CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production
© The Author(s) 2017
Received: 4 October 2016
Accepted: 27 December 2016
Published: 17 January 2017
Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-l-methionine (SAM or AdoMet).
Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen ‘Kaori-no-mai’ AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3′-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall.
An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.
Many plant natural products and polyphenols have been associated with a diverse array of health benefits, particularly in studies performed in vitro, but controversy surrounds many of these compounds with respect to their efficacy in humans despite the higher success rate of natural products versus other chemicals in Phase I testing . Nonetheless, industrial interest in these products has stagnated because of difficulty sourcing large quantities of complex natural products, primarily due to low abundance of these compounds in raw plant extracts. Complicated syntheses suffer from low yield due to the presence of multiple stereocenters, and, thus, offer little hope as a viable alternative to extraction . Despite these difficulties, the brightly colored plant pigments known as anthocyanins are intriguing candidates for continued investigation due to their antioxidant properties, reported health benefits, and high potential for use as natural food and beverage colorants  since they already pervade nearly all diets.
Anthocyanins are pigmented flavonoids that are classically considered to be naturally biosynthesized in plants through the subsequent conversion of flavanones to dihydroflavonols by flavanone 3β-hydroxylase (F3H), dihydroflavonols to leucoanthocyanidins by dihydroflavonol 4-reductase (DFR), and leucoanthocyanidins to anthocyanidins by the α-ketoglutarate- and Fe(II)-dependent anthocyanidin synthase (ANS). We have previously shown, however, that Escherichia coli expressing ANS are able to convert the flavan-3-ols afzelechin and catechin into the anthocyanidins pelargonidin and cyanidin, respectively . This affords the opportunity for one-step biotransformation of the cheap and abundant precursor (+)-catechin into the unstable compound cyanidin, which can be significantly stabilized by glycosylation with UDP-glucose: 3-O-glycosyltransferase (3GT) to form cyanidin 3-O-glucoside. Such a strategy enables extension and exploration of the heterologous microbial anthocyanin biosynthetic pathway in the genetically tractable host, E. coli.
Traditional microbial workhorse hosts like E. coli and Saccharomyces cerevisiae remain an attractive option for both exploration of natural enzymatic tailoring reactions and production of specialty natural products requiring several biosynthetic steps, primarily due to their well-characterized metabolisms and the wealth of advanced genetic tools [1, 5, 6]. Additionally, flavonoids and anthocyanins are convenient molecules for probing the capacity for co-option of specific endogenous microbial cosubstrates and cofactors; these polyphenols are easy to quantify, and most are readily permeable to cell membranes through passive and active diffusion (an important feature for substrate feeding, mutasynthesis [7–10], and co-culture studies  requiring inter-strain transport of pathway intermediates). Synthetic biology platforms developed for combinatorial heterologous gene expression in these hosts further facilitate rapid examination of genes from disparate sources , enabling augmented synthetic pathway flux due to either improved enzymatic properties (K m and k cat ), reduced inhibition from pathway intermediates and end products, or distinct heterologous expression capacities exhibited by divergent gene orthologs.
Prior to this work, our lab has successfully reconstituted many short flavonoid [11, 13, 14] and anthocyanin [3, 4, 15, 16] pathway segments in E. coli, which served as important testbeds to study endogenous cosubstrate or cofactor limitations [17, 18] toward the ultimate objective of de novo microbial production of complex polyphenols (starting with glucose and requiring construction of long pathways of 10–20 heterologous biosynthetic genes). Until now, however, we have not significantly extended these preliminary studies toward exploration of anthocyanin decoration. Instead, we have focused primarily on UDP-glucose improvement—by supplementation or overexpression of relevant genes in central carbon metabolism—for conversion of the anthocyanidins (aglycones) pelargonidin and cyanidin into the anthocyanins (glycosides) pelargonidin 3-O-glucoside and cyanidin 3-O-glucoside [3, 4, 15]. Other common anthocyanin modifications of interest besides hydroxylation include O-methylation  and C-methylation , aliphatic acylation (acetylation and malonylation) or aromatic acylation (cinnamoylation, hydroxycinnamoylation, hydroxybenzoylation, etc.) , and various glycosylations with distinct hexose or pentose donors at different carbon positions on the core anthocyanin (including diglycosides or linear disaccharide/trisaccharide extensions at a single carbon on the core structure) [22–24]. These decorations are desirable because they increase product stability and thus facilitate characterization and quantification in complex matrices like cell cultures. Additionally, such modifications change photochemical properties, making these tailored products attractive as natural pigments for the food, beverage, and cosmetics colorant industries. A platform strain enabling rapid screening of putative anthocyanin tailoring enzymes from various plant sources using (+)-catechin or other inexpensive and abundant substrates would also prove useful for plant scientists.
