Letter pubs.acs.org/synthbio
Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria Nicole E. Nozzi and Shota Atsumi* Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States S Supporting Information *
ABSTRACT: Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, longterm genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter PLlacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, PLlacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene. KEYWORDS: cyanobacteria, lac promoter, 2,3-butanediol
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things.14 It was observed in this work that though the three gene production pathway integrated into the S. elongatus genome was put under the control of an inducible promoter, full 23BD production was observed in the absence of any of the chemical inducer.13 This observation raised questions about the functionality of this system as a viable means of controlling gene expression in S. elongatus. The promoter used, PLlacO1, is a well characterized isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter derived from E. coli.15 IPTG inducible promoters have been utilized in cyanobacterial engineering with varying degrees of success.7,8,11,16,17 In Synechocystis sp. PCC 6803, tight regulation with the Lac promoter variant PA1lacO‑1 as well as several others has been demonstrated with induction ratios of 2 to 3 fold. However, the Ptrc variant resulted in constitutive expression only.17,18 Markley et al. successfully created IPTG inducible promoters with induction ratios ranging from 6 to 48 fold for Synechococcus sp. strain PCC 7002 via the addition of lac operator sites to constitutive promoters known to be successful in this strain.11 PLlacO1 specifically has been employed previously in S. elongatus engineering designs.19 However, a comparison of strain behavior with and without IPTG was only recently examined by Oliver et al. 2013 for a construct under the control of PLlacO1 in cyanobacteria.13 Tight control of gene expression
ising public awareness to dwindling petroleum resources and global warming has catalyzed research efforts toward the development of renewable methods for fuel and chemical production. The fermentation of food crops such as corn for the production of bioethanol demonstrates that the power of photosynthesis can be effectively harnessed to achieve not only renewable fuel production, but also a means to absorb the carbon dioxide produced by fuel combustion. However, the necessity of providing fermentable sugars for such microbial production has raised concerns of competition for arable land between food and fuel crops.1 To avoid this issue the use of photosynthetic microorganisms such as cyanobacteria is being explored as a potential biofuel production platform.2−6 As a photosynthetic organism with the ability to directly fix carbon dioxide, the need for fermentable sugars is eliminated. This fact could be a great competitive advantage for cyanobacteria over other potential production hosts if a reliable metabolic engineering toolbox could be established, as has been done for model organisms such as Escherichia coli and Saccharomyces cerevisiae. As it stands, engineered constructs introduced into nonmodel hosts like cyanobacteria still largely employ genetic control elements from model hosts in their design due to the lack of other available tools.7−11 However, these designs do not always behave in the same way in a new host.8,12 Previously we explored the construction of a biological pathway in the cyanobacterium Synechococcus elongatus PCC 7942 for the production of 2,3-butanediol (23BD) (Figure 1a),13 an important feedstock chemical employed for the production of rubber, solvents, and plastics among other © XXXX American Chemical Society
Special Issue: Genome Engineering Received: January 13, 2015
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Figure 1. (a) Heterologous enzymatic steps from pyruvate to 2,3-butanediol. ALS, acetolactate synthase; ALDC, acetolactate decarboxylase; ADH, alcohol dehydrogenase. (b) Example targeted vector design for recombination of a construct into the S. elongatus genome. (c) Fluorescence of sf GFP integrated into NSIII normalized by OD730 in S. elongatus in the absence and presence of 1 mM IPTG. (d) Fluorescence of sf GFP in E. coli normalized by OD600 5.5 h after induction with various concentrations of IPTG. (e) Fluorescence of sf GFP in S. elongatus normalized by OD730 24 h after induction with various concentrations of IPTG. Table below graph indicates average final OD730 for IPTG concentration above each column. Error indicates SD (n = 3).
