https://backend.orbit.dtu.dk/ws/files/189263285/1_s2.0_S1096717619301533_main.pdf

The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology.

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages Here we show that macrophages infected with S Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1 The changes in host metabolism promote intracellular replication of S Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S Typhimurium replication or virulence Salmonella Typhimurium establishes systemic infection by replicating in host macrophages Here, Jiang et al show that infected macrophages exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates that promote intracellular replication and virulence of S Typhimurium

/pdf/salmonella-typhimurium-reprograms-macrophage-metabolism-via-30t49kc7nh.pdf

Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence

Sustainable production of chemicals from renewable non-food biomass has become a promising alternative to overcome environmental issues caused by our heavy dependence on fossil resources Systems metabolic engineering, which integrates traditional metabolic engineering with systems biology, synthetic biology, and evolutionary engineering, is enabling the development of microbial cell factories capable of efficiently producing a myriad of chemicals and materials including biofuels, bulk and fine chemicals, polymers, amino acids, natural products and drugs In this paper, many tools and strategies of systems metabolic engineering, including in silico genome-scale metabolic simulation, sophisticated enzyme engineering, optimal gene expression modulation, in vivo biosensors, de novo pathway design, and genomic engineering, employed for developing microbial cell factories are reviewed Also, detailed procedures of systems metabolic engineering used to develop microbial strains producing chemicals and materials are showcased Finally, future challenges and perspectives in further advancing systems metabolic engineering and establishing biorefineries are discussed

/pdf/tools-and-strategies-of-systems-metabolic-engineering-for-3gsy6s5juj.pdf

Tools and strategies of systems metabolic engineering for the development of microbial cell factories for chemical production

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(19)30277-X.pdf

Metabolic Engineering of Escherichia coli for Natural Product Biosynthesis

High-Throughput Screening Technology in Industrial Biotechnology.

Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain's systems biology.

/pdf/adaptive-laboratory-evolution-of-a-genome-reduced-19uf18j6us.pdf

Adaptive laboratory evolution of a genome-reduced Escherichia coli

Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 105 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli, which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coliIMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli.

https://journals.asm.org/doi/pdf/10.1128/JB.00771-17

Minseob Yoo

Papers

Adaptive laboratory evolution of a genome-reduced Escherichia coli

Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12.