Construction of a nonnatural C60 carotenoid biosynthetic pathway

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Construction of a nonnatural C60 carotenoid biosynthetic pathway Ling Li, Maiko Furubayashi, Takuya Hosoi, Takahiro Seki, Yusuke Otani, Shigeko Kawai-Noma, Kyoichi Saito, and Daisuke Umeno ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00385 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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ACS Synthetic Biology

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Construction of a nonnatural C60 carotenoid biosynthetic pathway.

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Ling Li*, Maiko Furubayashi*#, Takuya Hosoi, Takahiro Seki, Yusuke Otani,

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Shigeko Kawai-Noma, Kyoichi Saito, Daisuke Umeno§

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Department of Applied Chemistry and Biotechnology, Chiba University, Japan

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Abstract

OPP

C15PP Wildtype CrtM

OPP

C30PP Engineered CrtM

CrtM

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Substrate cavity

C30-Phytoene

C60-Phytoene

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Longer-chain carotenoids have interesting physiological and electronic/photonic properties due

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to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-

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chain carotenoids in engineerable microorganisms will provide a platform to diversify and

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explore the potential of these molecules. We have previously reported the biosynthesis of

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nonnatural C50 carotenoids by engineering a C30-carotenoid backbone synthase (CrtM) from

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Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer

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C60 carotenoid pathways. Stepwise introduction of cavity-expanding mutations together with

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stabilizing mutations progressively shifted the product size specificity of CrtM toward efficient

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synthases for C60 carotenoids. By coexpressing these CrtM variants with hexaprenyl diphosphate

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synthase, we observed that C60-phytoene accumulated together with a small amount of C65-

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phytoene, which is the largest carotenoid biosynthesized to date. Although these carotenoids

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failed to serve as a substrate for carotene desaturases, the C25-half of the C55-phytoene was

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accepted by the variant of phytoene desaturase CrtI, leading to accumulation of the largest

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carotenoid-based pigments. Continuing effort should further expand the scope of carotenoids,

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which are promising components for various biological (light-harvesting, antioxidant, and

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communicating) and nonbiological (photovoltaic, photonic, and field-effect transistor) systems.

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Keywords: carotenoid, pathway engineering, enzyme engineering, protein stability 1 ACS Paragon Plus Environment

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Introduction

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Carotenoids are natural pigments covering yellow, orange and red colors that are biosynthesized

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by diverse plants, fungi, algae and microorganisms1, 2. Carotenoids possess essential biological

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functions, including light-harvesting, antioxidant activity or membrane fluidity control functions3.

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In addition to their established industrial value as food colorants, nutraceuticals, and animal

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feeds, interest has been growing in the use of carotenoids as pharmaceuticals or therapeutics in

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recent years4-7. Based on the demands in various fields, continuous efforts have been devoted to

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the discovery, chemical synthesis8, 9 and microbial engineering10-13 of novel carotenoid structures.

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One of the prominent properties of carotenoids is the high number of conjugated double

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bonds (CDBs), which are developed along the carotenoid skeleton. The color of the carotenoid is

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determined by the CDBs. Carotenoids with higher number of CDBs (such as lycopene or

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astaxanthin) show a deeper red color, have different photoproperties14, and are better

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antioxidants15. The growing recognition of carotenoids as possible device components or novel

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materials, including semiconductor elements (i.e., field-effect transistors (FETs)16-18),

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photovoltaic devices19, 20, and advanced photonic devices21, is derived from the properties of the

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CDB (polyene) structure of carotenoids. Thus, carotenoids with higher number of CDBs have

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promising potential and value for various fields, including biological, health and material

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applications.

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All carotenoids with known biosynthetic pathways are derived from C40 or C30

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backbones22. The C40 backbone (phytoene) is biosynthesized by the head-to-head condensation

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of two C20PPs (geranylgeranyl diphosphate) as precursors, whereas the C30 backbone (4,4’-

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diapophytoene; called C30-phytoene in this study) is biosynthesized similarly using two C15PPs

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(farnesyl diphosphate) (Figure 1). Some microorganisms produce carotenoids with C50

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carbons23-25, and many plants, fungi and cyanobacteria produce apocarotenoids (e.g. C14, C20 or

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C28 carbons1). Nonetheless, all non-C30/40 carotenoids are derived from the C30 or C40

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biosynthetic pathway using C30- or C40-phytoene as a backbone and by attaching carbons to or

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cleaving this core structure1. The natural C50 carotenoids have two C5 units attached on the C40

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backbones, but in a manner that they do not contribute to increase the number of CDBs.

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A potentially more extensive CDB structure of carotenoids could be realized by creating

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a longer backbone structure, which could be achieved by head-to-head condensation of even 2 ACS Paragon Plus Environment

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longer prenyl diphosphates (i.e., C25PP or C30PP). Previously, our lab constructed a nonnatural

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C50 backbone (C50-phytoene) pathway in Escherichia coli by engineering a C30-phytoene

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synthase (CrtM) from Staphylococcus aureus26, 27. Construction of the C50-phytoene pathway in

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E. coli enabled expansion of the C50 backbone carotenoid pathway into a range of C50 carotenoid

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pigments, including C50-lycopene, C50-β-carotene (also called decapreno-β-carotene28), and C50-

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astaxanthin (or decaprenoastaxanthin29) with a purple color and high antioxidant activity27. This

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success prompted our interest in biosynthesizing even larger carotenoids, including C60 or C70

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backbone carotenoid biosynthetic pathways.

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The present study describes our systematic efforts to create C60-phytoene, which is a C60

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carotenoid backbone synthase, by shifting the size specificity of the previously developed C50-

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phytoene synthase27. The obtained synthases were coexpressed with the C30PP synthase,

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resulting in the production of C60- and C65-phytoenes. Our engineered CrtIN304P desaturase

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resisted the conversion of these substrates and instead desaturated C55-phytoene into novel

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carotenoid pigments, thereby providing novel branching points for a diverse set of previously

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unexplored compounds with potential novel biological, photonic, and device properties.

