Yeast Pathway Kit: A Method for Metabolic Pathway Assembly withAutomatically Simulated Executable DocumentationFilipa Pereira,† Flavio Azevedo,† Nadia Skorupa Parachin,‡,§ Barbel Hahn-Hagerdal,‡

Marie F. Gorwa-Grauslund,‡ and Bjorn Johansson*,†

†CBMACentre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, Braga4710-057, Portugal‡Division of Applied Microbiology, Department of Chemistry, Lund University, SE-22100 Lund, Sweden

*S Supporting Information

ABSTRACT: We have developed the Yeast Pathway Kit (YPK) forrational and random metabolic pathway assembly in Saccharomycescerevisiae using reusable and redistributable genetic elements. Geneticelements are cloned in a suicide vector in a rapid process that omits PCRproduct purification. Single-gene expression cassettes are assembled in vivousing genetic elements that are both promoters and terminators (TP).Cassettes sharing genetic elements are assembled by recombination intomultigene pathways. A wide selection of prefabricated TP elements makesassembly both rapid and inexpensive. An innovative software toolautomatically produces detailed self-contained executable documentationin the form of pydna code in the narrative Jupyter notebook format tofacilitate planning and sharing YPK projects. A D-xylose catabolic pathwaywas created using YPK with four or eight genes that resulted in one of thehighest growth rates reported on D-xylose (0.18 h−1) for recombinant S.cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improvethe yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool.

KEYWORDS: metabolic engineering, Saccharomyces cerevisiae, D-xylose, synthetic biology, bioinformatics

Metabolic engineering of Saccharomyces cerevisiae has beenapplied for the production of a wide range of fuels and

chemicals (for review, see refs 1 and 2). Engineering ofproduction strains usually requires the expression of aconsiderable number of genes because it is rare for a singleenzyme to exert considerable control over a trait, such as fluxalong a metabolic pathway. The need to express multiple geneshas led to the application of techniques that allow thesimultaneous assembly of multiple promoters, genes, andterminators into metabolic pathways. Among the availabletechniques are the Gibson assembly protocol,3 which is ageneral technique for enzymatic assembly in vitro, and manyvariations of in vivo assembly by homologous recombinationbetween flanking sequences added by PCR.4,5 Gene copynumber,6 promoter strength,7 and positional effects ofindividual genes in a pathway8 may affect efficiency, butrationally designing and testing all permutations can beinfeasible for longer pathways. Alternatively, a pool of randomlyassembled pathways can be created from which the bestperforming pathways can be selected based on a screeningstrategy. Protocols for random pathway assembly engineeringbased on in vivo homologous recombination,9,10 Gibsonassembly methods,11 or both12 have also been described.Common for most protocols is that genetic parts such aspromoters and terminators are not easily shared and reused

because they are usually PCR products from chromosomalDNA or other sources and, as such, cannot be propagated.Most assembly protocols are “all or nothing” in the sense thatmultiple genes and regulatory sequences are joined in onereaction. Strategy or implementation errors such as a faultyPCR primer will yield little information to pinpoint the error asno pathway will be created. Furthermore, published pathwayassembly protocols are designed for either rational or randomassembly, but to do both requires reamplifying the genetic partswith new PCR primers.In this work, we have developed an alternative pathway

assembly approach, called the Yeast Pathway Kit (YPK). YPKdiffers from previous methods in that promoters, genes, andterminators are cloned in one of three closely spaced bluntrestriction sites in pYPKa, a highly efficient Escherichia colipositive selection vector designed for the rapid cloning ofunpurified PCR products. Episomal yeast single-gene ex-pression vectors are constructed from these basic elements byin vivo gap repair in S. cerevisiae three at a time in a promoter−gene−terminator configuration. This assembly is directed by

Received: November 25, 2015Published: February 25, 2016

Research Article

pubs.acs.org/synthbio

© 2016 American Chemical Society 386 DOI: 10.1021/acssynbio.5b00250ACS Synth. Biol. 2016, 5, 386−394

short overlaps of plasmid backbone sequences between thethree cloning sites.The single-gene expression vectors are subsequently joined

into pathways by homologous recombination betweenpromoters and terminators of each cassette. This can be doneby the pairwise use of the same sequence as a promoter orterminator in the two vectors. The YPK promoters andterminators are intergenic sequences from tandemly expressedS. cerevisiae genes that naturally serve both as terminators andpromoters (terminator−promoters or TPs) of the two adjacentgenes. This second stage of the assembly is directed by therelatively long TPs of each cassette (500−1300 bp). YPKprovides reusable genetic elements at several levels in theassembly process as there is an E. coli vector for each geneticelement that is easily verified, stored, propagated, anddistributed. The single-gene yeast expression vectors constitutea second level of reusable genetic elements as well as a way tostudy and verify each gene expression cassette separately.Assembly and verification of pathways can be performed usingonly two specific primers per gene together with a set of eightshort (<31-mer) standard PCR primers (Table S1). The TPand gene components can be randomly assembled by leavingout the single-gene expression vector assembly step, thusallowing the random combination of promoters genes andterminators into multigene pathways. Unique to the YPKpathway approach is an innovative specifically designedsoftware tool called ypkpathway that can automatically simulatethe assembly process and generate correct and completeexecutable documentation of complex constructs in thenarrative Jupyter notebook format.13 Jupyter notebooks areboth documentation and executable code containing asimulation of the assembly and cloning strategies usingpydna.14 The notebooks provide information such as thesequences of intermediate plasmids, automatically designedprimers, and simulated PCR conditions. The notebook formatpermits strategy changes (such as altered PCR primers) to beincorporated in existing documentation by editing andexecuting the notebook. This type of documentation allowsverification of the correctness of an assembled pathway and theefficient sharing and communication of strategies.The YPK method was validated by both rational and random

