Flexible and Versatile Strategy for the Construction of Large

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A Flexible and Versatile Strategy for Construction of Large Biochemical Pathways Yongbo Yuan, Erik Andersen, and Huimin Zhao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00117 • Publication Date (Web): 02 Sep 2015 Downloaded from http://pubs.acs.org on September 5, 2015

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ACS Synthetic Biology

Submitted to ACS Synthetic Biology, 7-17-2015

A flexible and versatile strategy for construction of large biochemical pathways

Yongbo Yuan1, Erik Andersen1, Huimin Zhao1,2,* 1

Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, 2

Departments of Chemistry, Biochemistry, and Bioengineering,

University of Illinois at Urbana-Champaign, Urbana, IL 61801

Keywords: synthetic biology, pathway engineering, ligase cycling reaction, DNA assembler, yeast homologous recombination

* To whom correspondence should be addressed. Phone: (217) 333-2631. Fax: (217) 333-5052. E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT Synthetic pathways and circuits have been increasingly used for microbial production of fuels and chemicals. Here we report a flexible and versatile DNA assembly strategy that allows rapid, modular and reliable construction of biological pathways and circuits from basic genetic parts. This strategy combines the automation friendly ligase cycling reaction (LCR) method and the high fidelity in vivo yeast-based DNA assembly method, DNA assembler. Briefly, LCR is used to pre-assemble basic genetic parts into gene expression cassettes or pre-assemble small parts into larger parts to reduce the number of parts, in which many basic genetic parts can be reused. With the help of specially designed unique linkers, all pre-assembled parts will then be directly assembled using DNA assembler to build the target constructs. As proof of concept, three plasmids with varying sizes of 13.4 kb, 24 kb, and 44 kb were rapidly constructed with the fidelity of 100%, 88% and 71%, respectively. The yeast strain harboring constructed 44 kb plasmid was confirmed to be capable of utilizing xylose, cellobiose and glucose to produce zeaxanthin. This strategy should be generally applicable to any custom-designed pathways, circuits, or plasmids.


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INTRODUCTION In metabolic engineering and synthetic biology, it is critical to rapidly and reliably construct a large number of metabolic pathways or genetic circuits of interest and screen for desired phenotypes1-3. Many DNA assembly methods have been developed to put basic genetic parts into gene expression cassettes4-6, however, the traditional restriction digestion and ligation based cloning method7 met with great difficulty in constructing complex and large DNA constructs containing multiple gene expression cassettes and intergenic components, especially in library constructions and design-build-test cycles8. Recently, a number of methods have been developed for one step assembly of multiple parts into pathways or circuits, such as restriction digestion based method9,

10

, and homologous recombination based Gibson assembly11,

12

and DNA

assembler method13. Although these methods have high assembly efficiency, they are not flexible and versatile because the basic genetic parts are all flanked by either restriction sites or homology arms, which often necessitates redesign of the same sequences for reuse in making different desired products. Therefore, there is an urgent need for a flexible and versatile strategy for DNA assembly in which most of the basic genetic parts are standardized and can be reused.

The newly developed ligase cycling reaction (LCR) method14 offers the opportunity to assemble basic DNA parts in a flexible and versatile manner. Designed DNA parts are linked in an ordered manner with the help of specific single strand bridging oligos which can be rapidly synthesized at low cost14. All DNA parts in LCR do not flank any additional sequences, so they can be repeatedly utilized in other pathway or circuit constructions without any modification. All of these basic standardized DNA parts, such as promoters, terminators, ribosome binding sites, and vector backbones could be massively pre-made and stored in a library. However, it is very



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difficult to construct plasmids of >20 kb using the LCR method because its assembly fidelity drop sharply when constructing plasmids of >12 kb14. In addition, LCR alone is not able to propagate the resulting assembled linear fragments or circular fragments. Thus, a PCR or E. coli transformation step is needed to amplify constructed DNAs. In contrast to the LCR method, the yeast homologous recombination based DNA assembler method has been demonstrated to be highly efficient and reliable in the construction of large pathways13 and even genomes11, 15-17. However, the DNA assembler method is not flexible and versatile in constructing large plasmids or circuits because a large number of basic genetic parts must be flanked by homology arms with their neighbors. In short, neither LCR nor DNA assembler alone is suitable for large-scale assembly of basic genetic parts into large plasmids of >20 kb.

