Dataset Information

Both levoglucosan kinase activity and transport capacity limit the utilization of levoglucosan in Saccharomyces cerevisiae.

ABSTRACT: Manufacturing fuels and chemicals from cellulose materials is a promising strategy to achieve carbon neutralization goals. In addition to the commonly used enzymatic hydrolysis by cellulase, rapid pyrolysis is another way to degrade cellulose. The sugar obtained by fast pyrolysis is not glucose, but rather its isomer, levoglucosan (LG). Here, we revealed that both levoglucosan kinase activity and the transportation of levoglucosan are bottlenecks for LG utilization in Saccharomyces cerevisiae, a widely used cell factory. We revealed that among six heterologous proteins that had levoglucosan kinase activity, the 1,6-anhydro-N-acetylmuramic acid kinase from Rhodotorula toruloides was the best choice to construct levoglucosan-utilizing S. cerevisiae strain. Furthermore, we revealed that the amino acid residue Q341 and W455, which were located in the middle of the transport channel closer to the exit, are the sterically hindered barrier to levoglucosan transportation in Gal2p, a hexose transporter. The engineered yeast strain expressing the genes encoding the 1,6-anhydro-N-acetylmuramic acid kinase from R. toruloides and transporter mutant Gal2p^Q341A or Gal2p^W455A consumed ~ 4.2 g L^-1 LG in 48 h, which is the fastest LG-utilizing S. cerevisiae strain to date.

SUBMITTER: Yang M

PROVIDER: S-EPMC9476349 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Both levoglucosan kinase activity and transport capacity limit the utilization of levoglucosan in Saccharomyces cerevisiae.

Yang Mengdan M Wei Tiandi T Wang Kai K Jiang Liqun L Zeng Dihao D Sun Xinhua X Liu Weifeng W Shen Yu Y

Biotechnology for biofuels and bioproducts 20220914 1

Manufacturing fuels and chemicals from cellulose materials is a promising strategy to achieve carbon neutralization goals. In addition to the commonly used enzymatic hydrolysis by cellulase, rapid pyrolysis is another way to degrade cellulose. The sugar obtained by fast pyrolysis is not glucose, but rather its isomer, levoglucosan (LG). Here, we revealed that both levoglucosan kinase activity and the transportation of levoglucosan are bottlenecks for LG utilization in Saccharomyces cerevisiae, a ...[more]

PMID: 36104808

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Project description:α-Ketoglutarate (αKG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae αKG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (≤ 5) showed that αKG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of αKG was further investigated under αKG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated αKG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of αKG. Consumption of αKG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative αKG transporter is repressed by glucose. Fully 13C-labeled αKG was introduced as a tracer during a glucose/αKG co-feeding chemostat to trace αKG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C αKG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular αKG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on αKG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of αKG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic αKG as precursor, both observed sinks should be minimized to increase the putative yields.

Dataset Information

Both levoglucosan kinase activity and transport capacity limit the utilization of levoglucosan in Saccharomyces cerevisiae.

Publications

Both levoglucosan kinase activity and transport capacity limit the utilization of levoglucosan in Saccharomyces cerevisiae.

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