assembly of several fungal D-xylose metabolic pathways. D-Xylose is the most abundant pentose sugar in lignocellulosicbiomass, but it cannot be directly metabolized by S. cerevisiae,so genetic engineering of S. cerevisiae to add this capacity15 hasbiotechnological importance. Using YPK, it was possible torationally assemble four or eight active D-xylose metabolic genesin one step. Executable Jupyter notebook documentation isprovided for each pathway, including all intermediary steps,facilitating the complete reproduction of the genetic constructs.Two pathways were also randomly assembled, resulting inpathways with different properties. Rationally assembledmetabolic pathways supported a specific growth rate of 0.18h−1 on defined medium with D-xylose as the carbon source,which is among the highest reported for recombinant S.cerevisiae cells expressing the fungal D-xylose pathway.16 Theyield of ethanol under oxygen-limited conditions reached 0.46 gethanol/g of D-xylose consumed, which is 90% of thetheoretical yield. YPK proved to be a practical, fast, andefficient protocol for pathway assembly.

■ RESULTS AND DISCUSSIONYPK Strategy. The YPK metabolic pathway assembly

strategy takes advantage of the observation that naturalintergenic sequences from genes expressed in tandem areboth terminators of the upstream gene and promoters for thedownstream gene. These intergenic sequences (designatedterminator−promoters or TPs) are used both for transcriptionregulation and for aiding the assembly of multiple genemetabolic pathways from single-gene expression cassettes. TPsfrom the intergenic sequences upstream of the genes TEF1(579), TDH3 (698), PGI1 (1302), FBA1 (630), PDC1 (955),RPS19b (626), RPS19a (544), TPI1 (583), and ENO2 (520)were PCR-amplified from S. cerevisiae chromosomal DNA(sizes given in parentheses). These TPs have been used for theexpression of heterologous proteins in S. cerevisiae, except forribosomal protein promoters RPS19b and RPS19a. The TPswere first cloned into the ZraI or EcoRV site of the pYPKasuicide vector (Figure 1A). Genes to be expressed were cloned

in the AjiI site of pYPKa. Promoters, genes, and terminatorswere amplified with primers specific to the pYPKa plasmidbackbone (Figure 1B). Single -gene expression cassettes wereformed by homologous recombination between the PCRproducts and the pYPK0 S. cerevisiae vector (Figure 1C,D).The single-gene expression cassettes can recombine intomultiple gene pathways, provided that they share a TP as apromoter in one cassette and a terminator in the other (Figure1E).

Promoter Activity Measurement. The relative strengthof the TPs cloned as promoters was evaluated by measuring the

Figure 1. Schematic representation of the YPK strategy for rationalassembly. The figure depicts the construction of a two-gene expressionpathway using YPK. Six DNA fragments are cloned into themulticloning site (MCS) of the pYPKa vector (A colored boxes),resulting in six different E. coli vectors (A). The unique restriction sitesZraI, AjiI, and EcoRV are separated by 50 bp (red box) and 31 bp(green box). The blue and yellow boxes represent areas ∼20 bpupstream of the ZraI site and downstream of the EcoRV site. Thestriped boxes represent plasmid backbone sequences upstream anddownstream of the MCS. Vector-specific primers (B) are used toamplify six PCR product, which recombine into two single-geneexpression vectors (C, D). The single-gene expression cassettes arejoined in a second recombination stage to form the final pathwayvector (E). The first and last cassettes are amplified with primersflanking the MCS so that extra plasmid backbone sequences are added(diagonally and vertically striped boxes). pYPKpw is a version ofpYPK0 that lacks the MCS to avoid unwanted recombination.