Microbial anthocyanin O-methylation is one straightforward target modification that has not been examined to date in the context of a heterologous biosynthetic pathway. Instead, most anthocyanin O-methyltransferases (AOMTs) have been characterized in planta or in vitro, following purification of heterologously-expressed enzyme from E. coli. Given the existence of S-adenosylmethionine (SAM or AdoMet, the cosubstrate used by AOMTs to methylate anthocyanins primarily at the 3′- and 5′-hydroxyls) in the natural metabolism of E. coli, coupled with the proven capability of E. coli to express active AOMTs, we posited that endogenous SAM pools might be sufficient to achieve in vivo anthocyanin O-methylation. Further supporting this hypothesis, E. coli has been utilized as a host for heterologous production of other natural compounds that require SAM as a cosubstrate such as vanillate , N-acyl-homoserine lactones , polyketides , and even for biotransformations of flavonoids to their O-methylated counterparts [28–30]. Until recently, however, there has been little effort aimed at improving SAM pools for co-option by exogenous, SAM-dependent methyltransferase reactions. Primary strategies have been more traditional, including supplementation of l-methionine  (the immediate and limiting SAM precursor) and overexpression of SAM synthase (metK) [27–29], but recent advances in the field of synthetic biology are expediting our ability to explore novel metabolic engineering strategies. In particular, the advent of dCas9-mediated transcriptional repression (also known as CRISPR interference, or CRISPRi) has enabled rapid assessment of metabolic engineering interventions at a rate that was previously inaccessible [32, 33], predominantly due to the ease and specificity of targeting the programmable synthetic transcription factor dCas9 with a guide RNA (gRNA) designed to be complementary to a ~20 bp target DNA site . Therefore, we turned to CRISPRi as a tool to quickly probe metabolic perturbations in E. coli for improved SAM availability and, as a result, increased anthocyanin O-methylation.
Anthocyanin O-methyltransferases from different organisms exhibit distinct in vivo production capacities
A common starting point for improving production of heterologous natural products in microbial hosts is screening of known and putative enzymes catalyzing the same reaction but isolated from different donor organisms . Although the outcomes of such screens do not necessarily shed light on the rationale for exceptional performance of a particular ortholog in the context of the reconstituted pathway, these screens often uncover a gene or combination of genes that perform significantly better than others in a specific expression context. Further investigation is often required to determine if an ortholog has better inherent enzymatic properties, is less sensitive to feedback inhibition, or is simply expressed at a more optimal level for production (not necessarily higher, but perhaps more balanced with the other heterologous pathways). With this in mind, we selected five previously validated anthocyanin O-methyltransferases (AOMTs) from the literature to test for extension of our synthetic heterologous pathway. AnthOMT (referred to in this work as SlAOMT for convenience) was originally characterized due to its role in anthocyanin diversification in tomato (Solanum lycopersicum) seedlings . CkmOMT2 was isolated as the major paralog—out of four in the genome of the fragrant cyclamen cultivar ‘Kaori-no-mai’—responsible for methylation of the anthocyanin delphinidin 3,5-diglucoside . VvAOMT1 from grape has been characterized as a flavonol and anthocyanin 3′, 5′ O-methyltransferase [37–39]. PhMF1 and PhMF2 are closely related paralogs from Petunia hybrida .
Gene dosage affects C3G and P3G titers
Given the low overall titers of both C3G and P3G obtained during the homolog screen, we hypothesized that substrate utilization and, thus, overall production would be improved by increasing expression of the upstream genes. We next evaluated the effect of gene dosage (mediated by plasmid copy number modulation) on P3G production. Expression of the upstream module composed of PhANS and At3GT was increased by transferring from the low-copy vector into high-copy vectors harboring one of the two best AOMTs (VvAOMT1 and CkmOMT2). In addition, an alternative upstream module composed of maltose binding protein (MBP)-tagged PhANS and At3GT was tested with each of the two best AOMTs in parallel. Although no strict hypothesis was posed as rationale for improved production using MBP tags, we had previously achieved high concentrations of soluble MBP-PhANS and MBP-At3GT during in vitro characterization (unpublished data), perhaps because cytosolic MBP (N-terminal membrane domain removed) is known to enhance solubility of its fusion partners . Moreover, modulation of protein translation rate through alteration of ribosome binding site (RBS) and N-terminal sequence (by fusion tag or alternative codon usage) is one of many methods used to balance expression of genes within a pathway [42, 43]. We thus speculated that even slight variation of upstream pathway expression due to the presence of N-terminal MBP tags could cause significant changes in production either through improved solubility of the upstream enzymes or through the resulting altered balance with the downstream AOMT.
