Download Yeast Pathway Kit: A Method for Metabolic

Research Article
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Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with
Automatically Simulated Executable Documentation
Filipa Pereira,† Flávio Azevedo,† Nadia Skorupa Parachin,‡,§ Bar̈ bel Hahn-Hag̈ erdal,‡
Marie F. Gorwa-Grauslund,‡ and Björn Johansson*,†
†
CBMACentre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, Braga
4710-057, 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) for
rational and random metabolic pathway assembly in Saccharomyces
cerevisiae using reusable and redistributable genetic elements. Genetic
elements are cloned in a suicide vector in a rapid process that omits PCR
product purification. Single-gene expression cassettes are assembled in vivo
using genetic elements that are both promoters and terminators (TP).
Cassettes sharing genetic elements are assembled by recombination into
multigene pathways. A wide selection of prefabricated TP elements makes
assembly both rapid and inexpensive. An innovative software tool
automatically produces detailed self-contained executable documentation
in the form of pydna code in the narrative Jupyter notebook format to
facilitate planning and sharing YPK projects. A D-xylose catabolic pathway
was created using YPK with four or eight genes that resulted in one of the
highest 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 improve
the 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
M
etabolic engineering of Saccharomyces cerevisiae has been
applied for the production of a wide range of fuels and
chemicals (for review, see refs 1 and 2). Engineering of
production strains usually requires the expression of a
considerable number of genes because it is rare for a single
enzyme to exert considerable control over a trait, such as flux
along a metabolic pathway. The need to express multiple genes
has led to the application of techniques that allow the
simultaneous assembly of multiple promoters, genes, and
terminators into metabolic pathways. Among the available
techniques are the Gibson assembly protocol,3 which is a
general technique for enzymatic assembly in vitro, and many
variations of in vivo assembly by homologous recombination
between flanking sequences added by PCR.4,5 Gene copy
number,6 promoter strength,7 and positional effects of
individual genes in a pathway8 may affect efficiency, but
rationally designing and testing all permutations can be
infeasible for longer pathways. Alternatively, a pool of randomly
assembled pathways can be created from which the best
performing pathways can be selected based on a screening
strategy. Protocols for random pathway assembly engineering
based on in vivo homologous recombination,9,10 Gibson
assembly methods,11 or both12 have also been described.
Common for most protocols is that genetic parts such as
promoters and terminators are not easily shared and reused
© 2016 American Chemical Society
because they are usually PCR products from chromosomal
DNA or other sources and, as such, cannot be propagated.
Most assembly protocols are “all or nothing” in the sense that
multiple genes and regulatory sequences are joined in one
reaction. Strategy or implementation errors such as a faulty
PCR primer will yield little information to pinpoint the error as
no pathway will be created. Furthermore, published pathway
assembly protocols are designed for either rational or random
assembly, but to do both requires reamplifying the genetic parts
with new PCR primers.
In this work, we have developed an alternative pathway
assembly approach, called the Yeast Pathway Kit (YPK). YPK
differs from previous methods in that promoters, genes, and
terminators are cloned in one of three closely spaced blunt
restriction sites in pYPKa, a highly efficient Escherichia coli
positive selection vector designed for the rapid cloning of
unpurified PCR products. Episomal yeast single-gene expression vectors are constructed from these basic elements by
in vivo gap repair in S. cerevisiae three at a time in a promoter−
gene−terminator configuration. This assembly is directed by
Received: November 25, 2015
Published: February 25, 2016
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DOI: 10.1021/acssynbio.5b00250
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■
Research Article
RESULTS AND DISCUSSION
YPK Strategy. The YPK metabolic pathway assembly
strategy takes advantage of the observation that natural
intergenic sequences from genes expressed in tandem are
both terminators of the upstream gene and promoters for the
downstream gene. These intergenic sequences (designated
terminator−promoters or TPs) are used both for transcription
regulation and for aiding the assembly of multiple gene
metabolic pathways from single-gene expression cassettes. TPs
from 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 the
expression of heterologous proteins in S. cerevisiae, except for
ribosomal protein promoters RPS19b and RPS19a. The TPs
were first cloned into the ZraI or EcoRV site of the pYPKa
suicide vector (Figure 1A). Genes to be expressed were cloned
short overlaps of plasmid backbone sequences between the
three cloning sites.
The single-gene expression vectors are subsequently joined
into pathways by homologous recombination between
promoters and terminators of each cassette. This can be done
by the pairwise use of the same sequence as a promoter or
terminator in the two vectors. The YPK promoters and
terminators are intergenic sequences from tandemly expressed
S. cerevisiae genes that naturally serve both as terminators and
promoters (terminator−promoters or TPs) of the two adjacent
genes. This second stage of the assembly is directed by the
relatively long TPs of each cassette (500−1300 bp). YPK
provides reusable genetic elements at several levels in the
assembly process as there is an E. coli vector for each genetic
element that is easily verified, stored, propagated, and
distributed. The single-gene yeast expression vectors constitute
a second level of reusable genetic elements as well as a way to
study and verify each gene expression cassette separately.
