Hernandez‑Arranz et al. Microb Cell Fact https://doi.org/10.1186/s12934-019-1204-z (2019) 18:152 Microbial Cell Factories RESEARCH Open Access Engineering Pseudomonas putida for isoprenoid production by manipulating endogenous and shunt pathways supplying precursors Sofía Hernandez‑Arranz, Jordi Perez‑Gil, Dominic Marshall‑Sabey and Manuel Rodriguez‑Concepcion* Abstract Background: The soil bacterium Pseudomonas putida is a promising platform for the production of industrially valu‑ able natural compounds. In the case of isoprenoids, the availability of biosynthetic precursors is a major limiting factor. In P. putida and most other bacteria, these precursors are produced from pyruvate and glyceraldehyde 3-phosphate by the methylerythritol 4-phosphate (MEP) pathway, whereas other bacteria synthesize the same precursors from acetyl-CoA using the unrelated mevalonate (MVA) pathway. Results: Here we explored different strategies to increase the supply of isoprenoid precursors in P. putida cells using lycopene as a read-out. Because we were not aiming at producing high isoprenoid titers but were primarily interested in finding ways to enhance the metabolic flux to isoprenoids, we engineered the well-characterized P. putida strain KT2440 to produce low but detectable levels of lycopene under conditions in which MEP pathway steps were not saturated. Then, we compared lycopene production in cells expressing the Myxococcus xanthus MVA pathway genes or endogenous MEP pathway genes (dxs, dxr, idi) under the control of IPTG-induced and stress-regulated promoters. We also tested a shunt pathway producing isoprenoid precursors from ribulose 5-phosphate using a mutant version of the Escherichia coli ribB gene. Conclusions: The most successful combination led to a 50-fold increase in lycopene levels, indicating that P. putida can be successfully engineered to substantially increase the supply of metabolic substrates for the production of industrially valuable isoprenoids. Keywords: DXS, Isoprenoids, Lycopene, MEP pathway, Pseudomonas putida Background increasing demand for novel isoprenoid structures with Isoprenoids (also known as terpenoids) constitute the new or improved bioactivities, has spurred many meta- largest class of natural products, with tens of thousands bolic engineering and synthetic biology projects focused of different structures found in all kingdoms of life [1]. on boosting isoprenoid production. Much effort has Many of them are high value compounds due to their been devoted towards their synthesis in microbial hosts, diverse range of applications in areas such as human mostly employing Escherichia coli and Saccharomyces nutrition and pharmaceutical industry [2]. Most eco- cerevisiae. However, interference with the endogenous nomically-relevant isoprenoids are typically found at low cell metabolism and toxic effects associated with the pro- levels in their natural sources. This, together with the duction of most isoprenoids, among others, are impor- tant issues that often prevent reaching commercially relevant yields [3]. *Correspondence: manuel.rodriguez@cragenomica.es In this context, the stress-resistant bacterium Pseu- Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Barcelona, Spain domonas putida is a promising alternative platform © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 2 of 14 for isoprenoid production and storage. Some P. putida strains can tolerate much higher level of isoprenoids than other microorganisms (e.g. its growth is not affected by concentrations of the monoterpene geranic acid that are toxic to E. coli or S. cerevisiae [4]). The high toler- ance to otherwise toxic compounds of P. putida cells is in part due to fine-tuning of lipid fluidity to adjust mem- brane functions, activation of a general stress-response system, increased energy generation and induction of efflux pumps [5]. Additionally, P. putida is amenable to targeted genetic modifications and it has been targeted in biofactories design and biodegradation studies due to its robust metabolism, among others characteristics. Several industrial processes based on this bacterium are being exploited for the production of fine chemicals (e.g. 2-qui- noxalinecarboxylic acid, 5-methylpirazine-2-carboxylic acid, chiral amines and 4-(hydroxypyridin-3-yl)-4-oxobu- tyrate), and the production of many different compounds, including isoprenoids, is under study [6]. P. putida has actually been used for the production of some isopre- noids using biotransformation approaches to obtain oxi- Fig. 1 Pathways for isoprenoid synthesis. The P. putida endogenous dation products of the plant monoterpene limonene [7] MEP pathway is shown in black. Exogenous pathways added to P. or de novo biosynthesis of the monoterpene geranic acid putida in this work are shown in color: shunt pathway from E. coli in [4] or the carotenoids zeaxanthin and β-carotene [8–10]. green, MVA pathway from M. xanthus in blue, and lycopene pathway Despite the low number of available studies, the achieve- from P. ananatis in red. Enzymes are marked in bold ment of substantial yields of isoprenoids such as geranic acid and zeaxanthin and the high tolerance of P. putida to some of these compounds, usually toxic for other micro- precursors. For example, the production of deoxyxy- bial models, illustrate the high potential of this bacterium lulose 5-phosphate (DXP) in the first and main rate- to become a platform of choice for the industrial produc- determining step of the MEP pathway can be achieved tion of valuable isoprenoids. An important conclusion of by different pathways [12]. The canonical enzyme, DXP these studies is that enhancing the supply of isoprenoid synthase (DXS), produces C5 DXP from C3 pyruvate precursors is crucial for success as the bacterium only and glyceraldehyde 3-phosphate (GAP), but several produces minor amounts of such precursors [7]. mutant versions of the E. coli ribB gene were found to All isoprenoids derive from two universal 5-car- encode enzymes with a “new DXS” (or nDXS) activity bon (C5) precursors: isopentenyl diphosphate (IPP) that made DXP using alternative substrates (Fig. 1) [13, and its double-bond isomer dimethylallyl diphosphate 14]. The wild-type ribB gene encodes 3,4-dihydroxy- (DMAPP). In nature there are two main pathways for 2-butanone-4-phospate (DHBP) synthase, which con- IPP and DMAPP production: the mevalonate (MVA) verts C5 ribulose-5-phosphate (Ru5P) into formate and pathway and the methylerythritol 4-phosphate (MEP) DHBP, the biosynthetic precursor of the xylene ring of pathway (Fig. 1). As most bacteria, P. putida only riboflavin [15]. Ru5P appears to be the substrate that harbors the MEP pathway. By contrast, other bacte- nDXS enzymes convert into DXP [13]. ria as well as archaea, fungi, and animal cells produce Here we have compared the performance of previ- their isoprenoids from MVA-derived precursors [11]. ously known and novel strategies to improve the sup- Because IPP and DMAPP availability is limiting for ply of IPP and DMAPP for isoprenoid biosynthesis in isoprenoid biosynthesis, the MVA and MEP pathways KT2440, the best characterized P. putida strain. Using have been the targets of many metabolic engineering as read-out the production of lycopene, a red carot- efforts to increase the supply of isoprenoid precur- enoid pigment with great commercial and nutritional sors in host cells [3]. A virtually unexplored alterna- importance, we increased isoprenoid precursor levels tive, however, is the use of non-canonical pathways to by introducing an exogenous MVA pathway for IPP and synthesize such precursors. Bacteria show a remark- DMAPP synthesis, by producing DXP via nDXS, and able plasticity for the production of their isoprenoid by expressing DXS and other MEP pathway enzymes under the control of different promoters. Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 3 of 14 Results Pseudomonas putida KT2440 can be engineered for lycopene production To use lycopene as a read-out of isoprenoid precursor availability in P. putida, a lycopene-producing operon (LYC) containing the crtE, crtB and crtI genes from Pan- toea ananatis [16], was expressed in the strain KT2440. crtE encodes the enzyme geranylgeranyl diphosphate (GGPP) synthase, which catalyzes the addition of three molecules of IPP to one DMAPP unit to yield GGPP, the immediate precursor for carotenoids. C20 GGPP is converted to C40 lycopene via phytoene by phytoene synthase and phytoene desaturase, encoded by crtB and crtI respectively (Fig. 1). Two strategies were used to introduce the LYC operon into P. putida. First, the Fig. 2 Cell density of P. putida strains producing lycopene. The operon containing the three genes and their own pro- indicated strains were grown in LB and their growth was measured as moter was cloned into the vector pSEVA421 [17]. The optical density at 600 nm ­(OD600). Values shown are the mean ± SD of plasmid obtained, p421-LYC, was introduced into the three independent assays strain KT2440 and its ability to produce lycopene was compared to that of cells carrying the empty vector. After 24 h growing in LB, cells were harvested for lyco- lower than those found in KT2440 cells containing the pene extraction and quantification by HPLC. While no plasmid p421-LYC (Table 1), perhaps due to differences lycopene was detected in empty vector control cells, in copy number or DNA context (genomic vs. plasmid- measurable levels of this red carotenoid (1.223 ng/ml ial). In any case, these results allowed to conclude that, of culture) were found in the strain expressing the LYC under our experimental conditions, the intermediate operon (Table 1). Cell growth estimated as optical density steps of the endogenous MEP pathway are not saturated at 600 nm ­(OD600) was not affected in any of the strains in strain KTLYC. We therefore selected this strain for (Fig. 2). exploring ways of increasing the supply of MEP-derived Once we confirmed the ability for lycopene synthe- isoprenoid precursors (IPP and DMAPP) in P. putida sis of P. putida cells, the crtE–crtB–crtI operon with its cells. own promoter was inserted into the Tn7 att site in the chromosome of KT2440, using a Tn7-based delivery Lycopene production in P. putida KTLYC is enhanced system [18], to yield the strain KTLYC. Strains KT2440 by expressing an exogenous MVA pathway and KTLYC were grown for 24 h in LB and lycopene As a first strategy to enhance the supply of metabolic accumulation was then measured by HPLC. Growth precursors for isoprenoid biosynthesis in P. putida, we of both strains was basically identical (Fig. 2a) whereas, introduced an exogenous MVA pathway (Fig. 1) in the as expected, lycopene was only detected in KTLYC KTLYC strain. The vector pMiS1-ges-MVA, contain- (Table 1). However, the amounts produced in this strain ing the bacterium Myxococcus xanthus genes required (0.106 ± 0.026 ng/ml culture) were an order of magnitude to convert acetyl-CoA into IPP and DMAPP under the control of the rhaPBAD promoter [4], was introduced into P. putida KTLYC. As control, the plasmid pMiS1- ges (similar to pMiS1-ges-MVA but without the MVA Table 1 Cell density (turbidity at 600 nm) and lycopene pathway genes) was also introduced into KTLYC. Cells accumulation (measured by HPLC) in cells cultured during 24 h in LB were cultured in LB medium and at the mid-exponential phase, expression of plasmid vector genes was induced by Strain Cell density ­(OD600nm) Lycopene L-rhamnose. After 24 h, cells were harvested for lycopene accumulation (ng/ml culture) extraction and quantification. Lycopene levels were esti- mated by measuring absorbance at 472 nm, normalized KT2440 5.417 ± 0.791 0.000 ± 0.000 to cell density (­OD600), and represented relative to con- KT2440(p421) 5.293 ± 0.623 0.000 ± 0.000 trols (Fig. 3). Lycopene production was about sevenfold KT2440(p421-LYC) 4.930 ± 1.191 1.223 ± 0.185 higher in the strain harboring the MVA pathway without KTLYC 6.157 ± 0.551 0.106 ± 0.026 this having an impact in cell growth (Fig. 3). These