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Results

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1. Discovery of C60-phytoene synthase activity

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The first committed step of the carotenoid pathway is synthesis of the phytoene-type backbone

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structure. CrtM is a C30-phytoene (usually known as 4,4'-diapophytoene or dehydrosqualene)

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synthase, which is an enzyme that uses two C15PPs as substrates (Figure 1). In our previous

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studies, we performed directed evolution of the S. aureus CrtM and obtained variants capable of

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C50-phytoene synthesis27, 30, 31. Coexpression with an engineered variant of the C15PP synthase

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(FDSY81A,I78G)32 from Geobacillus stearothermophilus, which is an enzyme that can produce

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larger prenyl diphosphates (i.e., C20PP, C25PP and C30PP) showed that three of the CrtM variants

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(CrtMF26A,F233S, CrtMW38A,F233S, and CrtMF26A,W38A,F233S) had weak activity up to C55-phytoene

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synthases27. This C55-phytoene was the largest product that we could produce.

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We were curious whether CrtM variants could synthesize even larger products when a

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product-specific C30PP synthase was adopted, thereby minimizing the C25PP level in the cell. For 3 ACS Paragon Plus Environment


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this purpose, hexaprenyl diphosphate synthase (HexPS) from Micrococcus luteus was

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coexpressed with the CrtM variants. HexPS has been reported to produce all-trans C30PP with

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high specificity in E. coli cell-free homogenates33 and is a heterodimeric enzyme encoded by two

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genes (hexa and hexb). We PCR-cloned these genes from the M. luteus genome into p15A vector,

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together with another gene called menG encoding demethylmenaquinone methyltransferase,

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which was flanked by hexa and hexb. We later confirmed that menG expression did not affect the

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amount and composition of the carotenoid accumulated (discussed below).

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cotransformed with the resultant HexPS- and CrtM variant-expressing plasmids (Figure 2).

E. coli was

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In the acetone-extracted fractions of cells expressing the three CrtM variants

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(CrtMF26A,F233S, CrtMW38A,F233S, and CrtMF26A,W38A,F233S), we observed a novel peak with a

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characteristic absorption maximum at 287 nm, which indicated the possession of three

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conjugated double bonds in the structure. The retention time (37.2 min) of the peak in reverse-

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phase HPLC was significantly higher than those of C55- and C50-phytoene (27.1 min and 22.5

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min, respectively), indicating that it has larger structure than any known phytoene-analog

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biosynthesized. The unique mass (m/z = 817) matching the exact corresponding molecular mass

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demonstrated that the new compound was C60-phytoene (C60H96). The cultures using CrtMF26A,

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CrtMW38A and CrtMF26A,W38A resulted in an accumulation of C40- or C45-phytoene (Figure 2). We

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speculate that these CrtM variants accepted the C20PP or C25PP byproduct produced by HexPS33,

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while they fail to synthesize C60-phytoene due to the lack of C30PP-accepting capacity.

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2. Identification of additional size-shifting substitutions

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Since none of the CrtM variants lacking F233S (CrtMF26A, CrtMW38A, and CrtMF26A,W38A)

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resulted in accumulation of C60-phytoene (Figure 1), it was apparent that F233S was the key for

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C60 activity. The tentative best producer of C60-phytoene, CrtMF26A,W38A,F233S (hereafter CrtMAAS),

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had a serine at the key 233 residue. We created CrtMF26A,W38A,F233A (CrtMAAA), which possessed

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a smaller and more hydrophobic amino acid (alanine) at residue 233.

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Substrate-size-controlling residues are known for various enzymes, and replacement of

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these residues with less bulky amino acids enables enzymes to accept large substrates34, 35. In the

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case of CrtM, two such mutations (F26A and F233S) were identified at bottom of the substrate

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cavity (Figure 3a). In this cavity, the two substrate binding positions are named as S1 or S236:

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the S1-bound substrates are the prenyl donors, whereas the S2-bound substrates are the allylic 4 ACS Paragon Plus Environment

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prenyl acceptors36. To enable CrtM to better accommodate C30PP and/or larger substrates, we

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selected ten bulky residues that could further increase the cavity size of CrtM upon alanine

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substitution (Figure S1), included four residues at the bottom of the cavity (L29, L141, L145,

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and I241), and six residues located on the "side" of the cavity (M15, F22, V37, Y41, V133, and

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L160) (Figures 3a and S2a)

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By using alanine-substituting primers targeted to each of the possibly size-shifting

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residues, CrtMAAA was subjected to site-directed mutagenesis, resulting in 10 quadruple mutants.

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Each CrtM variant was individually introduced into cells harboring HexPS to be scored as C60-

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phytoene producers. Most of the mutations at the S2 site or the bottom of the substrate cavity

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significantly elevated the C60 synthase activity (Figure 3b) compared to that of CrtMAAA.

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Among them, the best performing C60 synthase was L145A-containing CrtMAAA (hereafter,

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CrtM4A), which accumulated up to 222 µg/L (Figure 3b). In contrast, most of the residues

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residing on the side wall of the S2 did not increase the C60 synthase function (Figure S2b),

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presumably due to perturbation of either of the intermediate steps of the reaction cascade.

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Improvement of the C60 synthase activity by adding 4 size-shifting mutations reduced the

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C40 and C50 synthase activities. By coexpressing B. stearothermophilus FDSY81M or

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FDSY81A,T121A,V157A, the CrtM variants were selectively fed C20PP or C25PP, respectively27. With

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these test systems, we systematically scored the abilities of the CrtM mutants as a C40 or C50

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synthase (Figure 3c and d). All of the C60-improving mutations significantly decreased the C40

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or C50 synthase activity. Among the CrtM variants, the best performing C60-phytoene synthase

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was CrtMAAA-L145A, which was named CrtM4A.

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We also created a series of quadruple mutants based on CrtMAAS (Figure 3e-g). The

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majority of the size-shifting mutations exhibited similar and positive effects on this parent.

