Synthetic Multienzyme Complexes, Catalytic Nanomachineries for

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Synthetic Multienzyme Complexes, Catalytic Nanomachineries for Cascade Biosynthesis In Vivo Jiale Qu, Sheng Cao, Qixin Wei, Huawei Zhang, Rui Wang, Wei Kang, Tian Ma, Liang Zhang, Tiangang Liu, Shannon Wing-Ngor Au, Fei Sun, and Jiang Xia ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b03631 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

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Synthetic Multienzyme Complexes, Catalytic Nanomachineries for Cascade Biosynthesis In Vivo Jiale Qu,1 Sheng Cao,1 Qixin Wei,1 Huawei Zhang,2 Rui Wang,3 Wei Kang,1 Tian Ma,4 Liang Zhang,3 Tiangang Liu,4 Shannon Wing-Ngor Au,2 Fei Sun,5 Jiang Xia1,*

1

Department of Chemistry, the Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

2

School of Life Sciences, the Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

3

Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR,

China. 4

Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and

School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China. 5

Center for Biological Imaging, Core Facilities for Protein Science, Institute of Biophysics, CAS,

Beijing, China; University of Chinese Academy of Sciences, Beijing, China; National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

*Address correspondence to [email protected] Phone: (852) 3943 6165 Fax: (852) 2603 5057

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ABSTRACT Multienzyme complexes, or metabolons, are assemblies or clusters of sequential enzymes that naturally exist in metabolic pathways. These nanomachineries catalyze the conversion of metabolites more effectively than the freely floating enzymes by minimizing the diffusion of intermediates in vivo. Bioengineers have devised synthetic versions of multienzyme complexes in cells to synergize heterologous biosynthesis, to improve intracellular metabolic flux, and to achieve higher titer of valuable chemical products. Here we utilized orthogonal protein reactions (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag pairs) to covalently assemble three key enzymes in the mevalonate biosynthesis pathway and showed five-fold increase of lycopene and two-fold increase of astaxanthin production in E. Coli. The multienzyme complexes are ellipsoidal nanostructures with hollow interior space, uniform thickness and shapes. Altogether, intracellular covalent enzyme assembly has yielded catalytic nanomachineries that drastically enlarged the flux of carotenoid biosynthesis in vivo. These studies also deepened our understanding on the complexity of hierarchical enzyme assembly in vivo.

KEYWORDS covalent assembly, multienzyme complex, biosynthesis, SpyCatcher and SpyTag, carotenoids.

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Many cellular reactions in metabolic pathways are catalyzed by multienzyme complexes instead of enzyme mixtures with each enzyme being discrete and free-floating.1-4 Multienzyme complexes are formed through post-translational self-assembly of enzymes in a sequential biosynthetic cascade glued by protein−protein interactions. From the aspect of cascade catalysis, enzyme assembly increases the overall reaction rate, because the close proximity of the active sites facilitates intermediate transfer.5-7 In some complexes, nature built channels within the three-dimensional structures to transport intermediates from one active site to the next. In view of metabolic regulation, the advantages of enzyme assembly include (1) segregation of unstable intermediates from the bulk environment of cytoplasm to prevent their degradation and loss, (2) shielding the adverse effect of toxic intermediates to the host cell by rapid conversion to nontoxic products, and (3) sequestering common intermediates from competing reactions by other enzymes in the cytoplasm.8-11 Because of these benefits, multienzyme complexes are widely found in both eukaryotic and prokaryotic cells, such as tryptophan synthase, pyruvate dehydrogenase complex, the AROM complex, cellulosome complex, the purinosome complex etc.12-18

Metabolic engineers have constructed synthetic multienzyme complexes to increase the titer of heterologous biosynthesis. Various non-covalent biological interactions are used to achieve enzyme assembly. For example, cohesin/dockerin interaction pairs derived from the natural cellulosomes were used to assemble cellulases to generate “designer cellulosomes” to facilitate efficient degradation of cellulose.19 Signaling domains in eukaryotes like SH3, PDZ, GBD and their binding peptides were also favorable interaction pairs for enzyme assembly in prokaryotic cells because they pose minimal interference to the signaling of the prokaryotes.7, 20, 21 Protein−DNA interactions can also mediate enzyme assembly on DNA scaffolds like DNA origamis.22, 23These interactions have successfully driven enzyme assembly in heterologous biosynthesis, and significant titer increase of mevalonate, glucaric acid, resveratrol, catechin, butyrate and hydrochinone has been reported.24-26

Notwithstanding such a great success in practical use, how synthetic multienzyme complexes are assembled remain unclear as their structures have not been revealed ex vivo. A highly stable assembly however is the prerequisite to allow the complexes to withstand the purification step. Reversible protein−protein interactions that are popularly used in enzyme assembly (i.e. interactions with dissociation constants in micromolar range, on par with the concentration of the component enzymes ACS Paragon Plus 3 Environment

