Journal of Bacteriology, March 2004, p . 1531-1536, Vol . 186, No . 5

Evolution of a Pathway to Novel Long-Chain Carotenoids

Daisuke Umeno^* and Frances H . Arnold

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125

Received 5 June 2003/ Accepted 30 October 2003

 
Using methods of laboratory evolution to force the C₃₀ carotenoid synthase CrtM to function as a C₄₀ synthase, followed by furthermutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second [rearrangement] step of the two-step reaction . We usedthis information to engineer CrtM variants that can synthesizepreviously unknown C₄₅ and C₅₀ carotenoid backbones [mono- and diisopentenylphytoenes] from the appropriate isoprenyldiphosphate precursors . With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.

 
Carotenoids are natural pigments with important biological activities [4, 10, 16, 17] . Most are based on a 40-carbon [C₄₀] phytoene backbone produced by condensation of 2 molecules of geranylgeranyldiphosphate[GGDP; C₂₀PP], a reaction catalyzed by the carotenoid synthaseCrtB [Fig . 1] . The vast majority of the >700 known carotenoids[9] arise as a result of different types and levels of modificationof the C₄₀ backbone, catalyzed by promiscuous [downstream] carotenoid biosynthetic enzymes [5] . A few bacteria, notably Staphylococcusand Heliobacterium spp . [23, 24], have a C₃₀ pathway, whichstarts with the CrtM synthase-catalyzed condensation of 2 moleculesof farnesyldiphosphate [FDP; C₁₅PP] to form 4,4'-diapophytoene. Yet other bacteria [such as Corynebacterium and Halobacterium spp.] are known to accumulate C₅₀ carotenoids, but these longer-chainstructures are biosynthesized starting from the C₄₀ structureby the addition of 2 C₅ [isoprene] units [14] . Various longerisoprenyldiphosphates are made by different organisms [30] andare potential precursors for longer-chain carotenoids . Theyare precursors to other biosynthetic pathways, however, andno known carotenoids are derived from them . Thus, carotenoidsize is tightly controlled by the carotene synthase reaction[20, 27].

 
To create new pathways for the biosynthesis of carotenoids with backbones larger than C₄₀, we focused on engineering the carotenoid synthase to accept longer diphosphate substrates . Very little is known of the structure or basis for the specificity of these membrane-associated enzymes . Using random mutagenesis and a functional complementation screen for C₄₀ synthase activity, however, we identified single-amino-acid substitutions in theC₃₀ synthase CrtM [F26L or F26S] that confer the C₄₀ function [27] . By repeating this experiment with a random mutant librarythat was free from mutation at F26, we recently found two moreamino acid substitutions, W38C and E180G, that confer the same phenotype [26] . Upon further mutagenesis at these three residues,we show here that the specificity of the carotenoid synthaseCrtM is controlled at the second [rearrangement] step of its two-step reaction . Furthermore, we have engineered synthase variants that can make previously unknown C₄₅ and C₅₀ carotenoidbackbones [mono- and diisopentenylphytoenes] from the appropriateC₂₀ and C₂₅ isoprenyldiphosphate precursors . With this abilityto produce the new backbones in Escherichia coli comes the potentialto generate whole series of novel carotenoids upon additionof carotenoid-modifying enzymes to the engineered pathway.

 
Materials. The crtE [GGDP synthase], crtB [phytoene synthase], and crtI[phytoene desaturase] genes are from Erwinia uredovora, as describedelsewhere [21, 27] . crtM [diapophytoene synthase] and crtN [diapophytoenedesaturase] are from Staphylococcus aureus [25, 27] . Bacillusstearothermophilus farnesyldiphosphate synthase [BsFDS] wasPCR cloned from genomic DNA [ATCC 12980] according to the literature[13] . AmpliTaq polymerase [Perkin-Elmer, Boston, Mass.] wasused for mutagenic PCR, while Vent polymerase [New England Biolabs,Beverly, Mass.] was used for cloning PCR.

