A versatile active learning workflow for optimization of genetic and metabolic networks.

Optimization of biological networks is often limited by wet lab labor and cost, and the lack of convenient computational tools. Here, we describe METIS, a versatile active machine learning workflow with a simple online interface for the data-driven optimization of biological targets with minimal experiments. We demonstrate our workflow for various applications, including cell-free transcription and translation, genetic circuits, and a 27-variable synthetic CO-fixation cycle (CETCH cycle), improving these systems between one and two orders of magnitude.

A Modular In Vitro Platform for the Production of Terpenes and Polyketides from CO.

A long-term goal in realizing a sustainable biocatalysis and organic synthesis is the direct use of the greenhouse gas CO as feedstock for the production of bulk and fine chemicals, such as pharmaceuticals, fragrances and food additives. Here we developed a modular in vitro platform for the continuous conversion of CO into complex multi-carbon compounds, such as monoterpenes (C ), sesquiterpenes (C ) and polyketides. Combining natural and synthetic metabolic pathway modules, we established a route from CO into the key intermediates acetyl- and malonyl-CoA, which can be subsequently diversified through the action of different terpene and polyketide synthases.

Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in a minimal complex PURE system to directly run multistep reactions.

Light-powered CO fixation in a chloroplast mimic with natural and synthetic parts.

Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chemical energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets.

Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon Extension of Aldehydes to Chiral α-Hydroxy Acids.

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine-diphosphate-dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl-CoA thioester substrates through the release of the C -unit formyl-CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C -extensions of various aldehydes to yield the corresponding 2-hydroxyacyl-CoA thioesters.

Marine Proteobacteria metabolize glycolate via the β-hydroxyaspartate cycle.

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria, the further fate of this C metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the β-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago.

The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite.

Cells must cope with toxic or reactive intermediates formed during metabolism. One coping strategy is to sequester reactions that produce such intermediates within specialized compartments or tunnels connecting different active sites. Here, we show that propionyl-CoA synthase (PCS), an ∼ 400-kDa homodimer, three-domain fusion protein and the key enzyme of the 3-hydroxypropionate bi-cycle for CO fixation, sequesters its reactive intermediate acrylyl-CoA.

InhA, the enoyl-thioester reductase from forms a covalent adduct during catalysis.

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of and is a key target of antituberculosis drugs to combat multidrug-resistant strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC-MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle.

Combining Promiscuous Acyl-CoA Oxidase and Enoyl-CoA Carboxylase/Reductases for Atypical Polyketide Extender Unit Biosynthesis.

The incorporation of different extender units generates structural diversity in polyketides. There is significant interest in engineering substrate specificity of polyketide synthases (PKSs) to change their chemical structure. Efforts to change extender unit selectivity are hindered by the lack of simple screening methods and easily available atypical extender units.

In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme - Associated Bacterial Microcompartment.

Many bacteria encode proteinaceous bacterial microcompartments (BMCs) that encapsulate sequential enzymatic reactions of diverse metabolic pathways. Well-characterized BMCs include carboxysomes for CO-fixation, and propanediol- and ethanolamine-utilizing microcompartments that contain B-dependent enzymes. Genes required to form BMCs are typically organized in gene clusters, which promoted their distribution across phyla by horizontal gene transfer.

A synthetic pathway for the fixation of carbon dioxide in vitro.

Carbon dioxide (CO) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multicarbon compounds. We describe a synthetic cycle for the continuous fixation of CO in vitro.

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies.

Novel sulfated phosphoglycolipids from Natronomonas moolapensis.

Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol.

A comprehensive insight into the lipid composition of Myxococcus xanthus by UPLC-ESI-MS.

Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M.

Mutasynthesis-derived myxalamids and origin of the isobutyryl-CoA starter unit of myxalamid B.

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5-13) have been obtained by mutasynthesis from bkd mutants of M.