Experimental evidence and isotopomer analysis of mixotrophic glucose metabolism in the marine diatom Phaeodactylum tricornutum

Abstract

BackgroundHeterotrophic fermentation using simple sugars such as glucose is an established and cost-effective method for synthesizing bioproducts from bacteria, yeast and algae. Organisms incapable of metabolizing glucose have limited applications as cell factories, often despite many other advantageous characteristics. Therefore, there is a clear need to investigate glucose metabolism in potential cell factories. One such organism, with a unique metabolic network and a propensity to synthesize highly reduced compounds as a large fraction of its biomass, is the marine diatom Phaeodactylum tricornutum (Pt). Although Pt has been engineered to metabolize glucose, conflicting lines of evidence leave it unresolved whether Pt can natively consume glucose.ResultsIsotope labeling experiments in which Pt was mixotrophically grown under light on 100% U-13C glucose and naturally abundant (~99% 12C) dissolved inorganic carbon resulted in proteinogenic amino acids with an average 13C-enrichment of 88%, thus providing convincing evidence of glucose uptake and metabolism. The dissolved inorganic carbon was largely incorporated through anaplerotic rather than photosynthetic fixation. Furthermore, an isotope labeling experiment utilizing 1-13C glucose and subsequent metabolic pathway analysis indicated that (i) the alternative Entner-Doudoroff and Phosphoketolase glycolytic pathways are active during glucose metabolism, and (ii) during mixotrophic growth, serine and glycine are largely synthesized from glyoxylate through photorespiratory reactions rather than from 3-phosphoglycerate. We validated the latter result for mixotrophic growth on glycerol by performing a 2-13C glycerol isotope labeling experiment. Additionally, gene expression assays showed that known, native glucose transporters in Pt are largely insensitive to glucose or light, whereas the gene encoding cytosolic fructose bisphosphate aldolase 3, an important glycolytic enzyme, is overexpressed in light but insensitive to glucose.ConclusionWe have shown that Pt can use glucose as a primary carbon source when grown in light, but cannot use glucose to sustain growth in the dark. We further analyzed the metabolic mechanisms underlying the mixotrophic metabolism of glucose and found isotopic evidence for unusual pathways active in Pt. These insights expand the envelope of Pt cultivation methods using organic substrates. We anticipate that they will guide further engineering of Pt towards sustainable production of fuels, pharmaceuticals, and platform chemicals.

Modelling metabolism of the diatomPhaeodactylum tricornutum

“…The results also suggest that the Entner-Doudoroff and phosphoketolase pathways, uncommon in eukaryotes, have a potential role in diatom central carbon metabolism. These results are consistent with the work by Zheng et al [31], who also propose a role for these reactions based on 13 C-labelling investigations.…”

“…Although Entner-Doudoroff pathway has lower energy yield per glucose in comparison with EmbdenMeyerhoff-Parnass pathway, it has the advantage of lower protein cost and is thermodynamically less constrained [28]. Similarly, phosphoketolase, reported in bacteria and fungi [29,30] and involved in the degradation of C5 sugars to produce acetyl phosphate, has also been recently reported to be present in P. tricornutum [27,31], where again its physiological significance is unclear.…”

“…Though diatoms have reactions to produce serine, both from photorespiratory and non-photorespiratory pathways [36], Zheng et al [31] reported that a large proportion of serine and glycine was synthesized from glycolate through photorespiratory reactions, at least under mixotrophic conditions. However, there is no conclusive evidence for the photorespiratory pathways of higher plants existing in diatoms [17,18].…”

Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum

“…Simulations of heterotrophic or mixotrophic growth may be seen as tools to assess the metabolic capabilities of this organism in different genetic backgrounds. Some wild‐type P. tricornutum are able to utilize glucose when grown in the light (Zheng et al ., ), although different strains have different efficiencies of glucose metabolism, while some cannot use glucose at all (Huang et al ., ). Wild‐type P. tricornutum cannot grow on the sugar heterotrophically.…”

“…Mixotrophic growth conditions are of special interest for biofuel production (Wang et al ., ). The results of in silico predictions for the autotrophic case were validated by experimental evidence from previous studies, and results for the mixotrophic case were compared with data from a recent isotope labeling study (Zheng et al ., ). Our goal is to provide further knowledge of this system, as well as an interactive platform for integrating high‐throughput data in the future, which may provide insights into the optimal method to engineer diatoms for bioproducts.…”