In this work, we report a new strategy that combines LCR and DNA assembler for rapid construction of large biochemical pathways, circuits and plasmids. Four different pathways including the xylose utilizing pathway, cellobiose utilizing pathway, farnesyl pyrophosphate biosynthesis pathway (FPP module), and zeaxanthin biosynthetic pathway were evaluated individually and in combination. The LCR method was used to assemble basic genetic parts into gene expression cassettes or pre-assemble small DNA fragments into a bigger construct. The resulting gene expression cassettes or pre-assembled DNA fragments were then linked together by DNA assembler. Three plasmids with varying sizes of 13.4 kb, 24 kb, and 44 kb were rapidly constructed with the fidelity of 100%, 88% and 71%, respectively. All four pathways encoded in the 44 kb plasmid were confirmed to be functional.

RESULTS AND DISCUSSION


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Design of a new flexible and versatile strategy that combines LCR and DNA assembler In this study, we developed a flexible and versatile strategy for construction of large biochemical pathways. Common basic parts, like promoters and terminators, were pre-made and stored in a parts library. A set of helper vector backbones consisting of a pUC19 backbone that is only replicable in E. coli and flanked by a series of carefully designed unique linkers are also premade and stored in the parts library. The overall strategy comprises of the following main steps: (1) determine the genes and their order in a target pathway; (2) select an appropriate promoter/terminator pair from the pre-made library for each gene based on the strength of the promoter; (3) select a helper vector backbone flanked by two unique linkers (ULn-pUC19-ULn+1 combination) from the pre-made library based on the gene order in the target pathway. Once the helper vector for each gene expression cassette is designed, bridging oligos between parts will be determined and synthesized. The gene generated by PCR or gBlock must have two blunt ends and be 5’ phosphorylated. LCR is utilized to assemble each gene expression cassette which is typically smaller than 10 kb. PCR is then carried out to amplify the entire gene expression cassette by using the unique linkers as primers and the LCR product as template. All the resulting gene expression cassettes are then assembled to the final plasmid via the DNA assembler method (Figure 1, Table 1). In this study, totally 16 ULn-pUC19 backbone-ULn+1 combinations, 16 UL-pRS416 backbone-UL combinations, 16 yeast promoters, and 16 yeast terminators were pre-made and stored in the parts library (Table 1).

Evaluation of the assembly fidelity of LCR Because the DNA assembler method showed higher assembly fidelity14, 18 and we have extensive experience with the DNA assembler method13, 19-22, we decided to only evaluate the effectiveness



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of LCR for DNA assembly. A total of 9 arbitrarily selected DNA fragments of approximately 2 kb each and one 1.8 kb pUC19 vector backbone were used to construct a series of plasmids by varying the number of fragments to be assembled. As shown in Figure 2, the assembly fidelity was 100% when constructing plasmids smaller than 12 kb (6 fragments). The assembly fidelity was dropped sharply from 80% to 20% when 7 and 8 fragments were assembled. No correctly assembled plasmid was obtained in the construction of 18 kb and 20 kb plasmids (Figure S1ABC). In addition, when three gene expression cassettes from the xylose utilizing pathway (XR, XDH, XKS) were assembled with the pUC19 vector backbone to yield a plasmid of 13.3 kb, an assembly fidelity of 83% was obtained. Much lower assembly fidelities of 50 % and 17 % were observed when 2 and 3 more gene expression cassettes were added, respectively (Figure S2). These data show that LCR has near 100% assembly fidelity when constructing plasmids smaller than 12 kb but its fidelity dropped dramatically when constructing plasmids larger than 12 kb, which is consistent with the reported data14.