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Among them, CrtMAAS-F22A performed the best. As the producer of C60-phytoene, CrtMAAS-F22A

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was slightly lower in capacity than CrtM4A but exhibited the highest selectivity toward C60-

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phytoene.

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3. Further attempts to increase the production of larger carotenoids

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To further shift the size specificity toward larger carotenoids, we introduced other effective size-

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shifting mutations (F22A, L29A and/or I241A) into CrtM4A with various combinations. However,

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most of the resultant quintuple variants exhibited significantly lowered or almost no C605 ACS Paragon Plus Environment


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phytoene synthase activity (gray bars in Figure S3a). This may have been due to loss of stability;

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reports suggest that active site mutations is often destabilizing 37.

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Indeed, the Fold-X38, 39 calculation indicated that the addition of size-shifting mutations

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progressively decreased the overall stability of CrtM, as indicated by the increase in ΔΔGfold

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(Figure S3b). All variants that completely lost their C60 activities had ΔΔGfold values higher than

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+17 kcal/mol, whereas those for the variants retaining detectable C60 synthase activities had

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lower ΔΔGfold values. Thus, the performance of the C50 or C60 synthases are apparently limited

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by the stability.

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Many studies have suggested that the addition of a stabilizing substitution can rescue the 40, 41.

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effect of destabilizing substitutions

To increase enzyme stability and thereby rescue the

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activities, another mutation (E180G) was added to the CrtM variants (black bars in Figure S3a).

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This mutation was identified in two independent screening programs26, 30 and was shown to be a

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global suppressor mutation that could compensate for various destabilizing effects of the other

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mutations. The FoldX calculation indicated the high stabilizing effect of this mutation (ΔΔG = -

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2.39 kcal/mol) (Figure S3b). Indeed, the introduction of E180G doubled the cellular activity of

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CrtM4A, resulting in the production of approximately 500 µg/L of C60-phytoene. With the slight

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modification in culture conditions, CrtM4AG produced >1 mg/L of C60-phytoene without any

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reinforcement in the precursor supply (Figure S3c), which was comparable to the yield of

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natural C40 carotenoids27. E180G also improved the cellular activity of most tested CrtM variants

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(Figure S3a). However, we could not integrate other cavity-enlarging mutations any further

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without loss of protein integrity. The stabilizing effect of E180G (-2.39 kcal/mol in ΔΔGfold) was

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easily canceled by the destabilizing effects of the size-shifting mutations (3.6, 3.2 and 2.8

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kcal/mol of ΔΔGfold for the F22A, L29A and I241A mutations, respectively).

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To see how CrtM shifted its product specificity during the course of evolution of C60-

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phytoene synthase activity, we expressed representative variants in various contexts (FDS,

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FDSY81M, FDSY81A,T121A,V157A, and hexPS for the selective in-cell feeding of C15PP, C20PP, C25PP,

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and C30PP, respectively) (Figure 4). All of the tested variants showed activities in multiple

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contexts, and the size preference of CrtM gradually shifted with the number of mutations

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introduced. C60 synthase activity appeared at the later stages and progressively evolved over

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several rounds. Introduction of the 6th mutation (I241A) did not increase the C60 synthase activity

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but did prominently decrease the C40 synthase activity. 6 ACS Paragon Plus Environment

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CrtM4AG accumulated small but detectable amounts of C65-phytoene (m/z = 885,

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corresponding to C65H104) (Figure 5) as the condensation product of C30PP and C35PP,

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corresponding to the largest carotenoid biosynthesized to date. In vitro analysis of purified

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HexPS33 showed that the predominant product of this enzyme was C30PP. The produced C35PP

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accounted for only a tiny fraction of this enzyme, suggesting opportunity for improvement in

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C65-phytoene synthesis. However, coexpression of B. subtilis HepPS (C35PP synthase) with

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CrtM4AG did not result in the detectable production of C65-phytoene or C70-phytoene (Figure S5).

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Considering that the cellular activity of HepPS was strong enough to completely shift the size

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distribution of the ubiquinone side chains (Figure S6), the failure to synthesize C70-phytoene or

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C65-phytoene in reasonable quantities was largely ascribed to the inability of the CrtM mutants to

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accept or convert C35PP as a substrate.

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4. Biosynthesis of the largest carotenoid pigments

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Next, we attempted to convert C60-phytoene into C60 pigments by expanding the conjugated

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double bonds using carotenoid desaturases. To this end, the phytoene desaturase CrtI from

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Pantoea ananatis was selected42. The natural function of this enzyme is the conversion of

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phytoene (C40) into lycopene with 11 conjugated double bonds by catalyzing four-step

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desaturations and two-step isomerizations. P. ananatis CrtI is known to possess weak but

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measurable desaturase activity toward C50-phytoene, yielding several C50 carotenoid pigments43.

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Previously, we elevated this promiscuous activity by adding a single mutation (N304P) to

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acquire an efficient 6-step desaturase that could convert C50-phytoene into C50-lycopene with 15

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conjugated double bonds27 (Figure 1). This CrtIN304P variant did not lose or diminish the

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wildtype C40-phytoene desaturase activity.

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Here, we examined whether CrtIN304P could also act on C60-phytoene to produce a variety

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of C60 pigments. To construct a p15A-based plasmid for C60-phytoene synthesis, we made a

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modification on the HexPS operon (hexa-menG-hexb) by deleting the menG gene, since it did

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not affect C60-phytoene synthesis (Figure S7). The resultant plasmid (p15A plasmid with hexa,

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hexb and crtM4AG) was coexpressed with a pUC-based plasmid expressing CrtIN304P.

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Unfortunately, no sign of desaturated products of C60-phytoene was detected, and the

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accumulated C60-phytoene remained unchanged upon CrtIN304P coexpression. However, novel

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desaturated carotenoids with molecular masses of 745 (C55H84), 743 (C55H82) and 741 (C55H80) 7 ACS Paragon Plus Environment


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were observed (Peaks 2, 3 and 4 in Figure 6). The UV/VIS absorption spectra of 2, 3 and 4 were

2

almost identical to those of ζ-carotene, neurosporene and lycopene, respectively44. Based on

3

these results we concluded that they are C55 carotenoids with 7, 9, and 11 conjugated double

4

bonds, respectively. Considering the failure of desaturating C60-phytoene together with the

5

excellent capability of CrtIN304P as a C50-phytoene desaturase, we believe that polyene structures

6

were developed on the C25-side in the C55-phytoene (C25PP+C30PP) backbone, leading to the

7

formation of the putative structure shown in Figure 1.

