Optimization of Artificial Curcumin Biosynthesis in E. coli by

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Optimization of Artificial Curcumin Biosynthesis in E. coli by Randomized 5’-UTR Sequences to Control the Multi-enzyme Pathway Sun-Young Kang, Kyung Taek Heo, and Young-Soo Hong ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00198 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on September 1, 2018

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ACS Synthetic Biology

Optimization of Artificial Curcumin Biosynthesis in E. coli by Randomized 5’-UTR Sequences to Control the Multi-enzyme Pathway Sun-Young Kang,† Kyung Taek Heo,†,‡ and Young-Soo Hong,*,†,‡ †

Anticancer Agents Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanjiro, Ochang-eup, Cheongju-si, Chungbuk 28116, Korea

‡

Department of Biomolecular Science, KRIBB school of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34141, Korea KEYWORDS. Artificial biosynthesis, multi-enzyme pathway, multiplex automatic genome engineering, Curcumin

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ABSTRACT: One of the optimization strategies of an artificial biosynthetic metabolic flux with a multi-enzyme pathway is when the enzyme concentrations are present at the appropriate ratios rather than at their maximum expression. Thus, many recent research efforts have focused on the development of tools that fine tune the enzyme expression, and these research efforts have facilitated the search for the optimum balance between pathway expression and cell viability. However, the rational approach has some limitations in finding the most optimized expression ratio in in vivo systems. In our study, we focused on fine-tuning the expression level of a six-enzyme reaction for the artificial biosynthesis of curcumin by screening a library of 5’-untranslational region (UTR) sequence mutants made by a multiplex automatic genome engineering (MAGE) tool. From the screening results, a variant (6M08rv) showed about a 38.2-fold improvement in the production of curcumin compared to the parent strain, in which the calculated expression levels of 4-coumarate: CoA ligase (4CL) and phenyldiketide-CoA synthase (DCS), two of the six enzymes, were much lower than those of the parent strain.

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Synthetic biology has emerged as an engineering tool for the construction of artificial biological systems capable of producing fine or bulk chemicals. Two aims of synthetic biology are to easily assemble and artificially control complex biological elements and systems in vivo, such as genetic circuits, modularized expression of enzymes, and metabolic flux.1-4 Synthetic biology has been used in many studies to produce natural therapeutic products, such as alkaloids, terpenoids, polyketides, and modified peptides, by constructing artificial pathways in heterologous hosts.5-7 Synthetic biology is easily accessible due to the advent of more sophisticated genetic tools that enable the targeted manipulation of heterologous microbes, either to modify existing metabolic pathways or to introduce completely new artificial pathways.8-10 Most artificial biosynthetic pathways are multi-reaction pathways that require a balanced expression of several enzymes.11, 12

Furthermore, the well-established overexpression systems of these enzymes often result in an excessive metabolic burden