Evaluation of the unique linkers Next, we evaluated the effect of the length of the unique linkers on the overall assembly fidelity of our combined LCR and DNA assembler strategy. A first set of unique linkers with 60 bps were designed and used to construct a 13.4 kb plasmid containing the xylose utilizing pathway and a 24 kb plasmid containing the xylose utilizing pathway and zeaxanthin biosynthesis pathway. Totally 9 unique linkers (4 unique linkers were used in the xylose pathway construction) were designed using R2oDNA (http://www.r2odna.com/) and flanked at both ends of the pUC19 backbone by over-lap extension PCR (Table 1). Desired PCR products were obtained when using the LCR reaction mixture as template and the corresponding unique linkers as primers (Figures


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S3 and S4). Together with the pre-made target vector backbone flanking by two unique linkers (ULn+1)-pRS416-UL1), the final 13.4 kb and 24 kb plasmids were constructed via the DNA assembler method. For the 13.4 kb plasmid, an 85 % assembly fidelity was obtained, while for the 24 kb plasmid, only 10 % assembly fidelity was obtained (Figures S3 and S4). A second set of unique linkers with 90 bps was designed and used to construct the same two plasmids (Table 1). The assembly fidelity was increased to 100 % and 88 % for the 13 kb plasmid and the 24 kb plasmid, respectively (Table 2).

Construction of a 44 kb plasmid using the combined LCR and DNA assembler strategy To demonstrate the new strategy for construction of larger plasmids, a 44 kb plasmid encoding the xylose utilizing pathway (3 genes), cellobiose utilizing pathway (2 genes), zeaxanthin biosynthesis pathway (5 genes), and FPP module (6 genes) was constructed. It has been reported that the separation of the vector backbone (episome and selection marker) leads to increased assembly fidelity23, in this study, the pRS416 backbone was divided into two parts which were located far away from each other in the plasmid (Figure 2A). Nine additional unique linkers were designed and totally 18 linkers were utilized in this 44 kb plasmid construction (Figure 2A, Table 1). Except for two separate backbone parts, which were flanked by two pairs of linkers, UL18pRS416(ORI)-UL1 and UL9-pRS416(URA)-UL10, respectively, other 16 pairs of linkers were flanked two ends of pUC19 backbone for LCR construction of gene expression cassettes (Table 1). Totally 64 fragments, including 16 promoters, 16 gene ORFs, 16 terminators and 16 pUC19 with different linkers were prepared to construct 16 gene expression cassettes by LCR. After LCR, all 16 gene expression cassettes were amplified by PCR. 18 DNA fragments, including the above two separated pRS416 backbone parts and 16 constructed gene expression cassettes, were



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assembled by the DNA assembler method to build the final 44 kb plasmid (Table 2). The constructed plasmids were verified by restriction digestion. By using 90 bp long unique linkers, 71 % of assembly fidelity was observed.

Functional analysis of the constructed 44 kb plasmid We investigated the functionality of the assembled 44 kb plasmid by a sugar consumption assay and analysis of zeathanthin production. For the sugar consumption assay, yeast cells harboring the 44 kb plasmid were inoculated in SC-URA medium supplemented with glucose, cellobiose and xylose. Due to the low initial OD600, sugars were utilized very slowly in the first 15 hours, but after 15 hours, glucose was consumed very quickly and was depleted at 24 hours. The other two sugars, xylose and cellobiose, were consumed much slower than glucose during the entire fermentation (Figure S8).

To check the functionality of the zeaxanthin biosynthetic pathway and compare zeaxanthin productivity between the strain harboring the 24 kb plasmid (without FPP module) and the strain harboring the 44 kb plasmid (with FPP module) (Figure S7), three yeast strains were tested, including the wild type CEN.PK2-1C (negative control), the CEN.PK2-1C strain with the 24 kb plasmid, and the CEN.PK2-1C strain with the 44 kb plasmid. Three colonies for each strain were picked. Cell disruption and zeaxanthin extraction were performed as described in Materials and Methods. Both the cells with the 24 kb plasmid and the cells with the 44 kb plasmid produced zeaxanthin, while the negative control did not. Moreover, the cells without the FPP module (the 24 kb plasmid) had a normalized zeaxanthin concentration of 2.4 µg/mL while the cells with FPP module (the 44 kb plasmid) had a normalized zeaxanthin concentration of 0.93 µg/mL (Figure


