Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66

Abstract

Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.

Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum

“…Lipid were extracted as described earlier ( Bartosova et al, 2021a ) with modifications. The cell pellets were snap-freezed by submerging them in liquid nitrogen for about 10 s and lyophilized overnight.…”

“…The FA concentrations were determined by LC-MS ( Heggeset et al, 2019 ). For determining lipid classes and species, lipid extracts were analyzed by a non-target semiquantitative lipidomics method based on ultrahigh performance supercritical fluid chromatography (UHPSFC)-mass spectrometry (MS) ( Bartosova et al, 2021a , b ). Dichloromethane was used as a sample diluent.…”

A comparative study at bioprocess and metabolite levels of superhost strain Streptomyces coelicolor M1152 and its derivative M1581 heterologously expressing chloramphenicol biosynthetic gene cluster

“…The cold extraction was used to extract CoAs from the biomass pellets (Bartosova et al, 2021). The CoAs were extracted using the extraction solvent having a composition of Acetonitrile: Buffer (50 mM NH 4 Ac, pH 5): CH 3 OH (7:2:1) The isotope dilution strategy was used in all methods of LC-MS/MS.…”

Nutrient-depended metabolic switching during batch cultivation of Streptomyces coelicolor explored with absolute quantitative mass spectrometry-based metabolite profiling

“…Supernatants were discarded, and pellets were quickly quenched in N 2 ( l ) and stored at − 80 °C until further analysis. NAD and CoA metabolites extraction were optimized using S. coelicolor as a biological model system and then extracted following cold extraction and quantified as described previously (Bartosova et al 2021 ; Kumar and Bruheim 2021a ; Rost et al 2020b ). Cold extractions in 50 mM NH 4 Ac having a composition of acetonitrile:buffer:CH 3 OH (7:2:1) including 5% of a 13 C-labelled biological extract (see below), at pH 9 and 5, respectively, for NADs and CoAs.…”

“…We have developed a set of LC–MS/MS methods for the absolute quantitative metabolite profiling including the 13 C-isotope dilution approach for enhanced analytical accuracy (Supplementary Fig. S1) (Bartosova et al 2021 ; Kumar et al 2021 ; Rost et al 2020a ; Kumar and Bruheim 2021b ). The current study aimed to apply these methods for the quantification of CCMs of S. coelicolor M145 during the growth and antibiotic production phases in two nutrient-limited conditions (L-glutamate and phosphate limited).…”