on the heterologous host cell.13 In many instances, the biosynthetic metabolic flux will be optimal when the enzyme concentrations are present at an appropriate ratio rather than at their maximum expression.2, 14, 15 Thus, many recent research efforts have focused on the development of tools that fine tune the enzyme expression, and these research efforts have facilitated the search for the optimum balance between pathway expression and cell viability.12, 16 Recently, several studies have shown that the rational control of genetic elements such as the ribosomal binding sequences (RBS), promoter and intergenic regions of each gene improves the balance of multi-gene expression.2, 5, 13-15, 17 However, because it is difficult to rationally design the optimal expression strength for each gene in a multi-enzyme pathway, these attempts have been focused on creating and selecting variants that contain the best metabolic flux. In our study, we used the multiplex automatic genome engineering (MAGE) tool to optimize the expression ratios of multiple enzymes in an engineered host that had acquired an artificial biosynthetic pathways. For this approach, the biosynthetic pathway for curcumin, a plant-derived yellow compound synthesized by a six-enzyme reaction, was used as a model system to optimize the expression ratios of the enzymes. The biosynthesis of curcumin has attracted much attention by many researchers for decades due to its diverse therapeutic properties including its anti-Parkinson’s, anti-Alzheimer’s, antiinflammatory, and cholesterol-lowering activities.18, 19 Therefore, the expression ratios of the six genes in the artificial curcumin pathway were altered by randomly mutated single-stranded DNAs using MAGE, and the mutant strains with various yields of curcumin production were selected, and the quantitative changes in these enzymes were calculated. Finally, one mutant (6M08rv) showed about a 38.2-fold improvement in the curcumin yield, in which the expression levels of two enzymes (4-coumarate: CoA ligase (4CL) and phenyldiketide-CoA synthase (DCS)) were much lower than that of the parent strain. Thus, the results of this study show that the expression ratios of an artificial multi-enzyme pathway acquired by a heterologous host can be optimized by a 5’-UTR region mutation using the MAGE tool. Additionally, this result shows that biosynthetic metabolic flux can be more optimized when the concentrations of enzymes are present at a suitable ratio. Curcumin is synthesized through the collaboration of two type III polyketide synthases, DCS and curcumin synthase (CURS2) in the presence of feruloyl-CoA and malonyl-CoA, from Curcuma longa.20, 21 DCS and CURS2 participate in curcuminoid biosynthesis, which includes curcumin, using the abundantly present CoA esters in turmeric (Figure 1). To produce curcumin in the heterologous host E. coli from a simple sugar as a starter, plasmids were constructed to include the artificial biosynthetic pathways of curcumin which contained six genes: the optal, sam5, com, and 4cl2nt genes along with the dcs and curs2 genes. We previously reported on the construction of artificial biosynthetic pathways that can produce hydroxycinnamic acids and stilbenes, respectively, using the optal, sam5, com, and 4cl2nt genes.22, 23 Therefore, the optal, sam5, com, and 4cl2nt genes from these pathways were used along with the dcs and curs2 genes to construct the artificial pathway to pro-

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duce curcumin in E. coli (Figure S1). The curcumin artificial biosynthetic pathway was built with two plasmids each contain-

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To fine-tune the gene expression levels of the six genes for a more effective curcumin production, the 5’-UTR sequences

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ing three genes as previously described in the assembly methods.23, 24 The pET-BioCT5M vector contains the optal, sam5, and com genes, and the other pET22-LacZ4DU vector contains the 4cl2nt, dcs, and curs2 genes. Each biosynthetic gene has a T7 promoter, a RBS upstream of the gene and a T7 terminator downstream the gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain,25 which harbors the artificial biosynthetic gene cluster (pET-BioCT5M & pET22-LacZ4DU), was cultured for 40 h in minimal glucose medium (SM) containing kanamycin and ampicillin. The curcuminoid peaks, which included the curcumin peak, were detected by HPLC analysis of this culture extract from the co-transformed strain (Figure 2(b)). This result means that a de novo artificial curcumin biosynthetic pathway was engineered in E. coli containing the two vectors system, although some of the previously bio-engineering methods containing a precursor feeding have already been reported.26-30

of each of the six genes were randomly modified with the MAGE tool, which is a powerful technique to generate libraries of targeted mutations.10, 31-35 To facilitate this application, the two plasmids for the curcumin biosynthesis were inserted into the E. coli chromosome first. The pET-BioCT5M vector already contains two parts of the bioC gene sequence, and the pET22LacZ4DU vector also contains two parts of the lacZ gene sequences for homologous insertion into the E. coli chromosome (Figure S1). The two vectors were sequentially inserted into the corresponding genes loci in the L-tyrosine overproducing E. coli ∆COS1 strain using the RedET recombination systems. A detailed method is described as well as an illustration of the protocol scheme in the supplementary information (Figure S2), and the resulting strain was called COS6-T5M4DU. The T5M module for ferulic acid production was inserted into the bioC gene, which is the gene for a malonyl-acyl carrier protein (ACP) methyltransferase.36 The elimination of this reaction step inhibits the consumption of malonyl-CoA or malonyl-ACP for fatty acid biosynthesis.37 Sequentially, the 4DU module for curcumin production from ferulic acid was inserted between the lacZ gene, and the insertion of these modules was verified by PCR (Figure S3). The final COS6-T5M4DU strain produced the curcuminoid compounds in the same culture conditions without the antibiotics mentioned above (Figure 2(c)). A total of six peaks, including a peak with the same retention time (17.2 min) as the curcumin standard, were observed at an absorbance of 420 nm (Figure 2(c)). The molecular weights of each of the six peaks were analyzed by LC/MS (Figure S4). Peak 1 had an m/z equal to 341 [M+H]+, which is the molecular weight of 3,3’-dihydroxy-demethoxycurcumin. The molecular weight of peak 2 (m/z 325 [M+H]+) was comparable to that of 3’-hydroxy-bisdemethoxycurcumin, and peak 3 (m/z 355 [M+H]+) had a molecular weight comparable to that of 3’-hydroxy-demethoxycurcumin. The molecular weights of peaks 4 (m/z 309 [M+H]+), 5 (m/z 339 [M+H]+) and 6 (m/z 369 [M+H]+), correspond to expected metabolites as bisdemethoxycurcumin, demethoxycurcumin, and curcumin, respectively (Figure S4).20, 21, 27, 38 Therefore, these products were identified to be curcuminoids which can be produced by the reactions of the six enzymes in the artificial pathway. The production patterns of the six compounds were slightly different, depending on the culture conditions. Therefore, the COS6-T5M4DU strain is the first engineered strain to successfully yield curcuminoids by inserting the six biosynthetic genes into the E. coli chromosome. This strain will be useful in the fermentation process because it does not require any antibiotics for vector selection and maintenance. However, the use of the MAGE technique has been limited to modify E. coli strains such the EcNR2 strain which is a mutS gene mutant.10, 31, 34, 39 The inhibition of MutS, which is a mismatch repair protein, can dramatically increase the recombination efficiency. In this study, we used a strain-independent method for the genome engineering which involves introducing a