Assembly and verification of pathways can be performed using
only two specific primers per gene together with a set of eight
short (<31-mer) standard PCR primers (Table S1). The TP
and gene components can be randomly assembled by leaving
out the single-gene expression vector assembly step, thus
allowing the random combination of promoters genes and
terminators into multigene pathways. Unique to the YPK
pathway approach is an innovative specifically designed
software tool called ypkpathway that can automatically simulate
the assembly process and generate correct and complete
executable documentation of complex constructs in the
narrative Jupyter notebook format.13 Jupyter notebooks are
both documentation and executable code containing a
simulation of the assembly and cloning strategies using
pydna.14 The notebooks provide information such as the
sequences of intermediate plasmids, automatically designed
primers, and simulated PCR conditions. The notebook format
permits strategy changes (such as altered PCR primers) to be
incorporated in existing documentation by editing and
executing the notebook. This type of documentation allows
verification of the correctness of an assembled pathway and the
efficient sharing and communication of strategies.
The YPK method was validated by both rational and random
assembly of several fungal D-xylose metabolic pathways. DXylose is the most abundant pentose sugar in lignocellulosic
biomass, but it cannot be directly metabolized by S. cerevisiae,
so genetic engineering of S. cerevisiae to add this capacity15 has
biotechnological importance. Using YPK, it was possible to
rationally assemble four or eight active D-xylose metabolic genes
in one step. Executable Jupyter notebook documentation is
provided for each pathway, including all intermediary steps,
facilitating the complete reproduction of the genetic constructs.
Two pathways were also randomly assembled, resulting in
pathways with different properties. Rationally assembled
metabolic pathways supported a specific growth rate of 0.18
h−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 The
yield of ethanol under oxygen-limited conditions reached 0.46 g
ethanol/g of D-xylose consumed, which is 90% of the
theoretical yield. YPK proved to be a practical, fast, and
efficient protocol for pathway assembly.
Figure 1. Schematic representation of the YPK strategy for rational
assembly. The figure depicts the construction of a two-gene expression
pathway using YPK. Six DNA fragments are cloned into the
multicloning site (MCS) of the pYPKa vector (A colored boxes),
resulting in six different E. coli vectors (A). The unique restriction sites
ZraI, AjiI, and EcoRV are separated by 50 bp (red box) and 31 bp
(green box). The blue and yellow boxes represent areas ∼20 bp
upstream of the ZraI site and downstream of the EcoRV site. The
striped boxes represent plasmid backbone sequences upstream and
downstream of the MCS. Vector-specific primers (B) are used to
amplify six PCR product, which recombine into two single-gene
expression vectors (C, D). The single-gene expression cassettes are
joined in a second recombination stage to form the final pathway
vector (E). The first and last cassettes are amplified with primers
flanking the MCS so that extra plasmid backbone sequences are added
(diagonally and vertically striped boxes). pYPKpw is a version of
pYPK0 that lacks the MCS to avoid unwanted recombination.
in the AjiI site of pYPKa. Promoters, genes, and terminators
were amplified with primers specific to the pYPKa plasmid
backbone (Figure 1B). Single -gene expression cassettes were
formed by homologous recombination between the PCR
products and the pYPK0 S. cerevisiae vector (Figure 1C,D).
The single-gene expression cassettes can recombine into
multiple gene pathways, provided that they share a TP as a
promoter in one cassette and a terminator in the other (Figure
1E).
Promoter Activity Measurement. The relative strength
of the TPs cloned as promoters was evaluated by measuring the
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between promoters than were observed here.18 Some of this
difference may be attributed to the technical differences
between the methods used. Another source of the observed
differences could be the influence of the different terminator
sequences.19
Construction of a Four-Gene D-Xylose Metabolic
Pathway in S. cerevisiae. S. cerevisiae is able to use D-xylose
as the sole carbon source when expressing heterologous xylose
reductase (XR) and xylitol dehydrogenase (XDH) from
Scheffersomyces stipitis.20 Overexpression of endogenous xylulokinase (XKS1)21,22 and the pentose phosphate pathway enzyme
transaldolase (TAL1)23 further increased the D-xylose metabolic
rate. A four-gene metabolic pathway was assembled using S.
stipitis genes XYL1 (XR) and XYL2 (XDH) and S. cerevisiae
genes XKS1 (XK) and TAL1. Each of the four genes was first
assembled into single-gene expression vectors: pYPK0_TEF1_XR_TDH3, pYPK0_TDH3_XDH_PGI1, pYPK0_PGI1_XK_FBA1, and pYPK0_FBA1_TAL1_PDC1 (gene
name in bold) by the same principle as that depicted in Figure
1C,D. The single-gene cassettes were assembled into a pathway
vector 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. An
identical pathway but for a point mutation in the XYL1 gene
(N272D) was also assembled and designated pMEC1135. This
mutation results in a preference for NADH over NADPH for
XR.24 Both pathway vectors were verified by restriction
digestion. The frequency of correct recombination was
calculated for the final construct, and 85% of the tested clones
had the expected size and restriction pattern. A S. cerevisiae
strain carrying the vector with wild-type XR (pMEC1136)
showed a slightly faster specific growth rate of 0.18 h−1 than
that of the strain containing XR carrying a point mutation
(Table 1, two first rows). However, the strain with wild-type
expression level of the HIS3 reporter gene. Promoter strength
was deduced from the His3p protein level detected as resistance
to 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-type
background (Figure 2). Resistance was measured by spotting
Figure 2. Terminator−promoters assay. S. cerevisiae CEN.PK 113-11C
(ura3, his3) was transformed with the indicated constructs, except
where CEN.PK 113-5D (ura3, HIS3) is explicity stated. A series of
dilutions (1, 10, 100, and 1000×) from an initial OD640nm of 0.1 were
spotted on defined solid media with 20 g/L of glucose as the sole
carbon source that was supplemented with uracil containing 3-AT (0,
60, 150, or 200 mM) and incubated at 30 °C for 3 days. Solid media
without 3-AT that was supplemented with histidine was also used to
control cell growth (first column).
four different dilutions of cells at each 3-AT concentration. The
first eight rows of Figure 2 contain strains expressing the HIS3
gene expressed from TPs TEF1, TDH3, PGI1, FBA1, PDC1,
RPS19b, RPS19a, and TPI1. The strength of the promoters
showed little difference except for PGI1 and TPI1, which
showed slightly lower activity, as can be seen from the reduced
cell growth in the rightmost column at 200 mM 3-AT for these
promoters. The RPS19b and RPS19a promoters showed a
relative strength comparable to that of the widely used TDH3
and TEF1 promoters, indicating that these promoters are useful
for protein (over)expression in S. cerevisiae. The single wildtype HIS3 gene in S. cerevisiae CEN.PK 113-5D supported
growth only up to 60 mM 3-AT. Plasmids p426TDH3_HIS3
and p426TEF1_HIS3 (Figure 2) contain the HIS3 gene under
the control of the promoter indicated by the plasmid’s name
(TDH3 and TEF1, respectively). These vectors are from a
widely used vector set17 and show similar or lower activity
compared to that of the same promoters in the YPK single-gene
constructs (Figure 2, first two rows). This shows that the
plasmid backbone sequences that are incorporated between the
TP serving as promoter and the gene (Figure 1, red boxes) or
between the gene and the TP serving as terminator (Figure 1,
green boxes) do not seem to negatively affect gene transcription or translation levels. Low-copy-number vectors
(p413TEF and pYPK3_RPS19b_HIS3_RPS19a) show markedly 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 similar
levels of 3-AT resistance. pYPK0_TDH3_rev_HIS3_ ENO2
has an inverted promoter and a low activity compared to that of
pYPK0_TDH3_rev_HIS3_ PGI1, indicating the importance of
promoter orientation in the construct. Measuring promoter
strength by GFP fluorescence has produced larger differences
Table 1. Specific Growth Ratesa
vector
pMEC1135
pMEC1136
pMEC1138
pMEC1139
pMECRandom1
pMECRandom4
growth rate
(μmax; h−1)
pathway
XR(N272D), XDH, XK, and TAL1
XR, XDH, XK, and TAL1
XR(N272D), XDH, XK, TAL1, TKL1,
RPE1, RKI1, and GXF1
XR, XDH, XK, TAL1, TKL1, RPE1,
RKI1, and GXF1
XR, XK, XDH, and TAL1
XR, XK, XDH, and TAL1
0.17 ± 0.01
0.18 ± 0.02
0.16 ± 0.01
0.18 ± 0.01
0.18 ± 0.01
0.15 ± 0.01
a
Aerobic maximum specific growth rate (μmax) with 20 g/L xylose as
the sole carbon source in submerged culture for S. cerevisiae strain
CEN.PK 113-5D transformed with the indicated vector. The medium
was defined with complete amino acid supplement. Results are given
with standard deviation obtained from three independent experiments.
XR consumed less D-xylose and had lower ethanol and higher
xylitol yields (Table 2). pMEC1136 is genetically comparable
to strain TMB3014,23 which has the same four genes
overexpressed in constructs that are chromosomally integrated.