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expression level of the HIS3 reporter gene. Promoter strengthwas deduced from the His3p protein level detected as resistanceto 60, 150, or 200 mM 3-amino-1,2,4-triazole (3-AT) in a ura3,his3 strain background relative to that in the wild-typebackground (Figure 2). Resistance was measured by spotting

four different dilutions of cells at each 3-AT concentration. Thefirst eight rows of Figure 2 contain strains expressing the HIS3gene expressed from TPs TEF1, TDH3, PGI1, FBA1, PDC1,RPS19b, RPS19a, and TPI1. The strength of the promotersshowed little difference except for PGI1 and TPI1, whichshowed slightly lower activity, as can be seen from the reducedcell growth in the rightmost column at 200 mM 3-AT for thesepromoters. The RPS19b and RPS19a promoters showed arelative strength comparable to that of the widely used TDH3and TEF1 promoters, indicating that these promoters are usefulfor protein (over)expression in S. cerevisiae. The single wild-type HIS3 gene in S. cerevisiae CEN.PK 113-5D supportedgrowth only up to 60 mM 3-AT. Plasmids p426TDH3_HIS3and p426TEF1_HIS3 (Figure 2) contain the HIS3 gene underthe control of the promoter indicated by the plasmid’s name(TDH3 and TEF1, respectively). These vectors are from awidely used vector set17 and show similar or lower activitycompared to that of the same promoters in the YPK single-geneconstructs (Figure 2, first two rows). This shows that theplasmid backbone sequences that are incorporated between theTP serving as promoter and the gene (Figure 1, red boxes) orbetween the gene and the TP serving as terminator (Figure 1,green boxes) do not seem to negatively affect gene tran-scription or translation levels. Low-copy-number vectors(p413TEF and pYPK3_RPS19b_HIS3_RPS19a) show mark-edly lower activity compared to that of multicopy vectors(p423TDH3 and pYPK0_RPS19b_HIS3_RPS19), as expected.pYPK0_RPS19b_HIS3_RPS19a and pYPK0_RPS19b_-HIS3_TPI have different terminators, but they show similarlevels of 3-AT resistance. pYPK0_TDH3_rev_HIS3_ ENO2has an inverted promoter and a low activity compared to that ofpYPK0_TDH3_rev_HIS3_ PGI1, indicating the importance ofpromoter orientation in the construct. Measuring promoterstrength by GFP fluorescence has produced larger differences

between promoters than were observed here.18 Some of thisdifference may be attributed to the technical differencesbetween the methods used. Another source of the observeddifferences could be the influence of the different terminatorsequences.19

Construction of a Four-Gene D-Xylose MetabolicPathway in S. cerevisiae. S. cerevisiae is able to use D-xyloseas the sole carbon source when expressing heterologous xylosereductase (XR) and xylitol dehydrogenase (XDH) fromScheffersomyces stipitis.20 Overexpression of endogenous xylulo-kinase (XKS1)21,22 and the pentose phosphate pathway enzymetransaldolase (TAL1)23 further increased the D-xylose metabolicrate. A four-gene metabolic pathway was assembled using S.stipitis genes XYL1 (XR) and XYL2 (XDH) and S. cerevisiaegenes XKS1 (XK) and TAL1. Each of the four genes was firstassembled into single-gene expression vectors: pYPK0_-TEF1_XR_TDH3, pYPK0_TDH3_XDH_PGI1, pYPK0_P-GI1_XK_FBA1, and pYPK0_FBA1_TAL1_PDC1 (genename in bold) by the same principle as that depicted in Figure1C,D. The single-gene cassettes were assembled into a pathwayvector that was given the systematic name pYPK0_-TEF1_XR_TDH3_XDH_PGI1_XK_FBA1_TAL1_PDC1,where the order of the genes and TPs is indicated by the name.This vector was also given the shorter name pMEC1136. Anidentical pathway but for a point mutation in the XYL1 gene(N272D) was also assembled and designated pMEC1135. Thismutation results in a preference for NADH over NADPH forXR.24 Both pathway vectors were verified by restrictiondigestion. The frequency of correct recombination wascalculated for the final construct, and 85% of the tested cloneshad the expected size and restriction pattern. A S. cerevisiaestrain carrying the vector with wild-type XR (pMEC1136)showed a slightly faster specific growth rate of 0.18 h−1 thanthat of the strain containing XR carrying a point mutation(Table 1, two first rows). However, the strain with wild-type

XR consumed less D-xylose and had lower ethanol and higherxylitol yields (Table 2). pMEC1136 is genetically comparableto strain TMB3014,23 which has the same four genesoverexpressed in constructs that are chromosomally integrated.The strain with pMEC1136 consumed almost 2 times theamount of D-xylose as that of TMB3014 during thefermentation, suggesting that having a higher copy number ofone or more of the genes involved is beneficial for pathwayperformance.

Figure 2. Terminator−promoters assay. S. cerevisiae CEN.PK 113-11C(ura3, his3) was transformed with the indicated constructs, exceptwhere CEN.PK 113-5D (ura3, HIS3) is explicity stated. A series ofdilutions (1, 10, 100, and 1000×) from an initial OD640nm of 0.1 werespotted on defined solid media with 20 g/L of glucose as the solecarbon source that was supplemented with uracil containing 3-AT (0,60, 150, or 200 mM) and incubated at 30 °C for 3 days. Solid mediawithout 3-AT that was supplemented with histidine was also used tocontrol cell growth (first column).