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suicide plasmid (pRED1) carrying the λ Red recombination system into the mutS gene.33, 40 Accordingly, the mutS gene in

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To optimize the expression ratio for a more efficient curcumin production, the MAGE approach was used as a method of

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the chromosome of the COS6-T5M4DU strain was temporarily inactivated using the pRED1 vector, which can restore the functional MutS activity through screening in the absence of antibiotic selection.33 The pRED1 vector containing the λ Red recombination system and a sequence with some homology to the mutS gene was used to provide the recombination system in the chromosome and to knock-out the mutS gene, thereby constructing the MAGE platform strain COS6-T5M4DU∆mutS (Figure 3 & S2). The COS6-T5M4DU∆mutS strain also had a similar curcuminoid production pattern as that of the parent COS6-T5M4DU strain (Figure 2(d)).

random mutation for the 5’-UTRs of the six biosynthetic genes in the COS6-T5M4DU∆mutS strain (Figure 4). MAGE is based on an oligomer-mediated λ Red recombination system that introduces a mutation into the homology sequence of the E. coli genome.10 According to previous studies on issues with the MAGE efficiency, such as using the lagging strand41-43 and phosphorothioate binding oligomers,44, 45 we designed and used several single strand DNA oligomers in this study (Table S3). The oligomers were designed to be the same for the six genes which were 90-mers in size based on the 5'-UTR sequence of the pET28 expression vector (Figure 4). Each oligomer contained homology sequences (about 40 bases) at both ends of the mismatch region which contained six consecutive degenerate bases (NNNNNN) just in front of the RBS sites (Figure 4). The 5’-UTR containing the Shine–Dalgarno sequence and the AU-rich sequence can be an important target for tunable expressions because the ribosome binding affinity is directly modulated. Additionally, the SpeI or EcoRI restriction enzyme recognition site was included behind the lac operator binding sequences which enables the easy selection of a mutant from the MAGE library. Through successive cycles of MAGE, the chromosomal inserted 5’-UTR sequences of the six genes increasingly diverged away from the wild type ones. Thus, we can cumulatively generate more variants through continuous cycling by MAGE. The variant strains with enhanced curcumin production were directly screened by the yellow colored colonies from a randomized library through successive MAGE cycles. Moreover, several white colonies were chosen to make sure that the 5’UTR region of the six genes really were changed during each MAGE cycle in the colonies. The selection of 70 variants was followed by PCR amplification and digestion by restriction enzyme (SpeI or EcoRI) for which the digested oligomers had the recognition sequence inserted into the target region of one of the six genes (Figure S5). Finally, all 70 variants were recovered from the temporary mutS mutation by the λ Red recombination system excised from the chromosome by screening for loss of antibiotic resistance after growth in the absence of antibiotic selection. From these cell populations, the targeted 5’-UTR region in the 70 MAGE variants was sequenced which provided proof of the genotypic variation (Table S4). The sequencing data from the 70 variants revealed at least one mutation in the 5’-UTR sequences among the six genes and the recognition sequence ACTAGT (for SpeI) or GAATTC (for EcoRI) behind the lac operator. In addition, several variants were found to have a RBS sequence variation near the 6 degenerate bases; this is believed to have occurred due to errors resulting from the synthesis process for the long oligomers. The frequency of mutations in the 5’-UTR sequences of the six target genes in the 70 variants was as follows: optal, 19%; sam5, 12%; com, 11%; dcs, 12%; 4cl2nt, 32% and curs2, 14% (Table S4). These results showed that the 70 selected variants had a mutation occur at a relatively constant rate in the six genes. However, the presence of the higher ratio of the 4cl2nt mutants was due to the high curcumin-production variants used in the selection process because the 4cl2nt mutant was used as a template strain following several cycles of MAGE. The mutS-recovered variants and the parent strain (COS6-T5M4DU) were cultured in SM medium to compare the curcuminoid productivity. The quantity of the curcuminoid production was measured by comparing the peak areas of the standard


