Improved Production of Medium-Chain-Length Polyhydroxyalkanoates in Glucose-Based Fed-Batch Cultivations of Metabolically Engineered Pseudomonas putida Strains

Poblete‐Castro, Ignacio; Rodriguez, Andre Luis; Lam, Carolyn M.C.; Kessler, Waltraud

doi:10.4014/jmb.1308.08052

Cited by 48 publications

(46 citation statements)

References 35 publications

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“…Among a list of different genetic targets, elementary flux mode analysis suggested an inactivation of glucose dehydrogenase encoded by gcd to prevent undesired by-product formation and excretion. In agreement with the prediction from the in silico model the constructed P. putida Δgcd mutant strain showed an increased PHA production in batch [13] and Fed-Batch cultivations [29]. Here, we continued to experimentally verify the predictions of our in silico model for the optimization of mcl-PHA production in P. putida .…”

Section: Introductionsupporting

confidence: 65%

“…P. putida KT2440 can re-consume gluconate and 2-ketogluconate once secreted to the medium. We recently demonstrated that this process is not efficient to improve mcl-PHA synthesis [29], resulting in a lower volumetric PHA productivity as compared to the one shown by the Δ gcd P. putida mutant strain under the same growth conditions. Several investigations have shown that the volumetric productivity is the key parameter for the industrial production of PHAs and thus, any factor influencing this parameter affects the economics and cost competitiveness of PHAs synthesis via microbial fermentation [30-33].…”

Section: Resultsmentioning

confidence: 99%

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Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida

et al. 2014

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BackgroundPseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.ResultsA genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms.ConclusionProduction of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.

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Section: Introductionsupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida

et al. 2014

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“…It can be excluded that S. elongatus cscB is responsible for PHA formation since it lacks the corresponding genes for PHA synthesis [22]. Moreover, the distribution pattern of 3-hydroxyalkanoic acids is typical for P. putida (Table 2), with 3-hydroxydecanoic acid being the most abundant monomer [23]. Apparently, PHA was accumulated in only a small fraction of the cells, which can be deduced when comparing the CFU/mL to the cell counts of Nile red-stained cells.…”

Section: Resultsmentioning

confidence: 99%

“…Here, we reached a maximal concentration of ~150 mg/L PHA, which is quite low, compared to pure cultures. However, this was achieved with a small number of cells compared to the cell densities reached in pure cultures, which are normally cultivated in fed-batch, high-cell density processes [23]. Nevertheless, it is crucial for efficient downstream processing to obtain a high concentration of PHA.…”

Section: Resultsmentioning

confidence: 99%

Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB

Löwe

Hobmeier

Moos

et al. 2017

Biotechnol Biofuels

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BackgroundOne of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. Cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals, showing promising production rates when compared to crop-based feedstock. However, intrinsic capacities of cyanobacteria to produce biotechnological compounds are limited and yields are low.ResultsHere, we present an approach to circumvent these problems by employing a synthetic bacterial co-culture for the carbon-neutral production of polyhydroxyalkanoates (PHAs) from CO2. The co-culture consists of two bio-modules: Bio-module I, in which the cyanobacterial strain Synechococcus elongatus cscB fixes CO2, converts it to sucrose, and exports it into the culture supernatant; and bio-module II, where this sugar serves as C-source for Pseudomonas putida cscAB and is converted to PHAs that are accumulated in the cytoplasm. By applying a nitrogen-limited process, we achieved a maximal PHA production rate of 23.8 mg/(L day) and a maximal titer of 156 mg/L. We will discuss the present shortcomings of the process and show the potential for future improvement.ConclusionsThese results demonstrate the feasibility of mixed cultures of S. elongatus cscB and P. putida cscAB for PHA production, making room for the cornucopia of possible products that are described for P. putida. The construction of more efficient sucrose-utilizing P. putida phenotypes and the optimization of process conditions will increase yields and productivities and eventually close the gap in the contemporary process. In the long term, the co-culture may serve as a platform process, in which P. putida is used as a chassis for the implementation of synthetic metabolic pathways for biotechnological production of value-added products.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0875-0) contains supplementary material, which is available to authorized users.

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“…CO 2 production rate based pulse feeding, cumulative CO 2 production based continuous feeding, etc.) have been proposed in the literature [5][6][7][8][9] . However, most of these strategies are focused on the KT2440 strain, which is different in terms of mcl-PHAs production from the rifampicin-resistant variant KT2442…”

Section: Introductionmentioning

confidence: 99%

Strategies for Automated Control of the Bioproduction of Mcl-PHA Biopolymers

Hrnčiřík¹

2017

Chem.Biochem.Eng.Q.

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Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are polyesters synthesized by numerous bacteria as storage material. Despite being promising candidates for biodegradable materials of industrial interest and environmental value, their usage is still rather limited because of high production costs. One of the areas with considerable potential for further improvements is control of the production process. This paper deals with the experimental work related to the design of control strategies for mcl-PHA biopolymer production process (Pseudomonas putida KT2442 fed-batch cultivations). For this bioprocess, a set of five control strategies (two main and three auxiliary strategies) have been proposed, together with the proper sequence of their switching during the fedbatch part of the production process. The application of these strategies with octanoic acid as a sole carbon source resulted in intracellular PHA content (max. mass fraction 65 % of mcl-PHA in cell dry mass (g g -1) and PHA productivity (max. 0.89 g L -1 h -1 ) comparable to the best results reported in the literature for this type of strain and carbon substrate.

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Improved Production of Medium-Chain-Length Polyhydroxyalkanoates in Glucose-Based Fed-Batch Cultivations of Metabolically Engineered Pseudomonas putida Strains

Cited by 48 publications

References 35 publications

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida

Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB

Strategies for Automated Control of the Bioproduction of Mcl-PHA Biopolymers

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