Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis.

Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold-increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.

Signal Peptide Efficiency: From High-Throughput Data to Prediction and Explanation.

The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology and important industrial applications. Proteins are directed into the Sec pathway by a signal peptide at their N-terminus. Estimating the impact of physicochemical signal peptide features on protein secretion levels has not been achieved so far, partially due to the extreme sequence variability of signal peptides. To elucidate relevant features of the signal peptide sequence that influence secretion efficiency, an evaluation of ∼12,000 different designed signal peptides was performed using a novel miniaturized high-throughput assay. The results were used to train a machine learning model, and a post-hoc explanation of the model is provided. By describing each signal peptide with a selection of 156 physicochemical features, it is now possible to both quantify feature importance and predict the protein secretion levels directed by each signal peptide. Our analyses allow the detection and explanation of the relevant signal peptide features influencing the efficiency of protein secretion, generating a versatile tool for the de novo design and in silico evaluation of signal peptides.

Novel method for high-throughput colony PCR screening in nanoliter-reactors.

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20,000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100,000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.

Bioengineering novel in vitro metabolic pathways using synthetic biology.

Huge numbers of enzymes have evolved in nature to function in aqueous environments at moderate temperatures and neutral pH. This gives us, in principle, the unique opportunity to construct multistep reaction systems of considerable catalytic complexity in vitro. However, this opportunity is rarely exploited beyond research scale, because such systems are difficult to assemble and to operate productively. Recent advances in DNA synthesis, genome engineering, high-throughput analytics, model-based analysis of biochemical systems and (semi-)rational protein engineering suggest that we have all the tools available to rationally design and efficiently operate such systems of enzymes, and finally harvest their potential for preparative syntheses.

Isolation of monoclonal microcarriers colonized by fluorescent E. coli.

Microencapsulation gains increasing importance for processing of bacterial libraries and especially in high-throughput (HT) environments where >10(6) samples per day are studied. As a rule, a one-to-one relationship between an individual cell and analytical results is of key importance. Ideally, each microcarrier would therefore contain exactly one cell or colony. However, synthesis of larger numbers of capsules containing exactly one cell is not feasible as cells are randomly distributed during carrier-production. The dilemma is that high dilution conditions will yield a satisfactory degree of monoclonality, but also a very large fraction of empty compartments, whereas distribution under low dilution generates unacceptable numbers of polyclonal compartments for whose removal no satisfactory technologies exist. Hydrogel carriers with a volume of 35 nL were used as growth compartments for individual microbial colonies. E. coli cells expressing green fluorescent protein (GFP) were encapsulated at low dilution thereby intentionally producing a considerable amount of polyclonal microcarrieres. Empty and polyclonal microcarriers were then removed from the desired monoclonal fraction by a COPAS Plus particle analyzer. The results were compared with model predictions in order to investigate possible limitations in the analysis and sorting of monoclonal microcarriers by COPAS. Fluorescent E. coli cells (GFP) distributed randomly throughout the microcarrier population. Cells were successfully propagated to colonies in the microcarriers and enriched to 95% monoclonality by a COAPS sorter. Enrichment-efficiency was found to mainly depend on the colony diameter. With increasing colony size two contrary effects were observed: First, improved sorting efficiency due to increased fluorescence intensity and therefore higher detection efficiency, and second, deterioration of sorting efficiency due to occlusion occurring in polyclonal carriers. The combination of microencapsulation under low dilution conditions followed by HT sorting procedures is an efficient way for isolating larger amount of monoclonal carriers from bacterial libraries while concomitantly keeping the amounts of empty carriers at a moderate level.

Corrigendum: Identification and Characterisation of a pH-stable GFP.

This corrects the article DOI: 10.1038/srep28166.

Identification and Characterisation of a pH-stable GFP.

Green fluorescent proteins (GFPs) are invaluable tools for modern cell biology. Even though many properties of GFP have been successfully engineered, a GFP retaining brightness at low pH has not emerged. This limits the use of GFP in quantitative studies performed in fluctuating or acidic conditions. We report the engineering and characterisation of tandem dimer GFP (pH-tdGFP), a bright and stable GFP that can be efficiently excited and maintains its fluorescence properties in acidic conditions. Therefore, pH-tdGFP could act as a quantitative marker for cellular processes that occur at low pH, such as endocytosis, autophagy or starvation.

Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors.

Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

Chiral, Three-Dimensional Supramolecular Compounds: Homo- and Bimetallic Oxalate- and 1,2-Dithiooxalate-Bridged Networks. A Structural and Photophysical Study.

In analogy to the [M(II)(bpy)(3)](2+) cations, where M(II) is a divalent transition-metal and bpy is 2,2'-bipyridine, the tris-chelated [M(III)(bpy)(3)](3+) cations, where M(III) is Cr(III) or Co(III), induce the crystallization of chiral, anionic three-dimensional (3D) coordination polymers of oxalate-bridged (&mgr;-ox) metal complexes with stoichiometries [M(II)(2)(ox)(3)](n)()(2)(n)()(-) or [M(I)M(III)(ox)(3)](n)()(2)(n)()(-). The tripositive charge is partially compensated by inclusion of additional complex anions like ClO(4)(-), BF(4)(-), or PF(6)(-) which are encapsulated in cubic shaped cavities formed by the bipyridine ligands of the cations. Thus, an elaborate structure of cationic and anionic species within a polymeric anionic network is realized. The compounds isolated and structurally characterized include [Cr(III)(bpy)(3)][ClO(4)] [NaCr(III)(ox)(3)] (1), [Cr(III)(bpy)(3)][ClO(4)][Mn(II)(2)(ox)(3)] (2), [Cr(III)(bpy)(3)][BF(4)] [Mn(II)(2)(ox)(3)] (3), [Co(III)(bpy)(3)][PF(6)][NaCr(III)(ox)(3)] (4). Crystal data: 1, cubic, P2(1)3, a = 15.523(4) Å, Z = 4; 2, cubic, P4(1)32, a = 15.564(3) Å, Z = 4; 3, cubic, P4(1)32, a = 15.553(3) Å, Z = 4; 4, cubic, P2(1)3, a = 15.515(3) Å, Z = 4. Furthermore, it seemed likely that 1,2-dithiooxalate (dto) could act as an alternative to the oxalate bridging ligand, and as a result the compound [Ni(II)(phen)(3)][NaCo(III)(dto)(3)].C(3)H(6)O (5) has successfully been isolated and structurally characterized. Crystal data: 5, orthorhombic, P2(1)2(1)2(1), a = 16.238(4) Å, b = 16.225(4) Å, c = 18.371(5) Å, Z = 4. In addition, the photophysical properties of compound 1 have been investigated in detail. In single crystal absorption spectra of [Cr(III)(bpy)(3)][ClO(4)][NaCr(III)(ox)(3)] (1), the spin-flip transitions of both the [Cr(bpy)(3)](3+) and the [Cr(ox)(3)](3)(-) chromophores are observed and can be clearly distinguished. Irradiating into the spin-allowed (4)A(2) --> (4)T(2) absorption band of [Cr(ox)(3)](3)(-) results in intense luminescence from the (2)E state of [Cr(bpy)(3)](3+) as a result of rapid energy transfer processes.

Molecular-Based Magnetism in Bimetallic Two-Dimensional Oxalate-Bridged Networks. An X-ray and Neutron Diffraction Study.

Bimetallic, oxalate-bridged compounds with bi- and trivalent transition metals comprise a class of layered materials which express a large variety in their molecular-based magnetic behavior. Because of this, the availability of the corresponding single-crystal structural data is essential to the successful interpretation of the experimental magnetic results. We report in this paper the crystal structure and magnetic properties of the ferromagnetic compound {[N(n-C(3)H(7))(4)][Mn(II)Cr(III)(C(2)O(4))(3)]}(n)() (1), the crystal structure of the antiferromagnetic compound {[N(n-C(4)H(9))(4)][Mn(II)Fe(III)(C(2)O(4))(3)]}(n)() (2), and the results of a neutron diffraction study of a polycrystalline sample of the ferromagnetic compound {[P(C(6)D(5))(4)][Mn(II)Cr(III)(C(2)O(4))(3)]}(n)() (3). Crystal data: 1, rhombohedral, R3c, a = 9.363(3) Å, c = 49.207(27) Å, Z = 6; 2, hexagonal, P6(3), a = 9.482(2) Å, c = 17.827(8) Å, Z = 2. The structures consist of anionic, two-dimensional, honeycomb networks formed by the oxalate-bridged metal ions, interleaved by the templating cations. Single-crystal field dependent magnetization measurements as well as elastic neutron scattering experiments on the manganese(II)-chromium(III) samples show the existence of long-range ferromagnetic ordering behavior below T(c) = 6 K. The magnetic structure corresponds to an alignment of the spins perpendicular to the network layers. In contrast, the manganese(II)-iron(III) compound expresses a two-dimensional antiferromagnetic ordering.

Chemical and enzymatic routes to dihydroxyacetone phosphate.

Stereoselective carbon-carbon bond formation with aldolases has become an indispensable tool in preparative synthetic chemistry. In particular, the dihydroxyacetone phosphate (DHAP)-dependent aldolases are attractive because four different types are available that allow access to a complete set of diastereomers of vicinal diols from achiral aldehyde acceptors and the DHAP donor substrate. While the substrate specificity for the acceptor is rather relaxed, these enzymes show only very limited tolerance for substituting the donor. Therefore, access to DHAP is instrumental for the preparative exploitation of these enzymes, and several routes for its synthesis have become available. DHAP is unstable, so chemical synthetic routes have concentrated on producing a storable precursor that can easily be converted to DHAP immediately before its use. Enzymatic routes have concentrated on integrating the DHAP formation with upstream or downstream catalytic steps, leading to multi-enzyme arrangements with up to seven enzymes operating simultaneously. While the various chemical routes suffer from either low yields, complicated work-up, or toxic reagents or catalysts, the enzymatic routes suffer from complex product mixtures and the need to assemble multiple enzymes into one reaction scheme. Both types of routes will require further improvement to serve as a basis for a scalable route to DHAP.