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2CD). Contrary to what we expected, the addition of the FPP module lowered the zeaxanthin production, which might be due to the metabolic burden or some unknown regulatory mechanisms associated with the FPP module, or both. Because all of the promoter-geneterminator cassettes in this study were randomly selected and the zeaxanthin production was not the focus of the study, we did not further optimize the pathway for higher productivity.

A number of DNA assembly methods have been developed to construct biochemical pathways and genetic circuits. However it is extremely difficult or impossible to directly construct large DNA molecules (>20 kb) from basic genetic parts in a rapid, modular and reliable manner using any existing DNA assembly method. This is due to, at least partially, the large number of DNA fragments to be assembled. In our hierarchical DNA assembly strategy, many small basic genetic parts are pre-assembled into larger DNA fragments, which significantly reduces the number of DNA fragments, thereby resulting in a higher overall assembly fidelity. The LCR method is highly flexible, because the DNA fragments used are not flanked by any other sequences. Therefore, many commonly used basic genetic parts, like promoters, terminators, open reading frames, vector backbones, can be pre-made and stored in a parts library for repeated usage in other constructions. However, in other similar methods, these basic genetic parts are flanked by restriction sites6 or specific recombination sites12, 13. Therefore, they can be utilized only in the construction of a specific pathway. Two most recent publications combining the Golden-Gate method and yeast homologous recombination allow modular assembly of multiple genes into a pathway24, 25. However, the Golden Gate method depends on the use of BsaI restriction site, which limits its application to genes without BasI. In contrast, our strategy is applicable to any genes and the pre-made parts library can enable automated construction of metabolic pathways



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in the following steps: (1) design of a target pathway; (2) chemical synthesis of ORFs and the corresponding bridging oligos; (3) LCR reaction with pre-made DNA parts (helper vector backbones with linkers, promoters, terminators) and synthesized ORFs & bridging oligos; (4) PCR amplification of the gene expression cassettes; (5) direct integration of the gene expression cassettes into the genome or assembly of all cassettes into a plasmid;. (6) screen for desired phenotype (integration) or transform host then screen (plasmid).

The generation and optimization of the unique linkers is critical in this strategy. These unique linkers have three functions: 1) make the entire DNA assembly strategy modular; 2) serve as primers for PCR amplification of all transcriptional units after LCR; 3) serve as homologous regions in the DNA assembler step. In this study, two different lengths of unique linkers (also as PCR primers or homology regions), including 60 bps and 90 bps, were evaluated for their effect on the assembly fidelity. Using these unique linker sequences as primers in PCR amplification of the gene expression cassettes, both types of unique linkers produced correct PCR products (Figures S3 and S4). However, compared to the 60 bp unique linkers, the 90 bp unique linkers resulted in a significantly higher assembly fidelity in constructing the 13 kb plasmid (100% vs 80%) and the 24 kb plasmid (87.5 % vs 10 %). Therefore, the 90 bp linkers were utilized in the subsequent construction of larger plasmids.

LCR is an automation friendly method and the strategy developed in this study can be potentially applied in a high throughput manner. However, purification of target PCR products may reduce the throughput based on the results from this study. It was found that even though the primers have been optimally designed, multiple PCR products were generated when amplifying the five


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genes in the zeaxanthin biosynthetic pathway (Figure S4), which may affect the fidelity of the subsequent DNA assembler based DNA assembly. A variety of strategies were used to optimize the PCR reaction to generate a single product, such as using Plasmid-SafeTM ATP-dependent DNase (Epicentre Biotechnologies, Madison, WI) to remove linear DNA fragments in LCR products as well as optimizing PCR buffers and Tm. However, none of these methods was successful (data not shown). One potential solution is to integrate a device capable of high throughput selective purification of desired PCR products. Another potential solution is to utilize rare-cutting restriction enzymes such as I-SceI to flank outside of the unique linkers to generate the ULn-gene expression cassette-ULn+1 by restriction digestion. However, the latter solution has two potential limitations. First, the LCR products need to be transformed into E. coli to amplify the plasmid DNA, which is generally difficult to be automated and takes two additional days. Second, because rare-cutting restriction enzymes recognize long DNA sequences (18 nucleotides in the case of I-SceI), the remaining nucleotides after digestion at each end of the resulting DNA fragments might cause trouble in the subsequent homologous recombination step.

