Iterative Cycle of Widely Targeted Metabolic Profiling for The

Fathima et al. Biotechnol Biofuels (2018) 11:188 https://doi.org/10.1186/s13068-018-1187-8 Biotechnology for Biofuels RESEARCH Open Access Iterative cycle of widely targeted metabolic profling for the improvement of 1‑butanol titer and productivity in Synechococcus elongatus Artnice Mega Fathima1, Derrick Chuang2, Walter Alvarez Laviña1,3, James Liao2, Sastia Prama Putri1* and Eiichiro Fukusaki1 Abstract Background: Metabolomics is the comprehensive study of metabolites that can demonstrate the downstream efects of gene and protein regulation, arguably representing the closest correlation with phenotypic features. Hence, metabolomics-driven approach ofers an efective 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 forma- tion of acetyl-CoA to feed into the pathway. Results: In 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. Conclusions: In this study, the iterative cycle of genetic modifcation based on insights from metabolomics successfully resulted in the highest reported 1-butanol productivity for engineered Synechococcus elongatus PCC 7942. Keywords: Cyanobacteria, Butanol, Widely targeted metabolomics, Rate-limiting step, Strain improvement *Correspondence: [email protected]‑u.ac.jp 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2‑1 Yamadaoka, Suita, Osaka 565‑0871, Japan Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fathima et al. Biotechnol Biofuels (2018) 11:188 Page 2 of 12 Background BUOHSE relative to EL22 due to a more efective conver- 1-Butanol is considered an important commodity chemi- sion of butanoyl-CoA to butanal by the PduP enzyme. cal and advanced biofuel [1]; thus, the introduction of a Surprisingly, the metabolome data indicated that the CoA-dependent pathway to produce 1-butanol in various amount of butanoyl-CoA was comparable to that of host organisms such as Escherichia coli [2, 3], Saccharo- EL22. Further analysis using kinetic profling showed myces cerevisiae [4], Pseudomonas putida, Lactobacillus that the release of CoA resulting from the introduction brevis, Bacillus subtilis [5, 6] and Cyanobacterium Syn- of PduP in BUOHSE enabled free CoA regeneration that echococcus elongatus [7] has become a common strategy in turn led to the increased rate of acetyl-CoA synthe- for microbial production. In addition, direct conversion sis. Here, the release of free CoA facilitated the reaction of CO2 to produce valuable products using photosyn- from pyruvate to acetyl-CoA, which was then used in the thetic organisms has garnered signifcant interest for butanol pathway [18]. Tese facts strongly indicate that its prospective role in attaining a carbon neutral society the reductive reaction of butanoyl-CoA to butanal should [8–13]. Cyanobacteria, photosynthetic microorganisms, be modifed further to improve 1-butanol productivity generally exhibit fast growth and genetic tractability, in the engineered cyanobacterial strain. In this work, we thus making them more advantageous as cell factories worked on further optimizing the PduP enzyme to facili- over eukaryotic algae and plants [14]. In relation to this, tate a more efective conversion from butanoyl-CoA to a cyanobacterial strain capable of producing 1-butanol butanal. In addition, since the previous analysis was only was engineered by introducing a modifed Clostridial limited to the CoA-dependent pathway, there is a pos- CoA-dependent pathway [15, 16]. Initially, Synechococcus sibility that other rate-limiting steps are present within elongatus strain EL22 was engineered using an oxygen- the 1-butanol-producing strain. Terefore, in the present sensitive enzyme, CoA-acylating butanal dehydrogenase study, widely targeted metabolic profling was used to (Bldh), for conversion of butanoyl-CoA to butanal and broaden the coverage of the metabolites analyzed, allow- was able to produce a low titer of 29.9 mg/L of 1-butanol ing for the identifcation of new rate-limiting steps in the [15]. Since oxygen sensitivity of Bldh hindered 1-butanol 1-butanol biosynthesis pathway to ultimately improve production, it was replaced with an oxygen-tolerant both titer and productivity. enzyme (PduP) in the BUOHSE strain, which resulted in a signifcant increase in 1-butanol production [16]. Results and discussion In recent years, metabolomics-based approaches for Comparative metabolite analysis of EL22 and BUOHSE strain improvement are widely being employed. Tis identifed rate‑limiting steps in 1‑butanol biosynthesis rapidly growing feld, focusing on the whole metabolite In this work, relative quantifcation approach for metabo- profle of a biological system, provides valuable informa- lomics was chosen over absolute quantifcation. It uses tion that can be applied in numerous ways [3, 17–20]. an internal standard to normalize the metabolite sig- By means of rapid detection of relevant metabolic per- nal intensity. Since this procedure is experimentally turbations, metabolomics can identify specifc targets less complicated and laborious, it allows for simultane- for strain improvement that may include identifcation ous profling of a larger number of metabolites as com- of rate limiting steps in a production pathway, metabo- pared to absolute quantifcation, thereby broadening the lite or product toxicity, cofactor imbalances, or deple- metabolite coverage [21, 22]. By using ion-pair reserved- tion of metabolites consumed by alternative pathways phase liquid chromatography triple quadrupole mass [3]. Terefore, a deeper understanding of the metabolic spectrometer (IP-RP-LC/QqQ-MS) system, 74 metabo- state of 1-butanol-producing strains can facilitate strain lites (Additional fle 1: Table S3) belonging to the central improvement by further strain modifcations. metabolism of Synechococcus elongatus strains EL22 and Previously, a targeted metabolomics analysis focusing BUOHSE were successfully annotated. on the CoA-dependent pathway in 1-butanol produc- In order to confrm that the previous rate-limiting step ing S. elongatus strain was performed by our previous can also be detected by using widely targeted analysis, research group [18]. In this published work, strains that the data were initially compared to the absolute quan- difer in enzymes that convert butanoyl-CoA to butanal tifcation data from previously published results [18]. were compared. Te introduction of an oxygen tolerant As mentioned previously, quantitative target analysis of (PduP) enzyme to replace the oxygen sensitive (BldH) acyl-CoAs suggested the accumulation of butanoyl-CoA enzyme was intended to increase the activity of this in BUOHSE as the rate-limiting step in 1-butanol biosyn- reaction in strain BUOHSE from base strain EL22. As a thesis. Tus, the relative concentration of butanoyl-CoA result, a major increase in 1-butanol titer was observed in in BUOHSE in comparison with EL22 was investigated. BUOHSE compared to EL22. Consequently, we expected As shown in Fig. 1a, our results also showed compa- to observe a decrease in the amount of butanoyl-CoA in rable levels of butanoyl-CoA in EL22 and BUOHSE, Fathima et al. Biotechnol Biofuels (2018) 11:188 Page 3 of 12 Fig. 1 a Relative intensity of intracellular butanoyl-CoA measurements from widely targeted metabolic profling of EL22 and BUOHSE. The y-axis represents the relative intensity of the metabolites, which was normalized to an internal standard. b Absolute intracellular concentration of butanoyl-CoA [pmol/mg-dry

Load more