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Letter

Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli Min-Ji Heo, Hwi-Min Jung, Jaeyong Um, Sang-Woo Lee, and Min-Kyu Oh ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00134 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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ACS Synthetic Biology

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Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing

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for n-Butanol Production in Escherichia coli

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Min-Ji Heoa,#, Hwi-Min Junga,#, Jaeyong Uma, Sang-Woo Leea, and Min-Kyu Oha,*

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a

Department of Chemical & Biological Engineering Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul ,136-713, South Korea

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Graphical Table of Contents

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ACS Paragon Plus Environment


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ABSTRACT

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Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively

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produce n-butanol in a defined medium under micro-aerobic condition. The butanol synthetic pathway

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genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in

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metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol

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production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA.

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For this purpose, the 5′-untranslated region sequence of gltA encoding citrate synthase was designed

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using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9

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genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase

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expression produced more butanol and the citrate synthase activity was correlated with butanol

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production. These results demonstrate that redistributing carbon flux using genome editing is an

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efficient engineering tool for metabolite overproduction.

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Keywords: Escherichia coli; CRISPR/Cas9; genome editing; 5’-untranslated region; n-butanol

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Biobutanol is a good alternative to fossil fuels because its energy density (29.2 MJ/L) is 90%

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of that of gasoline (32 MJ/L)1. n-Butanol is naturally produced by Clostridium species by acetone–

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butanol–ethanol fermentation, which has been intensively studied and developed2,3. Although

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Clostridium species are good host strains for producing n-butanol, they have shortcomings due to

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limited genetic engineering tools and complex physiology. Therefore, other strains, such as

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Escherichia coli, have been developed as hosts to produce n-butanol4. As E. coli is not a natural

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producer, the n-butanol pathway must be introduced into the strain. The coenzyme A (CoA)

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dependent pathway or a modified pathway have been introduced from Clostridium5,6. Other pathways,

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such as the reverse β-oxidative7, and ACS-dependent pathway8, etc. have been successfully used to

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produce butanol. Meanwhile, metabolic engineering of the host strain is also required. For example,

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E. coli with deleted metabolic pathways to by-products, including acetate, lactate, ethanol, and

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succinate, was used to achieve 30 g/L butanol production with gas-stripping method9. Rebalancing

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intracellular redox state has also been proven as an efficient method to achieve high butanol

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productivity10,11.

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The expression levels of multiple genes must be controlled for the host strain to efficiently

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produce biofuel12. Knockout and knockin of multiple genes in the E. coli genome have been utilized

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to produce butanol7-11. However, recent advances in metabolic engineering have provided more

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diverse tools to search for optimal gene expression levels. For example, knockdown of single or

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multiple gene(s) using sRNA and Hfq protein allowed to optimize expression levels of metabolic

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genes to overproduce of tyrosine and cadaverine13. Another synthetic biology tool for regulating gene

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expression level, dCas914, has been used in metabolic engineering of Corynebactrium glutamicum15.

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Although these techniques for modulating gene expression are very useful in metabolic engineering,



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the expression level of the target gene is difficult to estimate and engineered strains must express

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additional protein components, such as Hfq or dCas9. Therefore, optimizing gene expression level by

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modifying the 5′- untranslated region (UTR) is a good alternative to control gene expression because

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efficient genome editing methods and gene expression predicting tools have been developed. A

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specific sequence on the E. coli genome can be precisely changed for gene expression control using

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the RNA-guided genome editing tool CRISPR/Cas system16,17. On the other hand, the correlation

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between expression level of a certain gene and the 5′-UTR sequence was evaluated using recently

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developed computational tools18,19. Combining these methods allows for more precise control of the

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expression level of a specific gene in the host genome.

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In preliminary experiments, we engineered E. coli to produce n-butanol using glucose

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minimal media. A butyrate-producing strain previously developed in our laboratory20 was modified.