Table 1. Specific Growth Ratesa

vector pathwaygrowth rate(μmax; h

−1)

pMEC1135 XR(N272D), XDH, XK, and TAL1 0.17 ± 0.01pMEC1136 XR, XDH, XK, and TAL1 0.18 ± 0.02pMEC1138 XR(N272D), XDH, XK, TAL1, TKL1,

RPE1, RKI1, and GXF10.16 ± 0.01

pMEC1139 XR, XDH, XK, TAL1, TKL1, RPE1,RKI1, and GXF1

0.18 ± 0.01

pMECRandom1 XR, XK, XDH, and TAL1 0.18 ± 0.01pMECRandom4 XR, XK, XDH, and TAL1 0.15 ± 0.01

aAerobic maximum specific growth rate (μmax) with 20 g/L xylose asthe sole carbon source in submerged culture for S. cerevisiae strainCEN.PK 113-5D transformed with the indicated vector. The mediumwas defined with complete amino acid supplement. Results are givenwith standard deviation obtained from three independent experiments.

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Construction of an Eight-Gene D-Xylose MetabolicPathway. An eight-gene D-xylose consumption pathway wasconstructed in order to further test the flexibility and capacity ofYPK. The pathway had the same initial genes and TPs in thesame order as those in the previously described four-genepathway. Additionally, S. cerevisiae genes TKL1, RPE1, andRKI1 encoding pentose phosphate pathway enzymes and theCandida intermedia GXF1 gene encoding a D-xylose transporterwere included as they all have been linked to enhanced D-xyloseutilization.23,25,26 The resulting pathway was given the system-atic name pYPK0_TEF1_XR_TDH3_XDH_PGI1_XK_F-BA1_TAL1_PDC1_TKL1_RPS19b_RPE1_RPS19a_RKI1_TPI1_GXF1_ENO2 and the shorter alias pMEC1139. Thesystematic name reflects the genes and TPs and the order inwhich they appear in the vector. An identical pathwaycontaining the mutant XR gene was named pMEC1138. Thelast four genes in the pathway were also joined together in theseparate vector, pYPK0_PDC1_TKL1_RPS19b_RPE1_RP-S19a_RKI1_TPI1_GXF1_ENO2, with the shorter aliaspMEC1137.An eight-gene pathway was also constructed and confirmed

by a different strategy. The pMEC1136 vector was linearizedafter the last TP using restriction endonuclease PacI (Figure1E) and recombined with a DNA fragment containing the fourgenes of pMEC1137. This shows that it is feasible to extendand combine pathways to circumvent, for example, localconstruction difficulties, adding flexibility to the YPK system.Growth rates, D-xylose consumption, and production of

biomass, glycerol, acetic acid, ethanol, CO2, and xylitol werealso evaluated for yeast strains carrying the eight-genepathways. The growth rates observed for strains with eight-gene pathways (pMEC1139 and pMEC1138) were comparableor slightly lower than the ones found for strains with four-genepathways (Table 1). The N272D mutation in XR caused aslight reduction in growth rate, as was also observed for thefour-gene pathways. No significant differences were observedfor D-xylose consumption or xylitol or ethanol production(Table 2) between the two strains with either the eight- or four-gene pathway expressing the same version of XR. These resultsindicated that the additional overexpression of RPE1, RKI1,

TKL1, or GXF1 is not necessary to reach the optimal growthrate and ethanol yields under the tested conditions and alsoindicated that the increased metabolic burden of the four extragenes does not seem to affect cell metabolism negatively.

Random Assembly of D-Xylose-Utilizing Pathways. Ahierarchical two-step assembly strategy was used to ensure theassembly of single-gene expression cassettes into a predefinedsequence. The assembly of several genes and TPs directlywould, in theory, allow a pool of vectors to be created thatdiffer in their gene identities, gene copy number, TP fragment,and gene order. This pool might contain better performingpathways that could be found by selection. This hypothesis wastested by constructing a randomly assembled D-xylose-utilizingpathway using the same building blocks as those for therationally designed four-gene pathway, i.e., four genes (XYL1,XYL2, XKS, and TAL1) and seven TPs (TPI1, TDH3, TEF1,PDC1, PGI1, FBA1, and RPS19b) (Figure 3). A total of 18DNA fragments (two PCR products per TP) were transformedtogether with linear pYPK0 vector. Transformants wereincubated for 4 h (short recovery) or 24 h (long recovery) innonselective liquid glucose medium to increase the chance ofrecovering transformants. One transformant was obtained aftera short period of recovery and 4 days of growth on mediumwith D-xylose as the sole carbon source, whereas several cloneswere obtained after a long period of recovery. Plasmid DNAfrom the largest colony from each recovery period wasextracted, transferred to E. coli, and designated pMECRandom1and pMECRandom4. Vector pMECRandom4 was analyzed bya combination of restriction digestion, DNA sequencing, andanalytical PCR. The sequence of its promoters, genes (in bold),and terminators was as follows: TDH3_XK_TEF1_XDH_PDC1_TAL1_PGI1_XR_PDC1_XR_PGI1. The pathway hadtwo copies of XR, controlled by the PGI1 and PDC1promoters, and the PDC1 promoter was present twice. Thepresence of multiple copies of XR might reflect a selectiveadvantage or result in subtle differences in the relative amountof DNA fragments in the recombination mixture. Interestingly,the TP present twice is the second longest of the TPs used.When it was transferred back to S. cerevisiae, pMECRandom1transformants had a specific growth rate equal to that of the