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Discussion

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In the present study, we engineered C50-phytoene producing CrtM variants into a decent

15

C60-phytoene synthase. We used the CrtMAAA or CrtMAAS variant, capable of producing C50-

16

phytoene we reported in our previous study, as the parent for the site-directed mutagenesis. With

17

the aid of structural information for CrtM36, 45, possible size-shifting mutations were tested. Out

18

of the ten tested mutations predicted to enlarge the reaction cavity of CrtM (Figure S1a), five

19

(F22A, L29A, L145A, L160A, and I241A) increased the C60-phytoene production (Figure 3).

20

This was similar to various other isoprenoid biosynthetic enzymes, including short-chain prenyl

21

transferases, medium-chain prenyl transferases34, and terpene cyclases46, which have also been

22

successfully engineered to accept and convert larger substrates by progressive (step by step)

23

removal of bulky residues from the cavity floor. With the aid of the E180G stabilizing mutation,

24

the production level of C60-phytoene exceeded 1 mg/L in flask culture (Figure S3c), which was

25

comparable to the production of natural C40 counterparts by E. coli.

26

The mutations we tried in this study can be divided into three groups based on their

27

location: bottom, S1-side or S2-side (red, blue or orange, respectively, in Figure 3a; also see

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Table S1). All of the bottom and S2-side mutations increased C60-phytoene activity (Figure 3b,

29

e), while the S1-side mutations did not (Figure S2). We observed several trends regarding the

30

substrate specificity. First, the mutations located on the bottom (L29A, I241A and L145A, red in

31

Figure 3a) all showed similar trends: higher C60-phytoene production and lower C50- and C408 ACS Paragon Plus Environment

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phytoene production (Figure 3b-g). Second, the mutations in S2-side (F22A, L160A) showed

2

somewhat different from the bottom mutations. The effect of F22A mutation was larger on

3

CrtMAAS compared to CrtMAAA, suggesting an interaction with the F233A/S residue. L160A

4

slightly increased C60-phytoene activity for CrtMAAA but not for CrtMAAS, and increased C40-

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phytoene activity for both CrtM parents. Third, all of the S1-side mutations we tried exhibited

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lower C60-phytoene production compared to the parent CrtMAAA or CrtMAAS (Figure S2). The

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substrate positioning in S1 is known to be crucial to initiate the reaction36,

8

understandable that the mutations at the "side" of S1 have highly negative impacts on the total

9

activity of the enzyme CrtM, possibly hindering the ionization process. That said, it should be

10

note that the performance of the mutants in this paper are presented as the carotenoid accumulted

11

in E. coli and they cannot be simply connected to the true, biochemical character of the mutants.

12

Still, observation herein represents the highly epistatic nature of size-shifting mutations.

47, 48.

Thus, it is

13

One of the motivations in synthetic biology is to develop novel biological materials with

14

functions far beyond the natural systems. The genetic parts for such require many mutations, and

15

accumulation of mutations results in destabilization and an eventual dead-end of engineering. In

16

case of CrtM, the cavity-expanding amino acid substitutions were difficult to integrate after

17

accumulating more than four substitutions, mostly due to their destabilizing nature. In fact, even

18

CrtMAAA (CrtMF26A,W38A,F233A), which was the parent variant used for the amino acid

19

substitutions, was highly unstable; when overexpressed, all CrtM variants with F233A appeared

20

mostly in the insoluble fraction (Figure S4). Further substitutions resulted in the further decrease

21

in stability (Figure S3b), resulting in the complete loss of total activities in the end (Figure S3a).

22

The importance of the stabilizing mutation has been suggested over the years for creating a novel

23

enzyme function by mutations, most of which are destabilizing40, 41. The E180G mutation, which

24

was a previously discovered stabilizing mutation26,

25

destabilizing active site mutations, allowing the efficient production of C60-phytoene. Although

26

we did not observe any C70-phytoene production in this work, identification of additional

27

stabilizing mutations should allow us a search for and accommodation of additional cavity-

28

enlarging mutations.

30,

partially relieved the effect of the

29

Although a general trend between cavity volume and product specificity was observed,

30

notable exceptions could be found. For instance, the larger active site (CrtMF26A,W38A,F233S) was

31

not as effective as a smaller site (CrtMW38A,F233S) in Figure 2. In addition, the effect of mutations 9 ACS Paragon Plus Environment


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turned out to be highly context-dependent. For instance, the addition of size-shifting mutations

2

altered the substrate/product specificity of CrtMAAA and CrtMAAS differently (Figure 3). Also, on

3

CrtMAAA-based variants (Figure 3b-d), F22A, L29A, I241A, and L145A all increased C60-

4

activity with the cost of C40- and C50-activities, while L160A increased C60-activity only with the

5

cost of C50-activity (C40-synthase activity was rather improved). These results could not simply

6

be explained by the mere stability change of the apo-protein. We believe the alteration in binding

7

energy between the enzymes and the substrates should be also taken into account. Considering

8

that most of the CrtM variants in question are highly insoluble, computational approaches to

9

calculate the binding energy with substrates could be a promising way to refine our capacity to

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explain the mutants’ performance on multiple contexts, as well as to predict how many more

11

size-shifting mutations we can introduce into this enzyme.

12

The amount of C65-phytoene production was small (Figure 5) and C70-phytoene could

13

not be detected. To examine if the endogenous C30PP and C35PP production was sufficient, we

14

characterized the UQs produced in the cells expressing HexPS, HepPS or IspB (Figure S6). E.