The yeast homologous recombination has been proven capable of constructing extremely large DNA molecules11, 15. By using a set of unique linkers with 90 bps, we have demonstrated that the combined LCR and DNA assembler strategy was able to construct 44 kb plasmid with a fidelity of 71 % from a total of 50 fragments (Table 2). DNA assembler relies on yeast to perform the homologous recombination, which takes longer time than E. coli in term of cell growth and plasmid amplification. However, unlike E. coli, which is difficult to transform and stably maintain extremely large DNA molecules (e.g. 100 kb), the S. cerevisiae host used for DNA assembler has been proven capable of handling plasmids over a few hundred kb in size.



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Summary A flexible and versatile DNA assembly strategy was developed, which allows rapid, modular and reliable construction of biological pathways and circuits from a large number of basic genetic parts. Although yeast is the preferred host, this strategy can be adapted to other organisms like bacterial and mammalian cells by extending the pre-made parts library. Once combined with automated robotic systems, this strategy will greatly accelerate the design-build-test cycle for high throughput pathway construction and optimization/refactoring. In the long term, it can be potentially applied in building designer chromosomes in a high throughput manner.

METHODS Strains, media, and cell cultivation Escherichia coli DH5α (Cell Media Facility, University of Illinois at Urbana-Champaign, Urbana, IL) and BW25141 (lacIq , rrnBT14, ∆lacZWJ16, ∆phoBR580, hsdR514, ∆araBADAH33, ∆rhaBADLD78, galU95, endABT333, uidA(∆MluI)∷ pir+ recA1, derived from E. coli K-12 strain BD792) were used for DNA manipulation. E. coli strain was cultured in Luria-Bertani broth (LB) (Fisher Scientific, Pittsburgh, PA) containing appropriate antibiotics at 37 °C and 250 rpm unless specified otherwise. Saccharomyces cerevisiae strain CEN.PK2-1C (MATa ura3-52 trp1-289 leu2-3,112 his3∆1 MAL2-8C SUC2) (EUROSCARF) was used to assemble DNA fragments and as a host of all constructed plasmids in zeaxanthin fermentation. Complex medium consisting of 2 % peptone, 1 % yeast extract supplemented with 2 % glucose (YPD) was used for seed culture. Synthetic complete medium (SC-URA) consisting of 0.17 % Difco yeast nitrogen base without amino acids, 0.5 % ammonium sulfate and 0.083 % amino acid drop out mixture without uracil


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(MP Biomedicals, Solon, OH), supplemented with 2% glucose or mixture of glucose, xylose and cellobiose was used for DNA homologous recombination and zeaxanthin fermentation. All chemicals were purchased from Sigma Aldrich or Fisher Scientific.

Ligase cycling reaction (LCR) A total of 10 DNA fragments, including 9 random ~2 kb fragments from various sources and a 1.8 kb pUC19 backbone, were used to determine the assembly fidelity of LCR. LCR condition: 25 µl reaction system containing 3 nM of each DNA part, 30 nM of each bridging oligo, 8% v/v DMSO (New England Biolabs, Ipswich, MA), 0.45 M betaine (Fluka 14290, Sigma, St. Louis, MO), and 7.5 U thermostable DNA ligase and buffer (Epicenter Biotechnologies, Madison, WI). The following temperature cycles were setup: 2 min at 94 °C, 50 cycles of 10 s at 94 °C, 30 s at 55 °C, 60 s at 66 °C, the assembly mix was then incubate at 4 °C. The bridging oligo was designed with both halves with melting temperature of 70 °C14. The pUC19 backbone plus a various number of random 2 kb fragments from one to nine were assembled by LCR. For plasmids of 10 kb, 5 µl of LCR reaction mixture was first concentrated by the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA) and then transformed into 50 µl NEB 10-beta electrocompetent E. coli cells (New England Biolabs, Ipswich, MA). After transformation, cells were spread on agar plates with 100 µg/mL ampicillin. Colonies were randomly picked to isolate plasmids. The fidelity of each LCR assembly was determined by the percentage of the colonies with correct plasmids versus the total number of colonies checked (Figure S1).