Table 2. Xylose Consumption and Product Formationa

yield (g/g of D-xylose consumed)

pathwayxylose consumed

(g/L) biomass ethanol xylitol glycerolaceticacid

ethanol(g/L)

% Crecovery

pMEC1135XR (N272D), XDH, XK, and TAL1 18.55 0.001 0.46 0.15 0.08 0.008 8.52 107pMEC1136XR, XDH, XK, and TAL1 17.57 0.001 0.30 0.37 0.11 0.008 5.18 103pMEC1138XR (N272D), XDH, XK, TAL1, TKL1, RPE1, RKI1 andGXF1

18.55 0.002 0.46 0.14 0.07 0.008 8.48 109

pMEC1139XR, XDH, XK, TAL1, TKL1, RPE1, RKI1, and GXF1 16.40 0.001 0.31 0.35 0.11 0.006 5.00 105pRandom1XR, XK, XDH, and, TAL1 14.15 0.001 0.38 0.16 0.13 0.007 5.35 105pRandom4XR, XK, XDH, and TAL1 18.90 0.004 0.42 0.23 0.06 0.012 6.29 106TMB3014XR, XDH, XK, and TAL1 9.86 0.002 0.29 0.39 0.04 0.010 2.82 96aXylose consumption and product formation are shown for recombinant S. cerevisiae strains after 206 h of oxygen-limited fermentations with 20 g/Lxylose as the sole carbon source in minimal medium. Displayed values are the average of biological triplicates.

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strain containing pMEC1136, whereas the strain withpMECRandom4 showed the slowest growth rate of all strainstested (Table 1). The pRandom4 transformants consumedmore D-xylose than any other strain, but they also producedmore xylitol (Table 2). The higher D-xylose consumption rateand xylitol yield are consistent with increased XR activity.8,27

The two randomly assembled pathways displayed both thehighest and lowest D-xylose consumption rates of all pathwaysconstructed, combined with relatively high xylitol and lowethanol yields. This result may reflect a limitation of theselection strategy since these pathways were selected foraerobic growth on D-xylose and not for ethanol productivity oryield. The random assembly strategy has a high degree offreedom in the way promoters, genes, and terminators cancombine. This increases the number of testable combinationsbut also leads to a low yield of pathways conferring the desiredphenotype, which is reflected in the low number of randompathways obtained.Ypkpathway Software and Executable Documenta-

tion. The planning required to create large multigeneassemblies is a daunting task regardless of the assemblyprotocol used. Often, dozens of PCR primers need to bedesigned, where one error will impede the entire construction.The ypkpathway software that accompanies the YPK can aid inthe planning of complex assembly projects using a text filecontaining the promoter, gene, and terminator sequences inFASTA or Genbank text format in the order in which theyshould be assembled (Figure 4). ypkpathway automaticallygenerates a series of Jupyter notebook13 files, which areinteractive computational environments that combine execut-able Python28 code, rich text, figures, and hyperlinks. TheJupyter notebook format is rapidly gaining traction for scientificcomputation as it makes code and analysis easily accessible.29

The pathway construction process is described in thesenotebooks using the recently developed pydna14 package forcloning and assembly simulation. Figure 5 shows the header ofa Jupyter notebook file describing a four-gene D-xylose

metabolic pathway generated by ypkpathway. Upon execution,each notebook simulates the construction of a pathway or anintermediate vector and produces metadata such as primersequences, PCR conditions, and a download link in the end ofthe document providing the final sequence. The nontrivialsimulation of homologous recombination is done entirelywithout assumptions other than the primary sequence of thefragments using the graph-theory-based algorithm provided bypydna.The files created by ypkpathway enable researchers to design

and carry out cloning simulations while creating interactive,reproducible protocols of the data, cloning steps, and thoughtprocesses underpinning the result. The rudimentary knowledgeof python needed to follow the cloning strategies is curentlypart of most relevant university curricula. These protocols canbe easily shared on the web as static versions, where they can beviewed using only a web browser.30 They can also be shared ascollections of interactive papers that let others repeat thecloning simulation for verification or even alter parameters orinput data to achieve different or improved results.All genetic constructs made as a part of this work were

documented in detail using this method and are available onGithub as a self-contained repository that can be forked ordownloaded as a compressed folder.31 The same files can beviewed online as static files through the free nbviewer Jupyternotebook rendering service.30

Figure 3. Schematic representation of random pathway assembly.Terminator−promoters (TPs) (A) similar to the ones described inFigure 1A are assembled directly without prior assembly into single-gene expression vectors (Figure 1B,C). (B) One of many possibleways that sequences can recombine together, resulting in differences ingene copy number, gene order, and combination between genes andTPs.

Figure 4. Graphical user interface of ypkpathway. ypkpathway uses alist of sequences as input and produces a folder containing sequencefiles and Jupyter notebooks.