CRISPRi-mediated deregulation of methionine and S-adenosyl methionine biosynthesis improves P3G titers
While we have previously shown that endogenous pools of UDP-glucose are limiting for production of the anthocyanins C3G and pelargonidin 3-O-glucoside (similar to C3G, but monohydroxylated at the para-position of the B ring) from their corresponding aglycones in E. coli , we sought in this work to explore the potential limitation of SAM availability for O-methylation of anthocyanins. Several approaches have been previously utilized to overproduce SAM in E. coli, but one surprisingly neglected strategy posed at the initiation of this work was deregulation of SAM and methionine biosynthesis by simply silencing metJ, the ligand-responsive transcriptional repressor that naturally regulates production of methionine and SAM in response to feedback from SAM accumulation (and, to a lesser degree, accumulation of other structurally similar ligands) . Although pathway deregulation is not always sufficient to engender accumulation of biosynthetic intermediates, it is often successful because of the coordinated response—in some cases simultaneous repression, de-repression, and activation of genes at key regulatory nodes—that has naturally evolved to drive flux through a pathway in the absence of a feedback regulator’s cognate ligand (or in the presence of the cognate ligand, depending on whether a transcription factor binds or releases DNA in response to association with the ligand).
To test the hypothesis that CRISPRi-mediated repression of metJ would improve SAM availability for O-methylation, compatible vectors bearing constitutive CRISPRi elements (dCas9, tracrRNA, and a minimal single-spacer CRISPR array) were co-transformed with the best vector from the gene dosage experiment (VvAOMT1 with MBP-tagged upstream module on high-copy vector). Interestingly, C3G titer was affected by addition of the non-targeting CRISPRi control vector to this strain. Although it is not clear why this occurred, we speculate that previously reported growth inhibition associated with expression of dCas9 and crRNA/gRNA  slightly altered the production capacity or induction optimum for this strain. Regardless, compared to the non-targeting spacer (previously used as a negative control in E. coli by other groups  and ours [32, 50]), both anti-metJ spacers improved production of P3G by approximately twofold (Fig. 5c), representing approximately 20-fold improvement over the best strain from the initial screen. Critically, the conversion of C3G to P3G by AOMT was also markedly improved by MetJ repression (Fig. 5d), which would likely be expected if AOMT’s cosubstrate SAM were limiting. Additionally, it is recommended that multiple gRNAs (or spacers) be selected against each CRISPRi target due to our current inability accurately predict gRNA (spacer) efficiency and specificity . In this work, testing more than one metJ dCas9-binding site demonstrated two important points. Targeting one site achieved slightly higher production than targeting the other site in a similar location, supporting the notion that non-intuitive changes in dCas9-binding site selection can affect production level. Secondly, similar improvements in C3G to P3G conversion for both metJ repression targets relative to the non-targeting control imply that improved production was indeed due to metJ repression; in other words, use of alternative dCas9-binding sites against a single target gene or promoter can serve as a specificity control for the assayed phenotype. Finally, the best metJ repression strain from this experiment (MetJ1) was used to demonstrate cultivation scale-up from 1 mL scale in multi-well plates to 25 mL working volume in 125 mL non-baffled shake flasks. Production improved slightly upon scale-up to a final titer of 56.3 ± 0.6 mg/L (mean ± SEM of three independent experiments), a 21-fold improvement over the best strain from the ortholog screen.
