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A Nitrate-Blind P. putida Strain Boosts PHA Production in a Synthetic Mixed Culture.

ABSTRACT: One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. In this context, cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals. Based on this, we recently presented a synthetic bacterial co-culture for the production of medium-chain-length polyhydroxyalkanoates (PHAs) from CO2. This co-cultivation system is composed of two partner strains: Synechococcus elongatus cscB which fixes CO2, converts it to sucrose and exports it into the culture supernatant, and a Pseudomonas putida strain that metabolizes this sugar and accumulates PHAs in the cytoplasm. However, these biopolymers are preferably accumulated under conditions of nitrogen limitation, a situation difficult to achieve in a co-culture as the other partner, at best, should not perceive any limitation. In this article, we will present an approach to overcome this dilemma by uncoupling the PHA production from the presence of nitrate in the medium. This is achieved by the construction of a P. putida strain that is no longer able to grow with nitrate as nitrogen source -is thus nitrate blind, and able to grow with sucrose as carbon source. The deletion of the nasT gene encoding the response regulator of the NasS/NasT two-component system resulted in such a strain that has lost the ability use nitrate, but growth with ammonium was not affected. Subsequently, the nasT deletion was implemented in P. putida cscRABY, an efficient sucrose consuming strain. This genetic engineering approach introduced an artificial unilateral nitrogen limitation in the co-cultivation process, and the amount of PHA produced from light and CO2 was 8.8 fold increased to 14.8% of its CDW compared to the nitrate consuming reference strain. This nitrate blind strain, P. putida?nasT attTn7:cscRABY, is not only a valuable partner in the co-cultivation but additionally enables the use of other nitrate containing substrates for medium-chain-length PHA production, like for example waste-water.

SUBMITTER: Hobmeier K

PROVIDER: S-EPMC7261876 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Nitrate-Blind <i>P. putida</i> Strain Boosts PHA Production in a Synthetic Mixed Culture.

Hobmeier Karina K Löwe Hannes H Liefeldt Stephan S Kremling Andreas A Pflüger-Grau Katharina K

Frontiers in bioengineering and biotechnology 20200525

One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. In this context, cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals. Based on this, we recently presented a synthetic bacterial co-culture for the production of medium-chain-length polyhydroxyalkanoates (PHAs) from CO<sub>2</sub>. This co-cultivation syst ...[more]

PMID: 32523942

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Project description:The inexhaustible nature and biodegradability of bioplastics like polyhydroxyalkanoates (PHAs) make them suitable assets to replace synthetic plastics. The eventual fate of these eco-friendly and non-toxic bioplastics relies upon the endeavors towards satisfying cost and, in addition, execution necessity. In this study, we utilized and statistically optimized different food (kitchen-/agro-) waste as a sole carbon/nitrogen source for the production of PHA at a reduced cost, indicating a proficient waste administration procedure. Seven different types of kitchen-/agro-waste were used as unique carbon source and four different types of nitrogen source were used to study their impact on PHA production by Bacillus subtilis MTCC 144. Among four different studied production media, mineral salt medium (MSM) (biomass: 37.7 g/L; cell dry weight: 1.8 g/L; and PHA: 1.54 g/L) was found most suitable for PHA production. Further, carbon and nitrogen components of MSM were optimized using one-factor-at-a-time experiments, and found that watermelon rind (PHA = 12.97 g/L) and pulse peel (PHA = 13.5 g/L) were the most suitable carbon and nitrogen sources, respectively, in terms of PHA (78.60%) recovery. The concentrations of these factors (sources) were statistically optimized using response surface methodology coupled with the genetic algorithm approach. Additionally, in order to enhance microbial PHA production, the interaction of citrate synthase, a key enzyme in the TCA cycle, with different known inhibitors was studied using in silico molecular docking approach. The inhibition of citrate synthase induces the blockage of the tricarboxylic cycle (TCA), thereby increasing the concentration of acetyl-CoA that helps in enhanced PHA production. Molecular docking of citrate synthase with different inhibitors of PubChem database revealed that hesperidin (PubChem compound CID ID 10621), generally present in citrus fruits, is the most efficient inhibitor of the TCA cycle with the binding score of -11.4 and warrants experimental validation. Overall, this study provides an efficient food waste management approach by reducing the production cost and enhancing the production of PHA, thereby lessening our reliance on petroleum-based plastics.

Dataset Information

A Nitrate-Blind P. putida Strain Boosts PHA Production in a Synthetic Mixed Culture.

Publications

A Nitrate-Blind <i>P. putida</i> Strain Boosts PHA Production in a Synthetic Mixed Culture.

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