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curcumin in the HPLC analysis. These variants showed varying levels of curcuminoid production as expected (Figure S6). Most of the 37 yellow colonies had more or similar productivity to the parent strain, and no curcuminoid production was detected in the 33 colonies under the same culture conditions. Finally, we isolated 25 variants that had an increased curcuminoid production relative to the parent strain (Figure 5). Only eight of the 25 variants exhibiting more than a 4-fold increase in total curcuminoid production relative to that of the COS6-T5M4DU parent strain (0.3 ± 0.1 mg/L) were selected shown in Figure 6. It was confirmed that the six variants (6M08rv, 6M18rv, 6M19rv, 9M08rv, 6M12rv, and 8M10rv) had a 10-fold increase in curcumin production compared to the parent strain (0.1 ± 0.01 mg/L). Their curcumin productivity was 3.8 ± 0.6, 2.0 ± 0.5, 1.6 ± 0.9, 1.5 ± 0.2, 1.1 ± 0.3 and 1.0 ± 0.3 mg/L, respectively (Figure 6). Notably, the total curcuminoid productivity of the 6M08rv strain (3.9 ± 0.8 mg/L) was increased by about 12.6-fold compared to that of the parent strain, and the production of curcumin alone was 38.2 times (3.8 ± 0.6 mg/L) higher than that of the parent strain. Interestingly, all of the top eight high producers generally had a mutation in the 5’-UTR sequence of the 4clnt gene, and the highest producer, the 6M08rv strain, had mutation each in both the 4clnt and dcs genes. Next, the translation efficiencies of the sequence changes were calculated with the RBSDesigner program46 (Figure 6 & Table S5) and then compared these calculated values with their curcuminoids productivity. The RBSDesigner program can predict the translation efficiency of existing mRNA sequences from the 5’-UTR region and the given coding sequence.46 It can assist in calculating the levels of expression for a designed synthetic RBS of a protein expression vector. The calculated values of the synthetic RBS sequences showed reasonable results with the actual protein expression level; however, the expression level of each of the other genes followed different genetic contexts which presented quite different quantitative patterns (unpublished results).9, 47 We already designed the 5’-UTR sequences of the six genes based on the RBSDesigner program for over-expressing the proteins of interest (190.7 ~ 213.3 × 10-3; Table S4). Thus, the calculated translation values of the 4cl2nt and dcs genes in the 6M08rv strain were 0.5 × 10-3 and 6.8 × 10-3, respectively. These values are much lower than the original designed 191.1 × 10-3 and 190.7 × 10-3 value for both 4cl2nt and dcs in the parent strain (Figure 6 & Table S5). Interestingly, all of the top eight high producers had lower translation values than that of the original 4clnt gene, and their mutations are predicted to all have lower translation levels than that of the original 4CL enzyme. The difference between 6M08rv and 6M12rv was only in the translation efficiency (6.8 × 10-3 and 22.2 × 10-3, respectively) of the dcs gene, but it had four times the difference in curcumin productivity. The 9M08rv, which has a lower translation efficiency (107 × 10-3) for curs2 compared with 6M08rv, had a curcumin productivity that was one third lower. Taken together, we could speculate that the curcumin pathway does not need much CoA ligase for effective metabolic flux. The eight strains which included the parent strain and the 6M08rv variant, were cultured under the same culture conditions for curcuminoid production, and the expression levels of the proteins were analyzed by SDS-PAGE after His-tagged purification of the denatured cell extracts (Figure S7). Each of the six genes in the parent strain, which have similar values for the designed translational efficiency determined by an in silico program (Figure 6; 191~210 × 10-3), has quite different protein expression levels (Figure S7B, Line 1). An in silico method also predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. It is well known that the expression level of each gene is affected by the codon usage, secondary structure and various other factors resulting in quite different expression patterns, even if the same vector and culture conditions are used.48 Of the six enzymes in the parent strain, the 4CL and COMT enzymes have very weak expression levels, while the others (Sam5, TAL, DCS and CUR2 enzymes) have strong ones (Figure S7B). Although Sam5 and TAL enzymes are similar in size, they cannot be distinguished by this SDS PAGE. As a result, the real expression level of each of the six enzymes in these variants exhibited similar up and down pat-