This report details microbial O-methylated anthocyanin production as a case study for anthocyanin decoration in a platform strain, and the results indicate that the endogenous E. coli SAM pool is sufficient for designer methylation reactions but can be improved by targeted genetic interventions. For example, although SAM availability in E. coli has been the focus of a few other recent engineering efforts for O-methylation of natural products, the methionine and SAM biosynthesis deregulation strategy described herein could also facilitate in vivo characterization of SAM-dependent methyltransferases catalyzing C-, N-, and S-dependent methylation of other natural products . While our lab has strived in the past to systematically extend and connect flavonoid and anthocyanin pathways by improving cofactor and cosubstrate availability, this work was instead intended to demonstrate that underexplored anthocyanin tailoring reactions can be rapidly tested in a model microbial system using the inexpensive and abundant substrate (+)-catechin. We anticipate that additional enzymatic reactions will be tested by others using a similar strategy, but consideration should be devoted to availability of cognate cosubstrates; if a dearth of putative orthologs exists for a reaction of interest, it is conceivable that low intracellular cosubstrate concentration will impede conversion and product characterization. In any case, CRISPRi is a quick and easy alternative to gene deletion to improve metabolite pools. A significant limitation to this platform strain is that products are constrained to C3G (3′-, 4′-hydroxylated) as a precursor when feeding (+)-catechin as a substrate. There are a vast number of tailoring reactions that should be compatible with this C3G, but methylated anthocyanidins like petunidin and malvidin and their glycosides are inaccessible unless an appropriate upstream substrate can be synthesized (difficult to control stereochemistry)  or biosynthesized (not achieved to date, and P450 hydroxylation is quite difficult in E. coli).
In addition to screening and characterizing anthocyanin tailoring reactions, the platform described here could ultimately prove feasible for industrial production of modified anthocyanins. Despite modest titers outlined here for the novel anthocyanin product P3G, very impressive and extensive work published during the preparation of this manuscript examined several solutions to SAM limitation in E. coli K-12 MG1655(DE3) lineages for production of vanillate, an O-methylated natural product that had been produced in E. coli previously but suffered from low titers . Combined deregulation of SAM biosynthesis through metJ deletion, feedback desensitization of enzymes catalyzing committed or important biosynthetic steps, and deregulation of SAM regeneration improved de novo vanillate titers by twofold to approximately 400 mg/L . These findings imply that SAM-dependent natural product biosyntheses in E. coli at industrially relevant titers will likely be achievable. Interestingly, and in contrast to our work with anthocyanins, deletion of metJ alone was not sufficient to improve vanillate titers. It is possible that this difference stems from disparities in metabolite pools or regulation between B (used in this work) and K (used in ) E. coli lineages; alternatively, this could be attributed simply to distinct enzyme properties between the selected AOMT and catechol OMT used for vanillate production. Other strategies outlined in literature that have been reported to augment SAM production include supplementation of media with methionine and improvement of precursor ATP pools by reducing ATP usage in other reactions . Another tactic that has not been tested but would likely prove synergistic with MetJ deletion in conjunction with these other strategies is overexpression of MetR, as it has been shown that the combination of SAM:MetJ shortage with overproduction of MetR induces overexpression of methionine synthase . Nonetheless, a combination of all the aforementioned interventions with the UDP-glucose improvements previously described by our lab could eventually lead to industrially relevant titers for these high-value, complex specialty anthocyanins.
Finally, although there are limitations to using CRISPRi rather than gene deletions (potential for incomplete repression, commonly constructed on plasmid-based systems requiring antibiotics and prone to instability, etc.), there are significant advantages that make CRISPRi the unsurpassed tool for targeted gene silencing that exists today. First, design of CRISPRi constructs is swift and straightforward. CRISPRi vectors can be cloned, transformed into production strains, and assessed with predictable results 2–3 days after target selection. If designed appropriately, inducible CRISPRi also enables complete repression (silencing) or tunable, partial repression of essential genes at any point of the growth phase, such as when sufficient biomass has accumulated; a related, highly attractive feature is the capacity to partially repress and somewhat predictably tune both essential and nonessential genes with CRISPRi. Multiplexing can even be achieved following rapid hierarchical assembly of gRNAs or CRISPR arrays, and spacers that have been designed against conserved sequences in multiple strains are portable—i.e. the vectors can be transformed into any host background, facilitating faster assessment of multiple downregulations in disparate strains than any other existing method. Finally, CRISPRi can be used to gently coax cells into a rewired state with little effort; specifically, metJ repression is a “soft” strategy that achieves widespread metabolic perturbation (de-repression of many genes in the same pathway, simultaneously) without the metabolic burden associated with high-copy overexpression of many heterologous genes at once . We anticipate that similar CRISPRi-mediated, systems-level interventions will expedite understanding of how new cosubstrates can be co-opted for novel anthocyanin decorations.
