Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions

Abstract

Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

Figure 1. Thioesterase sequence alignments.

Structure-based sequence alignment of FatA TEs from Arabidopsis thaliana (AtTE_A) and Brassica napus (BnTE_A), Chlamydomonas reinhardtii (CrTE), and FatB TEs from Cuphea hookeriana (ChTE_B1 and ChTE_B2) and Umbellularia californica (UcTE_B). The Cys-His-Asn catalytic triad is shown by an asterisk (*). Conserved residues are highlighted in red and similar residues in blue boxes.

Figure 2. Thioesterase modeling, docking of ACP-TE protein-protein interactions, and blind substrate docking of fatty acid substrate to C. reinhardtii TE.

(A) Docking of CrTE (grey) with Cr-cACP (blue) showing a <10 Å distance between Cr-cACP Ser₄₃ (orange) and the active site Cys₃₀₆His₂₇₀Asn₂₆₈ triad (magenta) of CrTE. (B) Docked complex of CrTE (grey) and ChTE (yellow) showing similar binding modes of Cr-cACP to both plant and algal thioesterases. (C) Surface representation of blind docking of stearyl-4′-phosphopantetheine to CrTE showing the thioester bond of the substrate in close proximity to the TE active site and stearate in the binding tunnel of CrTE.

Figure 3. Schematic of activity-based crosslinking between CrACP and TEs.

(A) Apo-CrACP is formed by treating holo-CrACP with ACP hydrolase from P. aeruginosa , removing the pantetheine moiety from the conserved serine of CrACP. Presence of apo-CrACP is confirmed using a one-pot fluorescent labeling method , detected by visualization of a resulting SDS-PAGE gel at 365 nm. (+) formation of fluorescent crypto-CrACP; (−) control reaction in which fluorescent pantetheine analogue 1 was omitted. (B) Activity-based crosslinking scheme. Apo-CrACP is incubated with 2 or 3, Sfp, ATP, CoA-A, CoA-D, and CoA-E to generate the corresponding crypto-CrACPs 4 and 5. Upon incubation of crypto-CrACP with TE, protein-protein interactions trigger a site-specific covalent crosslinking reaction with the chloroacrylamide in 4 or the α-bromoamide in 5, forming an ACP-TE crosslinked complex. (C) Predicted enzymatic mechanism of the hydrolytic release of a fatty acid by CrTE using a Cys-Asn-His catalytic triad. (D) Mechanism of irreversible crosslink between TE and crypto-CrACP containing a reactive bromide on the carbon α to the site of nucleophilic attack by the TE.

Figure 4. Activity-based crosslinking as a determinant of functional interaction with C. reinhardtii cACP.

(A) Activity-based substrate mimics used in crosslinking assay; (B) SDS-PAGE gel showing Cr-cACP/CrTE interaction. Apo-Cr-cACP was modified with pantetheine analogue 2 or 3 to generate the corresponding crypto-Cr-cACPs (4 and 5). Crypto-Cr-cACPs were incubated with CrTE and crosslinking was visualized by SDS-PAGE analysis following FLAG affinity purification of the CrACP/TE complex. The band observed at 50 kDa is the FLAG-tagged CrTE and the band detected at ∼75 kDa is the ACP/TE complex. During overnight incubation at 37°C, reduced CrTE shows spontaneous oxidation (bands at ∼100 kDa). Pantetheine analogues used in crosslinking reactions are noted under the gel. A red arrow illustrates the ACP-TE complex in (B) and (C). (C) Apo-Cr-cACP was reacted with either α-bromopalmitic pantetheine probe 3 or α-bromohexyl pantetheine probe 6 to generate crypto-Cr-cACPs with C16 and C6 acyl chains attached, respectively. Each crypto- Cr-cACP was incubated with CrTE and monitored for extent of crosslinking by SDS-PAGE analysis of Ni-NTA-purified reactions. (D) Apo-Cr-cACP was modified with α-bromopalmitic pantetheine analogue 3 to form crypto-Cr-cACP, which was incubated with UcTE (left 2 lanes) or ChTE (right 2 lanes). Crosslinking was measured by SDS-PAGE analysis following FLAG affinity purification. The bands at ∼50 kDa are plant TEs.

Figure 5. Thioesterase activity assay.

Activity of plant and algal thioesterases and porcine pancreas type II lipase were determined by monitoring the hydrolysis of para-nitrophenylhexanoate for 16 hours at 30°C. (A) pH 7, TEs expressed in E. coli; (B) pH 8, TEs expressed in E. coli; (C) pH 7, TEs expressed in C. reinhardtii; (D) pH 8, TEs expressed in C. reinhardtii.

Figure 6. Fatty acid analysis of C. reinhardtii strains expressing thioesterases.

Fatty acid composition of Cr strains was determined by GC/MS analysis and comparison to authentic standards. Peak areas were integrated and compared to an external standard for quantification. Bar graphs denote abundances of (A) Myristic acid (14:0), (B) Palmitic acid (16:0), and (C) Oleic acid (18:1), and labels on the Y-axis correspond to the percentages of these fatty acids of the total fatty acid content. (D) Full GC/MS chromatograms of Cr strains expressing CrTE (red), UcTE (Blue) and wildtype CrTE (black). Three separate cultures of each strain were analyzed for fatty acid content and composition, and data were recorded and averaged with a mean deviation of 7% in each experiment. Statistical analyses were performed using SPSS (v13.0), and for all data analysis, a p-value<0.5 was considered statistically significant.