15

coli endogenously biosynthesize C40PP by IspB to provide the prenyl side-chain of ubiquinone-8

16

(UQ-8; 8 isoprenyl units) (Figure 1). The C30PP and C35PP synthases used in this study, HexPS

17

from M. luteus and HepPS from B. subtilis, are both the functional equivalent for IspB, providing

18

the prenyl side-chain for quinones in their hosts to produce UQ-6 or UQ-7, respectively.

19

Previous studies have shown that expressing heterologous prenyl synthases (C35PP and C45PP

20

synthases) in E. coli results in the accumulation of UQs with different side chains49 (UQ-7 or

21

UQ-9). Indeed, the size distribution of the quinone side chain dramatically changed with the type

22

of prenyl transferase expressed, reflecting the product specificity of the prenyl transferase. These

23

results indicated that the C30PP or C35PP are abundantly produced in the cell. It also suggests that

24

the ubiquinone preny ltransferase, not IspB, might be a major potential competitor for the longer-

25

chain carotenoid pathway.

26

We previously engineered a CrtIN304P variant that accepts C50-phytoene as a substrate and

27

efficiently conducted six-step desaturation of C50-phytoene. In this study, we showed that this

28

CrtIN304P variant with single amino acid mutation was able to accomodate C40-C55 substrates

29

(Figure 6), revealing its remarkable plasticity. However, we could not observe any desaturated

30

product of C60-phytoene. Another round of directed evolution was conducted but failed to

31

identify mutants that could produce C60-based pigments (not shown). Whether C60-lycpoene, an 10 ACS Paragon Plus Environment

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1

eight-step desaturation product with up to 19 conjugated double bonds, is stable enough to be

2

detected under physiological conditions is unknown. Considering the amount of C60-phytoene

3

that was maintained with or without CrtIs expression, we believe that CrtIN304P does not have the

4

capacity to act on C60-carotenoids.

5

A previous structural study42 showed that the substrate-binding domain of CrtI was

6

largely disordered. In contrast, phytoene desaturase (PDS) from rice (Oryza sativa42) provides a

7

clear view of how this enzyme class exerts target specificity. PDS catalyzes the selective and

8

symmetric introduction of two double bonds (2-step desaturation) in the position adjacent to the

9

central triene structure sitting in the middle of the phytoene. The substrate-binding domain of

10

PDS has an extensive substrate tunnel that can accommodate the entire C40 (natural) carotenoid.

11

Interestingly, when one end of the phytoene molecule is inserted in the deepest possible location

12

of the cavity, the flavin of FAD exhibits the closest proximity to the single bond adjacent to the

13

central triene structure, which is the position at which desaturation occurs. CrtI alone is capable

14

of conducting 4-step desaturation all the way to lycopene, but its desaturation always starts from

15

the inner-most single bond adjacent to the growing polyene core. Because CrtI also possess a

16

dead-end tunnel floor similar to that of PDS, the single bonds neighboring the triene core of the

17

C60 carotenoid are most likely physically inaccessible to the reaction center (near flavin) of CrtI.

18

Because the wild-type CrtI enzyme exhibits weak but detectable C50-phytoene activity43,

19

its tunnel must be deep enough to accommodate C25 isoprene units (half the size of the C50-

20

phytoene) such that the single bond neighboring the center triene can be accessed by FAD. The

21

crystal structure showed that N304P was located in the FAD-binding domain, and superposition

22

on PDS with CrtI showed that the residue was close to the flavin and the center of the substrate

23

tunnel in PDS. A previous site-saturation study of the N304 position revealed that not only Pro

24

but also other small residues (i.e., Ala, Gly, Ser, Thr and Cys) increased the C50-desaturation

25

activity whereas larger residues (Phe and Tyr) did not increase the activity27. N304P may have

26

improved the C50-desaturation activity either by making the substrate tunnel near FAD more

27

flexible or by altering the geometry of flavin to allow better access to the first substrate at the

28

single bond adjacent to the triene chromophore. Either way, this CrtI mutant catalyzes up to a

29

four-step desaturation of C55-phytoene (Figure 6). Provided that the desaturation only occurs at

30

the most inner single bonds adjacent to the polyenes, the CDBs should have been extended only

31

on the C25 side (Figure 6). To evolve this enzyme to function as a C60-desaturase, the substrate 11 ACS Paragon Plus Environment


1

tunnel must be deepened to accommodate an additional isopropyl C5 unit (approximately 80 Å3),

2

so that FAD could act on the most-inner single bond neighboring polyene on C30-half.

3

In addition to their traditional and established values as natural colorants, antioxidants

4

and nutraceuticals, carotenoids are increasingly gaining attention as biomaterials for next-

5

generation photonic/electronic devices16-18. The characteristic long pi-conjugated polyene

6

structures along the molecular backbone of carotenoids provide unique characteristics, such as

7

the ultrafast optical response50, 51 or ultra-high third-order optical nonlinearity52, 53. Artificial and

8

nonnatural exotic carotenoids with different photonic/electronic properties would have great

9

interest for such applications. Although inclusion of nonnatural functional groups is effective in

10

manipulating the physical/optical properties of carotenoids52, extension of their backbone size54

11

would be highly effective in sophistication of next-generation biomaterial devices. In addition to

12

the antioxidant activity15 and maximum wavelength of light absorption, the nonlinear optical

13

properties (e.g., second hyperpolarizability) of a polyene tend to elevate with the increasing

14

conjugation length55. The structures and functions of carotenoids are diverse in nature, but their

15

range is limited to the existing natural context, history and evolution. The nonnatural C60-

16

phytoene that we produced in this study can accommodate a highest number of CDBs than any

17

natural carotenoids, and can be the start point to further extend the long-chain carotenoid

18

pathway, providing novel branching points for a diverse set of previously unexplored compounds

19

with potential novel biological, photonic, and device properties.

20 21 22

Methods

23 24

Strains and reagents. E. coli XL10-Gold was used for cloning, and E. coli XL1-Blue was used

25

for carotenoid production. All enzymes were purchased from New England Biolabs. The

26

chemicals used were Lennox-LB Broth Base (Life Technologies), BactoTM Yeast Extract and

27

BactoTM Tryptone (BD Biosciences). All other chemicals and reagents used were purchased from

28

Nacalai Tesque (Kyoto, Japan). The antibiotics used were carbenicillin (50 µg/mL) and

29

chloramphenicol (30 µg/mL).

