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

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. 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.Here, 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.These 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.

SUBMITTER: Lowe H

PROVIDER: S-EPMC5517840 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description: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.

Project description:BackgroundCyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required.ResultsGrowth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L-1 of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L-1 h-1 was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L-1 of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems.ConclusionsRemoval of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.

Project description:The demand for protein is increasing with an expanding world population and is influencing the rapid growth of fish and animal agriculture. These sectors are becoming a significant source of water pollution and need to develop environmentally sustainable techniques that are cost-effective, ideally with potential for downstream value-added production. This study investigated the potential of one of the fastest-growing cyanobacterial species, Synechococcus elongatus UTEX 2973, for bioremediation of mixed wastewater (combination of sturgeon and swine wastewater). Three different mixing ratios (25:75, 50:50, and 75:25 sturgeon:swine) were compared to find a suitable combination for the growth of S. elongatus as well as carbohydrate accumulation in biomass. The final biomass production was found to be 0.65 ± 0.03 g Dry cell Weight (DW)/L for 75%-25 %, 0.90 ± 0.004 g DW/L for 50%-50 %, and 0.71 ± 0.04 g DW/L for 25%-75 % sturgeon-swine wastewater combination. Cyanobacteria cultivated in 50%-50 % sturgeon-swine wastewater also accumulated 70 % total carbohydrate of DW, whereas 75%-25 % sturgeon-swine and 25%-75 % sturgeon-swine accumulated 53 % and 45 %, respectively. Subsequently, the S. elongatus cells were grown in a separate batch of 50%-50 % sturgeon-swine wastewater and compared with cells grown in BG11 synthetic growth media. Cultivation in BG11 resulted in higher biomass production but lower carbohydrate accumulation than 50%-50 % mixed wastewater. Final biomass production was 0.85 ± 0.08 g DW/L for BG11 and 0.65 ± 0.04 g DW/L for 50%-50 % sturgeon-swine wastewater. Total carbohydrate accumulated was 75 % and 64 % of DW for 50%-50 % sturgeon-swine mixed wastewater and BG11 growth media, respectively, where glycogen was the main carbohydrate component (90 %). The nutrient removal efficiencies of S. elongatus were 67.15 % for orthophosphate, 93.39 % for nitrate-nitrite, and 97.98 % for ammonia. This study suggested that S. elongatus is a promising candidate for enabling simultaneous bioremediation of mixed wastewater and the production of value-added biochemicals.

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

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