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expressed in E. Coli cytoplasm) do not meet this requirement, as the complexes may be too fragile ex vivo. Reversible assembly also may also result in complexes with different compositions, structures, or shapes.27 In view of this limitation, we envision that covalent, site-specific protein reactions may be advantageous for enzyme assembly and may maximize the structural integrity, consistency and homogeneity of the complexes ex vivo. Genetically encoded click reactions that enable the formation of covalently linked protein complexes in a site-selective manner are perfect tools.28 SpyCatcher and SpyTag, small protein split fragments from a bacteria adhesion protein, spontaneously crosslink by reconstituting a covalent isopeptide bond under mild physiological conditions when simply mixed. 29, 30

Therefore, we choose to use these two protein fragments and their siblings SnoopCatcher and

SnoopTag as protein tags to build covalently-linked multienzyme complexes. The heterologous biosynthesis pathway of tetraterpenoids, carotenoids more specifically, can serve as a model system to validate the covalent assembly.31-34 Heterologous biosynthesis of carotenoids has been realized by introducing two pathways in E. Coli: an upstream mevalonate (MVA) pathway that produces fivecarbons building blocks (also universal terpene precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) and a downstream pathway that converts IPP and DMAPP to carotenoids.35, 36 Although two carotenoid members lycopene and astaxanthin have been successfully produced by engineered E. Coli, there is still room to improve in the product titer. 37

In this report, using covalent, site-specific protein reactions, we covalently assembled the first three enzymes of MVA pathway, an acetoacetyl-CoA thiolase (ACAT; here we used the AtoB enzyme from E. Coli, UniProt Id P76461), a hydroxy-methylglutaryl-CoA synthase (HMGS; here we chose the ERG13enzyme from Saccharomyces cerevisiae, UniProt Id P54839) and a rate-limiting hydroxylmethylglutaryl-CoA reductase (HMGR; here we chose a truncated version of the NADPH-dependent tHMG1from Saccharomyces cerevisiae, UniProt Id P12683) to synergize the production of mevalonate.38


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RESULTS Split protein fragments as enzyme tags in a model system in vitro Covalent enzyme assembly requires protein reactions that can proceed under the native condition in the cytoplasm, preferentially being site-specific. To test whether the Catcher/Tag pairs can enable the formation of covalent enzyme complexes, we first examined the isopeptide bond reconstitution reactions in vitro. Two enzymes, the cascade enzymes MenD and MenH, were used as models, and the four reactive moieties SpyCatcher, SpyTag, SnoopCatcher and SnoopTag were fused to the Ctermini of MenD and MenH respectively to give MenD-SpyCatcher (D-SpyC), MenH-SpyCatcher (H-SpyC0, MenD-SpyTag (D-SpyT), MenH-SpyTag (H-SpyT), MenD-SnoopCatcher (D-SnoopC), MenH-SnoopCatcher (H-SnoopC), MenD-SnoopTag (D-SnoopT) and MenH-SnoopTag (H-SnoopT). We cloned, expressed and purified these eight fusion enzymes (Figure 1A and Table S1). The corresponding pairs were mixed to induce conjugation reactions, and their reaction patterns were monitored using denaturing SDS-PAGE. Higher molecular weight protein bands can be observed between the matching Catcher and Tag pairs, as a convenient indicator for the formation of covalent complexes. Specifically di-enzyme fusions D-SpyC-SpyT-H, H-SpyC-SpyT-D, D-SnoopC-SnoopTH, and H-SnoopC-SnoopT-D were synthesized (Figure 1B and 1C). SpyCatcher fused enzymes did not react with SpyTag fused ones, and SnoopCatcher fused enzymes did not react with SnoopTag fused ones.

To examine the orthogonality inside the cytoplasm of E. Coli, we co-expressed MenD and MenH enzymes each fused with the Catcher and Tag fragments in E. Coli, lysed the cells, and resolved the proteins in the supernatant by reducing SDS-PAGE to check for in vivo reactivity (Figure 1D and 1E). Similar as the in vitro reactions, the fusion enzymes reacted in matched pairs to generate covalent enzyme complexes when overexpressed together in E. Coli (red arrows): D-SpyC efficiently reacted with H-SpyT, and D-SnoopT efficiently reacted with H-SnoopC. Mixing denatured cell lysates of DSpyC and H-SpyT did not produce covalent adduct, and the H-SpyT band remained unchanged (Figure 1C and 1D, lanes marked with *). We also co-cultured E. Coli cells that individually express D-SpyC with the cells that individually express H-SpyT, and lysed the cells in denaturing buffer together. Again, no obvious covalent adducts were observed and the H-SnoopT band remained unchanged (Figure 1C and 1D, lanes marked with **). This experiment validated that the four split protein fragments can be used as fusion tags for enzyme assembly, the fusion tags can form covalent


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conjugates in the cytoplasm in matched pairs, and the two reacting pairs are orthogonal. We next examined whether covalently assembled complex D-SpyC-SpyT-H remained its catalytic activity. MenF, MenD and MenH are known to catalyze the conversion of chorismate to the product (1R,6R)2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) through intermediates isochorismate and SEPHCHC. The di-enzyme complex D-SpyC-SpyT-H (MenD-SpyCatcherSpyTag-MenH) showed comparable production rate of SHCHC as MenD + MenH-SpyTag, but higher than MenD-SpyCatcher + MenH and unmodified MenD + MenH (Figure S1). MenF was added in this system to provide the substrate isochorismate for MenD. This result indicates that the covalently conjugated MenD and MenH complex remained the catalytic activity of the enzymes in this in vitro system.


