The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture.

BackgroundGlycerol has attracted attention as a carbon source for microbial production processes due to the large amounts of crude glycerol waste resulting from biodiesel production. The current knowledge about the genetics and physiology of glycerol uptake and catabolism in the versatile industrial biotechnology production host Saccharomyces cerevisiae has been mainly based on auxotrophic laboratory strains, and carried out in the presence of growth-supporting supplements such as amino acids and nucleic bases. The latter may have resulted in ambiguous conclusions concerning glycerol growth in this species. The purpose of this study was to re-evaluate growth of S. cerevisiae in synthetic glycerol medium without the addition of supplements.ResultsInitial experiments showed that prototrophic versions of the laboratory strains CEN.PK, W303, and S288c did not exhibit any growth in synthetic glycerol medium without supporting supplements. However, a screening of 52 S. cerevisiae isolates for growth in the same medium revealed a high intraspecies diversity. Within this group significant variation with respect to the lag phase and maximum specific growth rate was observed. A haploid segregant of one good glycerol grower (CBS 6412-13A) was selected for detailed analysis. Single deletions of the genes encoding for the glycerol/H+ symporter (STL1), the glycerol kinase (GUT1), and the mitochondrial FAD+-dependent glycerol 3-phosphate dehydrogenase (GUT2) abolished glycerol growth in this strain, implying that it uses the same glycerol utilization pathway as previously identified in auxotrophic laboratory strains. Segregant analysis of a cross between CBS 6412-13A and CEN.PK113-1A revealed that the glycerol growth phenotype is a quantitative trait. Genetic linkage and reciprocal hemizygosity analysis demonstrated that GUT1 CBS 6412-13A is one of the multiple genetic loci contributing to the glycerol growth phenotype.ConclusionThe S. cerevisiae intraspecies diversity with regard to glycerol growth is a valuable starting point to identify the genetic and molecular basis of this phenotype. This knowledge can be applied for further rational strain improvement with the goal of using glycerol as a carbon source in industrial biotechnology processes based on S. cerevisiae as a production organism.

Organisms are either heterotrophic or autotrophic, meaning that they cover their carbon requirements by assimilating organic compounds or by fixing inorganic carbon dioxide (CO). The conversion of a heterotrophic organism into an autotrophic one by metabolic engineering is a long-standing goal in synthetic biology and biotechnology, because it ultimately allows for the production of value-added compounds from CO. The heterotrophic Alphaproteobacterium Methylobacterium extorquens AM1 is a platform organism for a future C1-based bioeconomy. Here we show that M. extorquens AM1 provides unique advantages for establishing synthetic autotrophy, because energy metabolism and biomass formation can be effectively separated from each other in the organism. We designed and realized an engineered strain of M. extorquens AM1 that can use the C1 compound methanol for energy acquisition and forms biomass from CO by implementation of a heterologous Calvin-Benson-Bassham (CBB) cycle. We demonstrate that the heterologous CBB cycle is active, confers a distinct phenotype, and strongly increases viability of the engineered strain. Metabolic C-tracer analysis demonstrates the functional operation of the heterologous CBB cycle in M. extorquens AM1 and comparative proteomics of the engineered strain show that the host cell reacts to the implementation of the CBB cycle in a plastic way. While the heterologous CBB cycle is not able to support full autotrophic growth of M. extorquens AM1, our study represents a further advancement in the design and realization of synthetic autotrophic organisms.

A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli.