synthase (Figure3). Pinene is a monoterpene with two structural isomers, α-pinene and β-pinene (IUPAC names: (2,6,6-trimethylbicyclo[3.1.1]hept-2-ene and 6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane, respectively). Both α-pinene and β-pinene have multiple commercial applications. They are commonly used as flavor compounds to produce herbal or earthy flavors in food [6]. Additional potential commercial applications include uses as a antimicrobial agent, potential use as a jet fuel alternative and functionality as a starting compound for the synthesis of other terpenoids [3,7,8]. This review will examine the potential industrial applications of pinene, the current approaches for microbial pinene production, and the suitability of whey permeate as a medium for microbial pinene production. Processes 2020, 8, 263; doi:10.3390/pr8030263 www.mdpi.com/journal/processes Processes 2020, 8, 263 2 of 15 Processesmicrobial2020, pinene8, 263 production, and the suitability of whey permeate as a medium for microbial pinene2 of 16 production. FigureFigure 1. 1.Mevalonate Mevalonate (MVA)(MVA) pathwaypathway forfor eukaryotes.eukaryotes. Processes 2020, 8, 263 3 of 16 Processes 2020, 8, 263 3 of 15 Figure 2. Methylerythritol phosphate (MEP/DOX) pathway. Figure 2. Methylerythritol phosphate (MEP/DOX) pathway. Processes 2020, 8, 263 4 of 16 Processes 2020, 8, 263 4 of 15 FigureFigure 3.3. PinenePinene biosynthesisbiosynthesis from isopentenyl pyrophosphate. 2.2. Applications Applications ofof PinenePinene PinenePinene isis aa compoundcompound withwith severalseveral currentcurrent andand potential applications in in medicine, medicine, as as a a flavorflavor/aroma/aroma agent, agent, andandas as aa precursorprecursor forfor fuelfuel production.production. α- and β--PinenePinene ha haveve several several potential potential applicationsapplications in in medicine. medicine. Firstly, Firstly, it it exhibited exhibited antimicrobial antimicrobial properties properties and and increased increased the the e ffeffectivenessectiveness of commercialof commercial antibiotics antibiotics against against methicillin methicillin resistant resistantStaphylococcus Staphylococcus aureus [7aure]. αus-pinene [7]. α also-pinene reversibly also inhibitedreversibly acetylcholinesterase, inhibited acetylcholinesterase, and therefore and has therefore potential has to treatpotential neurodegenerative to treat neurodegenerative diseases such asdiseases Alzheimer’s such as disease Alzheimer’s and Parkinson’s disease and disease Parkinson’s [1,9]. Additionally,disease [1,9]. Additionally,α-Pinene reduced α-Pinene the impactreduced of scopolamine-inducedthe impact of scopolamine cognitive-induced impairment cognitive in impairment mice and 6-hydroxydopamine-inducedin mice and 6-hydroxydopamine Parkinson’s-induced diseaseParkinson’s in rats disease [9,10]. in Pinene rats [9,10] itself. isPinene used asitself flavoring is used agent as flavoring and in cosmetics,agent and butin cosmetics, is also used but to is derive also theused additional to derive aromatic the additional compounds, aromatic verbenone compounds, and carvone, verbenone which and arecarvone, used in which perfumery are used and in as insectperfumery repellents and [as8,11 insect,12]. Collectively,repellents [8,11,12] this evidence. Collectively, indicates this that evidence pinene hasindicates flavoring that applications,pinene has potentialflavoring medical applications value,, potential or could bemedical used asvalue an ingredient, or could inbe nutraceuticalused as an ingredient products. in nutraceutical productDimerizeds. pinene also has a volumetric energy similar to that of commercial jet fuel (JP-10) [3]. AlthoughDimerized it is not pinene applied also commercially has a volumetric as a fuel energy at this similar time, increased to that ofavailability commercial of jet dimerized fuel (JP-10) pinene [3]. couldAlthough assist it airlines is not inapplied shifting commercially from reliance as on a fuel fossil at fuels this towardstime, increased biofuels. availability For dimerized of dimerized pinene to bepinene an economically could assist viableairlines fuel, in shifting a sustainable from reliance production on fossil method fue mustls towards be developed. biofuels. For The dimerized combined annualpinene production to be an economically volume of αviableand βfuel,-pinene a sustainable is approximately production 49 millionmethod litersmust [be13 ].developed. This is several The orderscombined of magnitude annual production less than volume what would of α and be needed β-pinene to is make approximately a tangible contribution49 million liters to the[13] jet. This fuel is several orders of magnitude less than what would be needed to make a tangible contribution to the market, considering that U.S. airline carriers consumed 67 billion liters of jet fuel in 2016 [14]. jet fuel market, considering that U.S. airline carriers consumed 67 billion liters of jet fuel in 2016 [14]. 3. Plant Pinene Biosynthesis and Purification 3. Plant Pinene Biosynthesis and Purification Pinene is a secondary plant metabolite produced in a wide variety of aromatic plants in small concentrations,Pinene is howevera secondary it is producedplant metabolite industrially produced during in the a wide paper variety making of process aromatic [15 plants]. Plant-derived in small isopentenylconcentrations diphosphate, however (IPP) it is andproduced dimethylallyl industrial diphosphately during the (DMAPP) paper making are the precursorsprocess [15] of. pinenePlant- andderived other i terpenoidssopentenyl [diphosphate16]. Geranyl (IPP) diphosphate and dimethylallyl (GPP), a monoterpenoid diphosphate (DMAPP) precursor, are is formedthe prec throughursors theof condensationpinene and other of IPP terpenoids and DMAPP, [16] which. Geranyl is catalyzed diphosphate by a terpene (GPP), synthasea monoterpenoid (GPPS). GPP precursor is cyclized, is formed through the condensation of IPP and DMAPP, which is catalyzed by a terpene synthase by pinene synthase (PS) to form α- or β-pinene (Figure3)[ 16]. IPP and DMAPP are biosynthesized in (GPPS). GPP is cyclized by pinene synthase (PS) to form α- or β-pinene (Figure 3) [16]. IPP and plants via the MVApathway or the MEP pathway [5]. The MVApathway is initiated by the condensation DMAPP are biosynthesized in plants via the MVA pathway or the MEP pathway [5]. The MVA of two acetyl coenzyme A (acetyl CoA) molecules (Figure1). The MEP pathway is initiated by the pathway is initiated by the condensation of two acetyl coenzyme A (acetyl CoA) molecules (Figure condensation of glyceraldehyde 3-phosohate and pyruvate (Figure2). Glyceraldehyde 3-phosohate, 1). The MEP pathway is