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terns with the expected patterns from the in silico analysis. The real expression levels of the 4CL and DCS enzymes in the

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Among the high curcumin producers, the 4CL enzyme commonly had a low expression level as well as a lower predicted

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highest curcumin producer (6M08rv) were meaningfully reduced as predicted by the in silico calculation (Figure S7B, Line 2). All the other variants also showed a reasonable change in the expression level corresponding to the 5’ UTR mutation. However, the 9M08rv variant had a different expression level from that of the in silico prediction: CURS2 had a stronger expression. However, the cause of the differences was not identified.

translation value. The reasons for the weaker expression of CoA ligase are difficult to ascertain. However, one possibility is that the produced CoA thioesters cause damage to the cell.49 Coenzyme A has a clearly defined role as a cofactor for a number of oxidative and biosynthetic reactions in intermediary metabolism. Carboxylic acids can also form CoA-thioesters, and the resulting acyl-CoA may contribute to the compound's toxicity.49 However, there was not much big difference in the rate of growth between the parent strains and 6M08rv variants. 6M08rv variants tended to grow a little faster (Figure S8). There is no way to ascertain whether these growth differences are caused by the produced CoA thioester. Moreover, we suggest that the next step enzymes in the pathway, DCS and CURS2, are not effective or have a harmonious relation with the expressed 4CL enzyme. Another possibility is that 4CL and DCS are relatively fast reactions that do not allow full conversion of L-tyrosine (or p-coumaric acid) into curcumin. As such, lowering the concentration of the 4CL and DCS enzymes can help to accumulate more ferulic acid through TAL, Sam5 (C3H) and COMT enzymes before being converted to curcumin, which can be seen in strain 6M08rv. These assumptions already have been proven by a ferulic acid feeding experiment for curcumin production.26, 38 In addition, several of the high producers (6M08rv, 9M08rv and 6M12rv) exhibited a reduced expression of the DCS or/and CURS2 enzyme, and this phenomenon implies that 4CL and DCS (or/and CURS2) may have a critical stoichiometric balance between these proteins in the condensation reaction of CoA thioesters for curcumin biosynthesis. These results verify that maximum translation does not mean maximum metabolic flux in a natural or synthetic metabolic pathway. The expression ratio of multi-enzymes in artificial systems needs to be further optimized to achieve high productivity. However, the previously rational approaches have limitations in finding the most optimized expression ratio in an in vivo system.7-9, 14 Our results show that the fine-tuning of a multi-enzyme pathway is possible through small changes (only 6 base pairs) of the 5’-UTRs of multiple genes by the MAGE tool, thereby obtaining various expression levels of the multiple genes. The combination of MAGE with high-throughput screen technologies will be very powerful for creating and optimizing multi-enzyme pathways. Thus, such an approach would be applied in combination with the optimizing expression of genes in the precursor pathway, which could be further increase the curcumin titers. Furthermore, the reverse genetic approach using this optimized expression ratio will be an effective synthetic biological method for successful optimization of de novo production of valuable compounds in heterologous hosts. We expect that this example could be further extended and applied to other artificial biosynthetic pathways for the production of useful compounds in heterologous hosts.