gene encoding superfolder green fluorescent protein22 (sfGFP) as a reporter (Figure 1b). However, when the function of sfGFP under the control of PLlacO1 in S. elongatus was tested, repression and induction upon the addition of IPTG was typical for genes under the control of an inducible promoter (Figure 1c). This test was further verified by induction tests with a gradient of IPTG concentrations. While the dynamic range of PLlacO1 in S. elongatus is smaller than the dynamic range of other IPTG inducible promoters tested in other cyanobacterial strains,11,17 in our hands the inducible range of PLlacO1 in S. elongatus is comparable to the range measured in E. coli (Figure 1d and 1e). Furthermore, the range of IPTG concentrations tested for PLlacO1 induction in S. elongatus showed no detrimental effect on growth. These experiments
is essential for an engineered host in order to maintain genetic stability over the course of long-term production. The issue of genetic stability is especially important for engineering cyanobacterial strains. This is highlighted with a number of recent examples of mutated strains losing chemical production capability after a relatively short time.20 A large part of this issue may be the very small number of inducible promoters known to function effectively in cyanobacterial strains.17,21 In this study our goal was to improve the function of the PLlacO1 inducible system in S. elongatus in order to improve the genetic stability of our production strain, as well as expand the engineering toolbox available for cyanobacteria. In order to screen inducible promoters for expression control, the 23BD pathway genes were replaced with the B
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ACS Synthetic Biology indicated that contrary to our initial hypothesis, the function of PLlacO1 in S. elongatus did not seem to be the cause of the unrepressed 23BD production. With the confirmation of tight repression of PLlacO1 in S. elongatus, the effect of the particular pathway genes chosen on pathway expression was explored next. The 23BD production pathway for S. elongatus constructed by Oliver et al. 2013 consists of three genes: acetolactate synthase (alsS) from Bacillus subtilis, acetolactate decarboxylase gene (alsD) from Aeromonas hydrophila, and a secondary alcohol dehydrogenase gene (adh) from Clostridium beijerinckii, in that order (Figure 1a).13 Since the acetolactate substrate for acetolactate synthase (ALS) forms naturally in cells during valine biosynthesis, we hypothesized that a hidden promoter could be present in alsS causing unrepressed expression of alsD and adh. To explore this possibility, strains were constructed with alsS upstream of sf GFP with or without PLlacO1 in front of the two gene operon in both E. coli (strain MG1655) and S. elongatus (Figure 2a). The observation of fluorescence for the construct with alsS upstream of sf GFP under the control of PLlacO1 with and without IPTG indicated leaky expression in the presence of alsS. The observation of fluorescence for the construct with alsS upstream of sf GFP in the absence of PLlacO1 strongly suggested the presence of a constitutive promoter in alsS (Figure 2b and 2c). To confirm the absence of a promoter upstream of alsS, the constructs were tested for ALS activity. The observation of ALS activity in the presence of IPTG for only the construct including both PLlacO1 and alsS (Figure 2d) demonstrated (again) that PLlacO1 functioned normally when present, that its presence was required for expression of the full alsS gene, and confirmed the absence of an upstream promoter. This reinforced our conclusion that expression of genes downstream of alsS must be due to the presence of a constitutive promoter in alsS. On the basis of this conclusion, gene order was reconstructed from alsS-alsD-adh to alsD-adh-alsS and adh-alsD-alsS in order to eliminate the influence of the alsS promoter by placing it at the end of the operon (Figure S1a). We hypothesized that this arrangement would allow for proper repression and induction of 23BD production under the control of PLlacO1. However, tests of these constructs in E. coli yielded high levels of acetoin in the absence of IPTG, indicating unrepressed expression of pathway genes (Figure S1b). Attempts to introduce these constructs into S. elongatus did not yield viable transformants and so production from these constructs could not be tested in cyanobacteria. Further studies are required to elucidate the cause of the toxicity. The persistence of unrepressed expression from the rearranged pathways seemed to indicate the presence of another hidden promoter in one or both of the other two pathway genes, alsD and adh. To explore this hypothesis we constructed the same designs as was done for alsS, with alsD or adh upstream of sf GFP (Figure 3a). Again, as with alsS, fluorescence was observed for constructs with adh upstream of sf GFP with and without IPTG and with and without PLlacO1, indicating the presence of a hidden promoter in adh as well (Figure 3b and 3c). A promoter in adh would explain the unrepressed acetoin production in the rearranged pathways. In the case of the alsD-adh-alsS construct, 2-acetolactate is known to undergo spontaneous conversion to acetoin at high levels of ALS expression.23 Constructs with alsD upstream of sf GFP did not exhibit strong unrepressed fluorescence in the absence of IPTG or PLlacO1 and only a moderate increase in fluorescence
Figure 2. (a) Construct designs tested. (b) Fluorescence of sf GFP normalized by OD600 in E. coli in the absence and presence of 1 mM IPTG. (c) Fluorescence of sf GFP normalized by OD730 in S. elongatus in the absence and presence of 1 mM IPTG. Constructs integrated into NSIII. (d) Activity of ALS in E. coli in the absence and presence of 1 mM IPTG. Error indicates SD (n = 3).