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Briefly, the N-terminals of three enzymes that convert acetoacetyl-CoA to butyryl-CoA, such as 3-

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hydroxybutyryl-CoA dehydrogenase (Hbd), 3-hydroxybutyryl-CoA dehydratase (Crt), and trans-

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enoyl-coenzyme A reductase (Ter), were modified to attach to a scaffold protein. For this purpose, the

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GBD, SH3, and PDZ ligands were translationally fused to Hbd, Crt, and Ter, respectively, and

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expressed with the pCDF-HCT vector (Table 1). The scaffold protein with GBD, SH3, and PDZ

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ligand binding sites at a 1:1:2 ratio was overexpressed by the pJD758 vector21. Endogenous

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acetoacetyl-CoA thiolase (atoB), alcohol dehydrogenase (adhE2) from C. acetobutylicun and formate

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dehydrogenase (fdh1) from C. boidinii22 were overexpressed by the pACYC-AEF vector (Figure 1).

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The three plasmids were transformed to the DSM01 strain to construct EMJ40. After a 96 h

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incubation in a flask under anaerobic conditions with M9G defined medium, 0.052 g/L butanol was

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synthesized. Most butanol production experiments have been performed with rich media supplied


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with glucose4-11. However, to understand carbon flux and calculate carbon yield exactly, producing

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butanol in a defined medium is necessary. The strain developed here produced only a noticeable

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amount of butanol in a defined medium with a minimum amount of yeast extract (0.5 g/l) needed for

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initial growth of the strain. This must be because the optical density of this culture was very low under

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anaerobic condition, which was suspected to be the main limitation to butanol production in minimal

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medium. When EMJ40 was cultured under micro-aerobic condition, butanol production decreased,

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but optical density increased significantly (Figure 2a). It was inferred that significant energy was

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required for microbial growth with expression of butanol pathway enzymes and scaffolding protein,

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which was not supplied enough in a defined medium in anaerobic condition. The energy limitation

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might be overcome by cultivating strains in microaerobic or aerobic condition. However, further

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engineering strategies were needed to improve n-butanol in aerobic condition.

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The CoA-dependent pathway of Clostridium species introduced in this strain is oxygen

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sensitive. In particular, aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum loses

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activity when exposed to oxygen23. An oxygen-tolerant pathway from butyryl-CoA to butanol was

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demonstrated with CoA-acylating propionaldehyde dehydrogenase (PduP) from S. enterica and

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alcohol dehydrogenase (AdhA) from L. lactis24. Therefore, the pACYC-APAF plasmid to expressing

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atoB, pduP, adhA, and fdh1 was constructed and transformed with pCDF-HCT and pJD758 into

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DSM01, and the resulting strain was named EMJ50 (Table 1). After 96 h of culture under micro-

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aerobic conditions with M9G medium, 0.82 g/l butanol was synthesized by strain EMJ50, which was

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much higher than the 0.014 g/l from EMJ40. Butanol yield also increased from 0.0029 to 0.068 g/g

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glucose (Figure 2b), indicating that oxygen-tolerant alcohol dehydrogenase was very helpful to

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increase butanol synthesis under micro-aerobic conditions. Although the butanol titer obtained under



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micro-aerobic conditions increased after using oxygen-tolerant PduP and AdhA, the carbon yield (g

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butanol/g glucose) was still slightly lower than that of anaerobic culture using AdhE2 (0.082 g/g

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glucose). This may have been caused by loss of acetyl-CoA to the citric acid cycle under micro-

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aerobic condition because acetyl-CoA is a precursor for both butanol and the citric acid cycle.

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Therefore, carbon flux to the citric acid cycle must be reduced to increase butanol yield.

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For this purpose, we knocked down the expression level of gltA by editing its 5′-UTR

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sequence (Figure 3). UTR Designer19, which predicts protein expression levels using the 5′-UTR

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sequence, has been used in metabolic engineering25. Therefore, four different gltA 5′-UTR sequences

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predicted to have 50, 30, 10, and 1% of the expression levels of wild-type gltA were designed with the

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program (Table 2 and Figure 3). CRISPR/Cas9 experiments were conducted to edit the gltA 5′-UTR

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sequence on the DSM01 genome, using a slightly modified experimental method developed by Jiang

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et al16. Four 5` UTR- engineered strains were constructed and transformed with three plasmids

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expressing butanol synthesis pathway genes and named EMJ51–EMJ54 (Table 1). We also

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constructed a gltA deleted mutant and transformed it with the same set of plasmids (EMJ55).