Plasmid construction. crtN was subcloned into the EcoRI/NcoI site of pUCmodII [21], resulting in pUC-crtN . crtB was removed from previously constructed pUC-crtE-crtB-crtI [27] to give pUC-crtE-crtI . From these twoplasmids, genes and promoters [lacP-crtN and lacP-crtE-crtI, respectively] were PCR amplified and subcloned into the SalI site of pACYC184, resulting in pAC-crtN and pAC-crtI-crtE, respectively.Carotene synthase genes [crtB and crtM] were cloned into theXbaI/XhoI site in pUC18Nmod [27], resulting in pUC-crtB andpUC-crtM . Plasmid pUC-BsFDS_Y81A was constructed by subcloningthe Y81A mutant of BsFDS [followed by a ribosome binding site]into the EcoRI/NcoI site of pUCmod . crtB or crtM was subclonedinto the XbaI/XhoI site of this construct, resulting in pUC-crtB-BsFDS_Y81A and pUC-crtM-BsFDS_Y81A.

Site-directed mutants. PCR-based site saturation or substitution mutagenesis was performedon F26 [TTT], W38 [TGG], and E180 [GAA] by using the ExSitemethod [Stratagene] . Some site-directed mutants were obtainedfrom the saturation mutagenesis library, but the majority weresynthesized using individual primers with the appropriate codonat the targeted site . Double and triple mutants were constructedby repeated site-directed mutagenesis . Selected mutants weresubcloned into the XbaI/XhoI site of pUC-BsFDS_Y81A to producepUC-[crtM]-BsFDS_Y81A [square brackets indicate a crtM mutant].

Evaluating the C₃₀ and C₄₀ activities of CrtM variants. To measure C₄₀ synthase activity, genes encoding CrtM and itsvariants were placed in the XbaI-XhoI site of pUC18Nm [27] andtransformed into XL1 cells harboring pAC-crtE-crtI . Similarly,C₃₀ synthase activity was evaluated upon transformation of pUC-crtM [or pUC-[crtM]] into XL1 cells harboring pAC-crtN. Colonies were lifted onto white nitrocellulose membranes [Pall, Port Washington, N.Y.] and grown at room temperature for an additional12 to 24 h . Colonies were picked and cultured overnight in 96-well deep-well plates, each well containing 0.5 ml of liquid Luria-Bertani [LB] medium supplemented with carbenicillin and chloramphenicol[50 µg/ml each], and were shaken for 12 h at 37°C.A portion [20 µl] from each preculture was inoculatedinto 2 ml of fresh Terrific Broth [TB] culture . After beingshaken for 36 h at 30°C, cells were harvested and extractedwith acetone [1 ml] . The highest peak [475 nm] in each UV/visiblespectrum was used to score C₄₀ activity, while 470 nm was usedfor C₃₀ activity . Values reported are averages from six independentexperiments.

Carotenoid production and HPLC analysis. Plasmids [pUCs] were transformed into HB101 cells and grownon agar plates [LB] with carbenicillin [50 µg/ml] for14 to 16 h . Fresh colonies were picked, inoculated into TB medium,and shaken for 12 h at 37°C . An aliquot [0.5 ml] of thispreculture was inoculated into 150 ml of TB medium [in a 750-mltissue culture flask; Falcon] and shaken at 30°C for 36 to 40 h . Wet cells were harvested from the culture, extracted with 20 ml of acetone, transferred to 10 ml of hexane, driedwith anhydrous MgSO₄, and concentrated in a rotary evaporator. An aliquot of extract was passed through a Spherisorb ODS2 column [250 by 4.6 mm; particle diameter, 5 µm; Waters, Milford,Mass.] and eluted with an acetonitrile-isopropanol mixture [60:40[vol/vol]] at a flow rate of 1 ml/min by using an Alliance-HPLC[high-performance liquid chromatography] system [Waters] equippedwith a photodiode array detector . For analysis of molecularmass, a Series 1100 LC/MSD [Hewlett-Packard/Agilent, Palo Alto,Calif.] coupled with an atmospheric pressure chemical ionization[APCI] interface was used . The amount of each carotenoid wasdetermined by comparing the HPLC chromatogram peak area [at286 nm] to that of a ß-carotene standard [at 450 nm].To obtain the molar quantity, the value thus obtained [ß-caroteneequivalent] was multiplied by _ß-carotene [138,900cm^-1 M^-1 at 450 nm] divided by _phytoene [49,800 cm^-1 M^-1 at286 nm] . Molar quantity was then converted to a weight valueby multiplying by its molecular weight . The weights were thennormalized to the dry cell mass of each culture.