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In addition, instead of random 2 kb DNA fragments, various gene expression cassettes, including XR, XDH, and XKS from the xylose utilizing pathway, and CrtE, CrtB, CrtI, and CrtY from the zeaxanthin biosynthetic pathway, were used to test the assembly fidelity of LCR. A 6 kb pRS416 backbone plus a different number of these cassettes ranging from one to seven were assembled by LCR (Figure S2).

Design of unique linkers (ULs) All ULs were designed using R2oDNA (http://www.r2odna.com/) with the following criteria: GC content 50%; no start codons, common multiple cloning sites (MCS) or assembly restriction sites; no bacterial promoter-like sequences; no similar sequences within E. coli and S. cerevisiae genome26. These ULs were flanked at both ends of pUC19 backbones and the pRS416 backbones in a designed order (Table 1) and the combinations were utilized in LCR and DNA assembler, respectively. Detailed sequences of 18 unique linkers utilized in this study were listed in Table S1.

Hierarchical DNA assembly by combining LCR and DNA assembler A series of sequential pairs of unique linkers (ULn, ULn+1) were flanked at both ends of the pUC19 backbone by PCR (Table 1). All of these pre-made linker-backbone-linker combinations were stored for later use. A list of promoters and terminators were PCR amplified and also stored for later use (Table 1). To construct each gene expression cassette, the target gene was obtained by either PCR or gBlock synthesis (Integrated DNA Technologies (IDT), Coralville, IA). Bridging oligos (normally four in one LCR reaction) between neighboring DNA fragments were designed and sent to IDT for synthesis. The target gene, four bridging oligos, one pre-made 14 ACS Paragon Plus Environment

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pUC19 backbone flanked by two linkers, one pair of designed pre-made promoter and terminator, were assembled by LCR. Subsequently, PCR was performed using 1 µl LCR reaction mixture as template and the sequences of the corresponding linkers were used as primers to amplify the corresponding gene expression cassettes. All the resulting gene expression cassettes with sequential linkers, together with a pRS416 backbone flanked by two linkers, U1 and Un+1 (Table 1), were then assembled by the DNA assembler method (Figure 1)13.

Using the above hierarchical DNA assembly strategy, three plasmids with various sizes were constructed, including a 13.4 kb plasmid with a xylose utilizing pathway (Figure S3 and S5); a 24 kb plasmid with a xylose utilizing pathway and a zeaxanthin biosynthetic pathway (Figure S4 and S6), and a 44 kb plasmid with a xylose utilizing pathway, a cellobiose utilizing pathway, a FPP module and a zeaxanthin biosynthetic pathway (Figure 2A).

The constructed plasmids were confirmed by restriction digestion. CEN.PK2-1C yeast competent cells were prepared following the protocol described in the literature27. 200 ng of each designed DNA fragment were transformed into CEN.PK2-1C yeast competent cells, competent cells were then spread on SC-URA agar plates and incubated at 30 °C until colonies were visible. For plasmids without the zeaxanthin biosynthetic pathway (13.4 kb plasmid), colonies were randomly picked up, while for plasmids with the zeaxanthin biosynthetic pathway (24 kb and 44 kb plasmids), yellow colonies (the color of zeaxanthin) were picked and the percentage of yellow colonies were counted as well for efficiency calculation. Plasmids were isolated using Zymoprep Yeast Plasmid Miniprep II (Zymo Research, Orange, CA), and then isolated plasmids were transformed into TOP 10 E. coli competent cells (for 13 kb plasmid and 24 kb plasmid) or



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BW25141 E. coli competent cells (for 44 kb plasmid) and spread on LB ampicillin agar plates. The E. coli colonies grown on the agar plates were then cultured for plasmid isolation. After isolation of plasmids from E. coli, the plasmids were then digested by corresponding restriction enzymes to calculate the assembly fidelity.