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Figure 1. Covalent reactions between Catcher and Tag fused enzymes. (A) The eight tagged enzymes. (B, C) Covalent reactions between purified tagged enzymes. (C, D) Covalent reactions of coexpressed fusion enzymes in the cytoplasm. The fusion enzymes were co-expressed in E. Coli and the cells were lysed in lanes 1, 2 and 3. In the lane marked with *, E. Coli cells expressing either protein individually were lysed in denaturing buffer separately, and the lysates were mixed before SDS-PAGE analysis. In lane **, E. Coli cells expressing either protein individually were mixed and lysed in denaturing buffer together before SDS-PAGE analysis. Red arrows indicate the position of the covalent conjugate. The red circle indicates the position of MenH-SpyTag or MenH-SnoopTag which significant decreased upon reaction in the cytoplasm but remained nearly unchanged when unreacted.

Synthetic multienzyme complexes increase the production of carotenoids After validating the covalent assembly strategy in vitro, we next assembled terpene biosynthetic enzymes inside E. Coli cells aiming to increase the production of terpenoid products, here carotenoids more specifically (Figure 2). Precursor supply has been known to pose a limitation to the production of terpenoids, because the level of mevalonate is generally limited by the rate-limiting enzyme hydroxyl-methylglutaryl-CoA reductase (HMGR). Dueber and coworkers assembled ACAT, HMGS and HMGR on a synthetic protein scaffold through non-covalent protein–protein interactions and observed a significant increase of the titer of glucaric acid. 20 Here we also envision that covalent assembly of ACAT, HMGS and HMGR will also boost the production of carotenoid biosynthesis. We designed fusion proteins based on the crystal structure of ACAT to avoid disruption to the catalytic activity. ACAT crystalized as a tetramer with the N-terminus buried and the C-terminus exposed outside the protein (Figure S2). So, the protein tag was fused at the C terminus to minimize the disturbance to the expression and catalytic activity of ACAT. Similarly, tags were fused to the C termini of HMGR and HMGS (Figure S3 and S4).


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Figure 2. Schematic illustration of the lycopene/astaxanthin biosynthesis pathways, including relevant plasmids used in this study.

Two copies of SpyTag and two copies of SnoopTag were linked in tandem and between flexible linkers to give a SpyTag-SpyTag-SnoopTag-SnoopTag scaffold. This sequence was then fused to the C-terminus of the ACAT enzyme to give an ACAT-1:2:2 scaffold. The scaffold will react covalently with co-expressed HMGS-SpyCatcher (HMGS-SpyC) and HMGR-SnoopCatcher (HMGR-SnoopC) fusion enzymes in 1:2:2 ratio to give a 1:2:2 assembly unit (assembly unit is defined as a single peptide chain that are linked together through covalent bonds including peptide bonds and isopeptide bonds) (Figure 3A). The assembly units will further self-assemble into complexes, which we call 1:2:2 assembly. Re-designing the scaffold allows us to change the ratio of ACAT, HMGS and HMGR enzymes in the complex. For example, a SpyTag-SnoopTag tandem in ACAT-1:1:1 scaffold will


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enable the formation of a 1:1:1 assembly unit. Similarly, two di-enzyme complexes, a 1:1:0 assembly unit and a 1:0:1 assembly unit were constructed by excluding the HMGR or HMGS enzyme from the complex respectively — namely, the HMGR or HMGS enzyme will exist as a free-floating enzyme without being assembled in the complex.

Three plasmids were co-transformed into E. Coli to effect carotenoid biosynthesis (Figure 2). The first plasmid contains the first three enzymes ACAT, HMGR and HMGS responsible for converting acetyl CoA to mevalonate. pJQ plasmid series harbor different assembly states of the three enzymes, modified from the base plasmid pMH1 in which all the enzymes are free floating without assembly. The second plasmid pFZ81 contains ERG12, ERG8, MVD1 and Idi which will convert mevalonate to IPP and DMAPP. The third plasmid pFZ112 contains three enzymes CrtE, CrtI and CrtB, which are responsible for converting IPP and DMAPP to lycopene. Co-transforming three plasmids into E. Coli produces lycopene-producing stains Lyco 1 to Lyco 6. Specifically, Lyco 1 is a base strain in which all the enzymes are in free floating form, without enzyme assembly, also called 1:0:0 strain. Lyco 2 (1:0:1 strain) shall form an ACAT-HMGS complex only without HMGR. Lyco 3 (1:1:0 strain) shall form an ACAT-HMGR complex only without HMGS. Lyco 4 (1:1:1 strain) shall form an ACATHMGR-HMGS complex with 1:1:1 ratio. Lyco 5 (1:2:2 strain) shall form an ACAT-HMGR-HMGS complex with 1:2:2 ratio. Lyco 6 was modified from Lyco 5, in which the isopeptide-forming lysine residue Lys 31 of SpyCatcher and asparagine residue Asn854 in SnoopCatcher were mutated to alanine,39, 40 leading to incapability of forming the covalent assembly products while keeping the backbone of all the tags. So, we called Lyco 6 1:2:2-AA strain (Table S1 and S2).