results Values shown are the mean ± SD for three independent assays show that promoting the supply of IPP and DMAPP from Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 4 of 14 increase in lycopene accumulation even in the absence of IPTG (Fig. 4b). The positive effect of DXS on lycopene production was even stronger when dxs gene expression was induced with IPTG (25-fold). The observation that KT2440 cells lacking the capacity to produce lycopene showed very similar growth curves when transformed with p424-DXS and incubated with IPTG (Fig. 5) sug- gested that the growth phenotype is not caused by the accumulation of toxic levels of lycopene but results from the overexpression of the dxs gene. GGPP required for lycopene production is formed by adding three IPP molecules to one DMAPP acceptor, whereas bacterial isoprenoids are most often derived from farnesyl diphosphate (FPP), that is formed from two IPP and one DMAPP [11]. Because an unbalanced production of isoprenoid precursors has been shown to be detrimental for bacterial growth [19], we next inves- Fig. 3 Lycopene production in P. putida KTLYC cells harboring the tigated whether enhancing the interconversion of IPP MVA pathway. KTLYC cells transformed with vectors either containing and DMAPP might positively impact cell growth and or not the MVA pathway were cultured in LB. Expression of the MVA lycopene accumulation in strains overexpressing DXS. pathway operon was induced 3 h after inoculation by adding 0.2% (w/v) L-rhamnose to the culture. After 24 h, cell growth was estimated The IPP/DMAPP ratio is regulated by the enzyme iso- as optical density at 600 nm ­(OD600) and lycopene accumulation pentenyl diphosphate isomerase (IDI) that interconverts was calculated by measuring the absorbance at 472 nm of acetone IPP and DMAPP molecules (Fig. 1). The idi gene from extracts. a ­OD600 values. b Lycopene levels normalized to cell E. coli was cloned into the p424-DXS vector and the density and represented relative to those in cells without the MVA resulting p424-IDI-DXS plasmid was introduced into the pathway. Values shown are the mean ± SD of three independent assays. The asterisk indicates statistically significant difference (T test, strain KTLYC. Positive transformants were grown with P-value < 0.05) or without IPTG as described above (Fig. 4c) and both cell growth and lycopene accumulation were recorded at 24 h (Fig. 4d). In the absence of IPTG, lycopene pro- acetyl-CoA by using an exogenous MVA pathway can duction tended to be slightly higher in cells harboring efficiently improve lycopene production. p424-IDI-DXS compared to p424-DXS, but the differ- ence was not statistically significant. Induction with increasing concentrations of IPTG did not improve cell Up‑regulation of endogenous DXS greatly improves growth or lycopene production on cells harboring p424- isoprenoid precursor supply in KTLYC cells IDI-DXS (Fig. 4d) compared to p424-DXS (Fig. 4b). On Up-regulation of the levels of several MEP pathway the contrary, growth of KTLYC(p424-IDI-DXS) cells enzymes has been reported to improve flux through the was reduced compared to that of IDI-lacking strains at pathway in bacteria, with the clearest effect typically IPTG concentrations higher than 0.1 mM. Addition of observed with DXS when overexpressed at moderate lev- 1 mM IPTG to the medium also reduced the capacity of els. To test whether DXS was also a rate-limiting enzyme the cells for lycopene accumulation (Fig. 4). In summary, in P. putida, the dxs gene was amplified from KT2440 IPP/DMAPP imbalance might influence lycopene bio- cells and cloned into the pSEVA424 plasmid [17] under synthesis but it appears not to be a major factor limiting the transcriptional control of the strong and IPTG-induc- lycopene yields in P. putida cells. While DXS activity is ible Ptrc promoter. After transformation, KTLYC cells much more relevant to regulate flux to isoprenoid bio- harboring either the generated vector (p424-DXS) or an synthesis, a fine control of its levels is critical to prevent empty plasmid were cultured in LB and 1 h after inocula- deleterious effect on cell growth. tion the medium was supplemented with different con- centrations of IPTG. While 0.1 mM IPTG had no impact on culture growth, higher concentrations of the inducer Use of the stress‑regulated promoter PkatA to express IDI (e.g. 0.5 mM and 1 mM) caused a much slower growth and DXS has a modest effect on lycopene production (Fig. 4a). Lycopene accumulation in KTLYC(p424) and To overcome the toxic effects associated with a too strong KTLYC(p424-DXS) cells was quantified after 24 h by DXS activity, we next looked for alternative promoters measuring absorbance at 472 nm (Fig. 4b). The presence that could be down-regulated when cell growth is com- of the plasmid p424-DXS caused a dramatic (15-fold) promised. As starting point, we studied the expression of Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 5 of 14 Fig. 4 Cell density and lycopene content of P. putida KTLYC cells overproducing DXS. Overnight cultures of KTLYC cells transformed with the indicated vectors were inoculated in LB. Expression of the cloned genes was induced 1 h after inoculation by adding the indicated amounts of IPTG (mM) to the culture. After 24 h, growth was estimated from optical density at 600 nm (­ OD600) and lycopene accumulation was calculated by measuring the absorbance at 472 nm of acetone extracts. a Cell density kinetics of KTLYC cells transformed with either p424-DXS or empty pSEVA424 plasmids, estimated from ­OD600 values. b Cell density and lycopene content of the strains shown in a at 24 h. c Cell density kinetics of KTLYC cells transformed with either p424-IDI-DXS or empty pSEVA424 plasmids, estimated from O ­ D600 values. d Cell density and lycopene content of the strains shown in c at 24 h. Values shown are the mean ± SD of three independent assays. Asterisks indicate statistically significant differences (T-test, P-value < 0.05 * or 0.01 **) relative to KTLYC(p424) controls