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Manufacturing Multienzymatic Complex Reactors In Vivo by SelfAssembly to Improve the Biosynthesis of Itaconic Acid in Escherichia coli Zhongwei Yang, Hongling Wang, Yuxiao Wang, Yuhong Ren, and Dong-Zhi Wei ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00086 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Synthetic Biology

Manufacturing Multienzymatic Complex Reactors In Vivo by Self-Assembly to Improve the Biosynthesis of Itaconic Acid in Escherichia coli

Zhongwei Yang, Hongling Wang, Yuxiao Wang, Yuhong Ren*, Dongzhi Wei*

* Correspondence to: Y. Ren and D. Wei (Tel.: +86 21 6425 2163, Fax: +86 21 6425 0068) E-mail addresses: [email protected] (Y. Ren), [email protected] (D. Wei). Address: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

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ACS Synthetic Biology 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

ABSTRACT: The self-assembly of multi-enzyme into bioreactors is of extensive interest to spatially regulate valuable reactions. Despite the important progresses achieved, methods to precisely manufacture multi-enzymatic complex reactors (MECRs) are still poorly proposed both in vivo and in vitro, particularly for more than three bio-catalytically relevant enzymes. Here, we developed a sequential self-assembly system to form multitude MECRs involving three enzymes in the itaconic acid (IA) pathway with two pairs of protein-peptide interactions. The MECRs were identified as nano-scale particle-like structures when self-assembled in vitro and produced higher IA production than the unassembled and linearly assembled systems when applied in vivo coupling with CRISPR-Cas9 based metabolic engineering. This work provides novel insights into the construction of multifarious multi-enzyme complex into bioreactors by the self-assembly strategy for multi-step cascades to sequentially control metabolic fluxes inside cells. KEYWORDS: self-assembly, PPIs, MECRs, itaconic acid, CRISPR-Cas9


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The design of multi-step cascades in prominently efficient enzymatic reactors via rational assembly is of extensive interest given the advantages of assembled cascade reactions such as controlled metabolic fluxes and co-factor pools.1,2 Inspired by the principles of spatial organization and the temporal control of multitude reactions via targeting subcellular compartments or compacting enzymes into agglomerates within a cell in nature,3,4 biochemists have focused on the spatial regulation of valuable metabolic cascades in efficient bioreactors.5 Various artificial multi-enzyme assembly strategies, such as direct enzyme fusion, scaffold-mediated co-localization and encapsulation of enzymes in engineered compartments, are available for the spatial arrangement of bio-catalytically relevant enzymes.6-8 However, a proper assembly strategy for the precise control of metabolic flux, particularly the prominent manufacturing of efficient multi-enzyme reactors in a sequential self-assembly pattern, has yet to be established as it should enhance the overall synthetic flux.6 In prokaryotic systems, such as in Escherichia coli, direct enzyme fusion is the simplest assembly method.9 Nevertheless, enzyme fusion may influence the expression level of enzymes or drastically disrupt enzymatic conformation

into

inactive

inclusion

bodies.

Cell

membrane-bound

and

scaffold-associated strategies for multi-enzyme cascades exhibit difficulty in implementing the precise location and stoichiometric arrangement of highly ordered reactors via the simple fusion of targeting signals or tethering non-specific scaffolds,



as these may produce unstable and disordered macro-enzyme complexes.1 In the present study we hypothesize that the facilitated substrate channeling in the spatial arrangement of multi-enzyme may significantly improve the overall yields of the cascade following the adjacent assembly of relevant synthetic multi-enzyme,10-13 which accelerate the processing of the intermediates and prevents these from escaping, thereby conserving a high local concentration.5-8 At present, the sequential arrangement of multi-enzyme has yet to be obtained given that efficient substrate channeling may not occur when the enzymes are not precisely operated together by the aforementioned randomized assembly strategies.14 Hence, better assembly methods for the precise control of multi-enzyme in ordered complex bioreactors are required. We recently accomplished the self-assembly of two enzymes in the itaconic acid (IA) biosynthetic pathway into E. coli cells in our previous research, the results of which exhibited enhanced IA production.15 To construct more devisable and efficient multi-enzyme devices, an additional enzyme citrate synthase (gltA, GA) was proposed as a crucial enzyme in the IA cascades cooperating with the aconitase (acnA, ACN) and cis-aconitate decarboxylase (cadA, CAD) enzymes.16 A sequential cascade for ordered multi-enzymatic architecture was developed accordingly. The present study aims to manufacture a sequential self-assembly system to form prominently efficient multi-enzymatic complex reactors (MECRs) involving three bio-catalytically relevant multi-enzyme, which consist of regularly clustered enzyme-complex repeating unit