We next evaluated the lycopene producing titer of the six strains. All these strains grew under the same conditions and were induced by IPTG at the same OD value. The growth profiles of all the strains were similar (Figure 3B), indicating that covalent enzyme assembly does not pose adverse effect on the growth rate of the bacterial cells. At different time points, we lysed the cells, extracted lycopene from the lysates by acetone, and quantified the amount of lycopeneby HPLC and ultraviolet–visible spectrometer. The yield of lycopene was calculated as milligram lycopene per gram dry cell weight (mg/gDCW). All the assembly strains showed elevated lycopene titers compared with Lyco 1 (Figure 3C). More specifically, the 1:2:2 strain Lyco 5 showed the highest lycopene titer among all six strains reaching 6 mg/gDCW after 18 hours, followed by the 1:1:1 strain Lyco 4, about ACS Paragon Plus 9 Environment

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4.5 mg/gDCW. The 1:1:0 strain Lyco 2 and 1:0:1 strain Lyco 3, with assembly of only two enzymes, reaching 2 mg/gDCW, only slight increase from Lyco 1 the unassembled control strain which gave about 1.2 mg/gDCW. Ablating the assembly property of the SpyCatcher and SnoopCatcher in 1:2:2AA strain Lyco 6 significantly decreased lycopene production to close to the basal level. The implication of this set of experiments are multifold. (1) Comparing Lyco 1 (1:0:0 strain), Lyco 4 (1:2:2 strain) and Lyco 6 (1:2:2-AA strain), we conclude that the titer increase is the result of the covalent assembly between the Catcher and Tag pairs, but not due to the fusion tags. Lyco 6 differs from Lyco 4 only in two Lys to Ala mutations, but these two residue-specific mutations ablated the assembly and led to a titer decrease of 3 folds. (2) The comparison of Lyco 1 (1:0:0 strain), Lyco 2 (1:0:1 strain), Lyco 3 (1:1:0 strain) and Lyco 4 (1:1:1 strain) shows that the di-enzyme assembly can increase the lycopene production about 100%, but only when all three enzymes were assembled together, a marked (about 400%) increase of the production titer was observed. This is consistent with previous report that non-covalent assembly of the three will facilitate intermediate transfer for higher supply of mevalonate. (3) Lyco 5, the 1:2:2 strain, showed 40% increase of the lycopene titer compared with Lyco 4, the 1:1:1 strain. This may due to the higher abundance of HMGR in the 1:2:2 assembly, consistent with that reported by Dueber and coworkers. 20 Altogether, these experiments show a direct correlation between the enzyme assembly states and the titers of lycopene in E. Coli strains. It has been reported that the accumulation of the intermediate HMG-CoA limited flux, showing that HMGR is a flux-limiting enzyme in the engineered pathway. 41 Covalently assembling ACAT with HMGS and HMGR streamlines the flux-limiting enzyme with non-limiting ones, which explains why the 1:2:2 assembly showed highest productivity among all the assembled strains.


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Figure 3. Synthetic multienzyme complexes increase the titer of lycopene biosynthesis. (A) Schematic illustration showing the design and construction of multienzyme complexes with different ratios. (B) Comparison of the growth profile of strains Lyco 1 to 6 strains. (C) Comparison of the lycopene production titers of strains Lyco 1 to 6.

We next asked whether this strategy will increase the production titer of a terpenoid, astaxanthin, the end cyclization and oxidation product of tetraterpenoid, and also the strongest antioxidant in nature. Plasmid pFZ153 was used to replace pFZ112 as the third plasmid in the co-transformation. pFZ153 introduces additional enzymes CrtY, CrtZ and CrtW to the biosynthetic pathway, responsible for the cyclization, hydroxylation and oxidation of the lycopene (Figure 4A). Comparing the assembly strain Ast 2 which has 1:2:2 assembly of ACAT, HMGS and HMGR with the un-assembled strain basal Ast 1, we observed a titer increase of more than 100% from 0.5 to 1 mg/gDCW (Figure 4B). The twofold increase of astaxanthin is relatively modest compared to the five-fold increase of lycopene. Lycopene needs to undergo cyclization, ketolation and hydroxylation to give astaxanthin, catalyzed


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by three membrane-bound enzymes CrtY, CrtZ and CrtW. A possible reason may be the incomplete conversions (due to the hydrophobic nature of the reactants and the membrane-bound nature of the enzymes) resulted in accumulation of carotenoid intermediates such as lycopene, canthaxanthin, zeaxanthin and its isomers, instead of converting all the lycopene to astaxanthin. Altogether, this result indicates that the covalent assembly of ACAT, HMGS and HMGR is a general strategy to increase the production of terpenoids (carotenoids for example here), and also consistent with the speculation that the synthetic multienzyme complex increases the supply of mevalonate for terpenoid biosynthesis.

