Iterative cycle of widely targeted metabolic profiling for the improvement of 1-butanol titer and productivity in Synechococcus elongatus

Abstract

BackgroundMetabolomics is the comprehensive study of metabolites that can demonstrate the downstream effects of gene and protein regulation, arguably representing the closest correlation with phenotypic features. Hence, metabolomics-driven approach offers an effective way to facilitate strain improvement. Previously, targeted metabolomics on the 1-butanol-producing cyanobacterial strain Synechococcus elongatus BUOHSE has revealed the reduction step from butanoyl-CoA to butanal, catalyzed by CoA-acylating propionaldehyde dehydrogenase (PduP), as a rate-limiting step in the CoA-dependent pathway. Moreover, an increase in acetyl-CoA synthesis rate was also observed in this strain, by which the increased rate of release of CoA from butanoyl-CoA was used to enhance formation of acetyl-CoA to feed into the pathway.ResultsIn the present study, a new strain (DC7) with an improved activity of PduP enzyme, was constructed using BUOHSE as the background strain. DC7 showed a 33% increase in 1-butanol production compared to BUOHSE. For a deeper understanding of the metabolic state of DC7, widely targeted metabolomics approach using ion-pair reversed-phase LC/MS was performed. Results showed a decreased level of butanoyl-CoA and an increased level of acetyl-CoA in DC7 compared to BUOHSE. This served as an indication that the previous bottleneck has been solved and free CoA regeneration increased upon the improvement of the PduP enzyme. In order to utilize the enhanced levels of acetyl-CoA in DC7 for 1-butanol production, overexpression of acetyl-CoA carboxylase (ACCase) in DC7 was performed by inserting the gene encoding an ACCase subunit from Yarrowia lipolytica into the aldA site. The resulting strain, named DC11, was able to reach a production titer of 418.7 mg/L in 6 days, compared to DC7 that approached a similar titer in 12 days. A maximum productivity of 117 mg/L/day was achieved between days 4 and 5 in DC11.ConclusionsIn this study, the iterative cycle of genetic modification based on insights from metabolomics successfully resulted in the highest reported 1-butanol productivity for engineered Synechococcus elongatus PCC 7942.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1187-8) contains supplementary material, which is available to authorized users.

Advances in the Direct Production of Biofuels via Photosynthesis

“…The engineered cultures were cultivated for a maximum of 30 days, because the cumulative 1-butanol titers had started to drop at day 28, while the cell density started to decrease already from day 12. In this long-term experiment, the authors report a maximum 1-butanol yield of 4.8 g L À1 , which is greater than 10 times higher than the previous record of 0.418 g L À1 , obtained after 6 days of cultivation with engineered Synechococcus elongatus PCC 7942, 6 another model cyanobacterium. Compared to heterotrophic systems, this yield is greater than 5 times higher than the highest yield of 0.835 g L À1 reported for engineered yeast cells.…”

Identification of Key Metabolites in Poly-γ-Glutamic Acid Production by Tuning γ-PGA Synthetase Expression

“…The created strains harboring newly generated promoter sequences with different strength were subjected to metabolome analysis to reveal key metabolites that are most correlated with γ-PGA synthesis. For metabolome analysis according to previous studies (Mitsunaga et al, 2016;Fathima et al, 2018), 93 metabolites were detected that are part of the central carbon metabolism including pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), and glycolysis intermediates as well as amino acids and nucleotides. The samples were taken after 8 h during exponential growth.…”

Genetic engineering of non-native hosts for 1-butanol production and its challenges: a review

“…Cyanobacteria is an excellent microbial cell factory that can utilize solar energy and convert atmospheric CO 2 to useful products. Additionally, cyanobacteria can grow rapidly, and its genetic manipulation is easy [62]. Owing to its advantages over heterotrophs, cyanobacteria were equipped with a synthetic pathway of 1-butanol, to produce sustainable and eco-friendly biofuels.…”

“…Using metabolomics strategy, Noguchi et al revealed that the reduction reaction of butanoyl-CoA to butanal catalyzed by acylating aldehyde dehydrogenase (PduP) is a possible rate-limiting step in the 1-butanol pathway in Synechococcus elongatus [105]. Therefore, by enhancing the activity of PduP and inserting the gene encoding the subunit of the ACCase from Yarrowia lipolytica into the aldA (encodes for alcohol dehydrogenase) site, S. elongatus recombinant strain DC11 was able to accumulate 418.7 mg/L of 1-butanol, which is almost 1.4-fold more than its parent strain BUOHSE after 6 days of fermentation [62].…”