was observed for the alsD-sf GFP construct under the control of PLlacO1 in the presence of IPTG, indicating that constructs containing alsD are well repressed (Figure 3b and 3c). These results led to the construction of a third rearrangement of the original design: alsD-alsS-adh (Figure 4a). We hypothesized that though the constitutive promoter in alsS is upstream of adh, in contrast to alsS, adh is not toxic when overexpressed. We also constructed an S. elongatus strain with each of the pathway genes integrated into a different genomic locus (Figure 5a). With this design we would be able to look at control of 23BD production without the influence of gene arrangement within the operon. C
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Figure 3. (a) Construct designs tested. (b) Fluorescence of sf GFP normalized by OD600 in E. coli in the absence and presence of 1 mM IPTG. (c) Fluorescence of sf GFP normalized by OD730 in S. elongatus in the absence and presence of 1 mM IPTG. Constructs integrated into NSIII. Error indicates SD (n = 3).
When the operon with the alsD-alsS-adh gene arrangement was tested in E. coli, almost no acetoin was produced in either induced or uninduced conditions (Figure 4b). 23BD production was inducible, though the level of induced production was lower than that of the original pathway design (Figure 4c). It is worth noting here that the original pathway design is well repressed in the absence of IPTG when expressed in E. coli. However, when the original pathway design was compared with the alsD-alsS-adh arrangement in S. elongatus, the behavior observed was very different from that in E. coli. In contrast to expression control of sf GFP with PLlacO1 in S. elongatus (Figure 1c), and in contrast to expression control of the original 23BD operon (alsS-alsD-adh) with PLlacO1 in E. coli (Figure 4), acetoin and 23BD production from the original 23BD operon (alsS-alsD-adh) with PLlacO1 do not appear to be repressed at all in S. elongatus in the absence of IPTG. However, the rearranged operon exhibits cleanly inducible expression in S. elongatus (Figure 5b and 5c). The lower production of 23BD from alsD-alsS-adh versus the original design can be attributed to the fact that if the original design is expressing genes constitutively, then it has been producing for a longer period of time than alsD-alsS-adh. While the acetoin and 23BD production results from E. coli discussed above (Figure 4) contradict the sf GFP tests (Figure 3b) pointing to the presence of hidden promoters in alsS and adh, the differences in carbon flux between E. coli and S. elongatus may explain this discrepancy. Previous work has demonstrated that the production of pyruvate from the Calvin cycle may be a bottleneck in production for engineered cyanobacteria strains.13,24 In S. elongatus, the rate of carbon flux
from the Calvin cycle is not enough to allow for a further acetoin and 23BD production increase, beyond the initial leaky expression, upon IPTG induction.13,24 However, in E. coli the rate of carbon flux from glycolysis is great enough to allow for a significant increase in acetoin and 23BD production levels with IPTG induction, indicating the ALS step is one of the bottlenecks in acetoin and 23BD production. The S. elongatus strain with each pathway gene integrated into a different genomic locus exhibited similar clean inducible behavior to that of the alsD-alsS-adh operon (Figure 5). The greatest difference between these two new designs was the relative increase in acetoin accumulated by the strain in which the genes were separated (Figure 5b). We hypothesize that alcohol dehydrogenase (ADH) levels are higher in the alsDalsS-adh operon construct due to the aforementioned presence of a constitutive promoter in alsS, and so excess acetoin is quickly turned over to 23BD, whereas when the genes are separated the expression of adh is induced only with the addition of IPTG and total ADH levels are lower. Assays to measure the activity of ALS and ADH in S. elongatus in strains B, C, and D in Figure 5 were then carried out in order to gain more insight into the expression changes for the different operon arrangements. Measurements of ALS activity confirmed that in all three arrangements ALS activity is repressed and then induced upon the addition of IPTG (Figure 5d). Also, ADH activity when adh is directly downstream of alsS (Construct C) is much higher than those of other constructs in the absence of IPTG (Figure 5e). Following these experiments, two methods were applied to locate the hidden promoter sites in alsS and adh. In the first D