Fermentation for zeaxanthin production The constructed 24 kb and 44 kb plasmids were transformed into fresh CEN.PK2-1C yeast cells, inoculated into 3 mL SC-URA medium with 2% glucose, and grown overnight. The resulting cultures were inoculated into 50 mL SC-URA medium supplemented with 2 % glucose in 500 mL flasks. CEN.PK2-1C without plasmid was utilized as negative control. All flasks were shaken with 250 rpm at 30 °C. Samples were collected at various time points.

HPLC analysis 25 mL of CEN.PK2-1C S. cerevisiae cells harboring 24 kb plasmid or 44 kb plasmid, as well as the negative control, were taken from the main cultures followed by centrifugation. Cell pellets were washed once by water and re-suspended in 500 µl solution 1 of Zymoprep with 40 U Zymolyase (Zymo Research, Irvine, CA). Cell suspensions were then incubated at 37 °C for 1 h and centrifuged for 5 min at 10,000 rpm. The cell debris was washed by water twice and 300 µl acetone was used to extract the zeaxanthin from cell debris. For HPLC quantification, 10 µl acetone extract was measured by an Agilent 1100 HPLC with a Phenomenex Gemini 3u C18 110A (100×2 mm, 3 µm) column (Phenomenex, Torrance, CA) and monitored at 450 nm. Two mobile phases: A (H2O with 0.1 % formic acid), B (acetonitrile with 0.1 % formic acid). The gradient program: 0.2 ml/min flow rate, 0-1 min, 100 % A and 100 % B; 1-5 min, linear gradient


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from 100 % A to 0 % A; 5-16 min, 0 % A and 100% B. Authentic zeaxanthin (Sigma, St Louis, MO) was used as a standard to quantify zeaxanthin concentration.

ASSOCIATED CONTENT Supporting information This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding author *E-mail: [email protected] Author Contributions Y.Y. and H.Z. designed the research. Y.Y. and E.A. performed all experiments. Y.Y. and H.Z. analyzed all data. Y.Y. and H.Z. wrote the paper. Notes The authors declare that they have no competing financial interests.

ACKNOWLEDGMENTS We thank Dr. Jiazhang Lian for providing the FPP pathway and Jonathan Ning for valuable discussion on the LCR method. We also thank Dr. Lucas Li in metabolomics center of UIUC for his help in analyzing zeaxanthin samples. This work was partly supported by Defense Advanced Research Program Agency (HR0011-14-C-0069), Roy J. Carver Charitable Trust (13-4257), and Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign.



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Figure 1. Scheme of constructing large plasmids using the combined LCR and DNA assembler strategy. Common individual parts, e.g. promoters, terminators, and the helper vector (pUC19) backbone with two unique linkers (shown in cyan with different numbers), are pre-made by PCR and stored in a parts library. LCR is used to prepare a series of helper plasmids containing gene expression cassettes each consisting of a promoter, a target pathway gene, and a terminator. These gene expression cassettes are then PCR amplified using the corresponding unique linkers as primers. The resulting DNA fragments are assembled into the target plasmid by the DNA assembler method.


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Figure 2. Characterization of the constructed 44 kb plasmid. (A) The map of the designed 44 kb plasmid. (B) Restriction digestion analysis of 20 colonies. Lane C is a plasmid control without restriction digestion. M represents 2-Log DNA ladder (NEB). An asterisk represents a correct digestion pattern. (C) HPLC analysis of the cell extracts from four different samples from up to bottom: zeaxanthin standard, yeast cells with the 24 kb plasmid (without the FPP module), yeast cells with the 44 kb plasmid (with the FPP module), and negative control. (D) Comparison of zeaxanthin production between yeast cells harboring the 24 kb plasmid (without the FPP module) and yeast cells harboring the 44 kb plasmid (with the FPP module).