Figure 5. Output of ypkpathway. ypkpathway generates a series ofJupyter notebooks in a folder that can be viewed using a web browser.The notebooks provide a narrative interface to the assembly strategy,where each assembly step is exposed as code cells. In particular, thenotebooks contain automatically generated figures describing homol-ogous recombination steps. The notebooks can be modified, executed,and distributed without the need for ypkpathway software.

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Comparison between YPK and Existing Methods. TheYPK protocol offers advantages over the previously described“DNA assembler” method5 and Gibson3 assembly for certainpurposes. YPK uses a set of short standard primers for PCRamplification prior to assembly, whereas DNA assembler andGibson assembly require PCR primers with long tails that haveto be specifically made for each promoter−gene−terminatorcombination. A recently described toolkit shares thehierarchical assembly of single-gene expression vectors intomultigene pathways with YPK.32 The MoClo restrictionenzyme strategy is used for assembly, which is faster becauseE. coli is used as the host for intermediate constructs instead ofS. cerevisiae. However, purified DNA is required for theassembly, which may be a disadvantage in some cases. Gibsonassembly was modified with shared linker sequences introducedby PCR using long PCR primers.11,12 The COMPACTERprotocol9 is a development of DNA assembler to join a fixedgene set where each gene is under the control of a differentpromoter from a promoter library. A helper plasmid is requiredfor each gene in the pathway, which takes two consecutive cut-and-paste cloning steps to construct. Standard cloning vectorswithout positive selection were used, which makes vectorconstruction considerably more laborious when compared withthat using positive selection vector pYPKa. Still, unlike YPK,this method does not produce variations in gene copy numberor gene order.Final Remarks. Strains carrying the YPK D-xylose pathways

display a growth rate comparable to the highest found in theliterature for D-xylose-adapted strains.16 Notably, the strainpresented in this work was not subjected to adaptation. Thepresent pathways differ from previous designs in that theymaintain a high copy number of all expressed genes, which mayexplain the high growth rates. Promoter, gene, and terminatorare each separated by 50 or 31 bp pYPKa-derived plasmidbackbone sequence in the single-gene expression constructs.These two sequences are each repeated once for every gene in amultigene pathway and could possibly provide sites ofintramolecular recombination, although such recombinationevents have not been observed for any pathway made withYPK.The provided executable documentation in the form of

Jupyter notebooks simulating the genetic constructionsdescribes every step in the cloning process and enables thecomplete reproduction of the genetic constructs, requiring noadditional information. The ypkpathway software can be usedto automatically generate such documentation for newpathways. Since the conclusion of this work, the TP libraryhas been expanded to 30 unique sequences, theoreticallyallowing the assembly of an equal number of genes. Pathwayswith up to 15 genes have been constructed by our group withthe same efficiency as that for the eight-gene pathwaysdescribed in this work. YPK proved to be a very efficient toolfor the generation of synthetic metabolic pathways that havealready beem used to create industrial D-xylose-fermenting yeaststrains.33

■ METHODSStrains, Plasmids, and Cell Cultivation. E. coli XL1-Blue

(Stratagene) was used to propagate plasmids except forplasmids with a CRPs gene,34 for which the cyaA mutant E.coli strain BW26356, obtained from Coli Genetic Stock CenterCGSC, was used. E. coli transformants were selected on solidlysogeny broth (LB) medium supplemented with 100 mg/L

ampicillin. The S. cerevisiae strains CEN.PK 113-5D (ura3) andCEN.PK 113-11C (ura3, his3) were used as host strains forgenetic engineering. Yeast cells were cultivated in YPD (20 g/Lpeptone, 10 g/L yeast extract, and 20 g/L glucose) medium ordefined medium containing 6.7 g/L Difco yeast nitrogen basewithout amino acids (YNB w/o aa) with 20 g/L glucose or 20g/L D-xylose. Different concentrations of 3-AT were added todefined medium for promoter activity assays. For physiologicalstudies and growth rate measurements, 2 g/L amino acid drop-out mix containing 20 amino acids (Formedium, UK) wasadded to the defined medium. The exact composition of thedrop-out mix is given in Table S2. For selection of dominantmarkers, YPD medium was supplemented with 300 mg/LGeneticin (kanMX4 marker), hygromycin (hphMX4 marker),or phleomycin (bleMX4 marker), as required. Yeast strains andbacterial strains were cultured at 30 and 37 °C, respectively.Liquid cultures were incubated on an orbital shaker at 200 rpm.Yeast DNA transformation was carried out using poly(ethyleneglycol), lithium acetate, and single-stranded carrier DNA.35

Final pathway vectors constructed in this work are listed inTable 3. All vectors used for construction are listed in Table S2.

Plasmids constructed as a part of this work are listed in TablesS4 and S5. The Supporting Information contains detaileddescriptions of the construction of selected vectors, and theJupyter notebook documentation contains details of theconstruction of all vectors.

