catalysts Article Enhanced Lycopene Production in Escherichia coli by Expression of Two MEP Pathway Enzymes from Vibrio sp. Dhg 1, 1, 1 1, Min Jae Kim y, Myung Hyun Noh y , Sunghwa Woo , Hyun Gyu Lim * and Gyoo Yeol Jung 1,2,* 1 Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Korea; [email protected] (M.J.K.); [email protected] (M.H.N.); [email protected] (S.W.) 2 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Korea * Correspondence: [email protected] (H.G.L.); [email protected] (G.Y.J.); Tel.: +82-54-279-2391 (G.Y.J.) These authors contributed equally to this work. y Received: 28 October 2019; Accepted: 26 November 2019; Published: 29 November 2019 Abstract: Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of Escherichia coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids. Keywords: metabolic engineering; lycopene; MEP pathway; 1-deoxy-d-xylulose-5-phosphate synthase; farnesyl diphosphate synthase; Vibrio sp. dhg 1. Introduction Carotenoids are natural pigments present in plants and microorganisms [1]. Some carotenoids are known to function as membrane protective anti-oxidants [2,3], which has led to a high demand for carotenoids for medical and pharmaceutical applications [4,5]. Lycopene is one of the most highly valuable carotenoids owing to its potent antioxidant [6] and disease prevention properties [7,8]. The lycopene market is growing at 3.5 percent annually and is expected to exceed 133 million dollars in 2023, thus supporting high demand and value [9,10]. The industrial production of carotenoids has conventionally been conducted through an extraction from many natural products, such as tomato and watermelon [11]. However, the extraction method does not meet the growing demand due to limited supply of natural products [12,13]. In addition, its stable production is difficult because of fluctuating lycopene content of natural products: 0.00540–1.50 g lycopene/kg of tomato paste [14], Catalysts 2019, 9, 1003; doi:10.3390/catal9121003 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 1003 2 of 12 Catalysts 2019, 9, x FOR PEER REVIEW 2 of 11 0.03–0.07 g lycopene/kg of watermelon [15]. Chemical synthesis could be considered as an alternative method; however, there are also several critical limitations, including high production cost, low method; however, there are also several critical limitations, including high production cost, low yield, yield, and quality [12,16]. To overcome those limitations, microbial production of lycopene has been and quality [12,16]. To overcome those limitations, microbial production of lycopene has been studied studied as a promising strategy because it enables stable production through a simple and sustainable as a promising strategy because it enables stable production through a simple and sustainable process process [17,18]. [17,18]. Lycopene can be biologically synthesized through condensation of two precursors, isopentenyl Lycopene can be biologically synthesized through condensation of two precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), and subsequent chain elongation pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), and subsequent chain elongation reactions (Figure1)[ 19,20]. The two precursors, IPP and DMAPP, are produced via two distinct reactions (Figure 1) [19,20]. The two precursors, IPP and DMAPP, are produced via two distinct pathways [21]. One of the pathways is the mevalonate (MVA) pathway, which is mainly present pathways [21]. One of the pathways is the mevalonate (MVA) pathway, which is mainly present in in eukaryotes and archaea. The MVA pathway utilizes three moles of acetyl-CoA to synthesize eukaryotes and archaea. The MVA pathway utilizes three moles of acetyl-CoA to synthesize one mole one mole of IPP, which is then isomerized to DMAPP by the activity of isopentenyl-diphosphate of IPP, which is then isomerized to DMAPP by the activity of isopentenyl-diphosphate delta- delta-isomerase (Idi). The other pathway is the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, isomerase (Idi). The other pathway is the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which