grown without IPTG the stress-regulated genes ccoN (PP4250), cioA (PP4651) of IPTG (Fig. 6a). The operons ccoN1O1Q1P1 and cioAB and katA (PP0481) in KTLYC(p424-IDI-DXS) and encode two terminal oxidases, the cytochrome cbb3-1 KTLYC(p424) cells exposed to different concentrations oxidase and the cyanide-insensitive oxidase (CIO), Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 6 of 14 well as the empty pSEVA424 vector, were cultured in LB without IPTG and lycopene accumulation levels were analyzed every 24 h during 5 days. After the first 24 h (Fig. 6b) KTLYC(pPkatA-IDI-DXS) cells showed four- fold higher levels of lycopene than KTLYC(p424) but half as much compared to KTLYC(p424-IDI-DXS). Neither KTLYC(pPkatA-IDI-DXS) nor KTLYC(p424-IDI-DXS) cells produced more lycopene at longer incubation times (Fig. 6c). It is therefore possible that the Ptrc promoter present in the p424-IDI-DXS construct leads to higher enzyme levels than the PkatA promoter even without the IPTG inducer, hence being more efficient to increase the MEP pathway flux. Depletion of pyruvate and GAP might contribute to the cell growth defects associated with DXS overexpression Fig. 5 Effect of DXS overproduction on KT2440 cell growth. P. When KT2440 cells are grown in the presence of glu- putida KT2440 cells harbouring the vector p424-DXS or the empty cose, they run a biochemical cycle that merges activi- pSEVA424 plasmid were inoculated in LB and, after 1 h, the medium ties belonging to the Entner–Doudoroff (ED) pathway, was supplemented with the indicated concentration of IPTG (mM). the Embden–Meyerhof–Parnas (EMP) pathway and Increase in cell growth was measured as optical density at 600 nm the pentose phosphate pathway that produces equimo- ­(OD600). Values shown are the mean ± SD of three independent assays lar amounts of pyruvate and GAP, favors NADPH for- mation, and helps the bacteria to cope from different types of environmental stress [6, 23]. To test whether respectively, and they are up-regulated under low-oxygen the defective growth phenotype observed in DXS-over- conditions [20]. The expression of the katA gene, encod- producing P. putida cells might result from metabolic ing one of the major peroxide-degrading enzyme directly imbalance caused by low pyruvate and GAP levels, we eliminating oxidants in the cell, is regulated by matric and grew KTLYC(p424-DXS) and KTLYC(p424) cells in LB oxidative stress [21]. The levels of ccoN, cioA and katA either supplemented or not with 50 mM glucose. IPTG transcripts in the two strains under different growth con- was added at high concentration (1 mM) at the begin- ditions were estimated by real time quantitative RT-PCR ning of the exponential phase and cell growth and lyco- and represented as relative fold-changes in KTLYC(p424- pene accumulation were analyzed after 24 h. As shown IDI-DXS) compared to KTLYC(p424) cells (Fig. 6a), No in Fig. 7, the sharp difference in cell growth between changes were observed in the case of ccoN whereas cioA KTLYC(p424-DXS) and KTLYC(p424) strains was com- mRNA levels were drastically reduced in KTLYC(p424- pletely abolished in glucose-supplemented medium. IDI-DXS) cells independent of IPTG concentration Glucose, but not other carbon sources such as citrate or (i.e. regardless of DXS levels). In the case of katA, simi- succinate, also rescued growth defects observed when lar transcript levels were found in the two strains when cells were grown in minimal M9 medium (Fig. 7). These growing without the inductor but were reduced in results support the conclusion that depletion of GAP and KTLYC(p424-IDI-DXS) when IPTG was added to the pyruvate when DXS levels are too high is a major cause medium to induce idi-dxs co-transcription (Fig. 6a). The for the growth defects observed in engineered P. putida reduction was moderate with 0.1 mM IPTG, a concentra- cells. Furthermore, the addition of glucose to LB medium tion that hardly impacts growth of this strain (Fig. 4c). increased lycopene accumulation per cell (Fig. 7), sug- When growth was inhibited at 0.5 mM IPTG, however, gesting that IPP and DMAPP supply for isoprenoid a clear sixfold reduction in katA expression was detected biosynthesis could be improved by increasing the avail- (Fig. 6a). We therefore concluded that the PkatA pro- ability of both GAP and pyruvate or by finding alternative moter might be repressed when DXS (and IDI) activities sources for DXP synthesis. are high enough to cause growth-repressing effects. It was previously reported that single mutations in the With the aim of adjusting enhanced isoprenoid pre- E. coli ribB gene, encoding DHBP synthase, allow the cursor supply to actual cell metabolism and growth, we mutant enzymes (nDXS) to produce DXP from Ru5P next used the PkatA promoter [22] to construct a syn- and hence complement the absence of the DXS enzyme thetic PkatA-idi-dxs operon. KTLYC strains harbor- in E. coli [13, 14]. Because DXP production by nDXS ing the plasmids pPkatA-IDI-DXS or p424-IDI-DXS, as enzymes does not directly consume pyruvate and GAP Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 7 of 14 Fig. 6 Test of the P. putida PkatA promoter. a Transcript levels of ccoN, cioA and katA in KTLYC(p424-IDI-DXS) cells grown in the presence of the indicated concentrations of IPTG. The graph represents the ratio of the mRNA levels in KTLYC(p424-IDI-DXS) cells relative to KTLYC(p424) controls grown under the same conditions. Error bars indicate SD. b Lycopene accumulation in the indicated strains grown for 24 h in LB without IPTG. Values shown are the mean ± SD of three independent assays. Asterisks indicate statistically significant differences (T-test, P-value < 0.05 * or 0.01 **) relative to KTLYC(p424) controls. c Lycopene accumulation in the indicated strains grown in LB without IPTG for the indicated times (in hours). Values are shown relative to 24 h samples and represent the mean ± SD of three replicates in two independent assays and it is less efficient compared to DXS, we reasoned