The strain with pMEC1136 consumed almost 2 times the
amount of D-xylose as that of TMB3014 during the
fermentation, suggesting that having a higher copy number of
one or more of the genes involved is beneficial for pathway
performance.
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Table 2. Xylose Consumption and Product Formationa
yield (g/g of D-xylose consumed)
pathway
pMEC1135
XR (N272D), XDH, XK, and TAL1
pMEC1136
XR, XDH, XK, and TAL1
pMEC1138
XR (N272D), XDH, XK, TAL1, TKL1, RPE1, RKI1 and
GXF1
pMEC1139
XR, XDH, XK, TAL1, TKL1, RPE1, RKI1, and GXF1
pRandom1
XR, XK, XDH, and, TAL1
pRandom4
XR, XK, XDH, and TAL1
TMB3014
XR, XDH, XK, and TAL1
xylose consumed
(g/L)
biomass
ethanol
xylitol
glycerol
acetic
acid
ethanol
(g/L)
%C
recovery
18.55
0.001
0.46
0.15
0.08
0.008
8.52
107
17.57
0.001
0.30
0.37
0.11
0.008
5.18
103
18.55
0.002
0.46
0.14
0.07
0.008
8.48
109
16.40
0.001
0.31
0.35
0.11
0.006
5.00
105
14.15
0.001
0.38
0.16
0.13
0.007
5.35
105
18.90
0.004
0.42
0.23
0.06
0.012
6.29
106
9.86
0.002
0.29
0.39
0.04
0.010
2.82
96
a
Xylose consumption and product formation are shown for recombinant S. cerevisiae strains after 206 h of oxygen-limited fermentations with 20 g/L
xylose as the sole carbon source in minimal medium. Displayed values are the average of biological triplicates.
Construction of an Eight-Gene D-Xylose Metabolic
Pathway. An eight-gene D-xylose consumption pathway was
constructed in order to further test the flexibility and capacity of
YPK. The pathway had the same initial genes and TPs in the
same order as those in the previously described four-gene
pathway. Additionally, S. cerevisiae genes TKL1, RPE1, and
RKI1 encoding pentose phosphate pathway enzymes and the
Candida intermedia GXF1 gene encoding a D-xylose transporter
were included as they all have been linked to enhanced D-xylose
utilization.23,25,26 The resulting pathway was given the systematic name pYPK0_TEF1_XR_TDH3_XDH_PGI1_XK_FBA1_TAL1_PDC1_TKL1_RPS19b_RPE1_RPS19a_RKI1
_TPI1_GXF1_ENO2 and the shorter alias pMEC1139. The
systematic name reflects the genes and TPs and the order in
which they appear in the vector. An identical pathway
containing the mutant XR gene was named pMEC1138. The
last four genes in the pathway were also joined together in the
separate vector, pYPK0_PDC1_TKL1_RPS19b_RPE1_RPS19a_RKI1_TPI1_GXF1_ENO2, with the shorter alias
pMEC1137.
An eight-gene pathway was also constructed and confirmed
by a different strategy. The pMEC1136 vector was linearized
after the last TP using restriction endonuclease PacI (Figure
1E) and recombined with a DNA fragment containing the four
genes of pMEC1137. This shows that it is feasible to extend
and combine pathways to circumvent, for example, local
construction difficulties, adding flexibility to the YPK system.
Growth rates, D-xylose consumption, and production of
biomass, glycerol, acetic acid, ethanol, CO2, and xylitol were
also evaluated for yeast strains carrying the eight-gene
pathways. The growth rates observed for strains with eightgene pathways (pMEC1139 and pMEC1138) were comparable
or slightly lower than the ones found for strains with four-gene
pathways (Table 1). The N272D mutation in XR caused a
slight reduction in growth rate, as was also observed for the
four-gene pathways. No significant differences were observed
for D-xylose consumption or xylitol or ethanol production
(Table 2) between the two strains with either the eight- or fourgene pathway expressing the same version of XR. These results
indicated that the additional overexpression of RPE1, RKI1,
TKL1, or GXF1 is not necessary to reach the optimal growth
rate and ethanol yields under the tested conditions and also
indicated that the increased metabolic burden of the four extra
genes does not seem to affect cell metabolism negatively.
