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

Localization of agrin domains necessary for heparin and alpha-dystroglycan binding

Thesis (B.S.)--Univeristy of Illinois at Urbana-Champaign, 1997.Includes bibliographical reference (leaf 11)U of I OnlyTheses restricted to UIUC community onl

Running title: PhaF-PhaD interplay

15 p.-6 fig.-2 tabPhasin 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.This work was supported by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 633962 (P4SB), the Community of Madrid (P2013/MIT2807, P2018/NMT4389) and the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R).Peer reviewe

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

Abstract 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

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

18 p.-6 fig.-5 tab.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.This research received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 633962 (P4SB), 814418 (SinFonia) and 870294 (MIX-up). This work was supported by the CSIC Interdisciplinary Thematic Platform (PTI+) Sustainable Plastics towards a Circular Economy (PTI-Susplast+), the Community of Madrid (P2018/NMT4389) and the Spanish Ministry of Science and Innovation under the research grant BIOCIR (PID 2020-112766RB-C21).Peer reviewe

La Charente

01 mars 19091909/03/01 (A38,N11609)-1909/03/01.Appartient à l’ensemble documentaire : PoitouCh