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(Figure 1). The in vivo catalytic efficiency of the designed MECRs was superior to that of the dissociative multi-enzyme cascades and the linearly self-assembled bioreactors. Using CRISPR-Cas9 based metabolic engineering in E. coli,17 MECRs were constructed in the genetically manipulated strains to enhance IA production. In the IA pathway, CAD is an essential enzyme for the conversion of cis-aconitic acid to IA. Two other enzymes, specifically GA and ACN, were observed upstream of the cascades, catalyzing to form citric acid and then reversibly transforming to cis-aconitic acid, respectively. Wild-type E. coli cannot produce IA without enzyme CAD.15 Therefore, we codon-optimized the cadA gene from Aspergillus terreus and constructed the plasmids following a recently published procedure.18 To enhance the upstream cascade fluxes, the gltA and acnA genes from Corynebacterium glutamicum were also synthesized with codon optimization. The strategy for the self-assembly of the predicted dimeric GA, monomeric ACN, and dimeric CAD in vitro and in vivo is schematically shown in Figure 1. Two pairs of protein-peptide interaction domains (mouse SH3 and PSD95/DlgA/Zo-1 domains) and ligands (SH3 ligand and PDZ ligand) were applied to design a regulation machinery and inserted into the E. coli cells.6 The self-assembly strategy by two different interaction domains and ligands provided a novel insight for a controllable design that recruited biocatalytic multi-enzyme with diverse binding elements. The element-modified enzymes were expected to sequentially connect to form MECRs in multiple manners with two specific PPI tags by co-transforming self-assembly



plasmids into E. coli for the enhanced IA production (Figure S1). The details of plasmid constructions are available in the Supporting Information. In an effort to improve the IA production, the pH values of the cultivation medium were varied from 6.2 to 7.5 in the unassembled strain co-expressing GA, ACN and CAD enzymes (uaCGA) (Figure S2A). As a result, a pH of 7.0 was selected for all future fermentations. The IA productions of designed self-assembly strains were plotted in the M9 minimal media at 30°C (Figure S2B).19 As compared to the uaCGA strain, a 3.84-fold increase was observed in the optimal self-assembly design (strain SAS-3). To further enhance the IA production, the carbon sources of the M9 minimal media were optimized by adding 1% glucose as the sole carbon source (optimized MM medium, Figure S3A), which resulted in an extremely high IA production of 1.48 g/L in the strain SAS-3 (Figure S3B). The western blot analysis indicated that the expression of the key enzyme CAD were slightly decreased in strain SAS-3 compared to that in strain uaCGA (data not shown). As expected, the IA biosynthetic efficiency was enhanced dramatically by the sequential recruiting of relevant enzymes to spatially arrange and control the multi-enzyme cascades in vivo as compared to the unassembled one. The location of the interaction domains exhibited significant impacts on their ability to increase the biocatalytic efficiencies.6 ER/K was prepared by gene synthesis and used to link target gltA gene to the interaction domain or between two interaction domains.13 The linker sequence (GGGGS)2 was used to connect the corresponding interaction ligands with ACS Paragon Plus Environment

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other two genes.13 Therefore, the self-assembly pattern in strain SAS-3, which fused (GGGGS)2-PDZlig, (GGGGS)2-SH3lig, and ER/K-PDZ-ER/K-SH3 elements to the C-terminus of the ACN, CAD, and GA enzymes (plasmids pAPl and pCSlGPS), respectively, was selected for the formation of MECRs in vivo for the further enhanced IA production. Next, we expressed these element-modified APl, CSl, and GPS enzymes in E. coli BL21 (DE3) as well as the identical unfused enzymes by inserting gene sequences into corresponding pET-28a expression vector sites using the BamHI and XhoI restriction enzymes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that except for enzyme GA, most CAD and ACN were present in the form of inclusion bodies (data not shown) possibly due to the heterogeneous expressions.15 A similar strategy was used to increase the soluble protein expressions according to our previous study.15 All the proteins were purified by nickel-chelating affinity chromatography after 20 h of induction by the addition of 0.2 mM of the final concentration of isopropyl-β-D-thiogalactoside at 18°C (Figure S4). The specific activities of the APl, CSl, and GPS enzymes were determined at 37°C, wherein equal amounts of purified unfused enzymes were employed as the controls (Figure 2A).15,16 The results indicated that the element-modified enzymes retained almost all of the specific activities in comparison with the respective unfused enzymes, thereby indicating that the application of elemental fusion did not appreciably influence the protein activities.