In this study, we have engineered a strain to serve as a platform for modular, rapid examination of anthocyanin tailoring enzymes. The strain only requires supplementation of the inexpensive and abundant precursor (+)-catechin to generate anthocyanins for downstream modification. We exemplify the utility of this strain by engineering microbial production of an O-methylated anthocyanin for the first time. Combining classical metabolic engineering principles and cutting-edge synthetic biology approaches, we significantly improved production of our target compound P3G and gained insight about co-opting limiting AOMT cosubstrate pools. First, we screened AOMTs from disparate sources to isolate candidates with improved in vivo production capacities, a tried-and-true approach that should be considered at the outset of any metabolic engineering project. Next, we transferred the upstream genes to a high copy plasmid. The result was significantly improved flux, suggesting that these genes benefit from increased copy but implying that the effect of gene dosage on product titer should be assayed to explore expression space. Finally, we found that coordinated, global perturbation of SAM biosynthesis was obtainable through a single CRISPRi intervention, leading to increased conversion through the anthocyanin O-methylation tailoring reaction. The metJ dysregulation strategy described in this and another recent report  should be useful for improving methylation of any natural product, but the general steps taken here should also be applicable for studying unique anthocyanin tailoring reactions that require co-option of distinct cosubstrates. Future efforts to integrate anthocyanin pathway genes into the genome of E. coli should also be undertaken to generate stable production strains lacking plasmids.
Strains and plasmids
Strains and plasmids used in this study
Strain or plasmid name
E. coli strains
E. coli DH5α™
F-Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK−, mK+) phoA supE44 λ− thi-1 gyrA96 relA1
E. coli BL21 Star™ (DE3)
F-ompT hsdS B (rB−mB−) gal dcm rne131 (DE3)
E. coli plasmids
ColE1(AmpR), Petunia hybrida anthocyanidin synthase (PhANS) with N-terminal cytosolic MBP tag
ColE1(AmpR), Arabidopsis thaliana 3-O-glucosyltransferase (At3GT) with N-terminal cytosolic MBP tag
ColE1(AmpR), ePathBrick feature, PhANS
ColE1(AmpR), ePathBrick feature, At3GT
ColE1(AmpR), ePathBrick feature, PhANS with N-terminal MBP tag
ColE1(AmpR), ePathBrick feature, At3GT with N-terminal MBP tag
ColE1(AmpR), ePathBrick feature, PhANS and At3GT in monocistronic configuration
pACYC184(CmR), ePathBrick feature, PhANS and At3GT in monocistronic configuration
ColE1(AmpR), ePathBrick feature, PhANS and At3GT with N-terminal MBP tags in monocistronic configuration
ColE1(AmpR), ePathBrick feature, Vitis vinifera anthocyanin O-methyltransferase (VvAOMT1)
ColE1(AmpR), ePathBrick feature, fragrant cyclamen ‘Kaori-no-mai’ anthocyanin O-methyltransferase 2 (CkmOMT2)
ColE1(AmpR), ePathBrick feature, Petunia hybrida anthocyanin O-methyltransferase 1 (PhMF1)
ColE1(AmpR), ePathBrick feature, Petunia hybrida anthocyanin O-methyltransferase 2 (PhMF2)
ColE1(AmpR), ePathBrick feature, Solanum lycopersicum anthocyanin O-methyltransferase (SlAOMT)
ColE1(AmpR), ePathBrick feature, VvAOMT1, PhANS, and At3GT in monocistronic configuration
ColE1(AmpR), ePathBrick feature, VvAOMT1 and N-terminal MBP-tagged PhANS and At3GT in monocistronic configuration
ColE1(AmpR), ePathBrick feature, CkmOMT2, PhANS, and At3GT in monocistronic configuration
ColE1(AmpR), ePathBrick feature, CkmOMT2 and N-terminal MBP-tagged PhANS and At3GT in monocistronic configuration
pACYC184(CmR), tracrRNA, cas9(D10A, H840A), non-targeting CRISPR spacer with two BsaI sites
pACYC184(CmR), tracrRNA, cas9(D10A, H840A), CRISPR spacer targeting MetJ promoter at position 1
pACYC184(CmR), tracrRNA, cas9(D10A, H840A), CRISPR spacer targeting MetJ promoter at position 2
Primers and oligonucleotides used in this study
Nucleotide sequence (5′ → 3′)
Cognate crRNA [Proto]spacer sequence (5′ → 3′)
Cognate PAM (5′ → 3′)
For cloning of individual genes, PCR amplicons and entry backbones were digested with the following restriction enzyme (FastDigest, Thermo Fisher Scientific) combinations: PhANS (XbaI/XhoI), At3GT (NdeI/XhoI), MBP-PhANS (XbaI/XhoI), MBP-At3GT (XbaI/XhoI), VvAOMT1 (NdeI/XhoI), CkmOMT2 (NdeI/XhoI), PhMF1 (NdeI/XhoI), PhMF2 (NdeI/XhoI), SlAOMT (NdeI/XhoI), pETM6 (NdeI/XhoI or XbaI/XhoI). Digested amplicons and backbones were gel purified (E.Z.N.A MicroElute Gel Extraction Kit, Omega Bio-tek) and ligated (Rapid DNA Ligation Kit, Thermo Fisher Scientific) as appropriate to construct the single-gene vectors listed in Table 1. Ligation products were transformed into DH5α and confirmed by Sanger sequencing (GENEWIZ, Inc.).