Random Assembly of D-Xylose-Utilizing Pathways. A
hierarchical two-step assembly strategy was used to ensure the
assembly of single-gene expression cassettes into a predefined
sequence. The assembly of several genes and TPs directly
would, in theory, allow a pool of vectors to be created that
differ in their gene identities, gene copy number, TP fragment,
and gene order. This pool might contain better performing
pathways that could be found by selection. This hypothesis was
tested by constructing a randomly assembled D-xylose-utilizing
pathway using the same building blocks as those for the
rationally 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 18
DNA fragments (two PCR products per TP) were transformed
together with linear pYPK0 vector. Transformants were
incubated for 4 h (short recovery) or 24 h (long recovery) in
nonselective liquid glucose medium to increase the chance of
recovering transformants. One transformant was obtained after
a short period of recovery and 4 days of growth on medium
with D-xylose as the sole carbon source, whereas several clones
were obtained after a long period of recovery. Plasmid DNA
from the largest colony from each recovery period was
extracted, transferred to E. coli, and designated pMECRandom1
and pMECRandom4. Vector pMECRandom4 was analyzed by
a combination of restriction digestion, DNA sequencing, and
analytical 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 had
two copies of XR, controlled by the PGI1 and PDC1
promoters, and the PDC1 promoter was present twice. The
presence of multiple copies of XR might reflect a selective
advantage or result in subtle differences in the relative amount
of 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, pMECRandom1
transformants had a specific growth rate equal to that of the
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Figure 4. Graphical user interface of ypkpathway. ypkpathway uses a
list of sequences as input and produces a folder containing sequence
files and Jupyter notebooks.
Figure 3. Schematic representation of random pathway assembly.
Terminator−promoters (TPs) (A) similar to the ones described in
Figure 1A are assembled directly without prior assembly into singlegene expression vectors (Figure 1B,C). (B) One of many possible
ways that sequences can recombine together, resulting in differences in
gene copy number, gene order, and combination between genes and
TPs.
strain containing pMEC1136, whereas the strain with
pMECRandom4 showed the slowest growth rate of all strains
tested (Table 1). The pRandom4 transformants consumed
more D-xylose than any other strain, but they also produced
more xylitol (Table 2). The higher D-xylose consumption rate
and xylitol yield are consistent with increased XR activity.8,27
The two randomly assembled pathways displayed both the
highest and lowest D-xylose consumption rates of all pathways
constructed, combined with relatively high xylitol and low
ethanol yields. This result may reflect a limitation of the
selection strategy since these pathways were selected for
aerobic growth on D-xylose and not for ethanol productivity or
yield. The random assembly strategy has a high degree of
freedom in the way promoters, genes, and terminators can
combine. This increases the number of testable combinations
but also leads to a low yield of pathways conferring the desired
phenotype, which is reflected in the low number of random
pathways obtained.
Ypkpathway Software and Executable Documentation. The planning required to create large multigene
assemblies is a daunting task regardless of the assembly
protocol used. Often, dozens of PCR primers need to be
designed, where one error will impede the entire construction.
The ypkpathway software that accompanies the YPK can aid in
the planning of complex assembly projects using a text file
containing the promoter, gene, and terminator sequences in
FASTA or Genbank text format in the order in which they
should be assembled (Figure 4). ypkpathway automatically
generates a series of Jupyter notebook13 files, which are
interactive computational environments that combine executable Python28 code, rich text, figures, and hyperlinks. The
Jupyter notebook format is rapidly gaining traction for scientific
computation as it makes code and analysis easily accessible.29
The pathway construction process is described in these
notebooks using the recently developed pydna14 package for
cloning and assembly simulation. Figure 5 shows the header of
a Jupyter notebook file describing a four-gene D-xylose
Figure 5. Output of ypkpathway. ypkpathway generates a series of
Jupyter 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, the
notebooks contain automatically generated figures describing homologous recombination steps. The notebooks can be modified, executed,
and distributed without the need for ypkpathway software.
metabolic pathway generated by ypkpathway. Upon execution,
each notebook simulates the construction of a pathway or an
intermediate vector and produces metadata such as primer
sequences, PCR conditions, and a download link in the end of
the document providing the final sequence. The nontrivial
simulation of homologous recombination is done entirely
without assumptions other than the primary sequence of the
fragments using the graph-theory-based algorithm provided by
pydna.
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 thought
processes underpinning the result. The rudimentary knowledge
of python needed to follow the cloning strategies is curently
part of most relevant university curricula. These protocols can
be easily shared on the web as static versions, where they can be
viewed using only a web browser.30 They can also be shared as
collections of interactive papers that let others repeat the
cloning simulation for verification or even alter parameters or
input 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 on
Github as a self-contained repository that can be forked or
downloaded as a compressed folder.31 The same files can be
viewed online as static files through the free nbviewer Jupyter
notebook rendering service.30
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Comparison between YPK and Existing Methods. The
YPK protocol offers advantages over the previously described
“DNA assembler” method5 and Gibson3 assembly for certain
purposes. YPK uses a set of short standard primers for PCR
amplification prior to assembly, whereas DNA assembler and
Gibson assembly require PCR primers with long tails that have
to be specifically made for each promoter−gene−terminator
combination. A recently described toolkit shares the
hierarchical assembly of single-gene expression vectors into
multigene pathways with YPK.32 The MoClo restriction
enzyme strategy is used for assembly, which is faster because
E. coli is used as the host for intermediate constructs instead of
S. cerevisiae. However, purified DNA is required for the
assembly, which may be a disadvantage in some cases. Gibson
assembly was modified with shared linker sequences introduced
by PCR using long PCR primers.11,12 The COMPACTER
protocol9 is a development of DNA assembler to join a fixed
gene set where each gene is under the control of a different
promoter from a promoter library. A helper plasmid is required
for each gene in the pathway, which takes two consecutive cutand-paste cloning steps to construct. Standard cloning vectors
without positive selection were used, which makes vector
construction considerably more laborious when compared with
that using positive selection vector pYPKa. Still, unlike YPK,
this method does not produce variations in gene copy number
or gene order.