Figure 4. Synthetic complexes increase the production of astaxanthin. (A) Oxidation of the lycopene gives astaxanthin. (B) Synthetic multienzyme complexes doubled the titer of astaxanthin biosynthesis. Ast2 is the assembly strain with 1:2:2 ratio of ACAT. Ast1 is the control strain without enzyme assembly.

Structural characterization of the covalent multienzyme complexes ex vivo We next examined how ACAT-SpyTag-SpyTag-SnoopTag-SnoopTag scaffold assembled with HMGS-SpyC and HMGR-SnoopC fusion enzymes. In order to purify the complexes with larger quantity for structural characterization, we overexpressed the three enzymes using pJQ6 (pJQ6 and


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pJQ4 have the same gene constructs, but pJQ6 uses pGKJE8 as backbone and the enzymes are overexpressed by the induction of tetracycline, whereas pJQ4 uses pBBR1MCS-1 as backbone plasmid and the enzymes are constitutively expressed but at lower levels), purified and characterized the complexes from the cell lysate for ex vivo characterization. The covalent complexes were visible on SDS-PAGE of the cell lysate (Figure S5A). A His-tag was installed at the N-terminal His-tag on HMGR allowed us to purify the complexes from cell lysate through metal affinity chromatography followed by size exclusion chromatography (SEC) (Figure S5B). The ACAT-HMGS-HMGR 1:2:2 assembly unit is calculated to have a molecular weight of 320 kDa (the molecular weights of individual enzymes are 40.4 kDa for ACAT, 55.0 kDa for HMGS and 56.8 kDa for HMGR), and a tetrameric form will have a molecular weight of 1280 kDa, exceeding the upper limit of SEC. The major peak on SEC was collected. Although multienzyme particles with satisfactory purify was acquired, the purification steps gave a very low yield, possibly because the His-tag has been deeply buried within the complex. Next we proved that the enzyme complex indeed contains ACAT, HMGR and HMGS. The complex was trypsinized, and the resultant peptide fragments were analyzed by mass spectrometry. All three enzymes together with SnoopCatcher were found in the mass spectra, confirming that the purified enzyme complex indeed contains all three enzymes and are assembled by Catcher/Tag reactions (Figure 5).


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Figure 5. Characterization of the synthetic multienzyme complexes ex vivo. (C) Catalytic conversion of acetyl CoA to mevalonate by the synthetic complex. The major peak at 1.922 min is the mevalonate. (D) Trypsinization of the complexes followed by peptide peak mass spectrometric analysis identified peptides belonging to ACAT scaffold, HMGS-SpyCatcher and HMGR-SnoopCatcher (shown in red). Three typical peptides were analyzed by MS/MS as examples.

We next characterized the structure of the multienzyme complexes by transmission electron microscope (TEM). The enzyme complex sample was negatively stained with 2% (w/v) uranyl acetate and examined under an electron microscope (Talos F200C) operated at 200kV with a nominal magnification of ×73,000 and a pixel size of 1.22 angstrom. Particles of different size are clearly seen from the origin image (Figure 6A). 9391 particles were initially picked from 352 micrographs. Twodimensional (2D) classification were used to sort out particles for final analysis and bad particles were discarded. After several rounds of picking, 8791 particles were divided into 27 2D classes (Figure 6B). The 2D classification result shows hollow ellipsoidal structures with different dimensions of the long axis and short axis. Because no side views of a planar ring were observed, we could deduce that the multienzyme complex has a hollow interior. The long and short axis diameters of the ellipse-like 2D classes were measured using EMAN2. Assuming a perfect ellipsoidal shape, the long axis diameters ranged from 229 Å to 442 Å and the short axis diameters from 220 Å to 428 Å respectively, and the cross-sectional area can be calculated (Figure 6C). The percentage of the particles in each class was also shown in Figure 5C. Although the sizes vary between different classes, the thickness of the wall of the hollow ellipsoidal structures was measured to be around 4.9nm in virtually all the particles (Figure 6D). The wall therefore is likely made of the three enzymes. The ratio of the long axis versus short axis remained nearly identical. Altogether, these results show that although the size varies, most of the particles have a certain degree of homogeneity.


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Figure 6. Structures of the synthetic multienzyme complexes characterized by TEM under negative staining condition. (A) A representative raw picture of the TEM image. (B) 2D classification of the particles into 27 classes, and the 2D cross-sections were fitted to perfect ellipses. (C) Ellipse areas of 𝑎

𝑏

the 27 classes calculated as A = 𝜋 × 2 × 2 , and the percentage of each class in the total number of the particles. (D) Thickness (d) of the hollow ellipses of the 27 2D classes. (E) Ratio of long axis over 𝑎

short axis (𝑏 ) of the 27 2D classes.