 
C₄₀ and C₃₀ carotenoid synthase activities of CrtM variants. To probe how modifications at residues 26, 38, and 180 of S.aureus CrtM allow this C₃₀ carotenoid synthase to condense twoC₂₀ precursors and function as a C₄₀ synthase, we performedsaturation mutagenesis at all three sites . Significant fractionsof the F26X [where X stands for any amino acid] and W38X libraries[ca . 65 and 50%, respectively] showed a pink hue [due to accumulatedlycopene] upon transformation into XL1 cells harboring pAC-crtE-crtI.In contrast, only 2 to 3% of the E180X library colonies showeda [weak] pink color, indicating that only a few amino acids[probably glycine alone] can positively contribute to the C₄₀synthase activity.

Eleven, 11, and 3 site-directed mutants were created at positions F26, W38, and E180, respectively [Fig . 2] . These were tested individually for their abilities to lead to pigment production in a C₃₀ and a C₄₀ pathway assembled in E . coli. To test C₄₀synthase performance, mutants were transformed into E . colicells expressing the E . uredovora GGDP synthase CrtE and theC₄₀ desaturase CrtI . Cells containing CrtM variants that haveacquired C₄₀ synthase activity accumulate lycopene . The pigmentationlevel was determined from the peak height [at 475 nm] of theacetone extract . Similarly, C₃₀ synthase performance was evaluatedfrom the pigmentation level of cells transformed with the genesfor CrtM and the S . aureus C₃₀ desaturase CrtN . Functional CrtMvariants led to production of 4,4'-diapophytoene, which wasquantified [470 nm] in the acetone extract . As shown in Fig.2, replacement of F26 or W38 by a smaller amino acid significantlyincreased the C₄₀ synthase activity of CrtM . C₃₀ performancewas the highest for wild-type CrtM and decreased with decreasingsizes of the amino acid residues at these positions . Thus, gainof C₄₀ function by mutation of CrtM came at a cost to its C₃₀ synthase activity.

 
CrtM generates 4,4'-diapophytoene [product 1] in two distinctsteps: [i] abstraction of a diphosphate group from a prenyldonor, followed by head-to-head condensation of the donor andacceptor molecules, and [ii] rearrangement of the cyclic intermediate,followed by removal of a second diphosphate and a final carbocationquenching process [Fig. 3a] . This mechanism is virtually identicalto that of squalene synthase [SqS], the enzyme that catalyzesthe first step in cholesterol biosynthesis . Indeed, when deprivedof NADPH, SqS produces product 1 as the main product [3, 11]. Carotene synthases are similar to SqS in sequence and predicted secondary structure; they probably share a common ancestor andhave virtually identical folds . Although detailed biochemicalinformation on SqS is available [3, 11], the basis of its specificityis also poorly understood . Mapped onto the crystal structureof human SqS [hSqS] [19], F26 and W38 appear in helices B andC . Both side chains point into the pocket that accommodatesthe second half-reaction [Fig . 3b] . We reasoned that wild-typeCrtM is able to perform the first half-reaction of phytoene[C₄₀] synthesis [condensation of 2 GGDP molecules to form prephytoenediphosphate]but that the reaction is prevented from going to completionby bulky residues which sterically inhibit the second, rearrangementstep . When F26 or W38 is replaced with smaller or more flexibleamino acids, the reaction can proceed, and phytoene is produced.

 
In both the C₃₀ and C₄₀ pathways, M_F26A/W38G [CrtM with theF26A and W38G mutations] and M_F26A/W38A performed more poorlythan M_F26A, and M_F26G/W38G and M_F26G/W38A performed more poorlythan M_F26G [Fig . 4] . Thus, the combination of mutations at F26 and W38 appears to be harmful for the general performance ofCrtM, probably due to perturbation of the reaction pocket, whichdecreases the overall catalytic activity.

