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.

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Abstract. Nuryandani E, Susandarini R, Indrianto A, Nuringtyas RT, Fathima AM, Subandiyah S. 2020. Short Communication: Variations of morphology, anatomy, and metabolite profiles of Citrus reticulata Blanco cv. Tawangmangu grafts produced by shoot tip grafting using several rootstocks. Biodiversitas 21: 4671-4676.  Cultivation of Citrus reticulata Blanco cv. Tawangmangu, one of leading mandarin cultivars in Indonesia, remains challenging due to the limitations of suitable rootstocks. The use of suitable rootstocks will greatly determine the success of grafting and affect the performance of the scion. This study aimed to evaluate the morphological, anatomical, and metabolic profiles of grafts between Tawangmangu and rootstocks from various species using shoot tip grafting. Observations of morphological, anatomical, and metabolite variation were carried out eight weeks after the beginning of the growth of the scions. The morphological characters were observed on leaves and stems of the scions. Anatomical data were obtained from the examination of longitudinal sections of grafts prepared using paraffin-embedded method. Analysis of metabolite profiles was performed using LC-MS. The results showed that for two of the three rootstocks used, C. ichangensis × C. reticulata cv. Austera (Japansche Citroen (JC)) and Citrus (Fortunella) japonica (Fortunella) scion growth was vigorous and resulted in a large number of leaves and long stems. Morphological variation was found in the leaf shape, as indicated by wider and wavy leaves of grafts with Fortunella, while narrower and straighter leaves were found in grafts with JC. The grafting onto the third rootstock, C. hystrix, induced dwarfing of the scions. Variation in the anatomical profiles was found in vascular vessels, and in exodermic and endodermic cell sizes. Differences in metabolic profiles of the grafts were found compared to scion and rootstocks. Results of this study provide an important contribution to the successful improvement of Tawangmangu.