Comparison of aldehyde-producing activities of cyanobacterial acyl-(acyl carrier protein) reductases

Abstract

BackgroundBiosynthesis of alkanes is an attractive way of producing substitutes for petroleum-based alkanes. Acyl-[acyl carrier protein (ACP)] reductase (AAR) is a key enzyme for alkane biosynthesis in cyanobacteria and catalyzes the reduction of fatty acyl-ACP to fatty aldehydes, which are then converted into alkanes/alkenes by aldehyde-deformylating oxygenase (ADO). The amino acid sequences of AARs vary among cyanobacteria. However, their differences in catalytic activity, substrate specificity, and solubility are poorly understood.ResultsWe compared the aldehyde-producing activity, substrate specificity, and solubility of AARs from 12 representative cyanobacteria. The activity is the highest for AAR from Synechococcus elongatus PCC 7942, followed by AAR from Prochlorococcus marinus MIT 9313. On the other hand, protein solubility is high for AARs from PCC 7942, Microcystis aeruginosa, Thermosynechococcus elongatus BP-1, Synechococcus sp. RS9917, and Synechococcus sp. CB0205. As a consequence, the amount of alkanes/alkenes produced in Escherichia coli coexpressing AAR and ADO is the highest for AAR from PCC 7942, followed by AARs from BP-1 and MIT 9313. Strikingly, AARs from marine and freshwater cyanobacteria tend to have higher specificity toward the substrates with 16 and 18 carbons in the fatty acyl chain, respectively, suggesting that the substrate specificity of AARs correlates with the type of habitat of host cyanobacteria. Furthermore, mutational analysis identified several residues responsible for the high activity of AAR.ConclusionsWe found that the activity, substrate specificity, and solubility are diverse among various AARs. Our results provide a basis for selecting an AAR sequence suitable for metabolic engineering of bioalkane production while regulating carbon chain length.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0644-5) contains supplementary material, which is available to authorized users.

Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases

“…Even though aldehydes inside the cells are usually rapidly metabolized, fatty aldehydes could potentially be secreted out from the cells and thus accumulate in the culture medium. Another possibility is the conversion of fatty aldehydes to fatty alcohols by endogenous enzymes [ 7 , 34 ]. The W1 cells overexpressing Aar or Acr1 were cultivated at 30 °C with 1 mM IPTG induction, and fatty aldehydes and fatty alcohols were analyzed from both the biomass and the supernatant by GC–MS.…”

“…The key enzymes in aldehyde synthesis are fatty acyl-CoA (or -ACP) reductases (FAR). Various such reductases have been studied, including Aar from Synechococcus elongatus PCC 7942 [ 6 ], Aar-homologs from other cyanobacteria [ 7 ], and Acr1 from Acinetobacter baylyi ADP1 [ 8 ]. Notably, reductases found in marine bacterium Marinobacter aquaeolei VT8 [ 9 , 10 ] or plants [ 11 ] further reduce the fatty aldehyde intermediate to fatty alcohol.…”

“…As fatty aldehydes are rapidly metabolized in the cells, their analysis is often limited to indirect detection methods, such as conversion to alkanes [ 7 ] or alcohols [ 30 ]. We have previously developed a specific tool for monitoring the aldehyde production in the cells in real-time [ 13 , 22 , 31 ].…”

Production of alkanes from CO2 by engineered bacteria

“…Acrl is an endogenous fatty acy1-CoA reductase from A. baylyi that is involved in the natural storage lipid (wax ester) production pathway. AAR from Synechococcus elongatus is a fatty acyl-ACP/CoA reductase employed in the alkane production pathway in cyanobacteria, and earlier studies indicate that it is the most efficient among the cyanobacterial homologs [22]. Ramo, on the other hand, is a putative short-chain dehydrogenase from the bacterium Nevskia ramosa.…”

“…Thus, efficient alkane production requires careful balancing of the enzyme activities in order to provide ADO with sufficient amount of substrate, while avoiding these negative effects of fatty aldehyde accumulation. Furthermore, while the individual components (especially the cyanobacterial AARs and ADOs) of the pathway have been thoroughly studied [22–24], less attention has been paid to the performance of synthetic pathways comprised of enzymes from different origins.…”