KTLYC cells producing nDXS showed a good correla- that using nDXS enzymes might overcome some of the tion between IPTG and lycopene levels (Fig. 8b). How- problems associated with DXS overexpression. Although ever, lycopene accumulation in KTLYC(p424-nDXS) cells several mutations in ribB allow DXP synthesis, for this at the highest IPTG concentration tested (1 mM) was work we used the nDXS version harboring a missense only threefold higher than that reached in the absence of mutation resulting in a G108S change [13, 14]. Follow- inducer (Fig. 8b). ing a similar strategy to that described for DXS, we gen- Ru5P, the substrate of nDXS, is an intermediate of the erated the plasmid p424-nDXS and used it to transform pentose phosphate pathway, which is not very active in KTLYC cells. The resulting strain and the KTLYC(p424) P. putida [6, 23]. In complete (LB) medium, the addition control were cultured in LB in the absence or presence of 50 mM glucose had no effect on lycopene produc- of IPTG (added 1 h after inoculation) and lycopene accu- tion when KTLYC(p424-nDXS) cells were induced with mulation was analyzed after 24 h. Unlike that observed 1 mM IPTG (Fig. 7). In minimal (M9) medium, similar for KTLYC(p424-DXS) and KTLYC(p424-IDI-DXS) levels of lycopene were produced with glucose, citrate, or strains (Fig. 4), growth of KTLYC(p424-nDXS) cells succinate. It is interesting, however, that whereas lyco- was not affected by IPTG at any of the concentrations pene accumulation in KTLYC(p424-DXS) cells was much tested (Fig. 8a). Also different to DXS-producing strains, lower in M9 than in LB medium, KTLYC(p424-nDXS) Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 8 of 14 together with DXS. DXR catalyzes the second step of the MEP pathway, i.e. the reduction of DXP to MEP (Fig. 1). After generating construct p424-DXS-DXR and trans- forming KTLYC cells, growth of the generated strain in LB medium was monitored upon adding IPTG 1 h after inoculation (Fig. 9). In agreement with our conclusion that cell growth defects observed when the MEP path- way flux is up-regulated are mainly due to depletion of pyruvate and GAP, growth of KTLYC(p424-DXS-DXR) cells was compromised even at low concentrations of IPTG such as 0.1 mM (Fig. 9), which had no impact on the growth of KTLYC(p424-DXS) cells (Fig. 4a). To test whether the expected up-regulation of the MEP path- way flux in KTLYC(p424-DXS-DXR) cells had a positive impact on lycopene accumulation, we next compared the levels of this carotenoid in KTLYC cells transformed with p424-DXS-DXR, p424-DXS, or with an empty vec- tor and treated with 0.1 mM IPTG 1 h after inoculation. After 24 h, cells harboring the p424-DXS-DXR con- struct accumulated 50-fold higher lycopene levels per cell that KTLYC(p424) control. When compared to cells overexpressing only one of the two MEP pathway genes, KTLYC(p424-DXS-DXR) cells produced twice as much lycopene as KTLYC(p424-DXS) (Fig. 10). Discussion Improving the supply of IPP and DMAPP for the bio- Fig. 7 Growth and lycopene levels of KTLYC cells producing technological production of value-added isoprenoids either DXS or nDXS. The strains KTLYC(p424), KTLYC(p424-DXS) and in microorganisms has been the target of many stud- KLYC(p424-nDXS) were cultured in the indicated mediums: LB, LB ies. However, many other factors need to be taken into supplemented with 50 mM glucose, or M9 minimal salt medium account to achieve an efficient production of isoprenoids containing 50 mM glucose, 30 mM citrate or 30 mM succinate as carbon source. Expression of genes encoding DXS or nDXS was without affecting the fitness of the cell. They include (A) induced with 1 mM IPTG 1 h after inoculation. After 24 h, growth depletion of metabolic substrates, (B) toxicity of accumu- was estimated as optical density at 600 nm ­(OD600) (upper graph) lated intermediates or end products, and (C) saturation and lycopene accumulation was calculated by measuring the of the cell storage capacity. Strategies to deal with the last absorbance at 472 nm of acetone extracts of the cells and normalized two problems are abundant, but less attention has been to cell density (lower graph). Values shown are the mean ± SD of three independent assays. Asterisks indicate statistically significant paid to the first, despite the negative growth impact of differences (T-test, P-value < 0.05 * or 0.01 **) relative to KTLYC(p424) diverting the pools of central metabolites such as pyru- controls vate and GAP to the synthesis of IPP and DMAPP. While most studies aimed to improve IPP and DMAPP supply cells showed similar lycopene levels in both media have been done with well-characterized organisms such (Fig. 7). We therefore conclude that the main factor lim- as E. coli (a model for bacteria that use the MEP pathway) iting isoprenoid production in the KTLYC(p424-nDXS) and S. cerevisiae (a model for yeasts, which only use the strain is not Ru5P availability but the capacity of nDXS to MVA pathway), other microorganisms that show addi- synthesize DXP. tional advantages for the production and accumulation of high isoprenoid titers remain scarcely explored. This is the case with P. putida, a bacterium that shows a very Combination of DXS and DXR further increases lycopene high tolerance to toxic compounds such as monoterpenes accumulation [4, 5]. An additional advantage of P. putida is that glucose As a last strategy to up-regulate the endogenous MEP is metabolized to produce balanced amounts of the MEP pathway and increase lycopene synthesis in P. putida pathway substrates pyruvate and GAP [6, 10, 23]. These KTLYC, the endogenous enzyme DXP reductoisomerase central metabolic substrates are produced in non-equiv- (DXR) was overproduced in this strain either alone or alent amounts in other bacteria such as E. coli, where this Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 9 of 14 ◂ Fig. 8 Growth and lycopene content of P. putida KTLYC cells overproducing nDXS. KTLYC cells transformed with the indicated vectors were cultured