A highly selective biosynthetic pathway to non-natural C[subscript 50] carotenoids assembled from moderately selective enzymes

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C[subscript 50]-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C[subscript 50] backbones produced. We then further extend the C[subscript 50] pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C[subscript 50] carotenoids, including ~90% C[subscript 50]-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.Japan Society for the Promotion of Science (Fellowship for Young Scientists

The effects of the expression of terpene synthase and prenyltransferase genes on the production level of C₃₀ and C₄₀- carotenoids in <i>E. coli</i>.

<p>The TPSs on pUC18m vectors were expressed in <i>E. coli</i> strain XL1-Blue harboring pAC-MN (yellow bars) or pAC-EBI (red bars) with TXS variants (<b>a</b>), TEAS variants (<b>b</b>), GES variants (<b>c</b>), and FDS variants (<b>d</b>). After 48 hours of culture, carotenoid production was analyzed by extracting the carotenoid pigments with acetone and measuring the absorbance. The bars indicate the average of four samples, and the error bars indicate the standard deviation. Cell pellets before acetone extraction are shown above each of the bars.</p

A High-Throughput Colorimetric Screening Assay for Terpene Synthase Activity Based on Substrate Consumption

<div><p>Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C₃₀ and C₄₀ carotenoid pathways.</p></div

Fitness landscape of the TEAS variant pools.

<p>(<b>a</b>) <i>E. coli</i> colonies harboring pAC-MN and TEAS variants (representative colonies from the TEAS low library are shown). The cells were plated on LB-agar topped with a nitrocellulose membrane to provide a white background for demonstrating the colony color. The left panel shows the raw image taken by the image scanner. This image was subdivided by the RGB channels, and the blue channel shown in the right panel was used directly to represent the “yellowness” of the colonies. (<b>b</b>) Fitness landscape drawn using the values from the blue channel intensity. The image at the right of panel <b>a</b> was analyzed using ImageJ. The grey scale image in 8 bit ranged from 0 (black) to 255 (white), in which the whiter colonies will show the higher scores. The ranges we defined as “wildtype-level” and “dead” are indicated in green and grey background, respectively.</p

Directed evolution of TEAS.

<p>(<b>a</b>) Procedure for the directed evolution of TEAS for higher cellular activity. (<b>b</b>) The carotenoid production level of <i>E. coli</i> cells harboring TEAS variants and pAC-MN-idi. (<b>c</b>) <i>E. coli</i> production of 5EA by TEAS variants. FDS and Idi are additionally and constitutively expressed.</p

N-terminal truncation of geraniol synthase.

<p>(<b>a</b>) N-terminal alignment of GES with spearmint 4<i>S</i>-limonene synthase (LS) and sage 1,8-cineole synthase (CS), as redrawn from Iijima et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093317#pone.0093317-Iijima1" target="_blank">[39]</a>. The residues of the aligned sequences are numbered according to the residue number in the GES. The RRX₈W motif of LS and CS is underlined. The truncated GES positions are indicated in red. (<b>b</b>) The modeled GES structure of limonene synthase (PDBID: 2ONG) by SWISS-MODEL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093317#pone.0093317-Arnold1" target="_blank">[53]</a>. The residues corresponding to the truncation points are indicated in red. (<b>c</b>) Carotenoid production of the cells harboring pAC-MN co-expressed with pUC-GESs. Bars indicate the average, and the error bars represent the standard deviation of four samples. The picture above the bar graph shows the colors of the cell pellets. (<b>d</b>) Geraniol production of the truncated GES variants after 8 h of culture. (<b>e</b>) Western blot of pUC-GES variants in the soluble fraction.</p

Carotenoid pathways compete with TPSs for diphosphate precursors.

<p>(<b>a</b>) <i>E. coli</i> biosynthesizes GPP and FPP using endogenous farnesyl diphosphate (IspA), which provides direct substrates for monoterpene and sesquiterpene synthesis, respectively. Using FPP as a starter molecule, <i>S. aureus</i> CrtM and CrtN biosynthesize diaponeurosporene, a yellow C₃₀ carotenoid. By expressing GGPP synthase (CrtE from <i>P. ananatis</i> or <i>Pantoea agglomerans</i> in this work), a large portion of endogenous FPP is converted to GGPP. GGPP can be fed either to diTPSs or to the pathway of C₄₀ carotenoid pigment lycopene (by CrtB, and CrtI from <i>P. ananatis</i> or <i>P. agglomerans</i>). Geraniol synthase (GES) from sweet basil, 5-<i>epi</i>-aristolochene synthase (TEAS) from tobacco, and taxadiene synthase (TXS) from Pacific yew were used to represent monoTPSs, sesquiTPSs, and diTPSs, respectively. <i>G. stearothermophilus</i> farnesyl diphosphate synthase (FDS) and its specificity-shifted variants (FDS_Y81A and FDS_Y81M) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093317#pone.0093317-Ohnuma1" target="_blank">[41]</a> were also tested. (<b>b</b>) Left panel: diTPS competes for GGPP with C₄₀ carotenoid enzymes. pAC-EBI harbors <i>crtE</i>, <i>crtB</i> and <i>crtI</i> genes from <i>P. ananatis</i> under the control of the <i>lac</i> promoter. Right panel: monoTPS or sesquiTPS competes for FPP with C₃₀ carotenoid enzymes. pAC-MN harbors <i>crtM</i> and <i>crtN</i> genes from <i>S. aureus</i> under the control of the <i>lac</i> promoter.</p

Purifying selection against deleterious mutations in TXS.

<p>(<b>a</b>) The <i>E. coli</i> colonies harboring pAC-LYC that were transformed with TXS libraries. (<b>b</b>–<b>d</b>) The distribution in the number of mutation in the randomized region (671–771 aa) of TXS variants isolated from naïve library (<b>b</b>), red colonies (<b>c</b>), or while colonies (<b>d</b>).</p