Final Remarks. Strains carrying the YPK D-xylose pathways
display a growth rate comparable to the highest found in the
literature for D-xylose-adapted strains.16 Notably, the strain
presented in this work was not subjected to adaptation. The
present pathways differ from previous designs in that they
maintain a high copy number of all expressed genes, which may
explain the high growth rates. Promoter, gene, and terminator
are each separated by 50 or 31 bp pYPKa-derived plasmid
backbone sequence in the single-gene expression constructs.
These two sequences are each repeated once for every gene in a
multigene pathway and could possibly provide sites of
intramolecular recombination, although such recombination
events have not been observed for any pathway made with
YPK.
The provided executable documentation in the form of
Jupyter notebooks simulating the genetic constructions
describes every step in the cloning process and enables the
complete reproduction of the genetic constructs, requiring no
additional information. The ypkpathway software can be used
to automatically generate such documentation for new
pathways. Since the conclusion of this work, the TP library
has been expanded to 30 unique sequences, theoretically
allowing the assembly of an equal number of genes. Pathways
with up to 15 genes have been constructed by our group with
the same efficiency as that for the eight-gene pathways
described in this work. YPK proved to be a very efficient tool
for the generation of synthetic metabolic pathways that have
already beem used to create industrial D-xylose-fermenting yeast
strains.33
ampicillin. The S. cerevisiae strains CEN.PK 113-5D (ura3) and
CEN.PK 113-11C (ura3, his3) were used as host strains for
genetic engineering. Yeast cells were cultivated in YPD (20 g/L
peptone, 10 g/L yeast extract, and 20 g/L glucose) medium or
defined medium containing 6.7 g/L Difco yeast nitrogen base
without amino acids (YNB w/o aa) with 20 g/L glucose or 20
g/L D-xylose. Different concentrations of 3-AT were added to
defined medium for promoter activity assays. For physiological
studies and growth rate measurements, 2 g/L amino acid dropout mix containing 20 amino acids (Formedium, UK) was
added to the defined medium. The exact composition of the
drop-out mix is given in Table S2. For selection of dominant
markers, YPD medium was supplemented with 300 mg/L
Geneticin (kanMX4 marker), hygromycin (hphMX4 marker),
or phleomycin (bleMX4 marker), as required. Yeast strains and
bacterial 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(ethylene
glycol), lithium acetate, and single-stranded carrier DNA.35
Final pathway vectors constructed in this work are listed in
Table 3. All vectors used for construction are listed in Table S2.
Table 3. Pathway Vectors Constructed in This Worka
plasmid
description
pMEC1135
pYPK0-TEF1-SsXYL1(N272D)-TDH3-SsXYL2-PGI1ScXKS1- FBA1-ScTAL1-PDC1, URA3, 2μ
pYPK0-TEF1-SsXYL1-TDH3-SsXYL2-PGI1-ScXKS1-FBA1ScTAL1-PDC1, URA3, 2μ
pYPK0-PDC1-ScTKL1-RPS19b -ScRPE1-RPS19a-ScRKI1- I1CiGXF1-ENO2, URA3, 2μ
pYPK0-TEF1-SsXYL1(N272D)-TDH3-SsXYL2- PGI1ScXKS1-FBA1-ScTAL1-PDC1-ScTKL1-RPS19b-ScRPE1RPS19a-ScRKI1-TPI1-CiGXF1-ENO2, URA3, 2μ
pYPK0-TEF1-SsXYL1-TDH3-SsXYL2-PGI1-ScXKS1-FBA1ScTAL1-PDC1-ScTKL1-RPS19b-ScRPE1-RPS19a-ScRKI1TPI1-CiGXF1-ENO2, URA3, 2μ
pMEC1136
pMEC1137
pMEC1138
pMEC1139
a
See Tables S3−S6 and the Jupyter notebook documentation for a
complete list of constructed vectors.