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Under micro-aerobic condition, the gltA deletion mutant grew very poorly. All the genome

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edited mutants did as well as the wild type except EMJ54 (Figure 4a). EMJ54 showed 24% reduced

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growth at the late exponential phase (48 h). Modifying the gltA 5′-UTR sequences was supposed to

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reduce the gltA expression level and activity of its product. We measured enzyme activity of each

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strain using a citrate synthase assay kit. The gradual decrease in specific citrate synthase activity was

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in accordance with the prediction, but the magnitude of the decrease was much less than that the

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predicted gene expression (Figure 4b). EMJ54 had 67% reduced citrate synthase activity compared to

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EMJ50. The growth pattern suggested that significant reduction of microbial growth was recognized


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in a defined medium when the citrate synthase activity was reduced more than 50%.

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After culturing the strains in M9G medium, titers and yields of butanol were measured. Clear

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correlations were found between citrate synthase activity and butanol titer/yield. As citrate synthase

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activity decreased, the n-butanol titer increased up to 1.3-fold in EMJ52 (Figure 4c). The maximal

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butanol titer was achieved in EMJ52, which has 55% citrate synthase activity compared to parental

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strain. However, butanol titer deteriorated when citrate synthase activity was below 46% (EMJ53,

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EMJ54, EMJ55). Also as the citrate synthase activity decreased, ethanol yield increased significantly

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(Table S1). It is expected that as the expression of citrate synthase diminished, availability of acetyl-

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CoA increased. Although major ethanol production pathway was removed, other alcohol

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dehydrogenase can convert acetyl-CoA to ethanol. Furthermore, introduced alcohol dehydrogenase,

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adhA, has substrate specificity not only to butyraldehyde but also to acetaldehyde26. Therefore, more

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ethanol can be formed as aceyl-CoA accumulated in cytosol.

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A linear relationship was observed between citrate synthase activity and butanol yield, but

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the relationship between citrate synthase activity and butanol titer was more complex due to cell

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growth. Although the highest yield was obtained from EMJ55, in which gltA was deleted, a low

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butanol titer was observed due to the low growth rate and EMJ55 hardly consumed glucose for 72 h

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cultivation (Figure 4c, Table S1). Therefore, gltA knockdown was chosen as a better strategy11.27. We

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attempted gltA knockdown using the genome editing method with CRISPR/Cas9, and the necessary

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sequences for genome editing of the 5′-UTR region were designed using a bioinformatics tool, UTR

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designer.

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Both the genome editing and 5′-UTR design methods worked effectively. The components

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developed for genome editing, such as CRISPR, tracrRNA, the crRNA targeted gltA 5′-UTR, and the



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141 bp double-stranded rescue DNA, effectively changed the genome sequence as designed after

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transformation. More than 10 colonies were obtained after each experiment and at least 30% of them

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provided properly edited ones. Because the sacB gene was added to the plasmids for the

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CRISPR/Cas9 experiment, the plasmids were eliminated after an overnight cultivation with sucrose28.

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The experiment to modify the host genome took less than 1 week. On the other hand, designing the 5′-

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UTR sequence was effective but should be improved. All of the designed gltA 5′-UTR sequences

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provided reduced citrate synthase activity after genome editing. However, UTR Designer expected

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those sequences to reduce gltA expression levels to 50, 25, 10, and 1% of that of the wild type, but

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citrate synthase activity was actually reduced to 75, 55, 46, and 34%, respectively, possibly because

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of the complex interactions among various factors at the levels of transcription and translation.

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Nevertheless, our results demonstrate that combining these tools was quite useful to modulate the

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expression levels of a specific enzyme, which is very useful to balance metabolic flux between the

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product and cell growth or host maintenance to overproduce a metabolite.

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In summary, expression of citrate synthase, the first enzyme in the citric acid cycle, was

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controlled by modifying the 5′-UTR sequence of gltA on the genome. The citric acid cycle competes

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with butanol pathway as they both use acetyl-CoA as a precursor. We designed four different 5′-UTR

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sequences with different predicted gltA expression levels and edited them on the genome using the

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CRISPR/Cas9 system. These modifications improved the butanol titer and yield, which were

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correlated with citrate synthase activity.