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In this work we explored the cause of the poor functioning of the 23BD pathway under the control of the IPTG inducible promoter system in S. elongatus. We discovered that contrary to our initial hypothesis, unrepressed pathway expression was due to the presence of hidden promoters in two of the pathway genes. Furthermore, PLlacO1 operates well as an inducible promoter in S. elongatus (Figure 1e) and should be added to the list of reliable genetic tools for cyanobacterial genome engineering. This work demonstrates that the arrangement and locus of pathway genes in genomic engineering designs can greatly influence both chemical production and strain stability, and that these effects can vary between hosts. Specific to the findings of this work, the identification of hidden promoters in alsS and adh will have implications for any previously reported chemical production pathways employing these genes.27,28
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METHODS Reagents. The chemicals 2,3-butanediol (23BD) and acetoin were obtained from Sigma-Aldrich. Pyruvic acid sodium salt was obtained from Acros Organics. NADPH was obtained from Calbiochem. Isopropyl-β-D-thiogalactoside (IPTG) was obtained from Promega. Phusion polymerase was purchased from New England Biolabs. CircLigase ssDNA Ligase was obtained from Epicenter. Gentamicin was purchased from Teknova. Kanamycin was purchased from IBI Scientific. Spectinomycin was purchased from MP Biomedicals. Oligonucleotides were synthesized by Eurofins Genomics. Culture Conditions. Unless otherwise specified, S. elongatus strains were cultured in BG-11 medium29 with the addition of 50 mM NaHCO3. Cells were grown at 30 °C with rotary shaking (100 rpm) and light (55 μmol s−1 m−2 photons in the PAR range) provided by two 86 cm 20 W fluorescent tubes above the cell cultures. Cell growth was monitored by measuring OD730. For sfGFP measurements in S. elongatus, cells in exponential phase were diluted to an OD730 of 0.1 in 5 mL of BG-11 medium including 50 mM NaHCO3 and 10 mg/L gentamicin in 30 mL culture tubes. 0, 0.01, 0.1, 1, or 10 mM IPTG was added as appropriate. Readings were taken after 24 h. For sfGFP measurements in E. coli, overnight cultures were diluted 1:100 into 5 mL of Luria Broth media containing 15 mg/L gentamicin. Cells were grown at 37 °C to an OD600 of 0.2−0.4 followed by the addition of 0, 0.01, 0.1, 1, or 10 mM IPTG as appropriate. Readings were taken after 2−6 h. For acetoin and 23BD production in S. elongatus, cells in exponential phase were diluted to an OD730 of 0.1 in 25 mL BG-11 medium including 50 mM NaHCO3, 10 mg/L thiamine pyrophosphate, and 10 mg/L gentamicin in 125 mL baffled shake flasks. One mM IPTG was added to half of the flasks. Ten mg/L kanamycin and 20 mg/L spectinomycin were added to flasks with the strain containing exogenous genes in three different neutral sites (AL2256). Every 24 h, 10% of the culture volume was removed, the pH was adjusted to 7.5 ± 0.4 with 10 N HCl, and volume was replaced with BG-11 containing 0.5 M NaHCO3, achieving a final concentration of 50 mM NaHCO3 in the culture. Production was run for 72 h. Cultures were grown in the same way for enzyme assays except for the addition of thiamine pyrophosphate. For acetoin and 23BD production in E. coli, overnight cultures were diluted 1:100 into 5 mL of modified M9 medium27 containing 50 g/L glucose, 5 g/L yeast extract, and 15 mg/L gentamicin in 30 mL culture tubes. Cells were grown at 37 °C to an OD600 of 0.2−0.4 followed by addition of 1 mM
Figure 4. (a) Construct designs tested. (b) Acetoin produced by E. coli normalized by OD600 in the absence and presence of 1 mM IPTG. (c) 23BD produced by E. coli normalized by OD600 in the absence and presence of 1 mM IPTG. Error indicates SD (n = 3).