Using the ePathBrick subcloning procedure , At3GT and PhANS were then assembled into monocistronic configuration (each gene flanked by its own T7 promoter and terminator) by ligation of restriction digestion fragments from plasmid pETM6-At3GT (NheI/SalI) and pETM6-PhANS (AvrII/SalI), yielding plasmid pETM6-At3GT-m-PhANS. MBP-At3GT and MBP-PhANS fusions were assembled into monocistronic configuration by ligation of restriction digestion fragments from plasmid pETM6-MBP-At3GT (NheI/SalI) and pETM6-PhANS (AvrII/SalI), yielding plasmid pETM6-MBP-At3GT-m-MBP-PhANS. For co-expression of pETM6-harbored AOMTs with PhANS and At3GT, the two upstream genes were excised as a single cassette from pETM6-At3GT-m-PhANS (AvrII/SalI) and subcloned into low-copy vector pACM4 (AvrII/SalI) to generate plasmid pACM4-At3GT-m-PhANS. Initial screening of AOMT orthologs was performed by co-electroporation of all five pETM6-based AOMTs with ePathBrick-compatible pACM4-At3GT-m-PhANS into BL21 Star™ (DE3).
Next, the two best AOMTs, VvAOMT1 and CkmOMT2, were combined with either PhANS-m-At3GT or MBP-PhANS-m-MBP-At3GT on the high-copy pETM6 backbone. Ligation of pETM6-VvAOMT1 (NheI/SalI) with pETM6-At3GT-m-PhANS (AvrII/SalI) or pETM6-MBP-At3GT-m-MBP-PhANS (AvrII/SalI) yielded plasmids pETM6-VvAOMT1-m-At3GT-m-PhANS and pETM6-VvAOMT1-m-MBP-At3GT-m-MBP-PhANS, respectively. Similarly, ligation of pETM6-CkmOMT2 (NheI/SalI) with pETM6-At3GT-m-PhANS (AvrII/SalI) or pETM6-MBP-At3GT-m-MBP-PhANS (AvrII/SalI) generated plasmids pETM6-CkmOMT2-m-At3GT-m-PhANS and pETM6-CkmOMT2-m-MBP-At3GT-m-MBP-PhANS, respectively.
Plasmids used for CRISPRi/dCas9-mediated transcriptional repression were assembled as described previously [32, 50]. All CRISPRi plasmids harbored dCas9, tracrRNA, and a minimal CRISPR array containing a single spacer. Each CRISPR element is transcriptionally controlled by native Streptococcus pyogenes constitutive promoters as in our previous work [32, 50]. Plasmid pdCas9, encoding a non-targeting spacer, was utilized as a negative control for CRISPRi interventions and as a base plasmid for cloning metJ repression spacers. For both metJ repression targets, two 35 bp complementary and slightly offset oligonucleotides (Integrated DNA Technologies) containing the spacer sequences for dCas9 targeting were phosphorylated with T4 polynucleotide kinase (PNK, New England Biolabs) and annealed (37 °C for 30 min, 98 °C for 5 min, ramp down to 25 °C over 15 min) to build inserts for each metJ-repressor variant. Complementary offset oligos possessed the following sequences: 5′-AAACN30G-3′ and 5′-N30CAAAA-3′, where N30 represents the genomic protospacer (target) sequence. Although spacer sequences in this CRISPR system are ~30 bp, it has previously been shown that ~10 bp are typically trimmed from the 5′ end of the spacer during crRNA-tracrRNA processing by RNAse III, yielding functional spacers of ~20 bp . It is possible, however, that certain crRNAs retain the full length spacer as shown possible in the Type I-E CRISPR system from E. coli , but the effect that this would have on dCas9-mediated transcriptional repression of distinct targets in E. coli is currently unknown.
