Heterologous constitutive production of short-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2440: the involvement of IbpA inclusion body protein.

Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain à la carte materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, P. putida KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a "deaf" PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from Rhodospirillum rubrum, Cupriavidus necator and Pseudomonas pseudoalcaligenes, demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from C. necator resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaCCn synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in P. putida KT2440.

Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, ß-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

Phasin interactome reveals the interplay of PhaF with the polyhydroxyalkanoate transcriptional regulatory protein PhaD in Pseudomonas putida.

Phasin PhaF, a multifunctional protein associated with the surface of polyhydroxyalkanoate (PHA) granules that also interacts with the nucleoid, contributes significantly to PHA biogenesis in pseudomonads. As a protein present on the surface of PHA granules, PhaF participates in granule stabilization and segregation, whereas its deletion has a notable impact on overall transcriptome, PHA accumulation and cell physiology, suggesting more extensive functions besides solely being a granule structural protein. Here, we followed a systematic approach to detect potential interactions of PhaF with other components of the cell, which could pinpoint unexplored functions of PhaF in the regulation of PHA production. We determined the PhaF interactome in Pseudomonas putida KT2440 via pull-down-mass spectrometry (PD-MS) experiments. PhaF complexed with PHA-related proteins, phasin PhaI and the transcriptional regulator PhaD, interactions that were verified to be direct using in vivo two-hybrid analysis. The determination of the PHA granule proteome showed that PhaI and three other potential PhaF interacting partners, but not PhaD, were granule-associated proteins. Analysis of the interaction of PhaF and PhaD with the phaI promoter by EMSA suggested a new role for PhaF in interacting with PhaD and raises new questions on the regulatory system controlling pha gene expression.

Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria.

BACKGROUND: There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration. RESULTS: Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration. CONCLUSIONS: We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.

Histone H3 Threonine 11 Phosphorylation Is Catalyzed Directly by the Meiosis-Specific Kinase Mek1 and Provides a Molecular Readout of Mek1 Activity in Vivo.

Saccharomyces cerevisiae Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in S. cerevisiae and Schizosaccharomyces pombe Molecular genetic experiments in S. cerevisiae confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation in vitro with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity in vivo Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.

A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation.

The nonrandom distribution of meiotic recombination influences patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in the accumulation of Spo11 protein covalently bound to small DNA fragments. By sequencing these fragments, we uncover a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore how DSB distribution is influenced by large-scale chromosome structures, chromatin, transcription factors, and local sequence composition. Our analysis offers mechanistic insight into DSB formation and early processing steps, supporting the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We also discuss implications for evolutionary dynamics of recombination hot spots.

Histone methylation sets the stage for meiotic DNA breaks.

Covalent post-translational modifications of histones have important functions in transcription, replication, repair, and other aspects of eukaryotic chromosome dynamics. Trimethylation of lysine-4 on histone H3 is enriched at actively transcribed loci in many organisms. The impact of this histone modification on transcription has been extensively studied, but less is known about its effects on other chromosomal processes. An intriguing new study in this issue of EMBO Journal demonstrates that H3 lysine-4 trimethylation is critical in budding yeast for formation of the programmed DNA double-strand breaks that initiate homologous recombination during meiosis. These findings have important implications for elucidating the previously recognized but little understood connections between meiotic break formation and transcriptional promoters in this organism.

Structure, function, and mechanism of the phenylacetate pathway hot dog-fold thioesterase PaaI.

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

Crystal structure and functional analysis of lipoamide dehydrogenase from Mycobacterium tuberculosis.

We report the 2.4 A crystal structure for lipoamide dehydrogenase encoded by lpdC from Mycobacterium tuberculosis. Based on the Lpd structure and sequence alignment between bacterial and eukaryotic Lpd sequences, we generated single point mutations in Lpd and assayed the resulting proteins for their ability to catalyze lipoamide reduction/oxidation alone and in complex with other proteins that participate in pyruvate dehydrogenase and peroxidase activities. The results suggest that amino acid residues conserved in mycobacterial species but not conserved in eukaryotic Lpd family members modulate either or both activities and include Arg-93, His-98, Lys-103, and His-386. In addition, Arg-93 and His-386 are involved in forming both "open" and "closed" active site conformations, suggesting that these residues play a role in dynamically regulating Lpd function. Taken together, these data suggest protein surfaces that should be considered while developing strategies for inhibiting this enzyme.

Structure of Escherichia coli uridine phosphorylase at 2.0 A.

The 2.0 A crystal structure has been determined for Escherichia coli uridine phosphorylase (UP), an essential enzyme in nucleotide biosynthesis that catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose-1-phosphate and uracil. The structure determination of two independent monomers in the asymmetric unit revealed the residue composition and atomic details of the apo configurations of each active site. The native hexameric UP enzyme was revealed by applying threefold crystallographic symmetry to the contents of the asymmetric unit. The 2.0 A model reveals a closer structural relationship to other nucleotide phosphorylase enzymes than was previously appreciated.

Structural analysis of Saccharomyces cerevisiae myo-inositol phosphate synthase.

The New York Structural Genomics Research Consortium has targeted highly conserved but uncharacterized enzyme families for structure determination. As part of this effort, the 2.65-A crystal structure has been determined for Saccharomyces cerevisiae myo-inositol 1-phosphate synthase (MIP), an essential enzyme that catalyzes critical steps in inositol biosynthesis. The structure determination of four independent monomers in the asymmetric unit (240 kDa) reveals atomic details and residue composition for the partially closed NAD-containing active sites in apo-configuration. The structure further reveals extensive interactions involved in tetrameric assembly of the enzyme complex.