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Strains, chemicals, and genes. There are lists of plasmids, strains and primers in Table S1 & S2. Curcumin was pur-

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chased from TCI (Japan), and used as a standard for the compound identification by high-pressure liquid chromatography (HPLC). A codon optimized TAL gene (optal) from Saccharothrix espanaensis, a 4-coumarate-3-hydroxylase gene (sam5) from S. espanaensis, a caffeic acid O-methyltransferase gene (com) from Arabidopsis thaliana, a codon optimized 4coumarate: CoA ligase gene (4cl2nt) from Nicotiana tabacum, a codon optimized diketide CoA synthase gene (dcs) and curcumin synthase gene (curs2) from Curcuma longa were synthesized. Construction of a curcuminoids-producing strain for MAGE. To construct an artificial curcumin biosynthetic pathway, each biosynthetic module (the T5M module for ferulic acid production containing the optal, sam5, and com genes and the 4DU module for curcumin production from ferulic acid containing the 4cl2nt, dcs, and curs2 genes) was cloned into a pET22 vector by In-fusion cloning kit (Takara Bio, USA) resulting in pET22-IF-T5Mp and pET22-IF-4DUp, respectively (Figure S1). Meanwhile, a kanamycin resistance gene (neo) was cloned into the two plasmids (pET-BioCH1neoH2 and pETLacZH1neoH2) with the bioC and lacZ homologous sequence, respectively (Figure S1). Subsequently, the pETBioCH1neoH2 and pET-LacZH1neoH2 were cut by PacI and, each module was inserted into the PacI sites resulting in the pET-BioCT5M and pET22-LacZ4DU vectors, respectively (Figure S1). Finally, the pET-BioCT5M vector contains the optal, sam5, com, and neo genes flanked two bioC gene homology sequences, and the pET22-LacZ4DU vector contains the 4cl2nt, dcs, curs2, neo genes flanked by two lacZ gene homology sequences (Figure S1). The co-transformed E. coli strain with the two pET-BioCT5M and pET22-LacZ4DU vectors produced curcuminoid compounds (Figure 2(b)). Next, after digested by NheI, the linearized pET-BioCT5M and pET22-LacZ4DU vectors were integrated into the chromosome of a Ltyrosine overproducing E. coli ∆COS1 strain with the λ RedET recombinase kit (Gene Bridges, German). The BioCT5M fragment was inserted into the bioC gene of the ∆COS1 strain resulting in the COS6-T5M::neo strain (Figure S2; Step1). Then, the neo gene from the COS6-T5M::neo strain was removed using 707-FLPe recombinase (Gene Bridges, German), resulting in the COS6-T5M strain (Figure S2; Step2). Subsequently, the linearized LacZ4DU fragment was inserted into the lacZ gene of the COS6-T5M strain using the λ RedET recombinase kit resulting in the COS6-T5M4DU::neo strain (Figure S2; Step3). Then, the neo gene from the COS6-T5M4DU::neo strain was removed using 707-FLPe recombinase, resulting in the COS6-T5M4DU strain (Figure S2; Step4). The COS6-T5M4DU strain also produce curcuminoid compounds (Figure 2(c)). To construct the MAGE platform strain, the pRED1 vector containing the λ Red module, chloramphenicol resistance gene and part of the mutS homology sequence was inserted into the mutS gene of the COS6-T5M4DU strain as previously described.33 As a result, the final strain, COS6-T5M4DU∆mutS (Figure S2; Step5), has a temporary inactivated mutS gene and an intracellular recombination system. The genome-integrated mutS inactivated MAGE colonies (COS6T5M4DU∆mutS) were preserved and grown on LB plates supplemented with chloramphenicol. All engineered strains were confirmed by PCR analysis (Figure S3). Design of the MAGE oligomers and MAGE experiments. The synthetic MAGE oligomers used in this study are listed in Table S3. The MAGE oligomers were designed as 90-mers based on the 5'-UTR sequence of the pET28(+) vector. Each oligomer has about a 40-mer homology sequence at both ends for binding to a target region and 6 consecutive degenerate bases (6N=A,C,G,T) in front of the RBS for random mutation (Figure 4). In addition, these oligomers included the SpeI or EcoRI recognition sites behind the lac operator so that the mutated genes can be easily selected from the mutant library. According to former studies on mutation efficiency issues of MAGE, four types of oligomers for both directions (“F” and “R”)