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CONCLUSIONS Covalent assembly of ACAT, HMGS and HMGR constructs a multidomain enzyme complex, a catalytic nanomachinery with three catalytic activities in one entity. Genetic fusion of the three enzymes in one peptide chain in head-to-tail manner is a convenient way of forming a multidomain and multifunctional enzyme. The limitation however is that different component units may affect each other and cause misfolding and even activity loss. Post-translational conjugation reactions thereby avoid such interference and ensure the integrity of each component unit. Similar as previous reports,20 here we show that an ACAT-HMGS-HMGR ratio of 1:2:2 give the highest product yield of carotenoids – 5-fold increase of lycopene after 8 reactions downstream of mevalonate and 2-fold increase of astaxanthin after 11 enzymatic reactions downstream. How the ACAT-HMGS-HMGR 1:2:2 covalent conjugate assembled into such a nanostructure is elusive. We speculate that the multimerization states of the three component enzymes are critical. The crystal structure of ACAT showed a tetramer state (Figure S2), HMGS is a dimer (Figure S3), and HMGR is a tetramer (Figure S4). All the enzymes have extensive protein − protein interaction interfaces >2000 Å2, suggesting that the combined effect of covalent protein − protein reactions and protein multimerization forces resulted in ellipsoidal structures with hollow interior and a thickness of 4.9 nm. Further efforts are needed to construct even more homogenous samples that meet the requirement of high-resolution structural determination by cryogenic EM analysis. From the aspect of catalytic kinetics, although the kinetic parameters of ACAT, HMGS and HMGR in the literature are measured under different conditions, the kcat value of ACAT measured in vitro is generally much higher than the other two enzymes.42-45 The evidence in vivo showed that the activity of HMGR is flux limiting. Therefore, the overall catalytic efficiency may be ascribed as the result of channeling effect, although other explanations may also exisit.46 The covalent protein complex therefore resembles multidomain synthases both in its structure and in the catalytic mechanism.

Despite all the merits, the covalent assembly strategy is not flawless as the covalent reactions still may not proceed to completion in cytoplasm at the expression level of our strains. Free, unassembled enzymes may still exist in our system. Incomplete assembly could be due to the steric hindrance that may jeopardize the reaction efficiency, or the intracellular environment that may pose adverse effect on the reaction efficiency, or the lack of an accurate control on the intracellular concentration of the enzymes. Altogether, although devising synthetic nanomachineries as cellular components as artful


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as the natural ones still remains a challenge, these efforts eventually will lead to a detailed understanding on the structure-activity relationship of the synthetic multienzyme complexes in vivo.


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MATERIALS AND METHODS Materials and instrument Plasmids containing lycopene and astaxanthin biosynthesis pathway were gifts from Tiangang Liu of Wuhan University. SpyCatcher and SpyTag genes were gifts from Sun Fei of Hong Kong University of Science and Technology. ACAT-SpyTag-SpyTag-SnoopTag-SnoopTag gene was synthesized by Beijing Genomics Institute (Shenzhen, China). PCR primers were also purchased from Beijing Genomics Institute (Shenzhen, China). Lysogeny broth and agarose were purchased from USB Corporation. Plasmid extraction was performed using Takara Plasmid Miniprep Kit or E.Z.N.A.® Plasmid Mini Kit I, (V-spin) from Omega Bio-tek company. PCR was carried out using Takara EmeraldAmp® GT PCR Master Mix or Phusion® High-Fidelity PCR Master Mix with HF Buffer from New England Biolabs (UK) Ltd, depending on fidelity requirement. DNA fragments were purified by gel electrophoresis using Bio-Rad DNA Electrophoresis Cells & Systems and extracted through TaKaRa MiniBEST Agarose Gel DNA Extraction Kit or Omega E.Z.N.A.® Gel Extraction Kit. Direct recovery of PCR fragments without gel electrophoresis was performed through Monarch® PCR & DNA Cleanup Kit (5 μg) from New England Biolabs Ltd. Fragments ligation was achieved by Thermo T4 DNA ligase or Takara T4 DNA ligase. T-Vector pMD19 (Simple) was bought from Takara Bio, Inc. Gibson Assembly® Master Mix and DpnⅠ were ordered from New England Biolabs Ltd. Recombinant plasmids were transformed into home-made E. Coli XL1-Blue competent cell. E. Coli culture plates were obtained from SPL Life Sciences. DNA sequencing service was provided by Beijing Genomics Institute. Lysogeny broth and chloramphenicol were purchased from USB Corporation (Ohio, United States). Kanamycin was ordered from Life Technologies while ampicillin was bought from Sigma-Aldrich. Organic solvent including ACN, methanol and acetone were ordered from Labscan Limited (Thailand). Speed-vac concentrator was bought from Hualida Instrument (China). Lycopene and astaxanthin was detected on LC-20A UFLC Shimadzu Corporation (Japan). Lycopene content was measured by Shimadzu UV-3600 Plus UV-VIS-NIR Spectrophotometer (Japan).