The in vitro self-assembly system was constructed by the mixture of the purified APl, CSl, and GPS enzymes, which triggered the formation of multitude MECRs. The dynamic kinetics of the self-assembly MECRs were detected by dynamic light scattering (DLS) measurements (Figure 2B-D). As controls, the size and size distribution of the individual enzymes were also analyzed (Figure 2B). Notably, the results showed that the enzyme with/without PPI elements exhibited approximate diameters except for the enzymes GA and GPS. The size differences of enzymes GA and GPS might be account for the fusion of two PPI domains and two α-helical linker ER/K. As presented in Figure 2C, the processes of the self-assembly MECRs were observed within several minutes and increased over time following the in vitro mixing of the three element-modified enzymes. In addition, the molar ratios of the APl, CSl and GPS enzymes were varied at the GPS enzyme concentration in the mixture of 1 µM, the results of which demonstrated consistent results with the self-assembly mechanism (Figure 1), thereby indicating an increase in the formation of MECRs at a APl, CSl, and GPS enzyme molar ratio of 2:1:1 (Figure 2D) as compared to other molar ratios. Although most of the three enzymes self-assembled following 30 min of in vitro mixing, a small amount of residuals did not interact or associate in the bulk environment possibly due to the low retaining enzyme concentrations. To further characterize the microstructure of the MECRs in vitro, scanning electron microscopy (SEM), field-emission scanning electron microscopy (FESEM), and atomic-force microscopy (AFM) were presented altogether. The acquired SEM


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images indicated that the APl, CSl, and GPS enzymes self-assembled as particle-like structures with sizes ranging from 50 nm to greater than 120 nm (Figure S5A). The obtained (2APl-CSl-GPS)n MECRs consisted of regularly clustered and sequential four-enzyme unit repeats (2APl-CSl-GPS) that connected together in groups at their bases and self-assembled into the particle-like microstructures. FESEM imaging provided a greater level of detail for the detection of the microstructures (Figure 3A). The protein aggregation behaviors were observed in Figure 3A and Figure S5B, which illustrated more complicated protein complexes that were constituted by numerous self-assembled MECRs,20,

21

as well as clear dispersive 50-120 nm diameter

multi-enzyme complex (Figure 3A, Figure S5C). Further details on the structures of the (2APl-CSl-GPS)n MECRs were researched according to the AFM method, which indicated that the height of the self-assembled multi-enzyme complex were around 13 nm and were consistent with the exact value of a single APl enzyme (Figure 3B,C and Figure S5D). Larger protein complexes were also observed by AFM imaging, which might be due to the natural aggregation under solution conditions in vitro (Figure S5E).20 The AFM analysis results agreed well with the SEM and FESEM test results. Interestingly, according to the assembly diagram in Figure 1, the MECRs containing multiple regularly clustered enzyme complex unit repeats (2APl-CSl-GPS) tended to construct larger and more non-homogeneous structures, which were not frequently observed in the mentioned detections (Figure S5A). The diameters of the assembled multi-enzyme complex ranged from 50 to 120 nm (or marginally larger diameters),



