Engineering a biotechnological microorganism for growth on one-carbon (C1) intermediates, produced from the abiotic activation of CO2, is a key synthetic biology step towards the valorization of this greenhouse gas to commodity chemicals. Here, we redesign the central carbon metabolism of the model bacterium Escherichia coli for growth on C1 compounds using the reductive glycine pathway. Sequential genomic introduction of the four metabolic modules of the synthetic pathway resulted in a strain capable of growth on formate and CO2 with a doubling time of ~70 hours and growth yield of ~1.5 gCDW / molformate. Short-term evolution decreased doubling time to less than 8 hours and improved biomass yield to 2.3 gCDW / mol-formate. Growth on methanol and CO2 was achieved by the expression of methanol dehydrogenase in the evolved strain. Establishing synthetic formatotrophy and methylotrophy, as demonstrated here, paves the way for sustainable bioproduction rooted in CO2 and renewable energy.

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SummaryBecause of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h−1, followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h−1 and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased.Funding Information No funding information provided.

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTSindependent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K s values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for L-lysine production were tested. The L-lysine yield by C. glutamicum DM1729, a rationally engineered L-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729⌬hpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729⌬hpr enabled L-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher L-lysine yield.The Gram-positive soil bacterium Corynebacterium glutamicum is used industrially for the production of more than 2,160,000 tons of L-glutamate and more than 1,330,000 tons of L-lysine per year (Ajinomoto, Tokyo, Japan). The amino acid production with C. glutamicum is focused on glucose, fructose, and sucrose as carbon sources, with a preference for glucose (30,31). However, C. glutamicum also can use sugars like ribose and maltose, the alcohols inositol and ethanol, and organic acids like acetate, propionate, pyruvate, L-lactate, citrate, and L-glutamate as carbon and energy sources (2, 6).At the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPON) (Fig. 1), the distribution of the carbon flux within the metabolism takes place and is especially important for amino acid synthesis (59). To improve L-lysine yields, the optimization of the PPON toward increased oxaloacetate supply is crucial (10, 46). Oxaloacetate utilized for L-lysine synthesis can be regenerated by PEP carboxylase (encoded by ppc) (13) or pyruvate carboxylase (pyc) (48). In terms of gluconeogenesis, oxaloacetate can be converted to either pyruvate by oxaloacetate decarboxylase (odx) (32) or to PEP by PEP carboxykinase (pck) (28). The irreversible conversion of PEP to pyruvate is catalyzed either by pyruvate kinase (pyk) with the formation of ATP (24) or by enzyme I (EI) of the PEP-dependent phosph...

Decoupling biorefineries from land use and agriculture is a major challenge. As formate can be produced from various sources, e.g., electrochemical reduction of CO, microbial formate-assimilation has the potential to become a sustainable feedstock for the bioindustry. However, organisms that naturally grow on formate are limited by either a low biomass yield or by a narrow product spectrum. The engineering of a model biotechnological microbe for growth on formate via synthetic pathways represents a promising approach to tackle this challenge. Here, we achieve a critical milestone for two such synthetic formate-assimilation pathways in Escherichia coli. Our engineering strategy involves the division of the pathways into metabolic modules; the activity of each module-providing at least one essential building block-is selected for in an appropriate auxotrophic strain. We demonstrate that formate can serve as a sole source of all cellular C1-compounds, including the beta-carbon of serine. We further show that by overexpressing the native threonine cleavage enzymes, the entire cellular glycine requirement can be provided by threonine biosynthesis and degradation. Together, we confirm the simultaneous activity of all pathway segments of the synthetic serine-threonine cycle. We go beyond the formate bioeconomy concept by showing that, under anaerobic conditions, formate produced endogenously by pyruvate formate-lyase can replace exogenous formate. The resulting prototrophic strain constitutes a substantial rewiring of central metabolism in which C1, glycine, and serine metabolism proceed via a unique set of pathways. This strain can serve as a platform for future metabolic-engineering efforts and could further pave the way for investigating the plasticity of metabolic networks.

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.

The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent L-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, L-lactate production by C. glutamicum was determined. The overexpression of sugR reduced L-lactate production by about 25%, and sugR deletion increased L-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative L-lactate dehydrogenase in C. glutamicum.Corynebacterium glutamicum, which was isolated as an Lglutamate-excreting soil bacterium (1, 39), is a predominantly aerobic, biotin-auxotrophic, gram-positive bacterium widely used for the industrial production of more than 2 million tons of amino acids per year, mainly L-glutamate and L-lysine (31,43). A general view of this nonpathogenic bacterium, which has become a model organism for the Corynebacterineae, a suborder of Actinomycetales that also comprises the genus Mycobacterium (54), can be found in two recent monographs (9, 17).C. glutamicum is able to grow on a variety of sugars, sugar alcohols, and organic acids as sole carbon and energy sources (64). As in many other gram-positive and gram-negative bacteria, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is the major sugar uptake system (15,37,45,47). The PTS-mediated glucose, fructose, and sucrose uptake in C. glutamicum operates by phosphoryl group transfer from phosphoenolpyruvate via EI (encoded by ptsI) and HPr (ptsH) to the sugar-specific permeases EII Glc , EII Fru , and EII Suc , respectively (ptsG, ptsF, and ptsS, respectively).

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