Plasmid construction HMGS-SpyCatcher gene was constructed through plasmid overlap extension PCR. Briefly, HMGS fragment was amplified. The PCR product was recovered by Monarch® PCR & DNA Cleanup Kit to ACS Paragon Plus 18 Environment

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obtain a high quality fragment. This fragment was then served as mega-primers to PCR with the template pET32-MenD-SpyCatcher. Having overlap region in the template, the mega-primer replaced part of the plasmid and inserted into the plasmid to form the final product pET32-MenD-HMGSSpyCatcher, similar as the site-directed mutagenesis process except that PCR product is used as mega primers instead of normal synthesized primers. After treated with DpnⅠto eliminate the plasmid template, the PCR solution was transformed into XL1-Blue competent cell. Single colonies were picked up and DNA sequencing was performed to screen out the correct construct. HMGRSnoopCatcher gene was similary obtained. Purified HMGR fragment was used as mega-primers to insert into plasmid pET32-MenD-SnoopCatcher to construct the final product pET32-MenD-HMGRSnoopCatcher.

Plasmid pJQ1 containing the modified MVA pathway was built through Gibson assembly strategy. Briefly, fragment 1 with the scaffold construct ACAT-SpyTag-SpyTag-SnoopTag-SnoopTag, fragment 2 containing the HMGS-SpyCatcher part, fragment 3 with the HMGR-SnoopCatcher gene, and fragment 4 containing the vector part of plasmid pMH1 were all obtained from PCR using corresponding primers. Overlap regions were designed between fragment 1 and fragment 2, fragment 2 and fragment 3, fragment 3 and fragment 4, fragment 4 and fragment 1. Finally, fragment 1,2,3,4 were ligated together through Gibson assembly to obtain the final construct pJQ1. Other plasmids were similarly constructed.

Lycopene production and quantification The lycopene producing strain L1 was obtained by co-electroporation of plasmids pMH1, pFZ81 and pFZ110 into E. Coli BL21 competent cell. Similarly, co-electroporation of plasmids pJQ1, pFZ81 and pFZ110 was performed to obtain production strain L2. Single colonies of each strain were picked up into 5mL LB supplemented with 50μg/mL kanamycin, 100μg/mL ampicillin, 34μg/mL chloramphenicol and grew overnight in 37℃ to serve as the start culture. 1mL start culture was then inoculated into conical flask containing 100mL LB supplemented with the antibiotics. Cells were allowed to grow until the optical density at 600nm (OD600) reached 0.6-0.7. IPTG was added to a final concentration of 0.1 mM to induce the protein overexpression and lycopene production. Cells culture continued to grow overnight in 37℃ to produce lycopene. After the overnight production, 6mL cell cultures were centrifuged at 10000g for 3min to obtain the cell pellet. The supernatant was ACS Paragon Plus 19 Environment

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discarded and the pellet was resuspended in 1mL DI water. The solution was centrifuged again at 10000g for 5min to harvest the cell pellet. The cell pellet was dried up using the speedvac concentrator supplemented with a vacuum pump. The dry cell pellet was then grinded into fine powder. The lycopene accumulated in the cell was extracted by acetone in 55℃ for 15min in dark. After centrifugation at 13000g for 30min, the clear supernatant containing the lycopene was collected for further analysis.

The production of lycopene was examined on the HPLC with an analytical column Waters XTerra MS C18 Column, 125Å, 5 µm, 3.9 mm X 150 mm. Before injecting into the HPLC, the supernatant was 1:1 (v/v) diluted by methanol. Water and ACN both containing 0.1% TFA (v/v) were used as mobile phase. Gradient running at 1mL/min was applied to elute the lycopene. Peaks was monitored at the corresponding absorption wavelength (474nm). The quantification of lycopene was conducted on the ultraviolet–visible spectrometer. The absorption of the supernatant was measured at 474nm, which is the maximum absorption wavelength of lycopene. The content of lycopene was calculated with the absorption coefficient ε474nm =178000 cm-1 M-1.

Astaxanthin production and quantification The astaxanthin production strain A1 was obtained by co-electroporation of plasmids pMH1, pFZ81 and pFZ153 into E. Coli BL21 competent cell. Similarly, co-electroporation of plasmids pJQ1, pFZ81 and pFZ153 was performed to produce the production strain A2 while strain A3 was obtained through co-electroporation of plasmids pMH1, pFZ81 and pJQ2.Single colonies of each strain were picked up into 5mL LB supplemented with 50μg/mL kanamycin, 100μg/mL ampicillin, 34μg/mL chloramphenicol and grew overnight in 37℃ to serve as the start culture. 1mL start culture was then inoculated into conical flask containing 100mL LB supplemented with the antibiotics. Cells were allowed to grow until OD600 reached 0.6-0.7. IPTG was added to a final concentration of 0.1mM to induce the protein overexpression and astaxanthin production. Cells culture continued to grow overnight in 37℃ to produce astaxanthin. After the overnight production, 6mL cell cultures were centrifuged at 10000g for 3min to obtain the cell pellet. The supernatant was discarded and the pellet was resuspended in 1mL DI water. The solution was centrifuged again at 10000g for 5min to harvest the cell pellet. The cell pellet was dried up using the speedvac concentrator supplemented with a vacuum pump. The dry cell pellet was then grinded into fine powder. The astaxanthin accumulated ACS Paragon Plus 20 Environment

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in the cell was extracted by acetone in 55℃ for 15min in dark. After centrifugation at 13000g for 30min, the clear supernatant containing the astaxanthin was collected for further analysis.