in LB. Expression of the cloned genes was induced 1 h after inoculation by adding the indicated amounts of IPTG (mM) to the culture. After 24 h, growth was measured as optical density at 600 nm ­(OD600) and lycopene accumulation was calculated by measuring the absorbance at 472 nm of acetone extracts. a Cell density kinetics of KTLYC cells transformed with either p424-nDXS or empty pSEVA-424 plasmids estimated from ­OD600 values. b Cell density and lycopene content of the strains shown in a at 24 h. Values shown are the mean ± SD of three independent assays. Asterisks indicate statistically significant differences (T-test, P-value < 0.05) relative to KTLYC(p424) controls grown without IPTG Fig. 9 Cell growth of P. putida KTLYC strains overproducing DXS and DXR. Overnight cultures of KTLYC cells transformed with the indicated plasmids were inoculated in LB and, after 1 h, the indicated concentration of IPTG (mM) was added to the medium. Graph shows the increase in cell density at 600 nm ­(OD600). Values shown are the mean ± SD of three independent assays supply of pyruvate and GAP is also important to upregu- late isoprenoid production while preventing major inter- ferences with overall microbial metabolism leading to defective growth. To investigate different ways of stimulating the meta- bolic flux through the endogenous MEP pathway to isoprenoid precursors, we generated a P. putida strain (KTLYC) with an exogenous 3-gene operon to produce lycopene as a read-out. By transforming this strain with imbalanced availability of pyruvate and GAP can limit the plasmids to either overexpress endogenous MEP path- activity of the MEP pathway [24]. Increasing the levels of way genes or incorporate alternative shunt pathways, both pyruvate and GAP in P. putida actually led to an we could multiply lycopene production (i.e. IPP and increase (1.3-fold) in carotenoid (β-carotene) production DMAPP supply) by 3 (with nDXS; Fig. 8), 7 (with the [10], suggesting that the supply of metabolic substrates MVA pathway; Fig. 3), 25 (with DXS; Fig. 4) and 50 (with limits the MEP pathway flux. Our data reported here DXS and DXR; Fig. 10). It is most likely, however, that further support the conclusion that ensuring a proper further optimization of at least some of these strategies Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 10 of 14 or a combination of several could result in even higher increases. Additive and synergistic effects could also be achieved by improving the supply of MEP pathway sub- strates. A recent example is the rewiring of the P. putida core metabolism for glucose assimilation from the native Entner–Doudoroff to the linear Embden–Meyerhof–Par- nas glycolysis, which improved the production of both pyruvate and GAP [10]. The metabolic plasticity shown by P. putida, which is able to metabolize a broad variety of carbon sources including fatty acids, glycerol and aro- matic compounds [25], provides an additional opportu- nity to redesign the pools of substrates required for the synthesis of IPP and DMAPP. Feeding the bacteria with different carbon sources might help accumulate higher pools of pyruvate, GAP (i.e. glucose) or acetyl-CoA (i.e. fatty acids) and the combination of different carbon sources could help release the competition of the pro- ductive (exogenous) pathways with the housekeeping (endogenous) metabolism. The hierarchical utilization of different carbon sources [26] might limit our ability to combine fully active metabolic pathways to simultane- ously feed productive and housekeeping pathways in the cell. However, our knowledge of the mechanisms control- ling this hierarchy are constantly improving [27, 28], pro- viding new engineering opportunities. Among the novel strategies tested here, the use of nDXS to produce DXP from Ru5P was most successful as it improved lycopene accumulation up to threefold with- out negatively affecting cell growth (Fig. 8). While this increase is still far from that achieved with the canonical DXS enzyme (Fig. 4), it is important to keep in mind that nDXS was identified as a mutant enzyme which acquired the ability to produce DXP as a result of a point mutation and it is not optimized for DXP synthesis. Our estima- tion is that only about 10% of the Ru5P substrate is trans- formed into DXP by nDXS. Increasing the DXS activity of the mutant enzyme should be feasible and even fast if using the new automatized platforms available for adap- tive laboratory evolution approaches [29, 30]. An opti- mized nDXS would allow a complementary strategy using the canonical DXS to push the MEP pathway using pyruvate and GAP up to the limits that show no effect on growth with an additional DXP input obtained from the transformation of Ru5P by nDXS. The availability of Fig. 10 Cell growth and lycopene accumulation in P. putida KTLYC the crystal structure of the wild-type ribB protein [31] cells overproducing DXS and DXR. Overnight cultures of KTLYC should allow to infer how the newly selected mutations cells transformed with the indicated vectors were inoculated in LB could affect the DXS activity of nDXS, an information and induced to express the cloned genes with 0.1 mM IPTG added that could be very valuable to eventually create syn- 1 h after inoculation. a Cell density kinetics of the indicated strains thetic enzymes able to produce DXP from Ru5P or from estimated from ­OD600 values. b Cell density and lycopene content of the strains shown in a at 24 h. Values shown are the mean ± SD of substrates that do not interfere with the endogenous three independent assays. Asterisks indicate statistically significant MEP pathway or other essential metabolic processes differences (T-test, P-value < 0.05 * or 0.01 **) relative to KTLYC(p424) in the bacteria. As a complementary strategy, the sup- controls ply of Ru5P might be enhanced by rewiring the pentose Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 11 of 14 phosphate pathway. Ru5P is a C5 substrate and hence the limiting role of DXS is released in P. putida (Fig. 4d) it is converted into DXP by nDXS without losing car- is also different to that observed in E. coli [29]. bon (unless the reaction catalyzed by DXS, which starts with two C3 precursors and involves a decarboxylation). Conclusion Together, the use of optimized versions of nDXS either Production of isoprenoids has been extensively engi- alone or in combination with other strategies might neered in classical microbial chassis such as E. coli and increase IPP and DMAPP production to new high levels. S. cerevisiae but much less effort has been made in P. Another novel strategy that we tested was the fine putida despite the unique characteristics that make regulation of DXS expression by using an endogenous this organism a very attractive host for the industrial promoter that responded to cell growth stress (Fig. 6). production of these metabolites. The present work Using the PkatA promoter we only achieved a fourfold analyzed different ways of enhancing the supply of iso- increase in lycopene accumulation compared to empty prenoid precursors (IPP and DMAPP) in P putida strain plasmid controls, suggesting that the transcriptional KT2440 using lycopene as read-out. The most success- activity of this promoter in the conditions tested is too ful combination led to a 50-fold increase of lycopene low to effectively boost the MEP pathway and hence to accumulation. Under our experimental conditions, the reach the stress conditions that could eventually repress production of limonene in P. putida cells engineered to transcription. It is likely that the use of stronger but synthesize this monoterpene was also boosted by the still stress-responsive promoters might work better. same MEP pathway constructs that improved lycopene The PkatA promoter might be engineered to increase levels, supporting the conclusion that the increases we its transcriptional activity while preserving its regula- observed in downstream isoprenoid products are indic- tory traits. Alternatively, analysis of the transcriptome ative of a major up-regulation of the metabolic flux to of KTLYC(p424-DXS) cells grown in the presence of dif- IPP and DMAPP. Our work therefore represents an ini- ferent concentrations of IPTG should allow to identify tial but important step towards engineering P. putida genes that are highly expressed when cells grow normally cells to produce high isoprenoid titers. The unique met- but are repressed when cell growth is compromised in an abolic plasticity of P. putida together with its astonish- IPTG-dependent fashion (i.e. when DXS activity is too ing capacity to tolerate high levels of toxic isoprenoids high). The promoters of such genes might be then used in such as monoterpenes and its amenability to genetic synthetic operons to increase the supply of DXS (and IDI, engineering make this bacterium a promising candidate DXR, nDXS, etc.) and hence the availability of isoprenoid to become a favorite microbial cell factory to produce precursors under optimal growth conditions but down- high-value isoprenoids. regulate them when growth-repressing effects appear. This will give time to the cells to readjust their metabo- lism and retake isoprenoid overproduction afterwards. Methods The more “classical” approaches that we tested, i.e. Bacterial strains and culture media the use of an exogenous MVA pathway to produce IPP Escherichia coli and P. putida cells were cultured at and DMAPP from acetyl-coA and the overexpression of 37 °C and 30 °C, respectively, with strong aeration. LB MEP pathway genes, led us to conclude that we cannot broth (10 g/l tryptone; 5 g/l yeast extract, 10 g/l NaCl) directly extrapolate the results obtained with one bacte- was used as complete growth medium. M9 minimal rium (e.g. E. coli) to engineer another bacterium (e.g. P. salts medium [36] was supplemented with trace ele- putida). For example, using the MVA pathway typically ments [37], and either 30 mM citrate, 30 mM succinate works better than DXS up-regulation in E. coli [32–34] or 50 mM glucose as the carbon source. When needed, but we observed the opposite behavior in P. putida. A antibiotic were added at the following concentrations: similar observation was previously reported in cyanobac- kanamycin 25 μg/ml, gentamicin 25 μg/ml, strepto- teria growing in photoautotrophic conditions to produce mycin 50 μg/ml (or 500 μg/ml when the host strain isoprene [35]. The presence of the bacterial MVA path- harboured a gentamicin-resistance determinant), ampi- way operon improved lycopene accumulation by seven- cillin 100 μg/m. Cell density was monitored by measur- fold (Fig. 3), similar to the tenfold increase reported to ing turbidity (optical density) at 600 nm ­(OD600). cause the same operon on monoterpene production in P. To generate strain KTLYC, a 3788 bp DNA fragment putida DSM 12264 cells [4]. By contrast, the presence of containing the P. ananatis genes crtE, crtB and crtI and a plasmid containing DXS was able to promote lycopene their own promoter was PCR-amplified from the vec- accumulation in KTLYC cells by 15-fold in the absence tor pACCRT-EIB [16] and cloned into the NotI site of of any induction (Fig. 4b). The almost complete lack of the Tn7-based delivery vector pTn7-M [18]. The gener- effect of IDI overproduction on lycopene synthesis when ated plasmid, named pTn7-M-LYC, was transferred to Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 12 of 14 P. putida strain KT2440 by tetra-partite mating using Table 2 Oligonucleotides used for real-time RT-PCR E. coli cc118λpir (pTnS1) as the supplier of Tn7 trans- Name DNA sequence posase and E. coli HB101 (pRK600) as the transfer function donor, as previously described [18]. The cor- RpoN-Fw TCG​ACC​CGG​AGC​TGG​ATA​ rect insertion of the transposon at the att Tn7 site of RpoN-Rv CGG​C TC​GAA​C TG​C TG​GAT​ the P. putida genome was checked by sequencing. Pp_katA-818 dir ACC​CAT​TCG​ACG​TGA​CCA​AG Pp_katA-899 rv GGG​T TA​CGG​T TG​AGC​TCC​AG Plasmids Pp_ccoN-452 dir AGC​GCA​ACA​CCA​AAC​ACA​TC Plasmid p421-LYC contains