Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation

Abstract

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FAfluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox-and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.

Use of Biofuel Industry Wastes as Alternative Nutrient Sources for DHA-Yielding Schizochytrium limacinum Production

“…Micronutrients are an important ingredient in microbial culture media influencing several biochemical processes. Although research on the effect of micronutrients on thraustochytrids is limited, it has been reported that under combined Fe, Cu, Zn and Mn limiting conditions, biomass productivity is impeded in several Schizochytrium and fungi species [66][67][68]. Consequently, the increase in biomass and lipid productivity with increasing concentrations of effluent in our study could be attributed to the increasing concentrations of trace elements, since nitrogen, phosphorous and potassium content was almost identical with negligible differences among the treatments.…”

Sweetpotato vines hydrolysate promotes single cell oils production of Trichosporon fermentans in high-density molasses fermentation

“…In addition, the high ME activity for SVH-added molasses culture may also have a relationship with the C/N ratio in the medium. In the assay of Chaisawang et al, the determination of ME activities under different C/N ratios showed that (Chaisawang et al, 2012) ME activity increased as the C/N ratio of media increased at the late lipogenic phase, which was highly consistent with the NADPH requirement. Unlike ME activity, there is no obvious relationship between the NADP + -IDH activity (Table 2) and the lipid accumulation (Fig.…”

Improved docosahexaenoic acid production in Aurantiochytrium by glucose limited pH-auxostat fed-batch cultivation

“…By analysis of its 18S rRNA gene sequence (GenBank accession number JF266572), B072 was most closely related to a companion isolate B013 (JF266573) with 98% sequence identity) (Chaisawang et al, 2012). Blast analysis revealed that the next most closely related record at GenBank was for a Thraustrochytrium species (DQ834733) at 94% identity suggesting that Aurantiochytrium B072 was most likely a new species in the genus Aurantiochytrium.…”

“…ATP-citrate lyase (ACL), carnitine acetyltransferase (CAT), glucose-6-phosphate dehydrogenase (G6PDH) and NADP-malic enzyme (NADP-ME) were assayed according to Chaisawang et al (2012). NAD-isocitrate dehydrogenase (NAD-IDH) was assayed according to Harris et al (2006) and pyruvate carboxylase (PC) according to Wynn and Ratledge (1997).…”

“…1. During this period, the major TFA product was in triacylglycerol (TAG) form and there were no major changes in the absolute levels of cellular protein and carbohydrate after exhaustion of the N-source (Chaisawang et al, 2012).…”