Plasmids constructed as a part of this work are listed in Tables
S4 and S5. The Supporting Information contains detailed
descriptions of the construction of selected vectors, and the
Jupyter notebook documentation contains details of the
construction of all vectors.
Construction of the pYPKa Vector. The positive
selection vector pCAPs34 has a toxic mutant version of the
CRP gene encoding the cyclic AMP receptor protein. The toxic
gene is disrupted when DNA fragments are cloned within the
open reading frame of the toxic gene. The pCAPs vector was
amplified with primers 567 and 568 (Table S1 and Figure 1B),
introducing a partial AjiI restriction site on each end of the
linear PCR product. The PCR product was treated with T4
polynucleotide kinase and T4 DNA ligase and subsequently
transformed into E. coli BW26356. In the resulting pYPKa
vector, the AscI site was silently replaced by an AjiI restriction
site between the ZraI and EcoRV restriction sites in the CRPs
gene. The structure of the vector was confirmed by restriction
analysis, and the toxicity of the uninterrupted CRPs gene was
confirmed by transforming sensitive E. coli XL1-Blue.
Cloning of Genetic Elements in pYPKa. Intergenic
regions from tandemly expressed genes in S. cerevisiae
containing TPs were chosen from a set commonly used for
protein expression in the literature (TEF1, TDH3, PGI1, FBA1,
PDC1, TPI1, and ENO2) as well as from two ribosomal protein
■
METHODS
Strains, Plasmids, and Cell Cultivation. E. coli XL1-Blue
(Stratagene) was used to propagate plasmids except for
plasmids with a CRPs gene,34 for which the cyaA mutant E.
coli strain BW26356, obtained from Coli Genetic Stock Center
CGSC, was used. E. coli transformants were selected on solid
lysogeny broth (LB) medium supplemented with 100 mg/L
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ACS Synthetic Biology
linearized pYPKpw vector for in vivo homologous recombination into a multigene pathway (Figure 1E).
Physiological Characterization. Cultures were inoculated
and grown for 12 h in 10 mL of defined medium with 20 g/L
glucose at 30 °C; cells were washed and used to inoculate 10
mL of defined medium with 20 g/L D-xylose. This culture was
grown at 30 °C for 12 h. Cells were then inoculated in 50 mL
of defined medium with 20 g/L D-xylose at an initial OD640nm of
0.05 in a 250 mL shake flask and incubated at 30 °C with
shaking at 200 rpm. The OD640nm was measured every hour,
and the maximum specific growth rate was calculated during
the exponential growth phase. Fermentations under oxygenlimited conditions were performed as follows: cells were
inoculated and grown in 50 mL of defined medium with 20 g/L
glucose at 30 °C and then washed and used to inoculate 100
mL of YNB medium with 20 g/L D-xylose at an initial OD640nm
of 1 in a 100 mL unbaffled shake flask that was incubated and
30 °C with shaking at 200 rpm. Every 24 h, 1.5 mL of culture
was collected for dry weight biomass measurement and HPLC
analyses 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. DXylose, xylitol, glycerol, acetic acid, and ethanol were quantified
using an HPLC column (Rezex RHM-H+, 300 × 7.8 mm or
Rezex ROA-H+ (8%), 300 × 7.8 mm) and UV (210 nm,
organic acids) detection. A mobile phase of 2.5 mM H2SO4 was
used at a flow rate of 0.5 mL/min, and the column temperature
was 60 °C. The mass of CO2 produced was calculated based on
the initial and final weights of each culture and took into
account the weight of samples that had been removed.
ypkpathway Software. The ypkpathway software is a
graphical tool specifically designed for planning YPK pathway
assembly experiments. The ypkpathway software is available for
the Windows, MacOSX, and Linux platforms, and a graphical
installer is provided. A manual with installation instructions and
examples is available in the Supporting Information. ypkpathway was implemented using the python programming language
together with the pydna14 software package, which in turn
depends on the widely used Biopython package.37 The
graphical user interface was implemented using the PyQt4
graphical user interface library.38 The Jupyter notebook
generation’s logic depends on the software packages Jupyter13
and notedown.39
genes, RPS19a and RPS19b. The TPs were amplified from
chromosomal DNA of S. cerevisiae CEN.PK 113-5D and ligated
in pYPKa digested with blunt restriction enzyme ZraI
(promoters) and EcoRV (terminators), resulting in two
separate vectors. Figure 1A shows an example of pYPKa vector
construction. Genes RPE1 and RKI1 were amplified from
chromosomal or plasmid DNA and ligated in vitro with pYPKa
previously cut with blunt restriction enzyme AjiI. All other
genes were amplified by PCR with tailed primers adding the
necessary homology (Figure 1, red and green boxes). Plasmids
derived from pYPKa were designated pYPKa_L_XYZN, where
L indicates the restriction enzyme used (Z, A, or E) and XYZN
indicates the name of the promoter, gene, or terminator. Table
S5 lists the constructed pYPKa vectors.