Construction of the pYPKa Vector. The positiveselection vector pCAPs34 has a toxic mutant version of theCRP gene encoding the cyclic AMP receptor protein. The toxicgene is disrupted when DNA fragments are cloned within theopen reading frame of the toxic gene. The pCAPs vector wasamplified with primers 567 and 568 (Table S1 and Figure 1B),introducing a partial AjiI restriction site on each end of thelinear PCR product. The PCR product was treated with T4polynucleotide kinase and T4 DNA ligase and subsequentlytransformed into E. coli BW26356. In the resulting pYPKavector, the AscI site was silently replaced by an AjiI restrictionsite between the ZraI and EcoRV restriction sites in the CRPs

gene. The structure of the vector was confirmed by restrictionanalysis, and the toxicity of the uninterrupted CRPs gene wasconfirmed by transforming sensitive E. coli XL1-Blue.

Cloning of Genetic Elements in pYPKa. Intergenicregions from tandemly expressed genes in S. cerevisiaecontaining TPs were chosen from a set commonly used forprotein expression in the literature (TEF1, TDH3, PGI1, FBA1,PDC1, TPI1, and ENO2) as well as from two ribosomal protein

Table 3. Pathway Vectors Constructed in This Worka

plasmid description

pMEC1135 pYPK0-TEF1-SsXYL1(N272D)-TDH3-SsXYL2-PGI1-ScXKS1- FBA1-ScTAL1-PDC1, URA3, 2μ

pMEC1136 pYPK0-TEF1-SsXYL1-TDH3-SsXYL2-PGI1-ScXKS1-FBA1-ScTAL1-PDC1, URA3, 2μ

pMEC1137 pYPK0-PDC1-ScTKL1-RPS19b -ScRPE1-RPS19a-ScRKI1- I1-CiGXF1-ENO2, URA3, 2μ

pMEC1138 pYPK0-TEF1-SsXYL1(N272D)-TDH3-SsXYL2- PGI1-ScXKS1-FBA1-ScTAL1-PDC1-ScTKL1-RPS19b-ScRPE1-RPS19a-ScRKI1-TPI1-CiGXF1-ENO2, URA3, 2μ

pMEC1139 pYPK0-TEF1-SsXYL1-TDH3-SsXYL2-PGI1-ScXKS1-FBA1-ScTAL1-PDC1-ScTKL1-RPS19b-ScRPE1-RPS19a-ScRKI1-TPI1-CiGXF1-ENO2, URA3, 2μ

aSee Tables S3−S6 and the Jupyter notebook documentation for acomplete list of constructed vectors.

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genes, RPS19a and RPS19b. The TPs were amplified fromchromosomal DNA of S. cerevisiae CEN.PK 113-5D and ligatedin pYPKa digested with blunt restriction enzyme ZraI(promoters) and EcoRV (terminators), resulting in twoseparate vectors. Figure 1A shows an example of pYPKa vectorconstruction. Genes RPE1 and RKI1 were amplified fromchromosomal or plasmid DNA and ligated in vitro with pYPKapreviously cut with blunt restriction enzyme AjiI. All othergenes were amplified by PCR with tailed primers adding thenecessary homology (Figure 1, red and green boxes). Plasmidsderived from pYPKa were designated pYPKa_L_XYZN, whereL indicates the restriction enzyme used (Z, A, or E) and XYZNindicates the name of the promoter, gene, or terminator. TableS5 lists the constructed pYPKa vectors.Construction of the pYPK0 E. coli/S. cerevisiae Shuttle

Vector and Its Derivatives. The plasmid pSU036 containsthe yeast 2μ origin of replication sequence and URA3auxotrophic selectable marker between the ampicillin resistance(amp) gene and the pUC origin of replication.36 The soleEcoRV site of this vector was removed, resulting in plasmidpMEC1030 (Table S4). This plasmid was used to makepYPK0, which is a yeast version of pCAPs34 containing the S.cerevisiae URA3 auxotrophic marker and the 2μ origin ofreplication using the strategy described for pSU0.36 The CRPs

gene was partially deleted, removing the ZraI, AjiI, and EcoRVrestriction sites and the surrounding sequence (colored boxesin Figure 1). Three different versions of the pYPK0 andpYPKpw E. coli/S. cerevisiae shuttle vectors were constructedcontaining a single- or multicopy origin of replication andhphMX4, kanMX4, and bleMX4 selection markers (Table S3).Vector construction details are given in the SupportingInformation and in notebook format.31

Construction of Single-Gene Expression Vectors.Genes and TPs in pYPKa were PCR-amplified with vector-specific standard primers (Table S1 and Figure 1B) flankingeach sequence. TPs cloned as promoters in ZraI andterminators in EcoRV and genes cloned in the AjiI site wereamplified with the primer pairs indicated in Table S1 andFigure 1B. Because the fragments were cloned in the samevector at slightly different positions, the PCR products share30−50 bp of flanking homology. The PCR products werecotransformed in yeast with pYPKpw that had been previouslylinearized with ZraI, FspAI, or EcoRV, which all cut within a 14bp region located in the CRP sequence derived from pYPKa.This resulted in a recombination among the four DNAfragments (Figure 1B,C). The resulting expression vectorswere confirmed by diagnostic colony PCR. The frequency ofcorrect assembly observed in this construction step was close to90% (results not shown).Rational Pathway Design. The construction of multigene