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Methods

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Strains, plasmids, and primers

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The strains, plasmids, and primers used in this study are listed in Tables 1 and 2. E. coli

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DH5α was used to construct plasmids. Escherichia coli MG1655 integrated with λDE3 was

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genetically modified to produce butanol. Four genes, such as frdA, ldhA, pta, and adhE, were deleted

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from MG1655(DE3) using P1 phage transduction and used as the host strain (DSM01)20,29.

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Additionally, gltA was deleted in the same manner.

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Genome editing experiment

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Four

different

gltA

5′-UTR

sequences

were

designed

using

UTR

designer

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(http://sbi.postech.ac.kr/utr_designer) to modulate the gltA expression level19. A protocol modified

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from a previous report was used for the genome editing experiment with CRISPR/Cas916. Briefly,

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cas9, crRNA, and the tracrRNA region were amplified by polymerase chain reaction (PCR) using the

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primers listed in Table 2 with the pCas9 plasmid (Addgene, Cambridge, MA, USA)16. sacB was

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amplified using genomic Bacillus subtilis DNA. The cas9 and sacB PCR products were cloned into

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pZA31MCS using Gibson assembly (New England Biolabs, Ipswich, MA, USA), and named pZA-

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Cas9 (Table 1). Similarly, crRNA, tracrRNA, and sacB were cloned into pZS21MCS and named pZS-

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CRISPR (Table 1). Both strands of the gltA 5'-UTR sequence (gltA crRNA-S and gltA crRNA-A,

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Table 2) were synthesized by Bioneer Inc. (Daejeon, Korea) to insert the gltA targeting crRNA

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sequence. Then, the synthesized DNA fragments were slowly annealed and ligated into pZS-CRISPR

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digested with BsaI, resulting in pZS-CRISPRgltA containing crRNA targeting gltA-5′-UTR. The



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rescue DNAs for homologous recombination after Cas9 nuclease digestion were prepared from pairs

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of synthesized oligomer DNAs with 22 bp overlapping sequence (Table 2). The oligomer pairs, such

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as gltA Rescue F and 0.6R, gltA Rescue F and 0.3R, gltA Rescue F2 and 0.5R, and gltA Rescue F2 and

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0.4R, respectively, were denatured at 96°C and annealed by slow cooling, followed by extended DNA

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synthesis using the Klenow fragment (Takara Bio, Shiga, Japan). We conducted a genome editing

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experiment using DSM01 as the host. First, pKD4630 and pZA-Cas9 was sequentially transformed

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into DSM01 using electroporation. Then, gltA Rescue DNA and pZS-CRISPRgltA were co-

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transformed into the strain and the transformants were screened on a LB plate containing ampicillin

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(50 µg/ml), kanamycin (50 µg/ml), and chloramphenicol (50 µg/ml).

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Mutant strains harboring the plasmids for CRISPR/Cas9 editing were incubated overnight at

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37°C in liquid LB medium containing 40 g/l sucrose and streaked on an LB agar plate. The colonies

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that lost all antibiotics resistance were selected on an agar plate. The genome-editing results were

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confirmed by PCR of the gltA 5′-UTR region using the gltA-5′-F and gltA-5′-R primers, followed by

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sequencing of the PCR products by CosmoGenetech (Seoul, Korea) using the gltA-5′-seq primer

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(Table 2).