The production of astaxanthin was examined on the HPLC with the Waters analytical column. Before injected into the HPLC, the supernatant was 1:1 (v/v) diluted by methanol. Water and ACN both containing 0.1% TFA (v/v) were used as mobile phase. Gradient running at 1mL/min was applied to separate the different products. Peaks was monitored at the corresponding absorption wavelength (470nm). The quantification of astaxanthin was conducted through standard curve method.

Expression and purification of the multienzyme complexes Plasmid pJQ3 for the overexpression of the Catcher/Tag scaffold induced covalent enzyme complex were transformed into home-made BL21 E. Coli competent cells and plated onto E. Coli cell culture agar plate supplemented with 34 μg/mL chloramphenicol. Single colony were picked up into 5mL LB supplemented with antibiotic and grew overnight in 37℃ to serve as the start culture. 4ml start culture was then inoculated into large conical flask containing 400mL LB supplemented with 34μg/mL chloramphenicol. Cells were allowed to grow until OD600 reached 0.6-0.8. Tetracycline was added to a final concentration of 2μg/mL to induce the complex overexpression. Cells culture continued to grow overnight in 16℃ to produce the desired complex.

Cells were harvested by centrifugation at 5000g for 8min. The supernatant was discarded and the pellet were fast frozen by liquid nitrogen before stored in -80℃ until further treatment. The pellet stored in -80℃ was resuspended using 25mL buffer A (50mM Tris, 300mM NaCl, 10mM imidazole, 2mM 2-mercaptoethanol, pH 8.0). The cell suspensions were lysed by ultrasonication for 1h and then the lysate were centrifuged at 20000g for 1h. The supernatant were collected and the debris were saved for later analysis. HisTrap HP column was first equilibrated with buffer A for 10 column volumes. After filtration, the supernatant was load onto the buffer A pre-equilibrated HisTrap HP column using peristaltic pump in 4℃.

The following steps were performed on an AKTA fast protein liquid chromatography (FPLC) instrument. The inlets and the pipeline of FPLC were first rinsed by filtered DI water and then the two inlets were rinsed by buffer A and buffer B (50mM Tris, 300mM NaCl, 500mM imidazole, 2mM ACS Paragon Plus 21 Environment

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2-mercaptoethanol, pH 8.0) respectively. Finally the whole system was equilibrated with buffer A. The sample loaded HisTrap HP column was applied onto the FPLC. 30mL buffer A was used to wash out nonspecific binding proteins. Gradient of buffer B was linearly increased to 100% in 100mL. Each peak was collected for later analysis. The column was further washed with 20mL buffer B and then 50mL filtered DI water. The whole FPLC system was washed with another 100mL filtered DI water. All peaks and fractions were examined using SDS-PAGE. Peaks containing target complex were concentrated and buffer exchanged to storage buffer (50mMTris, 200mM NaCl, pH 7.4) using Amicon® Ultra 15 mL Centrifugal Filters device. The complex was then further purified by size exclusion chromatography.

Particle analysis by transmission electron microscopy (TEM) Briefly, TEM grid was first treated to become hydrophilic on one side. Complex solution was stained by mixing with uranyl acetate. The hydrophilic side of the grid was placed onto the droplet of the solution. After sample loading, filter paper was used to absorb excess liquid on the grid. The grid was put into the sample holder of the TEM instrument. Finally the sample holder was inserted into the TEM instrument for complex imaging.


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AUTHOR CONTRIBUTIONS J. X. conceived the project, designed experiments and wrote the manuscript. J. Q., S. C., Q. W., H. Z., R. W., W. K., and T. M. carried out the experiments. L. S., T. L., S. A. and S. A. helped analyze the data.

SUPPORTING INFORMATION AVAILABLE The Supporting Information is available free of charge on the ACS Publications website at DOI: Plasmids, strains, and primers used in this study; strains and plasmids construction; DNA and protein sequence information of all the enzymes and proteins used in this study; comparison of the catalytic activity of the covalently assembled enzyme complexes with free enzymes; the structure of ACAT, atoB from E. Coli.; the structure of HMGS, ERG13 enzyme from Saccharomyces cerevisiae; the structure of HMGR from Saccharomyces cerevisiae; purification of the multienzyme complexes from an overexpression strain (PDF) ACKNOWLEDGMENTS This work was partially funded by National Key R&D Program of China (2018YFA0903204) and grants from University Grants Committee of Hong Kong (GRF Grants 14306317, N_CUHK422/18, and 14307218) and CUHK direct grant (4053278).


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TOC Figure

6 O OH

4 HO O

2

0

Unassembled


1:2:2 assembly

Synthetic Multienzyme Complexes, Catalytic Nanomachineries for

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