Construction of the pYPK0 E. coli/S. cerevisiae Shuttle
Vector and Its Derivatives. The plasmid pSU036 contains
the yeast 2μ origin of replication sequence and URA3
auxotrophic selectable marker between the ampicillin resistance
(amp) gene and the pUC origin of replication.36 The sole
EcoRV site of this vector was removed, resulting in plasmid
pMEC1030 (Table S4). This plasmid was used to make
pYPK0, which is a yeast version of pCAPs34 containing the S.
cerevisiae URA3 auxotrophic marker and the 2μ origin of
replication using the strategy described for pSU0.36 The CRPs
gene was partially deleted, removing the ZraI, AjiI, and EcoRV
restriction sites and the surrounding sequence (colored boxes
in Figure 1). Three different versions of the pYPK0 and
pYPKpw E. coli/S. cerevisiae shuttle vectors were constructed
containing a single- or multicopy origin of replication and
hphMX4, kanMX4, and bleMX4 selection markers (Table S3).
Vector construction details are given in the Supporting
Information and in notebook format.31
Construction of Single-Gene Expression Vectors.
Genes and TPs in pYPKa were PCR-amplified with vectorspecific standard primers (Table S1 and Figure 1B) flanking
each sequence. TPs cloned as promoters in ZraI and
terminators in EcoRV and genes cloned in the AjiI site were
amplified with the primer pairs indicated in Table S1 and
Figure 1B. Because the fragments were cloned in the same
vector at slightly different positions, the PCR products share
30−50 bp of flanking homology. The PCR products were
cotransformed in yeast with pYPKpw that had been previously
linearized with ZraI, FspAI, or EcoRV, which all cut within a 14
bp region located in the CRP sequence derived from pYPKa.
This resulted in a recombination among the four DNA
fragments (Figure 1B,C). The resulting expression vectors
were confirmed by diagnostic colony PCR. The frequency of
correct assembly observed in this construction step was close to
90% (results not shown).
Rational Pathway Design. The construction of multigene
pathways was done using a strategy similar to that for the
assembly of the single-gene cassettes. The first cassette in the
sequence was amplified with primers 577 and 778 (Figure 1B
and Table S1), where primer 577 anneals at a distance away
from the cassette to incorporate a stretch of plasmid backbone
in the 5′ part of the PCR product (Figure 1E, diagonally striped
box). Each subsequent cassette, excluding the last, was
amplified using vector specific primers 775 and 778 that anneal
very close to the cassette. Finally, the last cassette was amplified
with primers 775 and 578 that incorporate a stretch of the
plasmid backbone at the 3′ end (Figure 1E, vertically striped
box). Cassette PCR products were cotransformed in yeast with
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssynbio.5b00250.
Cloning strategies for vectors described in the text; tables
containing primers, plasmids, vectors, genes cloned, and
drop-out supplement mixture composition; and the
manual for ypkpathway software, including installation
instructions (PDF)
Example text files containing raw data for pathway
assembly (ZIP)
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +351-253-60-1517. Fax: +351-253-67-8980. E-mail:
[email protected].
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ACS Synthetic Biology
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§
(N.S.P.) Departamento de Biologia Celular, Instituto de
́
Ciências Biológicas, Universidade de Brasilia,
70790-190
́
Brasilia-DF,
Brazil.
Author Contributions
B.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 experiments. F.P., F.A., and N.P. conducted experiments. B.J. coded
the ypkpathway software. B.J., F.P., N.P., F.A., B.H.-H., and
M.F.G.-G. wrote the manuscript.
Notes
The authors declare no competing financial interest.
Contact the corresponding author to obtain pYPKa vectors.
■
ACKNOWLEDGMENTS
This work was supported by the Fundaçaõ para a Ciência e
Tecnologia Portugal (FCT) through Project MycoFat PTDC/
AAC-AMB/120940/2010. F.A. was supported by FCT fellowship SFRH/BD/80934/2011. CBMA was supported by the
strategic programme UID/BIA/04050/2013 (POCI-01-0145FEDER-007569) funded by national funds through the FCT
I.P. and by the ERDF through the COMPETE2020 - Programa
Operacional Competitividade e Internacionalizaçaõ (POCI).
The authors wish to thank to Dr. Paula Gonçalves for the
pGXF1 vector, Dr. Daniel Schlieper for the pCAPs vector, Dr.
Yukio Nagano for the pSU0 vector, Dr. Peter Kötter, University
of Frankfurt, Germany, for the S. cereivise CEN.PK strains, and
Dr. Nina Q. Meinander for the p4** vectors.
■
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