attempt cyclized RT-PCR was utilized to discover the transcriptional start site.25 However, PCR amplification repeatedly yielded a smear rather than a single band via gel electrophoresis. These results suggest the presence of more than one transcriptional start site. Next, we used a promoter prediction program as an alternative approach. The bacterial promoter prediction program BPROM via Softberry26 identified four potential promoter sites in alsS and three in adh (Table S3). These sequences, 30 base pairs upstream of the identified −35 box to 20 base pairs downstream of the identified −10 box, were cloned upstream of sf GFP (Figure 6a). None of these constructs showed any significant fluorescence except for the third promoter identified in adh. However, this sequence from adh cannot entirely account for the fluorescence level reached when the entire adh gene is placed upstream of sf GFP (Figure 6b). From these two approaches we assume that alsS and adh contain multiple promoter sites and that it is the combined effect of these that causes the unrepressed expression of downstream genes. E
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Figure 5. (a) Construct designs tested. A, B, and C are integrated into NSIII. In design D each gene is integrated into the NS indicated within the same strain. (b) Acetoin produced by S. elongatus normalized by OD730 in the absence and presence of 1 mM IPTG. (c) 23BD produced by S. elongatus normalized by OD730 in the absence and presence of 1 mM IPTG. (d) ALS activity in S. elongatus measured in terms of nmol of acetoin formed per minute per milligram of protein. (e) ADH activity in S. elongatus measured in terms of nmol of NADPH consumed per minute per milligram of protein. Error indicates SD (n = 3).
Figure 6. (a) Construct designs tested. For D, the small blue rectangle indicates a section of the alsS gene containing a predicted promoter sequence (see Table S3). For E, the small red rectangle indicates a section of the adh gene containing a predicted promoter sequence (see Table S3). (b) Fluorescence of sf GFP normalized by OD600 in E. coli. Error indicates SD (n = 3).
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Acetolactate synthase (ALS) activity was determined as previously described.23 The concentration of acetoin produced was measured by a standard curve using pure acetoin. One specific unit of ALS activity corresponds to the formation of 1 nmol of acetoin per milligram of protein per minute. Alcohol dehydrogenase (ADH) activity was determined by measuring the oxidation of NAD(P)H. The reaction mixture contained 50 mM 3-(N-morpholino)propanesulfonic acid (Mops) pH 7.0, 25 mM acetoin and 0.2 mM NAD(P)H. The consumption of NAD(P)H was monitored at 340 nm. One specific unit of ADH activity corresponds to the oxidation of 1 nmol of NAD(P)H per minute per milligram of protein. Background activity was determined for each sample by addition of water in place of substrate, and this rate was subtracted from final rates measured with substrate.