30

12 ACS Paragon Plus Environment

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1

Plasmid construction. pUC-hexPS was derived from a plasmid obtained from Dr. Alexander

2

Tobias56. The hexa-menG-hexb operon was amplified from the M. luteus genome using the

3

following

4

TTTGAATTCAGGAGGTAGATCAatgcgttatttacataaaattgaactagaattaaaccg-3'

5

TTTTCTAGAttaataaacacgttttaacattttttcgtggatttctt-3'. The amplicon was inserted into the EcoRI-

6

XbaI site of the pUCmod30 vector under the Plac promoter. pAC-hexPS was constructed by

7

amplifying

8

TTGACAGCTTATCATCGATAAGCTTaggtttcccgactggaaagcg-3'

9

GTGATAAACTACCGCATTAAAGCTTgtgaaataccgcacagatgcg-3'. The resulting amplicon was

10

inserted into the HindIII site of pACmod10 using SLIC. The pAC-FDS and pUC-CrtM variants

11

were derived from our previous report27. The new CrtM variants were constructed using these

12

pUC-CrtM variants. pAC-hexab2 was constructed by (1) replacing the upstream hexA in pAC-

13

hexPS with 5'-GAATTCaggaggtagatcaATG-3' and (2) deleting the menG gene and replacing

14

with the sequence 5'-TAAcgatgcatcttgcatacaagccgaaagaaaaatagaatggATG-3' (stop codon of hexA

15

and start codon of hexB capitalized). pUCara-crtIN304P was derived from a previous study27. pAC-

16

hexab2-crtM4AG was constructed by inserting the Plac-crtM4AG fragment amplified by pUC-

17

crtM4AG and inserted above the ClaI/HindIII restriction site of pAC-hexab2.

primers:

the

Plac-hexPS

region

using

5'-

the

following

and

primers: and

5'-

5'5'-

18 19

Culture conditions. Single colonies were inoculated into 2 mL of LB media with antibiotics in

20

culture tubes and shaken at 37 °C for 16 h. For the small-scale assays (Figure S3a), the cultures

21

were diluted 100-fold into 2 mL of fresh Terrific Broth in 48-well deep-well plates and shaken at

22

30 °C and 1000 rpm for 48 h. For the medium-scale assays (Figures 3 and S2), the cultures were

23

diluted 100-fold into 5 or 10 mL of fresh Terrific Broth in 50-mL Falcon tubes and shaken at

24

30 °C and 200 rpm for 48 h. For the large-scale assays (Figures 2, 4, and 6, and

25

Supplementary Figs. 1, 3b, 4, and 5), the 2-mL overnight culture was diluted 100-fold into 40

26

mL of fresh Terrific Broth in a 200-mL flask and shaken at 30 °C and 200 rpm for 48 h. For the

27

desaturated carotenoids (Figure 6), gene expression was induced by 0.2% (w/v) L-arabinose

28

(final concentration) 8 h after inoculation, followed by 40 h of culture before harvest.

29 30

Product extraction and purification. Each cell culture was centrifuged at 3,270 × g and 4 °C

31

for 15 min. The cell pellets were washed with 10 mL of 0.9% (w/v) NaClaq and repelleted by 13 ACS Paragon Plus Environment


1

centrifugation. Products were extracted by adding 10 mL of acetone, followed by vigorous

2

vortexing (for the extraction of desaturated carotenoids in Figure 6, 30 mg/L of butylated

3

hydroxytoluene in acetone was added as the antioxidant). Hexane (1 mL) and 1% (w/v) NaClaq

4

(35 mL) were added, and the samples were centrifuged at 3,250 × g for 15 min. The product-

5

containing hexane phase was collected. Hexane was evaporated by a vacuum concentrator. Then,

6

the extracts were dissolved in 15-50 μL of THF:methanol (6:4) for further analysis.

7 8

HPLC-MS analysis for compound identification and quantification. A 2-15 μL aliquot of the

9

final extract was analyzed using the Shimadzu Prominence HPLC-PDA or HPLC-PDA-MS

10

(APCI) system. The column used in Figures 2, 6 and S1 was a Waters Spherisorb ODS2

11

Analytical Column (4.6 x 250 mm, 5 µm, PSS831915). The remaining experiments were

12

performed using a Shim-pack FC-ODS column (75 mm L. x 2.0 mm I.D., 3.0 μm). The mobile

13

phase used was acetonitrile/isopropanol (6:4) in Figures 2, 6 and S1 and methanol/isopropanol

14

(6:4) at 0.25 mL/min in the remaining experiments. The detectors used were a photodiode array

15

(200-700 nm) and APCI-MS. Mass scans were measured from m/z 10 up to m/z 1000 (or 1300

16

for Figures S5 and S6) at a 300 °C interface temperature, 300 °C DL temperature, ±4500 V

17

interface voltage, and neutral DL/Qarray with N2 as the nebulizing gas. For carotenoid

18

quantification, β-carotene (purchased from Sigma) was used as a standard, since C60-phytoene

19

molecule was not commercially available. A standard curve was created for known amounts of

20

β-carotene and its HPLC peak area at 451 nm. The molar amount of C60-phytoene in each sample

21

was quantified by comparing its peak area with the β-carotene standard curve and then

22

multiplying by the ratio of molar extinction coefficients (εβ-carotene/εphytoene, where εβ-carotene is

23

138,900 M-1 cm-1 at 450 nm and εphytoene is 48,000 M-1 cm-1 at 286 nm57) The calculated

24

molecular weight of C60-phytoene (C60H96, 817.3 gmol-1) was used to calculate the mass (gram).

25 26 27


Page 14 of 27

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1

Author Information.

