The Effects of Carbon Source and Growth Temperature on the Fatty Acid Profiles of Thermobifida fusca

Abstract

The aerobic, thermophilic Actinobacterium, Thermobifida fusca has been proposed as an organism to be used for the efficient conversion of plant biomass to fatty acid-derived precursors of biofuels or biorenewable chemicals. Despite the potential of T. fusca to catabolize plant biomass, there is remarkably little data available concerning the natural ability of this organism to produce fatty acids. Therefore, we determined the fatty acids that T. fusca produces when it is grown on different carbon sources (i.e., glucose, cellobiose, cellulose and avicel) and at two different growth temperatures, namely at the optimal growth temperature of 50°C and at a suboptimal temperature of 37°C. These analyses establish that T. fusca produces a combination of linear and branched chain fatty acids (BCFAs), including iso-, anteiso-, and 10-methyl BCFAs that range between 14- and 18-carbons in length. Although different carbon sources and growth temperatures both quantitatively and qualitatively affect the fatty acid profiles produced by T. fusca, growth temperature is the greater modifier of these traits. Additionally, genome scanning enabled the identification of many of the fatty acid biosynthetic genes encoded by T. fusca.

Keywords: Actinomycete; Thermobifida fusca; branched chain fatty acids; fatty acid biosynthesis pathway; fatty acid synthase; gas chromatography- mass spectrometry; principal component analysis.

FIGURE 1

GC profiles of T. fusca fatty acids. Typical GC profiles of fatty acid methyl esters isolated from T. fusca cultures grown on cellobiose at the indicated growth temperatures. Fatty acids were identified by mass-spectrometry and by comparing retention time with commercial standards. a = iso-15:0; b = anteiso-15:0; c = n-15:0; d = iso-16:0; e = n-16:0; f = 10-methyl-16:0; g = iso-17:0; h = anteiso-17:0; i = n-17:0; j = unknown; k = 10-methyl-17:0; l = iso-18:0; m = unknown; n = n-18:0; o = 10-methyl-18:0; p = n-19:0.

FIGURE 2

Fatty acid yield generated by T. fusca in various growing conditions. Total accumulation of all identified fatty acid products (µmoles/g dry weight) when T. fusca was grown on glucose, cellobiose, cellulose, or Avicel as carbon source and cultured at either 37°C or 50°C, respectively. Fatty acid species are stacked in order of increasing chain length. Error bars represent standard error from three replicates.

FIGURE 3

PCA analysis. PCA analysis was conducted with Metaboanalyst software. An ellipse indicating 95% confidence regions for each heterotic group (37°C or 50°C growth temperature) is provided. Only one replicate of T. fusca supplemented with glucose at 37°C is depicted.

FIGURE 4

Fatty acid biosynthesis pathway. Acyl-CoA starting substrates for FAS are generated from glycolytic catabolism of glucose or from α-keto acids that can be produced via the catabolism of branched-chain amino acids or as the penultimate intermediates in the biosynthesis of branched chain amino acids. The malonyl-ACP substrate used for the elongation reaction of FAS is synthesized from acetyl-CoA by ACCases. The acyl-CoA and malonyl-ACP substrates are used by the FAS system to elongate iso-, ante-iso, and linear n-fatty acids, which can be modified to produce unsaturated and 10-methyl branched chain fatty acids.