pathways was done using a strategy similar to that for theassembly of the single-gene cassettes. The first cassette in thesequence was amplified with primers 577 and 778 (Figure 1Band Table S1), where primer 577 anneals at a distance awayfrom the cassette to incorporate a stretch of plasmid backbonein the 5′ part of the PCR product (Figure 1E, diagonally stripedbox). Each subsequent cassette, excluding the last, wasamplified using vector specific primers 775 and 778 that annealvery close to the cassette. Finally, the last cassette was amplifiedwith primers 775 and 578 that incorporate a stretch of theplasmid backbone at the 3′ end (Figure 1E, vertically stripedbox). Cassette PCR products were cotransformed in yeast with

linearized pYPKpw vector for in vivo homologous recombina-tion into a multigene pathway (Figure 1E).

Physiological Characterization. Cultures were inoculatedand grown for 12 h in 10 mL of defined medium with 20 g/Lglucose at 30 °C; cells were washed and used to inoculate 10mL of defined medium with 20 g/L D-xylose. This culture wasgrown at 30 °C for 12 h. Cells were then inoculated in 50 mLof defined medium with 20 g/L D-xylose at an initial OD640nm of0.05 in a 250 mL shake flask and incubated at 30 °C withshaking at 200 rpm. The OD640nm was measured every hour,and the maximum specific growth rate was calculated duringthe exponential growth phase. Fermentations under oxygen-limited conditions were performed as follows: cells wereinoculated and grown in 50 mL of defined medium with 20 g/Lglucose at 30 °C and then washed and used to inoculate 100mL of YNB medium with 20 g/L D-xylose at an initial OD640nmof 1 in a 100 mL unbaffled shake flask that was incubated and30 °C with shaking at 200 rpm. Every 24 h, 1.5 mL of culturewas collected for dry weight biomass measurement and HPLCanalyses of substrates and products.Samples were centrifuged for 3 min at 5000 rpm, and 1 mL

of the supernatant was filtered and analyzed by HPLC. D-Xylose, xylitol, glycerol, acetic acid, and ethanol were quantifiedusing an HPLC column (Rezex RHM-H+, 300 × 7.8 mm orRezex ROA-H+ (8%), 300 × 7.8 mm) and UV (210 nm,organic acids) detection. A mobile phase of 2.5 mM H2SO4 wasused at a flow rate of 0.5 mL/min, and the column temperaturewas 60 °C. The mass of CO2 produced was calculated based onthe initial and final weights of each culture and took intoaccount the weight of samples that had been removed.

ypkpathway Software. The ypkpathway software is agraphical tool specifically designed for planning YPK pathwayassembly experiments. The ypkpathway software is available forthe Windows, MacOSX, and Linux platforms, and a graphicalinstaller is provided. A manual with installation instructions andexamples is available in the Supporting Information. ypkpath-way was implemented using the python programming languagetogether with the pydna14 software package, which in turndepends on the widely used Biopython package.37 Thegraphical user interface was implemented using the PyQt4graphical user interface library.38 The Jupyter notebookgeneration’s logic depends on the software packages Jupyter13

and notedown.39

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssynbio.5b00250.

Cloning strategies for vectors described in the text; tablescontaining primers, plasmids, vectors, genes cloned, anddrop-out supplement mixture composition; and themanual for ypkpathway software, including installationinstructions (PDF)Example text files containing raw data for pathwayassembly (ZIP)

■ AUTHOR INFORMATION

Corresponding Author*Phone: +351-253-60-1517. Fax: +351-253-67-8980. E-mail:bjorn_johansson@bio.uminho.pt.

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Present Address§(N.S.P.) Departamento de Biologia Celular, Instituto deCiencias Biolo gicas, Universidade de Brasilia, 70790-190Brasilia-DF, Brazil.

Author ContributionsB.J. supervised and planned the overall work. F.P., N.P., B.H.-H., M.F.G.-G., and B.J. conceived the design of the experi-ments. F.P., F.A., and N.P. conducted experiments. B.J. codedthe ypkpathway software. B.J., F.P., N.P., F.A., B.H.-H., andM.F.G.-G. wrote the manuscript.

NotesThe authors declare no competing financial interest.Contact the corresponding author to obtain pYPKa vectors.

■ ACKNOWLEDGMENTSThis work was supported by the Fundacao para a Ciencia eTecnologia Portugal (FCT) through Project MycoFat PTDC/AAC-AMB/120940/2010. F.A. was supported by FCT fellow-ship SFRH/BD/80934/2011. CBMA was supported by thestrategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCTI.P. and by the ERDF through the COMPETE2020 - ProgramaOperacional Competitividade e Internacionalizacao (POCI).The authors wish to thank to Dr. Paula Goncalves for thepGXF1 vector, Dr. Daniel Schlieper for the pCAPs vector, Dr.Yukio Nagano for the pSU0 vector, Dr. Peter Kotter, Universityof Frankfurt, Germany, for the S. cereivise CEN.PK strains, andDr. Nina Q. Meinander for the p4** vectors.

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