 
Based on the SqS structure, E180 in CrtM is positioned outsidethe reaction pocket, closer to the location where the firsthalf-reaction occurs [Fig . 3b] . At this position, glycine isthe only amino acid that allows CrtM to exhibit measurable C₄₀ synthase activity . In contrast to the F26X and W38X mutants,which showed a marked decrease in C₃₀ performance, M_E180G showed a slight increase in C₃₀ synthase activity [data not shown]. Thus, the E180G mutation positively affects CrtM performancein both the C₃₀ and C₄₀ contexts . In fact, for all tested CrtMvariants [M_F26G, M_F26A, M_F26G/W38G, M_F26G/W38A, M_F26A/W38G,and M_F26A/W38A], addition of E180G enhanced pigmentation forthe C₃₀ and C₄₀ pathways [Fig . 4].

CrtM variants generate longer [C₄₅ and C₅₀] carotenoid backbones when supplied with the C₂₅ precursor FGDP. Isoprenyldiphosphates are ubiquitous building units for thousandsof natural products and cell components . Different isoprenyldiphosphate synthases catalyze the consecutive condensation of C₅ units to produce a wide range of isoprenyldiphosphates [C₁₀ to C_20,000]. Isoprenyldiphosphate synthases with different product size distributions are known, and the molecular basis of their product size determination is well understood [29] . BsFDS is very specific and producesFDP almost exclusively, both in vitro and in vivo [13] . Ohnumaet al . have shown, however, that the product specificity of BsFDS can be controlled by altering the size of the amino acid at position 81 [18] . The Y81A BsFDS variant produces farnesylgeranyldiphosphate[FGDP; C₂₅DP] as the main product in vitro, with small amountsof GGDP . We observed that E . coli HB101 cells harboring pUC-crtM_wt-BsFDS_Y81A produced almost no carotenoids [Fig . 5b], while those harboringpUC-crtM_wt-BsFDS_wt produced product 1 at a high level, 1.1 mg/g[dry cell weight [DCW]] [Fig . 5a] . The fact that no C₃₀ carotenoids were observed indicates that FDP is not supplied for C₃₀ carotenoidproduction, which we attribute to its redirection toward thelonger isoprenyldiphosphates, catalyzed by BsFDS.

 
When coexpressed with BsFDS_Y81A, several CrtM variants with substitutions at positions 26 and 38 generated C₃₅ [product 2] and C₄₀ [product 3] carotenoids along with two novel carotenoids,products 4 and 5 [Fig . 5c] . Based on their mass analysis [M⁺ at m/e = 612 for product 4 and 680 for product 5], elution time,and characteristic absorption spectra [maximum peak at 286 nm],we conclude that products 4 and 5 are 16-isopentenylphytoene[the C₄₅ carotenoid backbone, C₂₀ plus C₂₅] and 16,16'-diisopentenylphytoene[C₅₀ backbone, C₂₅ plus C₂₅] . The distribution of the different carotenoid backbones varied, depending on the synthase . Among the single mutants, M_F26A produced the highest levels of product 4 [ca . 130 µg/g [DCW]] and product 5 [78 µg/g [DCW]][Fig. 6] . Combining mutations at positions 26 and 38 usually decreased the total carotenoid production . For example, HB101 cells harboring pUC-M_F26A/W38A-BsFDS_Y81A produced lower levelsof carotenoids than cells with pUc-M_F26A-BsFDS_Y81A . However,the extent of decrease was negligible for products 4 and 5, while it was significant for products 2 and 3 . Thus, in this system, more than half the carotenoids were longer-chain structures, products 4 [35%] and 5 [22%] . This "shifted" size specificitywas further confirmed by analyzing the products in the C₃₀ pathway: when pUC-M_F26A/W38A was transformed into HB101 cells, a verysmall amount of product 1 was accumulated along with a smalleramount of product 2 [Fig . 5f] . In contrast, cells harboringwild-type pUC-crtM and pUC-M_F26A accumulated a high level ofproduct 1 [Fig . 5d and e].