Phosphorylated and annealed inserts were then cloned into the recipient vector, pdCas9, at two adjacent BsaI sites in the minimal, single-spacer CRISPR array using a one-pot Golden Gate reaction with BsaI (New England Biolabs) and T7 DNA ligase (New England Biolabs). Plasmid pdCas9 was a gift from Luciano Marraffini (Addgene plasmid #46569). See Table 2 for a list of oligonucleotides used for construction of CRISPRi plasmids and their corresponding protospacer sequences.
To facilitate rapid screening of constructs and expression conditions, all experiments were performed in 1 mL cultures in polypropylene deep 48-well plates (5 mL, VWR) covered with breathable rayon film (VWR). A semi-rich defined media known as AMM, supplemented with 2% glucose and appropriate antibiotics (80 μg/mL ampicillin and 25 μg/mL chloramphenicol, as necessary), was utilized for all liquid culture growth, including overnights and production experiments. AMM has been described previously  and is composed of 3.5 g/L KH2PO4, 5.0 g/L K2HPO4, 3.5 g/L (NH4)2HPO4, 2 g/L casamino acids, 100 mL 10× MOPS mix (83.72 g/L MOPS, 7.17 g/L tricine, 28 mg/L FeSO4·7H2O, 29.2 g/L NaCl, 5.1 g/L NH4Cl, 1.1 g/L MgCl2, 0.48 g/L K2SO4, and 0.2 mL micronutrient stock), 1 mL 1 M MgSO4, 0.1 mL 1 M CaCl2, and 1 mL 0.5 g/L thiamine HCl. Micronutrient stock contains 0.18 g/L (NH4)6Mo7O24, 1.24 g/L H3BO3, 0.12 g/L CuSO4, 0.8 g/L MnCl2, and 0.14 g/L ZnSO4.
For production experiments, glycerol stocks of BL21 Star™ (DE3) expression strains containing anthocyanin pathway plasmids and, in some cases, CRISPRi plasmids were first streaked onto LB agar plates containing appropriate antibiotics. Following overnight growth on solid media, cells were inoculated into a single well of a polypropylene deep 48-well plate containing 1 mL liquid media. After 14 h growth in liquid culture at 225 rpm and 37 °C, all strains were subcultured by 50× back-dilution into fresh, room temperature media (20 μL overnight culture into 1 mL AMM), and incubated at 225 rpm and 30 °C. Next, 10 μL 100× substrate (1 g/L (+)-catechin final concentration) stock dissolved in dimethylformamide:ethanol (8:2, v/v) and 10 μL 100× inducer (1 mM IPTG final concentration) stock were added sequentially by multichannel pipette at the optimal cell density as measured on a Biotek Synergy 4 Microplate Reader, and cultures were further incubated at 225 rpm and 30 °C to allow conversion of (+)-catechin to P3G. Cultures were sampled for anthocyanin quantification 18–22 h post-induction with the exception of sampling timecourse studies. All production experiments were performed in biological triplicate, and reported production values represent mean and SEM as quantified by HPLC.
Anthocyanin quantification by HPLC
BFC, TDA, JYS, RJL, and MAGK conceived of the study. BFC designed experiments, analyzed data, and performed experiments with assistance from QDL. BFC, QDL, and DCK constructed plasmids and strains. BFC drafted the manuscript. All authors read and approved the final manuscript.
The authors would also like to thank Dr. Eun Ji Joo and Hila Dvora for their earlier unpublished efforts to express and characterize MBP-tagged PhANS and At3GT in vitro.
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting our findings can be found in the main paper and its additional file.
This research was supported by the National Science Foundation through an Early-concept Grant for Exploratory Research (EAGER, Grant No. MCB-1448657) and by the United States Department of Agriculture-Agricultural Research Service (USDA-ARS).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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