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and complement sequences (“On” and “Un”) containing phosphorothioate linkages were synthesized. Phosphorothioate bonds located at the terminal bases may increase the replacement efficiency by preventing in vivo degradation of the synthetic oligonucleotide molecules by endogenous exonucleases in the cell. The two kinds of combination strands (“On-F” and “On-R” or “Un-F” and “Un-R”) were used alternately in each MAGE cycle. The MAGE experiments were carried out manually according to the method previously reported.31, 34 Screening of curcumin-overproducing variants. During each MAGE cycle, these cultures were streaked on an MALB plates (containing 1 mM IPTG and 15 g/L glucose in LB agar), and cultured at 37°C for 1-2 days. Potential curcuminoverproducing mutants were first screened visually by a yellow color on the MALB plate and followed cleavage pattern analysis by SpeI or EcoRI restriction enzyme for the six genes. An overall of approximately five thousands colonies were screened for increased production of the yellow pigment by visual inspection. Finally, various nucleotide exchanges were detected in the 5’-UTR region sequences of one or more of the six genes among the selected 70 variants by cleavage pattern analysis (Table S4). Before the mutS recovery experiments, we confirmed that most of the yellow colonies had more or similar productivity to the COS6-T5M4DU∆mutS strain and that there was no detected curcuminoids production in the white colonies under the same culture conditions (Figure S6). Therefore, we isolated variants with a curcumin production capacity through MAGE up to 40 cycles. The confirmed variants were cultured in 3 ml of LB medium at 37 °C for about 3-7 days, and then, the cultures were streaked on LB plates. The colonies were picked randomly on LB plates supplemented with/without chloramphenicol, and each mutS-recovered variant was finally selected by screening colonies that have no resistant to chloramphenicol. All of the selected 70 variants had the mutS gene recovered which was used to compare the curcuminoids productivity. Finally, curcuminoids production in 37 of the 70 variants was detected by HPLC (Figure 5). Quantification of curcuminoid production in the selected variants. The mutS-recovered mutant was grown at 37 °C in LB. The overnight culture was inoculated into fresh LB, and it was grown at 37 °C to an OD600 of 0.4. IPTG was added to the final concentration of 1 mM for induction of inserted six curcumin biosynthetic genes. After the culture was incubated for 5 h at 26 °C and harvested by centrifugation, the harvested cell was suspended in the modified synthetic medium25 (SM; 3 g/L KH2PO4, 7.3 g/L K2HPO4, 8.4 g/L MOPS, 2 g/L NH4Cl, 0.5 g/L NaCl, 0.1 ml/L Trace elements, 5 g/L MgSO4, 5 g/L (NH4)2SO4, 15 g/L glucose and supplemented with 1 mM IPTG) and further incubated for 40 hour at 26 °C. A lower culture temperature (26 °C) is better for curcumin productivity compared with that of 37°C (data not shown). Additionally, the cotransformed (pET-BioCT5M & pET22-LacZ4DU) E. coil strain was cultured conditions with antibiotics (50 µg/mL kanamycin and 50 µg/mL ampicillin) under the same as mentioned above. The cultures were extracted with an equal volume of ethyl acetate (EA). These were dried and dissolved in methanol. The extracts were analyzed by the high performance liquid chromatography (HPLC) system using SunFire™ C18 column (250 × 4.6 mm, 5 µm; Waters, USA) at 20-80% acetonitrile [CH3CN–H2O (containing with 0.05% trifluoroacetic acid)] for 20 min at 1 mL/min conditions. The LC-MS/MS analysis were carried out according to the manual of the Xcalibur system (version 2.2 SP1.48; Thermo Scientific). Quantification of the curcuminoids was calculated by comparison with curcumin standard using the peak areas at 420 nm absorbance of the HPLC analysis. The quantification data was generated from triplicate independent experiments. Calculation of the translation efficiency of each gene in the parents strain and mutants. RBSDesigner program46was used to calculate the translation efficiency by nucleotide sequence exchange on the 5’-UTR sequence for each of the six genes. It can estimate the translation efficiency of a user-specified mRNA sequence.46 The region used to calculate the translation efficiency was total 394 bp sequence from AGATCT (BglII site in front of the promoter region on pET28a) to 227 bp


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of each coding region of six genes (Figure 4). The ATG codon in front of the His-tag sequence was commonly selected as a translational initiation site.