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which revealed that the repeating number of the single 2APl-CSl-GPS unit was probably between two and four. A possible reason for this phenomenon was that self-assembly probability of the free enzymes markedly decreased following an increase in steric hindrance among the larger complexes, where the complete accessibilities of those interaction proteins were not guaranteed beyond the requisite distances. The self-assembled multitude (2APl-CSl-GPS)n,(n=2-4) MECRs built a series of substrate channelings by proximity in step with the desired sequential reactions, thereby forcing the metabolic fluxes towards the targeting production to enhance the overall cascade efficiencies (Figure 1). Following the measurements of the in vitro multi-enzyme self-assembly system, we attempted to visualize and identify the self-assembly in the experimental strain SAS-3 in vivo by the mIrisFP-based three-fragment fluorescence complementation (TFFC) system.22 The TFFC system was constructed by splitting the photoactivatable fluorescent protein mIrisFP into three nonfluorescent fragments (mIN150, mIN(151−165), and mIC166). The green and red fluorescence signals inside the cells were only detected when three nonfluorescent fragments were successfully associated into an united protein structure by the specific PPIs. Thus we fused three nonfluorescent fragments in an appropriate manner to yield the plasmids of pCSl-mIN150-GPS-mIC166

and

pA-mIN(151-165)-PDZlig,

respectively,

with

pCAD-mIN150-GA-mIC166 and pACN-mIN(151-165) plasmids as the controls (Figure S1). The two plasmids were co-transformed in E. coli for TFFC detection.


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The red and green fluorescence imaging results by confocal laser scanning microscopy are presented in Figure 4A and Figure S6. The results revealed that the fluorescence signals of the split mlrisFP were stimulated by the PPIs of the PDZ (SH3) domain and PDZlig (SH3lig), which successfully associated and brought these fragments together into an united protein structure in vivo. The fluorescence intensities of the fragment-fused self-assembly strain increased rapidly with time, wherein the strain expressing protein mIrisFP was taken as the control, and reached its peak values after about 18 h (Figure 4B, C), thereby indicating that the self-assembly was completed. Based on the time taken for expression and self-assembly, the catalytic efficiency of strain SAS-3 improved from around 18 h. In the TFFC system, the in vivo self-assembly were precisely identified, which further demonstrated the formation of the (2APl-CSl-GPS)n MECRs. Apart from the sequential self-assembly system, we also designed a linear self-assembly system for identical biosynthesis enzymes by fusing sole interaction proteins (PDZ domain and PDZlig). As shown in Figure S7A, the enzymes were transformed into CAD-(GGGGS)2-PDZlig (CPl), CAN-ER/K-PDZ (AP), and GA-ER/K-PDZ (GP), and then self-assembled in an indefinite linear manner in E. coli. The mechanism indicated that either the linear multi-enzyme complexes, which contained diverse repeats of the (CPl-GP) inserting unit, or the simple assembly complex, specifically AP-CPl-AP, which lacked the GP enzyme, was present in the linear self-assembly system. The absence of the GP enzyme in the complex or other



variable assembly resulted in the disorder of the sequential reactions. Thus the substrate channeling did not occur between two relevant enzymes, which caused a lower cascade efficiency as compared to the sequential self-assembly system (Figure S7B). These results demonstrated that the rational self-assembly strategy was a powerful tool for enhancing multi-enzyme catalytic efficiencies in living cells. The metabolic engineering method was used to further increase the productivity using a CRISPR-Cas9 system-based genomic modification tool.23 Given that the IA biosynthetic pathway was integrated into the downstream of cis-aconitate, various genes exhibited impacts on the metabolic fluxes towards IA production, including the glyoxylate shunt pathway, TCA cycle, and byproduct pathways (Figure 5A). All selected genes for deletion are presented in Table S2.18,24-26 Every gene deleting operation increased the final concentrations of the excreted IA except for the ptsG gene (glucose phosphotransferase) (Figure 5B). The deletion of the ptsG gene may prevent the uptake of the glucose. Therefore, the ptsG gene was conserved in the next genetic strains to further control the metabolic flux towards the IA biosynthesis (strains ∆4, ∆5, and ∆6, Table S1). Given that the icd gene is essential in the TCA cycle of E. coli, this deletion resulted in a slower growth and an auxotrophy for 2-ketoglutarate (2-KG), which was added to 4 g/L of 2-KG during the cell cultivation process (Figure S8).18 Accordingly, five positive self-assembly strains were eventually constructed by the co-expression of pCSlGPS and pAPl, wherein the unassembled uaCGA strain was employed as a control. The fermentation results confirmed the


