Functional plasticity of HCO3- uptake and CO2 fixation in Cupriavidus necator H16

Justin Panich; Emili Toppari; Sara Tejedor-Sanz; Bonnie Fong; Eli Dugan; Yan Chen; Christopher J Petzold; Zhiying Zhao; Yasuo Yoshikuni; David F Savage; Steven W Singer

doi:10.1016/j.biortech.2024.131214

Functional plasticity of HCO₃^- uptake and CO₂ fixation in Cupriavidus necator H16

Bioresour Technol. 2024 Oct:410:131214. doi: 10.1016/j.biortech.2024.131214. Epub 2024 Aug 9.

Authors

Affiliations

¹ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: [email protected].
² Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
³ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Advanced Biofuel and Bioproducts Process Development Unit, Lawrence Berkeley NationalLaboratory, Emeryville, CA 94608, USA.
⁴ Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA, 94720, USA.
⁵ The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, CA 94720, USA.
⁶ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, CA 94720, USA; Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, CA94720, USA.
⁷ Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA, 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.

PMID: 39127361
DOI: 10.1016/j.biortech.2024.131214

Abstract

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO₂ similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO₃^- uptake and CO₂ fixation in C. necator, providing new pathways for CO₂-based biomanufacturing.

Keywords: Biomanufacturing; C1 metabolism; CO(2) conversion; CRAGE; Carbonic anhydrase; DAB2; Gas fermentation; Genome engineering; Rubisco.

MeSH terms

Autotrophic Processes
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Bicarbonates / metabolism
Carbon Cycle / physiology
Carbon Dioxide* / metabolism
Carbonic Anhydrases / metabolism
Cupriavidus necator* / genetics
Cupriavidus necator* / metabolism
Halothiobacillus / metabolism
Ribulose-Bisphosphate Carboxylase / metabolism

Substances

Carbon Dioxide
Bicarbonates
Carbonic Anhydrases
Bacterial Proteins
Ribulose-Bisphosphate Carboxylase