Microbial electrosynthesis from carbon dioxide feedstock linked to yeast growth for the production of high-value isoprenoids.

The difficulty in producing multi-carbon and thus high-value chemicals from CO2 is one of the key challenges of microbial electrosynthesis (MES) and other CO2 utilization technologies. Here, we demonstrate a two-stage bioproduction approach to produce terpenoids (>C20) and yeast biomass from CO2 by linking MES and yeast cultivation approaches. In the first stage, CO2 (C1) is converted to acetate (C2) using Clostridium ljungdahlii via MES. The acetate is then directly used as the feedstock to produce sclareol (C20), ß-carotene (C40), and yeast biomass using Saccharomyces cerevisiae in the second stage. With the unpurified acetate-containing (1.5 g/L) spent medium from MES reactors, S. cerevisiae produced 0.32 ± 0.04 mg/L ß-carotene, 2.54 ± 0.91 mg/L sclareol, and 369.66 ± 41.67 mg/L biomass. The primary economic analysis suggests that sclareol and biomass production is feasible using recombinant S. cerevisiae and non-recombinant S. cerevisiae, respectively, directly from unpurified acetate-containing spent medium of MES.

Defining the cytosolic pathway of glutathione degradation in Arabidopsis thaliana: role of the ChaC/GCG family of γ-glutamyl cyclotransferases as glutathione-degrading enzymes and AtLAP1 as the Cys-Gly peptidase.

Glutathione homoeostasis is critical to plant life and its adaptation to stress. The γ-glutamyl cycle of glutathione biosynthesis and degradation plays a pre-eminent role in glutathione homoeostasis. The genes encoding two enzymatic steps of glutathione degradation, the γ-glutamyl cyclotransferase (GGCT; acting on γ-glutamyl amino acids) and the Cys-Gly dipeptidase, have, however, lacked identification. We have investigated the family of GGCTs in Arabidopsis thaliana. We show through in vivo functional assays in yeast that all three members of the ChaC/GCG subfamily show significant activity towards glutathione but no detectable activity towards γ-glutamyl methionine. Biochemical characterization of the purified recombinant enzymes GGCT2;2 and GGCT2;3 further confirmed that they act specifically to degrade glutathione to yield 5-oxoproline and Cys-Gly peptide and show no significant activity towards γ-glutamyl cysteine. The Km for glutathione was 1.7 and 4.96 mM for GGCT2;2 and GGCT2;3 respectively and was physiologically relevant. Evaluation of representative members of other subfamilies indicates the absence of GGCTs from plants showing significant activity towards γ-glutamyl-amino acids as envisaged in the classical γ-glutamyl cycle. To identify the Cys-Gly peptidase, we evaluated leucine aminopeptidases (LAPs) as candidate enzymes. The cytosolic AtLAP1 (A. thaliana leucine aminopeptidase 1) and the putative chloroplastic AtLAP3 displayed activity towards Cys-Gly peptide through in vivo functional assays in yeast. Biochemical characterization of the in vitro purified hexameric AtLAP1 enzyme revealed a Km for Cys-Gly of 1.3 mM that was physiologically relevant and indicated that AtLAP1 represents a cytosolic Cys-Gly peptidase activity of A. thaliana. The studies provide new insights into the functioning of the γ-glutamyl cycle in plants.