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strong correlations among the glyoxylate shunt, TCA, byproduct, and IA biosynthetic pathways, which may have directly resulted in an elevated metabolic flux towards the desired IA biosynthesis, thereby strongly enhancing the IA production up to 3.06 g/L in the strain ∆6-SAS-3 and significantly decreasing the concentrations of the byproducts (Figure 5B and Table S6). In conclusion, we developed the self-assembly system in a novel fashion to rationally design sequential multi-enzymatic complex reactors (MECRs) in E. coli. The obtained MECRs presented characteristics of regularly clustered enzyme complexes with diameters from 50 to 120 nm, which were composed of a multitude of repeating units of four self-assembled enzymes (2APl-CSl-GPS) to sequentially control the cascades towards the final production, thus resulting in an extremely high overall biocatalytic efficiency. Along with CRISPR-Cas9 system-based genomic engineering, we demonstrated that the rational and integrative engineering approaches resulted in improved IA production in E. coli as compared to the unassembled and linearly self-assembled strains. The application of the sequential self-assembly strategy to MECRs provided insights into complicated multi-enzyme cascades with spatially controlled metabolic fluxes in living cells.

METHODS Construction of plasmids and transformation. All plasmids were constructed with a normal enzymatic assembly method in our previous study.15 All three target acnA (NCBI reference sequence ID: NP_600755.1), gltA (ID: NP_600058.1) and



cadA (ID: BAG49047.1) genes were synthesis with condon-optimized (Generay, Shanghai). Detailed information about plasmids and primers used in this study can be found in Supporting Information Table S1 and Table S3. Relevent plasmids were co-transformed into E. coli wild-type MG1655 strain for further shake flask fermentation experiments using the described method.27 Ampicillin (100 mg liter-1) and chloramphenicol (34 mg liter-1) were used where appropriate. Media and cultivations. The E. coli transformants were grown on solid or liquid LB medium (10 g/L Bacto tryptone, 5 g/L Yeast extract, 10 g/L NaCl) at 37°C. To determine the production and growth behaviors, the strains harboring two expression plasmids were further transferred to M9 minimal medium.18 Before cultivation, M9 minimal medium was supplemented with 1000 × trace metal mix (2.86 g liter-1 H3BO3, 1.81 g liter-1 MnCl2·4H2O, 0.222 g liter-1 ZnSO4·7H2O, 0.39 g liter-1 Na2MoO4·2H2O, 0.079 g liter-1 CuSO4·5H2O, 0.049 g liter-1 Co(NO3)2·6H2O). Carbon sources of the M9 minimal medium were optimized with varied concerntrations of glucose with/without 1% glycerol.19 For the strains ∆2, ∆3, ∆4, ∆5, and ∆6, 4 g/L of 2-KG was supplemented to the optimal MM medium at 30°C. All shake flask cultivations were conducted as triplicates. Individual protein expression and purification. Another expression vector pET-28a was used for individual protein expression and further perification. The sequences of acnA, aPl, cadA, cSl, gltA and gPS genes were amplified by PCR from pA and pCG series, respectively, and digested with the BamHI and XhoI restriction


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enzymes. Fragments were inserted into pET-28a which was digested with the same restriction enzymes in the multiple cloning sites. Each construted plasmid was transformed into E. coli BL21 (DE3) resulting in 6 recombinant strains. Expression strains were cultured in LB medium containing 50 mg liter-1 kanamycin and grown at 37°C with shaking. After reaching the optical density at 600 nm (OD600) of 0.5-0.6, 0.2 mM of the final concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to LB, and then E. coli were cultivated at 18°C for 20 h. The purification process was described in our recent publication by nickel-chelating affinity chromatography.15 The bradford protein assay kit (Solarbio, China) was selected for the protein quantitation according to the manufacturer's instruction. Enzymatic activity assay. The enzymatic activities of CAD series (CAD; CSl) and ACN series (ACN; APl) were determined according to the method described in the previous study.15 The activities of the citrate synthase (GA; GPS) were measured spectrophotometrically as reported previously.16 One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzd the conversion of 1 mmol of substrate per min into specific products at 37°C. The normalization treatments of specific activity of each modified enzyme with corresponding unfused enzyme was showed in Figure 2A. Metabolites and glucose quantification. Extracellular metabolite concentrations of IA, acetate, formate, and lactate were detected by reversed phase high-pressure liquid chromatography (RP-HPLC; Agilent 1100 series) using an Inertsil ODS-3