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1

ASSOCIATED CONTENT

2 3

Supporting Information. This PDF file contains Supplementary Tables S1-S5, Figures S1-S8 with legends and their refer-

4

AUTHOR INFORMATION

5

Corresponding Author

6

* E-mail: [email protected], Tel.: +82 43 240 6144; fax: +82 43 240 6169.

7

Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the

ences. This material is available free of charge via the Internet at http://pubs.acs.org.

manuscript.

10

Notes

11

The authors declare no competing financial interest.

12

ACKNOWLEDGMENT

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This work was supported in part by the KRIBB Research Initiative Program and by the Next-Generation BioGreen 21 Program (SSAC, PJ001108401) funded by the RDA, Republic of Korea. We would like to express our gratitude to Prof. S.K. Lee (UNIST) for kindly providing us with pRED1 vector. We thank to Mr. B.S. Lee (KRIBB) for his help with processing LC-MS/MS data.

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Figure 1. Biosynthetic pathway for heterologous curcuminoids production. Dashed box represents the six genes in the curcuminoids pathway: optal (tyrosine ammonia lyase; TAL), sam5, (coumarate-3-hydroxylase; C3H), com (caffeic acid O-methyltransferase; COMT), 4cl2nt (4-coumarate: CoA ligase; 4CL), dcs (diketide CoA synthase; DCS) and curs2 (curcumin synthase; CURS) gene. These six genes were inserted into the engineered E. coli chromosome. Brackets represent proposed curcuminoid structure.

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Figure 2. De novo production of curcuminoids in an engineered E. coli. HPLC profile of the standard curcumin (a), the culture extract of the co-transformed (pET-BioCT5M & pET22LacZ4DU) E. coil strain (b), COS6-T5M4DU strain (c), MAGE platform strain (COS6T5M4DU∆mutS) (d), and the optimized variant (6M08rv) strain (e). Each peak number indicated in the HPLC corresponds to the number of the proposed curcuminoid structure in Figure 1.

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Figure 3. MAGE platform strain with the artificial curcumin biosynthetic pathway. To fine-tune the gene expression level of the six genes for the curcumin production, sequentially, the six genes were inserted into the L-tyrosine producer, E. coli ∆COS1 which was engineered on the genome for also the feedback resistance of the chorismate mutase (aroGfbr) and prephenate dehydrogenase (tyrAfbr) genes in a tyrosine-mediated repression knockout (∆tyrR) strain background. The T5M module (optal, sam5, and com genes) for ferulic acid production was inserted into the bioC gene, which is a malonyl-acyl carrier protein (ACP) methyltransferase. The 4DU module (4cl2nt, dcs, and curs2 genes) for curcumin production from ferulic acid was inserted between the lacZ gene. Finally, the portable λ recombination system was located at the mutS locus on the genome.

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Figure 4. Diversification of the 5’-UTR sequence for each curcumin biosynthetic gene from the MAGE oligomers. Yellow box highlights the synthesized 90-mer oligo sequence originating from the pET28 expression vector, which is equally present in the front of the coding sequences of the six genes. Dashed box represents the partial sequences among the 90-mer oligos containing the six degenerate bases (red color) and SpeI or EcoRI cutting sites (under lined). Red line box shows the changed sequences of the corresponding gene in the variants. See supplementary data for the detailed 90-mer oligo sequences (Table S3) and changed sequences of the variants (Table S4).

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Figure 5. Curcumin and the other curcuminoids production of the MAGE variants. Comparisons of the curcumin (yellow)

and total curcuminoids (red) productivity in each of the 37 variants after a 40 h cultivation in modified minimal medium (SM). Star (*) indicates COS6-T5M4DU as the parental strain. Each error bar indicates the standard deviations between the measurements from triplicate cultures.

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Figure 6. Comparison of the in silico calculated translation efficiency and curcuminoids productivity of the top eight selected curcuminoids overproducing variants. The numbers in the boxes represent the calculated translation efficiency of the 5’UTR sequences using the RBSDesigner software. Each gene of the parent strain was designed to be under the control of the expression system with a strong promoter and the 5’-UTR sequence. Colored boxes denote confirmed 5’-UTR mutations at the corresponding genes of each variants. In this figure, the data show against selected top eight curcuminoids overproducing variants, the other variant (9M01rv) and parent strain used a control.

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Optimization of Artificial Curcumin Biosynthesis in E. coli by

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