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Plasmid construction for butanol production

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The pCDF-HCT vector constructed by Beak et al. (2013)20 was used to convert acetoactyl-

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CoA to butyryl-CoA (Figure 1). The genes of three enzymes, such as Hbd, Crt, and Ter, attached with

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the GBD, SH3, and PDZ ligands, respectively, were expressed using the pCDF-HCT vector3. The

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scaffold protein (GBD1SH31PDZ2) was expressed by pJD75821 to increase pathway efficiency. Other


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enzymes, such as atoB, adhA, adhE2, and fdh1, were amplified from genomic DNAs of E. coli

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MG1655, Lactococcus lactis, Clostridium acetobutylicum, and Candida boidinii, respectively, using

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the primers listed in Table 2. The codon-optimized pduP gene of Salmonella enterica was synthesized

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by Bioneer Inc. and amplified using the primers listed in Table 2. Four genes (atoB, pduP, adhA, and

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fdh1) were ligated into the corresponding sites of the pACYCDuet vector and named pACYC-APAF

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(Table 1). Similarly, three genes (atoB, adhE2, fdh1) were ligated into pACYCDuet and named

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pACYC-AEF. Either pACYC-APAF or pACYC-AEF was co-transformed with pCDF-HCT and the

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pJD758 using electroporation to produce butanol.

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Media and culture conditions

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Strains were selected and cultured on LB medium (10 g/l tryptone, 5 g/l yeast extract, and 10

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g/l NaCl) supplied with 50 µg/ml ampicillin, 50 µg/ml chloramphenicol, 50 µg/ml kanamycin, and

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100 µg/ml spectinomycin. Engineered E. coli was cultured in M9G medium (12.8 g/l Na2HPO4, 3 g/l

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KH2PO4, 0.5 g/l NaCl, 1 g/l NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 10 mg/l thiamine, 25 g/l glucose,

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0.5 g/l yeast extract, and 1,000× trace elements (27 g/l FeCl3·6H2O, 2 g/l ZnCl2·4H2O, 2 g/l

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CaCl2·2H2O, 2 g/l Na2MoO4·2H2O, 1.9 g/l CuSO4·5H2O, and 0.5 g/l H3BO3)) supplemented with 50

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µg/ml ampicillin, 50 µg/ml chloramphenicol, and 100 µg/ml spectinomycin5. E. coli was pre-cultured

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in 5 ml M9G medium overnight with appropriate antibiotics to produce butanol. The culture broth

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was transferred to 50 ml M9G medium in 250 ml flasks. All flasks were sealed with rubber stoppers to

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create micro-aerobic conditions and incubated at 37°C with shaking at 250 rpm, and 0.05 mM IPTG

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and 108 nM anhydrotetracycline (aTc) were added as inducers at 6 h.



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Citrate synthase activity assay

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E. coli was cultured in M9G minimal medium for 32 h to measure citrate synthase activity.

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Strains from 50 mL culture broth were harvested and washed twice by centrifugation at 35,000 × g for

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10 min followed by resuspension. The pellet was suspended in 10 mL of 120 mM Tris HCl (pH 8.0),

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containing 10 mM MgCl2, 0.1 M KCl, and 1 mM EDTA. The cells were mechanically disrupted by

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sonication on ice for 30 min. After centrifugation at 35,000 × g for 10 min, the supernatant was used

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to measure enzyme activity31. Citrate synthase activity was measured with a citrate synthase assay kit

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(Sigma-Aldrich, St. Louis, MO, USA). The enzyme reaction occurred with 30 mM acetyl-CoA, 10

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mM oxaloacetate, and 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) in citrate synthase assay

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buffer. Enzyme activity was determined by conversion of CoA-SH with DTNB to form

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thionitrobenzoic acid (yellow) per min in a 1 ml cuvette.

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Total protein concentration in cell lysates was determined by the Bradford based Bio-Rad

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Protein Assay Dye Reagent Concentrate with bovine serum albumin as the standard (Bio-Rad,

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Hercules, CA, USA). Citrate synthase activity was normalized to total protein in cell lysates to obtain

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specific citrate synthase activity (U/mg protein).

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Analytical methods

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Cell growth (OD600) and protein concentration (595 nm) were monitored with a UV-visibility

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spectrophotometer (DU Series 700; Beckman Coulter, Inc., Brea, CA, USA). Citrate synthase activity


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was measured at 412 nm using a multimode microplate reader (InfiniteⓇ-200PRO; Tecan, Hõganãs,

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Sweden). Glucose and butanol were analyzed by high-performance liquid chromatography (ACME-

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9000; Younglin Instrument, Seoul, Korea) with a Sugar SH1011 column (Shodex, Tokyo, Japan). The

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Column temperature was 60°C, and 5 mM sulfuric acid was used as the mobile phase at a flow rate of

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0.6 ml/min.