column (RP-18 100A, 250 × 4.6 mm) with a DAD detector (Spectrasystem UV1000, λ=210 nm). The column temperature was maintained at 40°C and 10 µL injection volume was used in a mobile phase composed of 0.1 M NH4H2PO4 (pH 2.6) with a flow rate of 1.2 mL/min. The concentrations of glucose in fermentation liquid at different time were quantified by the glucose oxidase peroxidase method using a Glucose Assay kit (Robio, China) with a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at 505 nm according to the manufacturer's instruction. Dynamic light scattering assay. Dynamic light scattering (DLS) analysis was performed to determine the particle sizes of individual pure enzyme (1 µM) and assembled MECRs at various time or molar ratios in aqueous solution using a DynaPro NanoStar instrument (Wyatt Technology, Santa Barbara, CA, USA). All protein samples were firstly filtered with a 0.22 µm syringe filter (MILLEX-GP) before self-assemblies in order to remove any supramolecular particles in the protein solutions and the measurements of each sample repeated 5 times at 25°C. Scanning electron, field-emission scanning electron and atomic force microscopy. For the microstructure observations, an FEI Nova Nano-SEM450 field emission scanning electron microscope (FE-SEM) and a Hitachi S-3400N SEM were presented altogether. The images collecting by FE-SEM were taken at an accelerating voltage of 3 kV and a working distance of 5.2 or 5.5 mm (Figure 3A). The operating condition of an accelerating voltage of 10 kV and a working distance between 4 and 6


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mm was used in SEM experiment (Figure S5A). Atomic force microscopy (AFM) was applied for the further detection of microstructure of MECRs using a tapping-mode scanning probe microscope (Veeco, Plainview, NY, USA). Samples were prepared on freshly cleaved mica (0.8 × 0.8 cm). The images was conducted using the Nanoscope v. 1.20 software. To reduce effects of soluble salt in aqueous solution, all protein samples were dialyzed to remove saline ions prior to analysis. Samples were filtered with a 0.22 µm syringe filter (MILLEX-GP) before self-assemblies in vitro. Fluorescence complementation assay. The mIrisFP coding sequence of TFFC system was divided into mIN150, mIN(151-165), and mIC166 fragments, which were amplified from the synthetic vector pRSFDuet1-mIrisFP (Generay, Shanghai).20 For the

construction

of

plasmids

pCSl-mIN150-GPS-mIC166

and

pA-mIN(151-165)-PDZlig (Figure S1), fragment (GGGGS)2-mIN150 was fused to the C-terminus of CSl enzyme, fragment (GGGGS)2-mIN(151-165)-(GGGGS)2 was inserted between ACN and PDZlig, and fragment (GGGGS)2-mIC166 was fused to the C-terminus of GPS enzyme, respectively, using the normal enzymatic assembly method and ClonExpress™ II One Step Cloning Kit (Vazyme, China). The two plasmids were co-transformed in E. coli MG1655, then cells were collected by centrifugation at various time and resuspended in 10 mM PBS for the imaging of TFFC using an A1R confocal laser scanning microscope (Nikon, Tokyo, Japan). The green flurescence of mIrisFP was excited at 488 nm and the red signal was excited at



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561 nm after irradiating with a 405nm laser light for 1 minute. As a control, fusing three fragments individually to the C-terminus of CAD, GA and ACN enzymes generated

CAD-(GGGGS)2-mIN150,

GA-(GGGGS)2-mIC166

and

ACN-(GGGGS)2-mIN(151-165), respectively. Enzyme mIrisFP was transformed into E. coli as a positive control. The fluorescence intensities were measured using a Cytation™ 3 (Biotek Instruments Inc., Winooski, VT) with standardized treatments (Figure 4B, C). CRISPR-Cas9 mediated Genome editing. The two-plsmid based CRISPR-Cas9 system in the study, pCas and pTargetF, was used for consecutive gene knockouts which were purchased in Addgene (62225 and 62226).17 In genome editing cases, the 20-bp complementary region (N20) with the requisite NGG PAM matching genomic loci of interest was programmed online (http://crispr.mit.edu/) according to the E. coli MG1655 genome assembly (GCA_000005845.2). For example, in the first deletion process of aceA gene, pTargetF-N20aceA was constructed with specific N20 sequence hanging at the 5’ end of primer aceA-N20-F1 by one-step cloning method. The donar DNA had about 550-bp sequence homologous to each side (upstream or downstream) of

