Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast

Abstract

One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO₂ and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO₂ in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.

Keywords: carbon labeling; glycine cleavage system; metabolic engineering; one-carbon metabolism; synthetic biology; tetrahydrofolate.

Figure 1

Reductive glycine pathway and a selection scheme for its activity in yeast. (A) The “metabolic engine” of the reductive glycine pathway: condensation of C1-moieties into the C2 compound glycine. Substructure of tetrahydrofolate (THF) is shown in brown. Lipoic acid attached to the H-protein of the glycine cleavage/synthase system (GCS) is shown in green. (B) Gene deletions (marked in red) required for the construction of a glycine auxotroph strain, which we used to select for glycine biosynthesis from the activity of the reductive glycine pathway; pathway enzymes are shown in green.

Figure 2

Three plasmids harboring genes encoding for different subsets of the enzymes of the reductive glycine pathway. pJGC1 harbors only the gene that encodes for MIS1, a trifunctional enzyme that converts formate to methylene-THF. pJGC2 harbors the genes encoding for the subunits of the GCS (the gene encoding for dihydrolipoamide dehydrogenase, LPD1, was not overexpressed since we reasoned its native expression would suffice as it participates in other complexes in the mitochondria, i.e., pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase). pJGC3 harbors the genes encoding for MIS1 and the enzymes of the GCS. Each gene was regulated by a different strong, constitutive promoter as shown in the figure. Each plasmid was based on the pL1A-lc vector backbone as explained in the Methods section.

Figure 3

Formate-dependent growth. (A) Growth of the glycine auxotroph strain harboring the pJGC3 plasmid using different concentrations of formate, 2% glucose and 10% CO₂. “No OE” refers to the negative control, i.e., a glycine auxotroph strain without a plasmid, while “No OE + glycine” refers to the positive control, i.e., a glycine auxotroph strain without a plasmid where glycine was added to the medium. Each curve represents the average of three replicates, which were not different by more than 10%. Growth curves were cut after reaching stationary phase. (B) Calculated growth rate as a function of formate concentration. Growth rate increases with increasing formate concentration up to 1 mM, remains rather stable up to 500 mM, and then sharply decreases with higher concentrations. .

Figure 4

¹³C-labeling experiments confirm glycine production from formate. Fraction of labeling of different amino acids in different strains and labeled feedstocks is shown. “G” corresponds to glycine, “S” to serine, “A” to alanine, “M” to methionine, and “T” to threonine. Complete labeling of glycine in the glycine auxotroph strain harboring pJGC3 upon feeding with ¹³C-formate confirms that glycine biosynthesis occurs only via the reductive glycine pathway. Partial labeling of glycine with ¹³C-CO₂ is attributed to the high production rate of unlabeled CO₂ in the mitochondria. See main text for a detailed discussion on the labeling pattern of these amino acids.