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Author Information

259 260

Corresponding Author

261

*E-mail: [email protected]

262 263

Author Contributions

264

#

265

wrote the manuscript. M.J.H., H.M.J. J.Y.U. and S.W.L. performed experiments.

M.J.H., H.M.J. contributed equally to this work. M.J.H., H.M.J. and M.K.O. conceived the idea and

266 267

Notes

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The authors declare no financial conflicts of interest.

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Acknowledgement

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This study was supported by a National Research Foundation of Korea funded by the Korean

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Government (2012M1A2A2026560 and 2014R1A2A2A03007094) and New & Renewable Energy



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Program of the Korea Institute of Energy Technology Evaluation and Planning (No.

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20133030000300).

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390

Figure legends

391 392

Figure 1. Butanol synthetic pathway map constructed in EMJ50. Red crosses on the arrow denote

393

deleted pathway genes in the host genome. Blue bold characters are overexpressed pathway genes,

394

and their sources are presented in parenthesis. EC: Escherichia coli, CA: Clostridium acetobutylicum,

395

TD: Treponema denticola, CB: Candida boidinii, SE: Salmonella enterica, and LL: Lactococcus

396

lactis

397 398

Figure 2. Butanol production related to oxygen sensitive and tolerant pathways. (a) Optical

399

density (gray bar) and butanol titer (white bar) of EMJ40 after 96 h cultivation in M9G medium. (b)

400

Butanol production (gray bar) and butanol yield (black circle) in EMJ50 and EMJ40. Error bars

401

represent standard deviations of three independent experiments.

402 403

Figure 3. Scheme for the change in E. coli metabolic flux after editing the gltA 5′-untranslated

404

region (UTR) sequence. The sequences include the PAM sequence (red), the gltA 5′-UTR sequence

405

(underlined), the ribosome binding site (bold), and the translational start codon (blue).

406 407

Figure 4. Results of citrate synthase activity and butanol production (a) Optical density of

408

engineered E. coli cultivating for 72 h in M9G medium. Symbols represent EMJ50 (●), EMJ51 (○),

409

EMJ52 (▲), EMJ53 (△), EMJ54 (■) and EMJ55 (□). (b) Citrate synthase activity of the genome-

410

edited E. coli strains. (c) Correlations between citrate synthase activities and butanol titer (●) or yield

411

(□) of the engineered E. coli after cultivating for 72 h in M9G medium. Error bars and dashed lines


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412

represent standard deviations of three independent experiments and regression results, respectively.

413 414

Table 1 Strains and plasmids used in this study

Name

Relevant characteristics

Source

DSM01

MG1655(DE3)△frdA::FRT△pta::FRT△ldhA::FRT△adhE::FRT

Baek et al. (2013)20

EMJ30

DSM01 △gltA

This work

EMJ31

DSM01, but GltA activity adjusted to 75%

This work

EMJ32


This work

EMJ33


This work

EMJ34

DSM01, but GlA activity adjusted to 34%

This work

EMJ40

DSM01/pJD758, pCDF-HCT, pACYC-AEF

This work

EMJ50

DSM01/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ51

EMJ31/pJD758, pCDF-HCT, pACYC-APAF

This work

EMJ52


This work

EMJ53


This work

EMJ54


This work

EMJ55


This work

Expression vector, CmR, p15A ori

EXPRESSYS

Strains

Plasmids pZA31MCS pZS21MCS pACYCDuet

R

Expression vector, Km , pSC101 ori

EXPRESSYS

R

Expression vector, Cm , p15A ori

pKD46

Red recombinase expression vector, Amp

pZA-Cas9

pZA31MCS, but PLtetO-1::cas9-PsacB::sacB

Novagen Datsenko and Wanner et al. (2000)32 This work

pZS-CRISPR

pZS21MCS, but TracerRNA-crRNA-PsacB::sacB

This work

R

pZS-CRISPRgltA pZS21MCS, but TracerRNA-crRNA(gltA)-PsacB::sacB

415 416 417 418 419

R

This work

pJD758

ColE1 ori, Amp , Ptet::scaffold protein

Dueber et al. (2009)27

pCDF-HCT

CDF ori, SmR, Plac::hbdGBDL-crtSH3L-terPDZL

Beak et al. (2013)20

pACYC-AEF

pACYCDuet, but Plac::atoB-adhE2-fdh1

This work

pACYC-APAF

pACYCDuet, but Plac::atoB-pduP-adhA-fdh1

This work

AmpR, ampicillin; CmR, chloramphenicol; KmR, kanamycin; SmR, spectromycin resistance, respectively.