aceA

gene

in

the

genome,

which

were

amplified

by

EcoRI-onestep-aceA-up-F-opt/onestep-aceA-up-R-opt

primers and

onestep-aceA-down-F-opt/HindIII-onestep-aceA-down-R-opt, respectively, to form a complete

editing

templates

inserting

into

the

EcoRI-HindIII

digested

pTargeF-N20aceA. The obtained pTargeT-∆aceA was further transformed into E. coli


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MG1655 harboring pCas for the first round of gene knockouts. Protocols for the preparation of competent cells and transformation by electroporation were described previously.17 Transformants of strain MG1655 ∆aceA (strain ∆1) were identified by PCR and DNA sequencing methods. Two plasmids of pCas and pTargeT-∆aceA in strain ∆1 were cured before the next round of gene deletion for icd gene. Using the above methods, the icd, poxB, pflB, ldhA genes were consecutively knocked out to construct strains MG1655 ∆aceA-∆icd (strain ∆2), MG1655 ∆aceA-∆icd-∆poxB (strain ∆4), MG1655 ∆aceA-∆icd-∆poxB-∆pflB (strain ∆5), and MG1655 ∆aceA-∆icd-∆poxB-∆pflB-∆ldhA (strain ∆6), respectively, while strain ∆3 was constructed by deleting ptsG gene in strain ∆2 (Table S1). Spectinomycin(50 mg liter-1), kanamycin (25 mg liter-1), ampicillin (100 mg liter-1) or chloramphenicol (34 mg liter-1) were used where appropriate. All primers and N20 sequences were listed in Table S4 and Table S5.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figure S1-S9, protein sequences, Table S1-S6, supporting methods.

Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 21778018) and the National Special Fund for the State Key Laboratory of Bioreactor Engineering (2060204).



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Figure 1. Strategy for the sequential self-assembly of multi-enzymatic complex reactors of the heterogeneous dimeric GA, monomeric ACN, and dimeric CAD using protein-peptide interaction domains and ligands (mouse SH3 and PDZ domains/ligands). The scheme on the right presents the putative self-assembly mechanism for the conversion of citric acid to itaconic acid in the presence of a sequential catalytic flux (green arrows).


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Figure 2. Characteristics of the purified enzymes and self-asemblies in vitro. (A) Specific activities with standardized treatments. (B) Dynamic light scattering (DLS) analysis of the hydrodynamic diameters of the purified enzymes. (C, D) Self-assembled MECRs at (C) various times and (D) molar ratios (enzyme GPS concentration in the mixture of 1 µM). The measurements of each sample were repeated five times at 25°C.



Figure 3. Microstructures of the (2APl-CSl-GPS)n MECRs in vitro. Representative images of the MECRs from (A) field-emission scanning electron microscopy (FESEM) and (B, C) atomic-force microscopy (AFM). Height assay on the right (white line); the multi-enzyme complex had a height of approximately 13.3 nm. Scale bars are presented in the images.


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Figure 4. CSl, APl, and GPS enzymes self-assembled in vivo and visualized using a mIrisFP-based three-fragment fluorescence complementation (TFFC) system. (A) The green and red mIrisFP TFFC signals were detected in E. coli due to the interaction proteins. (B, C) Quantitative analyses of the TFFC efficiency in the self-assembled strain based on the reconstructed green (B) and red (C) fluorescence intensities in comparison with the control strain expressing the mIrisFP protein with standardized treatments. Each data point represents the mean ± standard deviation of three measurements.



Figure 5. Cooperating metabolic engineering strategy with the sequential self-assembly system to increase the biocatalytic efficiencies. (A) Schematic representation of the metabolic networks integrating the itaconate pathway in E. coli. (B) The IA productions were measured in all genetically manipulated strains with the unassembled strain uaCGA employed as the control. Each data point represents the mean ± standard deviation of three measurements.


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Manufacturing Multienzymatic Complex Reactors ... - ACS Publications

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