# Pentose bisphosphate pathway can act in central metabolism for nucleoside-dependent growth of Thermococcus kodakarensis strains

## TL;DR

A modified strain of Thermococcus kodakarensis can grow using nucleosides via the pentose bisphosphate pathway, even without sulfur or pyruvate.

## Contribution

The study shows the pentose bisphosphate pathway functions as a central metabolic module for nucleoside-dependent growth in hyperthermophilic archaea.

## Key findings

- A laboratory-evolved strain of Thermococcus kodakarensis can grow without sulfur or pyruvate by utilizing nucleosides.
- Mutations in the RuBisCO gene and a nucleoside transporter subunit (NupD) are key to nucleoside-dependent growth.
- The pentose bisphosphate pathway supports CO2 fixation and cell growth when nucleosides are available.

## Abstract

A laboratory-evolved strain of Thermococcus kodakarensis was found to grow in a nutrient-rich medium without elemental sulfur (S0) or pyruvate, unlike the wild type. The growth of the evolved strain was stimulated by nucleoside supplementation, and the cells consumed nucleosides, accompanied by accumulation of nucleobases in the medium, indicating utilization of the ribose moiety by the cells for growth. The H2/CO2 production ratio was approximately 2:1, and the total amount of acetate and alanine produced was consistent with the amount of nucleosides consumed. These observations support the assimilation of the ribose moiety from nucleosides through the pentose bisphosphate (PBP) pathway employing the CO2-fixing form III RuBisCO. The evolved strain utilized pyruvate derived from nucleosides as both an energy source and an acceptor of an amino group to sustain peptides/amino acids catabolism even in the absence of S0 and pyruvate, of which the ability has been lacking in the wild-type strain. Whole-genome re-sequencing of the evolved strain identified mutations both upstream of the RuBisCO gene and within the coding region of a substrate-binding periplasmic component of a putative nucleoside-transporter subunit (NupD) with a domain organization distinct from previously characterized components. The mutation in the upstream region of the RuBisCO gene led to the upregulated expression, and those in nupD possibly enhanced nucleoside uptake by the NupD-containing transporter. The strain into which these mutations were reconstituted was able to grow in the nucleoside-supplemented medium without S0 or pyruvate.

Thermococcus kodakarensis usually requires S0 or pyruvate for the processing of the amino group when grown on peptides/amino acids. This study demonstrated that adaptive laboratory evolution enabled this archaeon to utilize nucleosides as a growth substrate via the carboxylating PBP pathway. Two mutations, one leading to higher expression of the CO2-fixing RuBisCO and the other enhancing nucleoside uptake, were found to be key modifications for the nucleoside-dependent growth. The results also indicated that NupDABC, containing NupD with a unique primary structural property, was the archaeal nucleoside-specific transporter. These findings demonstrated that the PBP pathway is not only a nucleoside salvage module but also potentially acts as a central metabolic module supporting cell growth when nucleosides are available. This expands our understanding of metabolic flexibility in hyperthermophilic archaea and suggests potential applications of the carboxylating PBP pathway in biotechnological processes involving CO2 fixation.