2

Corresponding Author

3

§E-mail:

[email protected]

4 5

Present Address

6

#Synthetic

7

Technology, Cambridge MA, USA

Biology Center, Department of Biological Engineering, Massachusetts Institute of

8 9

Author contributions

10

*L.L. and M.F. contributed equally to this work. L.L., M.F., and D.U. designed the experiments.

11

L.L, T.H., M.F. and T.S. conducted the experiments. L.L., M.F., Y.O, and T.S. performed the

12

data analysis. S. K-N., K.S., and D.U. supervised the projects. M.F., L.L., and D. U. wrote the

13

manuscript.

14 15

Acknowledgements

16

This work was supported by the Grant-in-Aid for Scientific Research from the Ministry of

17

Education, Culture, Sports, Science and Technology [JSPS KAKENHI Grants 15H04189,

18

15K14228, and 16H06450], the Hamaguchi Foundation for the Advancement of Biochemistry,

19

the Futaba Electronics Memorial Foundation, and the Shorai Foundation for Science and

20

Technology. L.L. is supported by a JSPS fellowship for young scientists [15J07486].

21



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52.

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56. 57.

Marder, S. R., Torruellas, W. E., Blanchard-Desce, M., Ricci, V., Stegeman, G. I., Gilmour, S., Brédas, J.-L., Li, J., Bublitz, G. U., and Boxer, S. G. (1997) Large molecular third-order optical nonlinearities in polarized carotenoids, Science 276, 1233-1236. Garito, A., Heflin, J., Wong, K., and Zamani-Khamiri, O. (1988) Recent studies on the nonlinear optical properties of conjugated linear chains and rigid rod polymers, In Nonlinear Optical Properties of Organic Materials, pp 2-11, International Society for Optics and Photonics. Zeeshan, M., Sliwka, H.-R., Partali, V., and Martínez, A. (2012) The longest polyene, Org. Lett. 14, 5496-5498. Hermann, J., and Ducuing, J. (1974) Third‐order polarizabilities of long‐chain molecules, J. Appl. Phys. 45, 5100-5102. Tobias, A. V. (2005) Directed evolution of biosynthetic pathways to carotenoids with unnatural carbon backbones. Doctoral Dissertation, California Institute of Technology, http://resolver.caltech.edu/CaltechETD:etd-08232005-174620. Britton, G. (1995) UV/visible spectroscopy, In Carotenoids, Spectroscopy, vol. 1B (Britton, G., Liaaen-Jensen, S., and Pfander, H., Eds.), pp 13-62, Birkhäuser Verlag, Basel.

20



1 2

Figure 1. Biosynthetic pathway for C55, C60, and C65 carotenoids.

3

E. coli endogenously biosynthesizes C15PP (farnesyl diphosphate), which can be used as the

4

precursor for the heterologously expressed carotenoid pathway. To construct carotenoids with

5

larger carotenoids, plasmids harboring precursor enzymes (FDS variants or HexPS), CrtM

6

variants, and desaturases (CrtI or variants) were introduced into E. coli in various combinations.

7

CrtM, C30-phytoene synthase from S. aureus. CrtB, C40-phytoene synthase from P. ananatis. CrtI,

8

C40-phytoene desaturase from P. ananatis. FDS, farnesyl diphosphate synthase from G.

9

stearothermophillus. HexPS, hexaprenyl diphosphate synthase from M. luteus. HepPS,

10

heptaprenyl diphosphate synthase from B. subtilis.

11 12

Figure 2. Coexpression of CrtM size variants with HexPS.

13

(a) HPLC analysis of the cell extracts of E. coli harboring eight CrtM variants as previously

14

reported27 and HexPS using absorption at 287 nm. The asterisk indicates the noncarotenoid peak

15

(quinone) identified by characteristic absorbance at 207 and 276 nm. (b) The absorption

16

spectrum of the novel peak appears at 37.2 min.

17 18

Figure 3. Site-directed mutagenesis of CrtM for improved C60-phytoene production.

19

(a) Amino acid residues forming the substrate cavity of CrtM (PDB ID: 3W7F). The co-

20

crystalized S1- and S2-bound C15PP substrate analogs are indicated by yellow and green sticks,

21

respectively. Green sphere indicates Mg2+. The CrtM variants with increased C60-phytoene

22

activity are shown in b-g, while the other CrtM variants with decreased activity are summarized

23

in Figure S2. (b-d) The amount of C60-, C50-, and C40-phytoene accumulated in E. coli

24

expressing CrtMAAA variants coexpressed with HexPS, FDSY81A,T121A,V157A, and FDSY81M,

25

respectively. (e-g) C60-, C50-, and C40-phytoene production by CrtMAAS variants coexpressed

26

with HexPS, FDSY81M and FDSY81A,T121A,V157A, respectively.

27 28

Figure 4. Progressive shift in size specificity of CrtM upon step-by-step incorporation of

29

beneficial mutations for C60 synthase activity.

30

Selected CrtM variants were coexpressed in the following four different contexts: E. coli

31

harboring wild-type FDS (for exclusive feeding of C15PP), FDSY81M (for exclusive feeding of 20 ACS Paragon Plus Environment

Page 20 of 27

Page 21 of 27 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


1

C20PP), FDSY81A,T121A,V157A (for selective feeding of C25PP) and HexPS (selective C30PP

2

synthase). HPLC-MS chromatograms of the carotenoids extracted from each transformant cell

3

are shown for each combination.

4 5

Figure 5. E. coli production of C65-phytoene.

6

HPLC-MS chromatogram of the carotenoid fraction extracted from E. coli coexpressing HexPS

7

and CrtM4AG. The inset shows the 10-fold magnification of the chromatogram, indicating C65-

8

phytoene. Note that this figure shows the duplicate/magnified version of data shown in Figure 4

9

(in the second box on the right at the bottom).

10 11

Figure 6. Desaturation of C55-phytoene.

12

Two plasmids (pAC-hexab2-crtM4AG and pUCara-crtIN304P) expressing HexPS, CrtM4AG, and

13

CrtIN340P were introduced into E. coli, and the carotenoid fraction of the resultant strain was

14

analyzed by reverse-phase HPLC-PDA-MS. The PDA (a) and MS (b) chromatograms of the

15

same sample are shown.

