Dataset Information

Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered Synechococcus elongatus.

ABSTRACT:

Background

Cyanobacterial 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.

Results

Growth 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.

Conclusions

Removal 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.

SUBMITTER: Kato A

PROVIDER: S-EPMC5452621 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered <i>Synechococcus elongatus</i>.

Kato Akihiro A Takatani Nobuyuki N Ikeda Kazutaka K Maeda Shin-Ichi SI Omata Tatsuo T

Biotechnology for biofuels 20170531

<h4>Background</h4>Cyanobacterial 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 <i>Synechococcus elongatus</i> 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 d ...[more]

PMID: 28580015

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Project description:The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.

Project description:BackgroundConverting carbon dioxide (CO2) into value-added chemicals using engineered cyanobacteria is a promising strategy to tackle the global warming and energy shortage issues. However, most cyanobacteria are autotrophic and use CO2 as a sole carbon source, which makes it hard to compete with heterotrophic hosts in either growth or productivity. One strategy to overcome this bottleneck is to introduce sugar utilization pathways to enable photomixotrophic growth with CO2 and sugar (e.g., glucose and xylose). Advances in engineering mixotrophic cyanobacteria have been obtained, while a systematic interrogation of these engineered strains is missing. This work aimed to fill the gap at omics level.ResultsWe first constructed two engineered Synechococcus elongatus YQ2-gal and YQ3-xyl capable of utilizing glucose and xylose, respectively. To investigate the metabolic mechanism, transcriptomic and metabolomic analysis were then performed in the engineered photomixotrophic strains YQ2-gal and YQ3-xyl. Transcriptome and metabolome of wild-type S. elongatus were set as baselines. Increased abundance of metabolites in glycolysis or pentose phosphate pathway indicated that efficient sugar utilization significantly enhanced carbon flux in S. elongatus as expected. However, carbon flux was redirected in strain YQ2-gal as more flowed into fatty acids biosynthesis but less into amino acids. In strain YQ3-xyl, more carbon flux was directed into synthesis of sucrose, glucosamine and acetaldehyde, while less into fatty acids and amino acids. Moreover, photosynthesis and bicarbonate transport could be affected by upregulated genes, while nitrogen transport and assimilation were regulated by less transcript abundance of related genes in strain YQ3-xyl with utilization of xylose.ConclusionsOur work identified metabolic mechanism in engineered S. elongatus during photomixotrophic growth, where regulations of fatty acids metabolism, photosynthesis, bicarbonate transport, nitrogen assimilation and transport are dependent on different sugar utilization. Since photomixotrophic cyanobacteria is regarded as a promising cell factory for bioproduction, this comprehensive understanding of metabolic mechanism of engineered S. elongatus during photomixotrophic growth would shed light on the engineering of more efficient and controllable bioproduction systems based on this potential chassis.