IPTG. Production was continued on a rotary shaker (250 rpm) for 24 h. Plasmid Construction. Plasmids used and constructed are listed in Table S1. All plasmids were constructed using sequence and ligase independent cloning (SLIC).30 Primers for constructions are listed in Table S2. A neutral site located between Synpcc7942_0893 (903,564− 904,283 bp) and Synpcc7942_0894 (904,845−905,417 bp) in the S. elongatus chromosome, designated herein as NSIII31 was used for insertion of expression cassettes. A neutral site located between 2,577,474 and 2,579,801 bp in the S. elongatus chromosome, designated herein as NSI,32 and a neutral site located between 81,106 and 83,759 bp in the S. elongatus chromosome, designated herein as NSII,33 were used for insertion of expression cassettes for strain AL2256 (Table S1). Transformation of S. elongatus. Transformation of S. elongatus was performed as previously described.34 Complete chromosomal segregation for the introduced fragments was achieved through propagation of multiple generations on a selective agar plate and verified by colony PCR. Correct recombinants were confirmed by PCR to verify integration of target genes into the chromosome. The strains used and constructed are listed in Table S1. Neutral sites I, II, and III as described above on the S. elongatus chromosome were used as targeting sites for recombination in this study. Quantification of sfGFP Expression. For sfGFP fluorescence experiments, 488 nm was used for excitation, and emission was measured at 530 nm using a Microtek Synergy H1 plate reader (BioTek). Acetoin Quantification. Acetoin was quantified by the method of Voges and Proskauer35 as optimized by Westerfield36 and adapted to small volume on 96-well plates. Samples were diluted in H2O to 100 μL initial volume. To this mixture was added 100 μL of a solution, prepared at the time of use, consisting of one part 5% (wt/vol) naphthol dissolved in 2.5 N NaOH and one part 0.5% (wt/vol) creatine in water. Readings were taken after 30 min using a Microtek Synergy H1 plate reader (BioTek). Triplicate measurements of no fewer than three standards, including at least one value each above, below, and within the desired range, were included in every assay. Quantification of 23BD. Supernatant samples from cultures were analyzed with a gas chromatograph (GC) (Shimadzu) equipped with a flame-ionization detector and an HP-chiral 20b column (30 m, 0.32 mm internal diameter, 0.25 mm film thickness; Agilent Technologies). Samples were prepared by mixing nine parts supernatant (diluted as necessary in H2O) with one part internal standard. For each analysis the GC oven temperature was held at 105 °C for 1 min, increased with a gradient of 20 °C min−1 until 225 °C, and held for 3 min. Ultrahigh purity Helium was used as the carrier gas. The temperature of the injector and detector were set at 225 and 260 °C respectively. Enzyme Assay. E. coli cells were collected 9 h after induction by centrifugation and resuspended in 300 μL BugBuster Protein Extraction Reagent (Novagen) and incubated at room temperature for 20 min for cell lysis. S. elongatus cells were collected 72 h after induction via centrifugation, washed with phosphate buffer pH 7.5 and resuspended to 300 μL in the same buffer. Crude extracts were prepared with 200 μL of 0.1 mm glass beads and a vortex mixer. The total protein determination was performed using Advanced Protein Assay Reagent from Cytoskeleton.
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ASSOCIATED CONTENT
* Supporting Information S
Strains and plasmids used in this study (Table S1), oligonucleotides used in this study (Table S2), predicted promoter sequences (Table S3) and acetoin and 23BD production in the E. coli strains with alsD-adh-alsS or adhalsD-alsS (Figure S1). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.5b00057.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
N.E.N. and S.A. designed research; N.E.N. performed the experiments; N.E.N. and S.A. analyzed data; and N.E.N. and S.A. wrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Asahi Kasei Corporation and the National Science Foundation CBET-1349663. N.E.N. was supported by a US National Institutes of Health Biotechnology Training Grant Fellowship (T32-GM008799)
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DOI: 10.1021/acssynbio.5b00057 ACS Synth. Biol. XXXX, XXX, XXX−XXX