is mostly present in bacteria and consists of a series of reactions initiated by the condensation which is mostly present in bacteria and consists of a series of reactions initiated by the condensation of pyruvate and glyceraldehyde-3-phosphate (G-3-P). Although the MEP pathway requires more of pyruvate and glyceraldehyde-3-phosphate (G-3-P). Although the MEP pathway requires more energy (ATP) and cofactors (NADPH) to synthesize IPP than the MVA pathway [13], the direct use energy (ATP) and cofactors (NADPH) to synthesize IPP than the MVA pathway [13], the direct use of the two C-3 glycolytic intermediates enables MEP pathway to have a 1.48-fold higher IPP yield of the two C-3 glycolytic intermediates enables MEP pathway to have a 1.48-fold higher IPP yield (0.83(0.83 C-moleC-mole/C-mole)/C-mole) thanthan thethe MVAMVA pathwaypathway (0.56(0.56 C-moleC-mole/C-mole)/C-mole) [13[13,22].,22]. SinceSince mostmost bacteriabacteria showing fast growth inherently possess the MEP pathway for synthesizing their building blocks [21], showing fast growth inherently possess the MEP pathway for synthesizing their building blocks [21], thethe MEPMEP pathwaypathway hashas beenbeen widelywidely utilizedutilized inin recentrecent studiesstudies forfor lycopenelycopene productionproduction [ [10,23].10,23]. FigureFigure 1.1. Microbial lycopene production pathway.pathway. Lycopene Lycopene production could be significantlysignificantly enhancedenhanced byby heterologousheterologous overexpressionoverexpression ofof CrtEBICrtEBI andand keykey metabolic enzymes, Dxs and IspA. Dxs, 1-deoxy-1-deoxy-dD-xylulose-5-phosphate-xylulose-5-phosphate synthase; synthase; IspA, farnesylIspA, farnesyl pyrophosphate pyrophosphate synthase; CrtE, synthase; geranylgeranyl CrtE, pyrophosphategeranylgeranyl synthase;pyrophosphate CrtB, phytoenesynthase; synthase;CrtB, phytoene CrtI, phytoene synthase; desaturase. CrtI, phytoene desaturase. SignificantSignificant achievementsachievements have have been been made made in lycopene in lycopene production production over the decadesover the by engineeringdecades by MEPengineering pathway. MEP Heterologous pathway. Heterologous expression ofexpression geranylgeranyl of geranylgeranyl diphosphate diphosphate synthase (crtE) synthase, phytoene (crtE), synthasephytoene ( crtBsynthase), and phytoene(crtB), and desaturase phytoene ( crtIdesaturase) could enable (crtI) Escherichiacould enable coli Escherichiato produce coli lycopene to produce using inherentlycopene MEP using pathway inherent [24 MEP]. Since pathway then, substantial[24]. Since increasethen, substantial in lycopene increase production in lycopene has been production reported byhas overexpression been reported ofby key overexpression metabolic enzymes of key metabolic [7,25]. The enzymes condensation [7,25]. of The pyruvate condensation and G-3-P of pyruvate has been knownand G-3-P as ahas rate-limiting been known step as ofa rate-limiting the MEP pathway, step of andthe MEP thus overexpressionpathway, and thus of dxs overexpression gene (encoding of 1-deoxy-dxs gene (encodingd-xylulose-5-phosphate 1-deoxy-D-xylulose-5-phosphate synthase) could enhance synthase) lycopene could enhance production lycopene by to production 3.5-fold [25 by]. Similarly,to 3.5-fold the [25]. catalytic Similarly, activity the catalytic of Dxs isactivity known of to Dxs be is subjected known to to be a negativesubjected feedback to a negative regulation feedback by IPPregulation and DMAPP by IPP [ 26and], andDMAPP expression [26], and of ispA expression(encoding of ispA farnesyl (encoding diphosphate farnesyl synthase) diphosphate could synthase) improve lycopenecould improve production lycopene by more production than 2-fold by more [27]. Onthan the 2-fold other [27]. hand, On there the haveother been hand, several there attempts have been to increaseseveral attempts lycopene to production increase lycopene by balancing production the expression by balancing levels the of precursors,expression pyruvate,levels of precursors, and G-3-P. Farmerpyruvate, and and Liao