 
Because the E180G substitution increases overall synthase activityin the C₃₀ and C₄₀ pathways [Fig . 4], and because it is farfrom F26 or W38 [Fig . 3b], we anticipated that introductionof E180G to M_F26A/W38A would enhance carotenoid production withoutaltering the preference for larger [C₄₅ or C₅₀] structures.Indeed, the highest production of products 4 [215 µg/g[DCW]] and 5 [147 µg/g [DCW]] was attained with HB101cells harboring pUC- M_{F26A/W38A/E180G}-BsFDS_Y81A [Fig . 6].

 
In previous work we showed that wild-type CrtM can produce aC₃₅ carotenoid backbone in the presence of high levels of theC₂₀ precursor GGDP [25] . Various downstream enzymes [desaturases and cyclases] from the C₃₀ and C₄₀ carotenoid pathways werefunctional on this nonnatural substrate, which led to the productionof a series of novel C₃₅ carotenoids . Using directed evolution,with screening for altered pigment production, we were ableto generate pathways for every possible C₃₅ desaturation product.Thus, it appears that once a carotenoid backbone structure iscreated, downstream enzymes, either natural or engineered, canaccept the new substrate, and whole series of novel carotenoidscan be produced . With the action of carotenoid-modifying enzymes,including desaturases, cyclases, hydroxylases, and cleavage enzymes, on these new extended backbones, it should be possible to double or even triple the diversity of the carotenoid kingdom.

It is argued that, over evolutionary time scales, secondary metabolic pathways explore chemical diversity via gene duplication and mutation of biosynthetic enzymes and thereby discover compounds that confer fitness advantages [28] . Secondary metabolic pathwaysin fact seem to have evolved features that facilitate efficientexploration of new chemical structures [8] . For example, thebiosynthetic enzymes often accept a range of substrates and/orproduce a variety of products from a single substrate [11, 22];this "promiscuous" behavior allows whole series of novel metabolitesto emerge upon minimal change in an existing pathway [1, 2,7, 12, 15, 25] . In contrast, some enzymes, frequently thosein key positions at the start of a pathway, show considerablestringency in the substrates they accept [6, 20, 27] . This upstream specificity serves to limit the production of unwanted by-products. For carotenoids, the highly specific synthase reaction appearsto be the major point of control over product diversity . Otherisoprenoid pathways are similar to the carotenoid pathways inthat the first pathway-specific enzymes are very specific intheir substrate selection and thereby channel the entire pathwayto a particular product [6] . Engineering of these specificity-controlling enzymes is likely to be the most efficient way to expand these other isoprenoid biosynthetic pathways to create new metabolites.

We do not know why the new carotenoid pathways that we generated in the laboratory are not seen in nature . Combination of two engineered enzymes, with as little as one amino acid substitution each, led to the production of novel carotenoid backbones, unambiguously showing that whole new carotenoid pathways can emerge and can do so rapidly . It is likely that C₄₅ and C₅₀ carotenoid pathwayshave been invented by nature but that we have not yet discoveredthem . Perhaps they have been invented and then discarded, becausethe producing organisms did not benefit . The benefits to humaninventors of these pathways, however, may be significant . In addition to the expected higher antioxidant activity [2] andpossible hormonal effects [4, 10], larger chromophores for carotenoids[19 conjugated double bonds for C₅₀ carotenoids, 23 for C₆₀]will extend the color range of these natural pigments . A varietyof isoprenyldiphosphate synthases that produce isoprenyldiphosphatesof different sizes [e.g., C₃₀DP, C₃₅DP, C₄₀DP, C₄₅DP, C₅₅DP, and natural rubber] are available [30]; these compounds could,in principle, serve as substrates for engineered synthases.

Although impressive in number, the known products of secondary metabolic pathways account for only a tiny fraction of the structures that could be produced . Engineering the upstream biosyntheticenzymes to accept new substrates allows us to generate wholenew pathways and access very large numbers of secondary metabolitesthat are not known in nature but should be chemically, and biologically,possible.

 
This research was supported by the U.S . National Science Foundation [BES-0118565] and Maxygen, Inc.

 
* Corresponding author . Mailing address: Division of Chemistry and Chemical Engineering, California Institute of Technology, 210-41 1200 E . California Blvd, Pasadena, CA 91125 . Phone: [626] 395-4162 . Fax: [626] 568-8743 . E-mail: umeno@cheme.caltech.edu.

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