2019 Artnice Mega Fathima

LC/MS/MS-based metabolic profiling for the improvement of Synechococcus elongatus 1-butanol-producing strains

Laboratory of Bioresource Engineering (Fukusaki Lab)

Artnice Mega Fathima


Chapter 1: General introduction

1-Butanol is considered an important commodity chemical and advanced biofuel. To allow 1-butanol production in a sustainable manner, recently photosynthetic organisms, such as cyanobacteria, have become attractive model microorganism for their ability to convert carbon dioxide to valuable products. Cyanobacteria, specially Synechoccous elongatus PCC 7942, generally exhibit fast growth and genetic tractability, thus making them more advantageous as cell factories over eukaryotic algae and plants. Microbial 1-butanol is natively produced via a natural metabolic CoA-dependent pathway in selected species of Clostridia. In relation to this, a cyanobacterial strain, capable of producing 1-butanol was engineered by introducing a modified Clostridial CoA-dependent pathway. Initially, Synechococcus elongatus strain EL22 was engineered using an oxygen-sensitive enzyme, CoA-acylating butanal dehydrogenase (Bldh), for conversion of butanoyl-CoA to butanal and was able to produce a low titer of 29.9 mg/L of 1-butanol. Since oxygen sensitivity of Bldh hindered 1-butanol production, it was replaced with an oxygen-tolerant enzyme, CoA-acylating propionaldehyde dehydrogenase (PduP) in the BUOHSE strain. As a result, a major increase in 1-butanol titer was observed in BUOHSE compared to EL22. However, cyanobacterial 1-butanol production is still considered to be low compared to other microbial hosts.

In recent years, metabolomics-based approaches for strain improvement are widely being employed, which enables rapid detection of important metabolites and rate-limiting steps in the production pathway. Metabolomics. a comprehensive study of metabolites, is able to demonstrate the downstream effects of gene and protein regulation, arguably representing the closest correlation with phenotypic features. Therefore, a deeper understanding of the metabolic state of Synechococcus elongatus 1-butanol producing strains offers an effective way to facilitate strain improvement. In this study LC/MS/MS-based metabolic profiling was applied, allowing for the identification of the possible target for pathway modification in the CoA-dependent pathway to ultimately improve both titer and productivity.


Chapter 2: Improvement of butanoyl-CoA to butanal reaction in 1-butanol biosynthesis pathway results in higher production of 1-butanol

Firstly, a comparative metabolome analysis of BUOHSE with the low producing strain (EL22) was carried out to identify target in 1-butanol biosynthetic pathway that required further improvement. By using ion-pair reserved phase liquid chromatography triple quadrupole mass spectrometer (IP-RP-LC/QqQ-MS) system, 74 metabolites belonging to the central metabolism were annotated. Briefly, the data suggested that the reduction reaction from butanoyl-CoA to butanal, catalyzed by PduP, was suggested as a possible target for strain improvement in the CoA-dependent pathway. Subsequently, modification based on the finding was performed in BUOHSE by replacing the original RBS upstream of pduP. This new strain, named DC7, outperformed 1-butanol titer of BUOHSE (319 mg/L) by 33% with a final titer of 426.75mg/L in 12 days after IPTG induction. To validate the result, butanoyl-CoA level and PduP enzyme activity of DC7 in comparison with BUOHSE was further investigated. Results showed a decreased level of butanoyl-CoA was realized and a 1.4 fold increase in the PduP enzyme activity was observed in DC7 compared to BUOHSE. This served as an indication that optimization of PduP activity effectively led to an improved conversion of butanoyl-CoA into butanal in DC7, which lead to the increased titer of 1-butanol.


Chapter 3: Optimization of acetyl-CoA to malonyl-CoA reaction enables high titer and productivity of 1-butanol

To gain a deeper understanding of the overall effect of the increased PduP activity in DC7 and to identify other targets for strain engineering, widely targeted metabolic profiling strategy was carried out to compare BUOHSE and DC7. Metabolome comparison of BUOHSE and DC7 has shown that acetyl-CoA was highly accumulated in the latter. Accordingly, to utilize this enhanced level of acetyl-CoA for 1-butanol formation in DC7, acetyl-CoA carboxylase (ACCase) was selected as a target for next improvement. Genetic modification involved two main strategies: disruption of aldA gene, encoding for alcohol dehydrogenase, to eliminate any unwanted consumption of acetyl-CoA and introduction of accase gene from Yarrowia lipolytica into the disruption site. This engineering strategy resulted in a strain, named DC11, which 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. In addition, a maximum 1-butanol titer per day of 117 mg/L per day between days 4 and 5, outperforming the first reported high 1-butanol producing strain BUOHSE by 57% (74.5 mg/L per day). Taken together all these data suggested that the iterative cycle of genetic modification based on insights from metabolomics successfully resulted in the highest reported 1-butanol titer and productivity for engineered Synechococcus elongatus PCC 7942.


Chapter 4: Conclusions and future perspectives

Metabolomics-assisted strain improvement employed in the Chapter 2 and Chapter 3 demonstrated the utility of metabolomics to effectively find targets for improvement in a biosynthesis pathway. The resulting strain developed in this study shows promise for future application in photosynthetic microbial based-1-butanol production. Lastly, in order to make cyanobacterial 1-butanol production system feasible in the future, a more comprehensive metabolome analysis, integration with other omics technologies, and optimization of the downstream process will may help to further design advanced cyanobacterial production system in the future.


List of publication:

Mega Fathima A, Chuang D, Laviña WA, Liao J, Putri SP, Fukusaki E. Iterative cycle of widely targeted metabolic profiling for the improvement of 1-butanol titer and productivity in Synechococcus elongatus. Biotechnol Biofuels. 2018;11:188. doi:10.1186/s13068-018-1187-8.