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Table 1. List of DNA parts used in this work, including the pre-made parts and structural genes from various pathways.

Pre-made parts (reusable)

Constructed for zeaxanthin pathway Structural genes

pRS416-Backbone with linker

Helper vector backbone with linkers

Promoter

Terminator

UL2-pRS416-UL1

UL1-pUC19-UL2

ADH1p

ADH1t

XR

UL3-pRS416-UL1

UL2-pUC19-UL3

PGK1p

CYC1t

XDH

UL4-pRS416-UL1

UL3-pUC19-UL4

PYK1p

ADH2t

XKS

UL5-pRS416-UL1

UL4-pUC19-UL5

TEF1p

PGI1t

CrtE

UL6-pRS416-UL1

UL5-pUC19-UL6

ENO2p

TPI1t

CrtB

UL7-pRS416-UL1

UL6-pUC19-UL7

TEF2p

FBA1t

CrtI

UL8-pRS416-UL1

UL7-pUC19-UL8

FBA1p

ENO2t

CrtY

UL9-pRS416-UL1

UL8-pUC19-UL9

PDC1p

TDH2t

CrtZ

UL9- pRS416(URA)-UL10

UL10-pUC19-UL11

HXT7p

PGK1t

NC801

UL11-pRS416(ORI)- UL1

UL11-pUC19-UL12

GPM1p

GPM1t

NCbg


UL12-pUC19-UL13

GDPp

PDC1t

ERG10


UL13-pUC19-UL14

PGI1P

GPDt

ERG20


UL14-pUC19-UL15

TDH2p

HXT7t

ERG8


UL15-pUC19-UL16

ACH1p

TEF1t

ERG13


UL16-pUC19-UL17

HXT5p

TEF2t

ERG12


UL17-pUC19-UL18

TPI1p

PYK1t

tHMGR



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Table 2. Evaluation of the overall assembly fidelity. Plasmids with a small size (13 kb), a medium size (24 kb) and a large size (44 kb) are used as models. Size (Kb)

Number of genes

Number of basic parts in LCR1

Number of unique linkers

Number of basic parts in DNA assembler2 10

Colony number per 1µg DNA of each fragment3 7830

13 kb

3

12

4

24 kb

8

32

44 kb

16

64

Overall assembly fidelity4 100 ± 0 %

9

25

1360

87.5 ± 4.5 %

18

50

284

71 ± 4 %

1. Include all basic genetic parts, e.g. promoters, coding sequences, terminators, and help vector backbones, in the LCR reaction 2. Include gene expression cassettes from PCR and pRS416 backbones. 3. Amount of DNA transformed into yeast for recombination. 4. The percentage of the number of correct plasmids among the total number of plasmids checked. Data were obtained from 3 biological triplicates.



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Scheme of constructing large plasmids using the combined LCR and DNA assembler strategy. Common individual parts, e.g. promoters, terminators, and the helper vector (pUC19) backbone with two unique linkers (shown in cyan with different numbers), are pre-made by PCR and stored in a parts library. LCR is used to prepare a series of helper plasmids containing gene expression cassettes each consisting of a promoter, a target pathway gene, and a terminator. These gene expression cassettes are then PCR amplified using the corresponding unique linkers as primers. The resulting DNA fragments are assembled into the target plasmid by the DNA assembler method. The authors developed a flexible and versatile DNA assembly strategy, which allows rapid, modular and reliable construction of biological pathways and circuits from a large number of basic genetic parts. Although yeast is the preferred host, this strategy can be adapted to other organisms like bacterial and mammalian cells by extending the pre-made parts library. Once combined with automated robotic systems, this strategy will greatly accelerate the design-buildtest cycle for high throughput pathway construction and optimization/refactoring. In the long term, it can be potentially applied in building designer chromosomes in a high throughput manner.

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