Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli , yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell–cell interactions, and the relationships between environmental and metabolic constraints.

Background: For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait. Results: Heme- (and menaquinone) stimulated aerobic growth was observed for several species and genera of lactic acid bacteria. These include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacilllus brevis, Lactobacillus paralimentarius, Streptococcus entericus and Lactococcus garviae. The increased biomass production without further acidification, which are respiration associated traits, are suitable for high-throughput screening as demonstrated by the screening of 8000 Lactococcus lactis insertion mutants. Respiration-negative insertion-mutants were found with noxA, bd-type cytochrome and menaquinol biosynthesis gene-disruptions. Phenotypic screening and in silico genome analysis suggest that respiration can be considered characteristic for certain species. Conclusion: We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species.

We observed that Lactobacillus reuteri JCM1112 produces B ₁₂ and folate. However, the folate/B ₁₂ mass ratio found was far below that desired for human consumption (∼170:1). We used metabolic engineering applying genetic and physiological approaches to improve this ratio and developed a generic and natural process that significantly increases folate production.

The coenzyme B12 production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B12 gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQOP, sirA, hemACBL, and cobUSC, hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B12 itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B12 pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B12 biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B12 biosynthesis and reuterin production, which might be implicated in Lb. reuteri's success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B12.

The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that (i) the PHB synthesis pathway becomes deregulated, and/or (ii) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve. The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232 mg L− 1, ~ 12% (w/w) of the dry biomass weight, and a productivity of 7.3 mg L− 1 d− 1; are to the best of our knowledge, the highest ever reported for PHB production directly from CO2

Summary: Protein investment costs are considered a major driver for the choice of alternative metabolic strategies. We tested this premise in Lactococcus lactis, a bacterium that exhibits a distinct, anaerobic version of the bacterial Crabtree/Warburg effect; with increasing growth rates it shifts from a high yield metabolic mode [mixed‐acid fermentation; 3 adenosine triphosphate (ATP) per glucose] to a low yield metabolic mode (homolactic fermentation; 2 ATP per glucose). We studied growth rate‐dependent relative transcription and protein ratios, enzyme activities, and fluxes of L. lactis in glucose‐limited chemostats, providing a high‐quality and comprehensive data set. A three‐ to fourfold higher growth rate rerouted metabolism from acetate to lactate as the main fermentation product. However, we observed hardly any changes in transcription, protein levels and enzyme activities. Even levels of ribosomal proteins, constituting a major investment in cellular machinery, changed only slightly. Thus, contrary to the original hypothesis, central metabolism in this organism appears to be hardly regulated at the level of gene expression, but rather at the metabolic level. We conclude that L. lactis is either poorly adapted to growth at low and constant glucose concentrations, or that protein costs play a less important role in fitness than hitherto assumed.