16


CrtM

OPP

C30-Phytoene

FDSY81M

CrtM variant

OPP

CrtM variant

OPP

Lycopene

Nonnatural long-chain carotenoids C50-Phytoene

CrtIN304P C50-Lycopene

CrtM variant

OPP

CrtI

Phytoene

CrtM variant

C55-Phytoene (1)

CrtIN304P 2 3

C60-Phytoene (5)

4 C55-desaturated carotenoids (putative)

CrtM variant

C65-Phytoene (6)

Precursor synthase

OPP

ACS Paragon Plus Environment O

Ubiquinone-8

O

OPP

O

1 C 20 PP 2 FDSY81A, 3 V157A 4 5 C 25 PP 6 7 8 HexPS 9 10 11 C 30 PP 12 HepPS 13 (HexPS) 14 15 C 35 PP 16IspB 17 C 40 PP 18

Natural carotenoids (C30, CPage 40) 22 of 27


O

C 15 PP

crtM variants

Desaturase

CrtM

400 200 0 400

C40

200 0

C40

400 200

C45

0 400

C40

200 0 400 200 0

*

800 400

C60

0

b

800 400

C60

0

400

C60

fds C50

0

200

crtM

Y81A,T121A V157A

400

F26A,W38A, F233S

200

ACS Paragon Plus Environment 0 10

15

20

25 30 35 Retention time (min)

40

287 nm

Absorbance

1 2 WT 3 4 5 F26A 6 7 W38A 8 9 F26A 10 W38A 11 12 13 F233S 14 15 F26A 16 F233S 17 18 W38A 19 F233S 20 F26A 21 W38A 22 F233S 23 24 25 26 27 28


hexPS

Absorbance (a.u.)

a

Page 23 of 27 variants

45

50

250

300

350

Wavelength (nm)

400

S2-Side Bottom

S1-Side

0.2 0.1

0

0

1.0

0.5

0.5

0

0

ACS Paragon Plus Environment

Mutation added to CrtMAAA

crtM variants

I241A

1.5

1.0

L145A

1.5

2.0

L29A

g

L160A

Amino acid position color coding

Y81M

0.3

0.1

2.0

fds

0.4

0.2

L145A

F233 L145 L29

0.3

L29A

F26A L141

f

0.4

I241A

L241

V37

0

L160A

L160

d

0.1

0

F22A

F22

M15

0.2

0.1

F22A

Y81A, T121A, V157A

V133 S1 Y41

S2

fds

0.3

0.2

CrtMAAA

c

0.3

C40-phytoene (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

CrtM

Page 24 of 27

0.4

CrtMAAS

hexPS

e

ACS Synthetic Biology 0.4 C60-phytoene (mg/L)

b

C50-phytoene (mg/L)

a

Mutation added to CrtMAAS

crtM variants

Page 25 of 27

crtM AAA

F26A, W38A, F233A

crtM

crtM 4AG

ACS Synthetic4ABiology F26A, W38A, F233A L145A

x105 6

crtM 4AG-I241A

F26A, W38A, F233A L145A, E180G

F26A, W38A, F233A L145A, E180G, I241A

5 4 3

C30

2 1 0

0

5

10

15

20 0

5

8

10

15

20 0

5

10

15

20 0

5

10

15

20 0

5

10

15

20

15

20 0

5

10

15

20 0

5

10

15

20 0

5

10

15

20

20 0

5

10

15

20 0

5

10

15

20 0

5

10

15

20

20

25 0

C40

6 4 2 0

0

5

10

15

20 0

5

10

5

C50

4 3 2 1 0

Intensity (a.u.)

fds 1 2 wildtype 3 4 5 6 fds 7 Y81M 8(C20PP synthase) 9 10 11 fds 12 Y81A,T121A, 13 V157A (C25PP synthase) 14 15 16 hexPS 17 18 (C30PP synthase) 19 20 21

crtM wildtype

0

8

5

10

15

20 0

*

6

5

10

15

C60

*

4 2

ACS Paragon Plus Environment

0 0

5

10

15

20

25 0

5

10

15

20

25 0

5

10

15

20

25 0

Retention time (min)

5

10

15

5

10

15

20

25

m/z 409 545 681 817 885

C30H48 C40H64 C50H80 C60H96 C65H104

x105 10

1 2 crtM4AG 3 W38A, F233A 4 F26A, L145A, E180G 5 6 7 8

Intensity (a.u.)

hexPS

x10


Page 26 of 27

8 6

m/z

4

681 (C50H80) 749 (C55H88)

2 0 0

ACS Paragon Plus Environment 5

10

15


20

817 (C60H96) 25

30

885 (C65H104) 953 (C70H112)

Absorbance (a.u.)

Intensity (a.u.)

1 2 3 5 b4 x10 5 6 7 8 9 10 11 12

ACS Synthetic Biology C55 desturated carotenoids

6 4

4 3

2

2

5

C55-phytoene

1

crtImut

crtM4AG

F26A,W38A,F233S L145A, E180G

C60-phytoene

0

Wavelength 286 nm 400 nm (x5) 435 nm (x5) 463 nm (x5)

m/z

4 3

4

2

3

2

*

1

1

5

0

681 (C50H80) 677 (C50H76) 675 (C50H74) 673 (C50H72) 749 (C55H88) 745 (C55H84) 743 (C55H82) 741 (C55H80) 817 (C

0

10

20

30

H

)

60 96 813 (C60H92) ACS Paragon Plus Environment 50 60

40


4 3 2 1 0

N304P

811 (C60H90) 809 (C60H88)

Absorbance (a.u.)

hexPS

aPage x105 27 8 of 27

40

400

2 300

400 436

20 0

415

300

40

400

500 465

600

3

500

600

C50-2 step

20

4

C55-4 step (457, 488)

0 300

400

500

Wavelength (nm)

600

Construction of a nonnatural C60 carotenoid biosynthetic pathway

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