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420

Table 2 Oligonucleotide sequences used in this study Name

Sequence (5’-3’)

cas9 F cas9 R tracrRNA F tracrRNA R crRNA F crRNA R

aaaagtcgacATGGATAAGAAATACTCAATAGGCT aaaactgcagTCAGTCACCTCCTAGCTGAC ataaaagcttTTACGAAATCATCCTGTGGAG taatggatccTTTTGCCTCCTAAAATAAAAAGTT aattggtaccAGTATATTTTAGATGAAGATTATTTCTTA attaaagcttATCACACTACTCTTCTTTTGCCTA

gltA crRNA S

aaacAGGTTGATGTGCGAAGGCAAATTTAAGTTCg

gltA crRNA A

aaaacGAACTTAAATTTGCCTTCGCACATCAACCT

sacB F

ataagcagcatcgcctgtTACCTGCCGTTCACTATTATTTAG

sacB R

cacatagacagcctgaATCGGCATTTTCTTTTGC

gltA Rescue Uni F

ccaaataacaaacgggtaaagccaggttgatgtgcgaaggcaaatttaagttcccgcagtcttacgctgtaggttaaaag gagcat

gltA Rescue 0.6 R

tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatctctgatgctccttttaacctacagcg

gltA Rescue 0.3 R

421 422 423 424

tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatcaacgatgctccttttaacctacagcg ccaaataacaaacgggtaaagccaggttgatgtgcgaaggcaaatttaagttcccgcagtcttacgcggctggtgtaaa gltA Rescue Uni F2 ggagcat gltA Rescue 0.5 R tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatggccgatgctcctttacaccagccgcg gltA Rescue 0.4 R tcaacagctgtgtccccgttgagggtgagttttgcttttgtatcagccatctcagatgctcctttacaccagccgcg gltA-5’-F AAAGTTGTTACAAACATTACCAGGAA gltA-5’-R TTCACCATTCAGCAGGATGTA gltA-5’-seq TACCCAGGTTTTCCCCTCTT atoB-F tatagtcgacaaggagatataATGAAAAATTGTGTCATCGTC atoB-R tatagcggccgcTTAATTCAACCGTTCAATCA adhE2-F ggagatatacatatggcaATGAAAGTTACAAATCAAAAAGAAC adhE2-R atatctccttTTAAAATGATTTTATATAGATATCCTTAAG fdh1-F aatcattttaaaaggagatataATGAAGATCGTTTTAGTCTTATATG fdh1-R cggtttctttaccagacTTATTTCTTATCGTGTTTACCG adhA-F tataagaaggagatatacaATGAAAGCAGCAGTAGTAAGAC adhA-R gatcttcattatatctccttTTATTTAGTAAAATCAATGACCATTC pduP-F tataggatccaaggagatataATGAATACTTCTGAACTCGAAACC pduP-R tatagagctcTTAGCGAATAGAAAAGCCGTTG Underlined letters indicate sequences used to increase efﬁciency of homologous recombination with E. coli MG1655 genomic DNA. Overlapping sequence for annealing two oligomers are shown in italic. Sequence which is binding to DNA template are shown in capital.

425 426 427 428


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429 430 431

Figure 1.

432 433



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434

(a)

435 436

(b)

437 438

Figure 2.

439 440


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441 442 443

Figure 3.

444 445 446 447 448 449 450 451 452 453 454 455 456



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

457

(a)

458 459

(b)

460 461

(c)

462 463

Figure 4.


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Controlling Citrate Synthase Expression by ... - ACS Publications

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