the crtE, crtB and crtI genes Pp_ccoN-534 rv GTT​GAC​GAT​GTG​CAG​CAG​TG and their own promoter from P. ananatis. To generate Pp_cioA-306 dir ACT​GAC​TGC​GTT​C TT​CCT​CG this vector, the same 3788 bp DNA fragment inserted in xPp_cioA-400 rv TCA​CCG​TGG​AGA​AGA​AGT​GC the genome of the strain KTLYC was cloned in the NotI site of the vector pSEVA421 [17]. The pMiS1-ges-mva vector [4] contains the MVA pathway from M. xanthus HPLC in an Agilent 1200 series HPLC system (Agilent under the control of the promoter rhaPBAD; transcrip- Technologies), as previously described [39]. tion from this promoter can be induced by addition of L-rhamnose. To overproduce P. putida or E. coli enzymes involved in IPP and DMAPP synthesis, genes coding for RNA purification these enzymes were cloned downstream of the IPTG- The KTLYC(p424-IDI-DXS) strain was grown in LB inducible promoter Ptrc in the vector pSEVA424 [17]. To medium at 30 °C in aerated flask and after 1 h the indi- construct the p424-DXS vector, a DNA fragment includ- cated IPTG concentration was added. At the start of the ing the rbs region of the vector pET23 and the coding stationary phase ­(OD600 = 2.2), 1 ml of samples were col- region of P. putida dxs gene was cloned between the sites lected, harvested by centrifugation and immediately fro- EcoRI and HindIII of plasmid pSEVA424. Plasmid p424- zen at − 70 °C. RNA was purified from cell pellet using nDXS was made in a similar way using a version of the RNeasy RNA Purification Kit (Qiagen) following the E. coli ribB gene harboring the G108S mutation [14]. manufacturer’s instructions. Purified RNA was treated The p424-DXS plasmid was used as a backbone to clone with RNase-free DNase I (Ambion), as specified by the the idi gene from E. coli (upstream of dxs, in the EcoRI manufacturer. RNA was quantified using a NanoDrop site, rendering plasmid p424-IDI-DXS) or the P. putida (Thermo Scientific) and its integrity was analyzed by dxr gene (downstream of dxs, between HindIII and SpeI, agarose gel electrophoresis. The absence of DNA was rendering p424-DXS-DXR). The Ptrc promoter in the confirmed by real-time PCR using primers for rpoN, as vector p424-IDI-DXS was replaced with the P. putida previously described [40]. PkatA promoter after PCR amplification using primers katA-PacI-F (5′-T G T T A A T T A A G A C G T T C T Real‑time RT‑PCR G T C T G C T A C) and katA-AvrII-R (5′-C T C C T A Real-time RT-PCR assay was performed using total RNA G G G C C G G C T A A T C G G) and cloning between preparations obtained from three independent cultures the PacI and AvrII sites to create plasmid pPkat-IDI-DXS. (three biological replicates). RNA was reverse-tran- All plasmid constructs were sequenced and the plas- scribed into cDNA using the first-strand cDNA synthesis mids transferred to P. putida by triparental mating using kit (Roche) according to the manufacturer’s instructions. pRK600 as a donor of transfer functions. Real-time PCR was performed using LightCycler 480 SYBR Green I Master (Roche) and 0.3 μM of each primer on a LightCycler 480 Real-Time PCR System (Roche). Quantification of lycopene accumulation RNA levels were analyzed by relative quantization as An overnight culture of the strain of interest was diluted previously described [40]. The primers used are listed in to a final turbidity (­OD600) of 0.05 in fresh LB medium. Table 2. The indicated amount of IPTG or L-rhamnose was added to induce transcription from promoters Ptrc or Pram Acknowledgements respectively. Cells were allowed to grow at 30 °C with We thank Victor de Lorenzo for pSEVA vectors, Jens Schrader for plasmids pMiS1-ges and pMiS1-ges-MVA and Fernando Rojo for the P. putida strains. vigorous aeration. After 24 h, cell growth was meas- Technical support from Mª Rosa Rodríguez Goberna is greatly acknowledged. ured ­(OD600) and 5 ml aliquots were taken to lycopene extraction with acetone and quantification by measuring Authors’ contributions SHA, JPG and MRC conceived the whole study and designed the research; absorbance at 472 nm, as described at Rodríguez-Villalón MRC supervised the study; SHA, JPG and DMS performed experiments; SHA, et al. [38]. Three independent assays were performed. JPG, DMS and MRC analyzed and discussed data; SHA and MRC wrote the When indicated, lycopene levels were also measured by paper and JPG revised it. All authors read and approved the final manuscript. Hernandez‑Arranz et al. Microb Cell Fact (2019) 18:152 Page 13 of 14 Funding 15. Richter G, Krieger C, Volk R, Kis K, Ritz H, Götze E, et al. Biosynthesis of This work was funded by Grants PCIN-2015-103 and BIO2017-90877-REDT riboflavin: 3,4-dihydroxy-2-butanone-4-phosphate synthase. Methods from the Agencia Estatal de Investigación and 2017SGR-710 from Generalitat Enzymol. 1997;280:374–82. de Catalunya to MRC. We also thank the financial support of the MINECO 16. Cunningham FX, Chamovitz D, Misawa N, Gantt E, Hirschberg J. Clon‑ Severo Ochoa Programme for Centres of Excellence in R&D 2016-2019 (SEV- ing and functional expression in Escherichia coli of a cyanobacterial 2015-0533) and the Generalitat de Catalunya CERCA Programme to CRAG. JPG gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis was supported by a Marie Curie International Outgoing Fellowship within the of beta-carotene. FEBS Lett. 1993;328:130–8. 7th European Community Framework Programme (Project Number 627639). 17. Martínez-García E, Aparicio T, Goñi-Moreno A, Fraile S, de Lorenzo V. 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Llorente B, D’Andrea L, Ruiz-Sola MA, Botterweg E, Pulido P, Andilla J, Publisher’s Note et al. Tomato fruit carotenoid biosynthesis is adjusted to actual ripening Springer Nature remains neutral with regard to jurisdictional claims in pub‑ progression by a light-dependent mechanism. Plant J. 2016;85:107–19. lished maps and institutional affiliations. Ready to submit your research ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations • maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions