Abstract:BackgroundMost bacteria are grown in a binary fission way meaning a bacterial cell is equally divided into two. Polyhydroxyalkanoates (PHA) can be accumulated as inclusion bodies by bacteria. The cell division way and morphology have been shown to play an important role in regulating the bacterial growth and PHA storages.ResultsThe common growth pattern of Escherichia coli was changed to multiple fission patterns by deleting fission related genes minC and minD together, allowing the formation of multiple fissi… Show more
“…5B,E). In addition, the morphology and number of PHA granules was also affected in the engineered JBOi strain, a feature found in many recombinant PHA-producing strains with impaired growth 28, 29 .Figure 5Transmission electron micrographs of P . putida KT2440 ( A , B , C ) and the engineered JBOi strain 24 h after induction ( D , E , F ).…”
Cell lysis is crucial for the microbial production of industrial fatty acids, proteins, biofuels, and biopolymers. In this work, we developed a novel programmable lysis system based on the heterologous expression of lysozyme. The inducible lytic system was tested in two Gram-negative bacterial strains, namely Escherichia coli and Pseudomonas putida KT2440. Before induction, the lytic system did not significantly arrest essential physiological parameters in the recombinant E. coli (ECPi) and P. putida (JBOi) strain such as specific growth rate and biomass yield under standard growth conditions. A different scenario was observed in the recombinant JBOi strain when subjected to PHA-producing conditions, where biomass production was reduced by 25% but the mcl-PHA content was maintained at about 30% of the cell dry weight. Importantly, the genetic construct worked well under PHA-producing conditions (nitrogen-limiting phase), where more than 95% of the cell population presented membrane disruption 16 h post induction, with 75% of the total synthesized biopolymer recovered at the end of the fermentation period. In conclusion, this new lysis system circumvents traditional, costly mechanical and enzymatic cell-disrupting procedures.
“…5B,E). In addition, the morphology and number of PHA granules was also affected in the engineered JBOi strain, a feature found in many recombinant PHA-producing strains with impaired growth 28, 29 .Figure 5Transmission electron micrographs of P . putida KT2440 ( A , B , C ) and the engineered JBOi strain 24 h after induction ( D , E , F ).…”
Cell lysis is crucial for the microbial production of industrial fatty acids, proteins, biofuels, and biopolymers. In this work, we developed a novel programmable lysis system based on the heterologous expression of lysozyme. The inducible lytic system was tested in two Gram-negative bacterial strains, namely Escherichia coli and Pseudomonas putida KT2440. Before induction, the lytic system did not significantly arrest essential physiological parameters in the recombinant E. coli (ECPi) and P. putida (JBOi) strain such as specific growth rate and biomass yield under standard growth conditions. A different scenario was observed in the recombinant JBOi strain when subjected to PHA-producing conditions, where biomass production was reduced by 25% but the mcl-PHA content was maintained at about 30% of the cell dry weight. Importantly, the genetic construct worked well under PHA-producing conditions (nitrogen-limiting phase), where more than 95% of the cell population presented membrane disruption 16 h post induction, with 75% of the total synthesized biopolymer recovered at the end of the fermentation period. In conclusion, this new lysis system circumvents traditional, costly mechanical and enzymatic cell-disrupting procedures.
“…Thus, MreB was also tested for the enhanced production of poly(3HB). [159,168,169] Overexpressing mreB in an mreB deletion mutant under inducible expression of sulA, the shape of E. coli changed from rod-type to larger spherical shapes with enhanced poly(3HB) accumulation. [159] Through the finely controlled repression of ftsZ and mreB using the CRISPRi tool, the poly(3HB) production could be improved by having longer and larger cell shapes.…”
Microorganisms produce diverse polymers for various purposes such as storing genetic information, energy, and reducing power, and serving as structural materials and scaffolds. Among these polymers, polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized and accumulated intracellularly as a storage material of carbon, energy, and reducing power under unfavorable growth conditions in the presence of excess carbon source. PHAs have attracted considerable attention for their wide range of applications in industrial and medical fields. Since the first discovery of PHA accumulating bacteria about 100 years ago, remarkable advances have been made in the understanding of PHA biosynthesis and metabolic engineering of microorganisms toward developing efficient PHA producers. Recently, nonnatural polyesters have also been synthesized by metabolically engineered microorganisms, which opened a new avenue toward sustainable production of more diverse plastics. Herein, the current state of PHAs and nonnatural polyesters is reviewed, covering mechanisms of microbial polyester biosynthesis, metabolic pathways, and enzymes involved in biosynthesis of short‐chain‐length PHAs, medium‐chain‐length PHAs, and nonnatural polyesters, especially 2‐hydroxyacid‐containing polyesters, metabolic engineering strategies to produce novel polymers and enhance production capabilities and fermentation, and downstream processing strategies for cost‐effective production of these microbial polyesters. In addition, the applications of PHAs and prospects are discussed.
“…Moreover, the representative Class II PHA synthase, Ser325Thr/Gln481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 (encoded by phaC1), which possesses broad substrate specificity ranging from C3-C12 carbon atoms (Matsusaki et al 2000;Taguchi et al 2008), was also chosen to be expressed in Y. lipolytica.…”
Section: Construction Of Integrative Phb Expression Vectorsmentioning
Strains of Yarrowia lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35 g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.
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