An Escherichia coli system was engineered for the heterologous production of itaconic acid via the expression of cis-aconitate decarboxylase gene (cad), and then maximal itaconic acid levels produced by engineered E. coli were evaluated. Expression of cad in E. coli grown in Luria-Bertani (LB) medium without glucose in a test tube resulted in 0.07 g/L itaconic acid production after 78 h at 20 C. To increase itaconic acid production, E. coli recombinants were constructed by inactivating the isocitrate dehydrogenase gene (icd) and/or the isocitrate lyase gene (aceA). Expression of cad and inactivation of icd resulted in 0.35 g/L itaconic acid production after 78 h, whereas aceA inactivation had no effect on itaconic acid production. The intracellular itaconate concentration in the icd strain was higher than that in the cad-expressing strain without icd inactivation, which suggests that the extracellular secretion of itaconate in E. coli is the rate-determining step during itaconic acid production. pH-stat cultivation using the cad-expressing icd strain in LB medium with 3% glucose in a jar fermenter resulted in 1.71 g/L itaconic acid production after 97 h at 28 C. To further increase itaconic acid production, the aconitase B gene (acnB) was overexpressed in the cad-expressing icd strain. Simultaneous overexpression of acnB with the expression of cad in the icd strain led to 4.34 g/L itaconic acid production after 105 h. Our findings indicate that icd inactivation and acnB overexpression considerably enhance itaconic acid production in cad-expressing E. coli.Key words: aconitase; cis-aconitate decarboxylase; isocitrate dehydrogenase; isocitrate lyase; itaconic acid; metabolic engineering IntroductionOver the past several decades, there has been substantial interest in itaconic acid as a dicarboxyvinyl monomer produced by microbial fermentation of biomass (Kobayashi and Nakamura, 1964;Okabe et al., 2009;Willke and Vorlop, 2001). Owing to its useful properties, itaconic acid is used in the manufacturing of synthetic polymers, such as plastics and resins (Milson and Meers, 1985;Tate, 1981). Graft polymers with an itaconic acid-based main chain and oligolactate side chain have been synthesized and characterized in our laboratory Okada et al., 2012). The polymers consisting of itaconic acid had high biomass content and are therefore thought to contribute to carbon emission reduction.Itaconic acid is industrially produced from sugars, such as glucose, by the fungus Aspergillus terreus via submerged fermentation (Bonnarme et al., 1995). To date, many re searchers have reported increased productivity of a native itaconic acid producer, A. terreus, by using techniques associated with mutation breeding (Yahiro et al., 1995) and fermentation conditions (Okabe et al., 1993;Park et al., 1994;Riscaldati et al., 2000;Träger et al., 1989;Yahiro et al., 1997). Specifically, the spontaneous mutant A. terreus TN-484 is used as a target microorganism for optimizing the fermentation process. In a previous study, a typical yield of 82....

Highlights d We examine the state changes of hESCs by overexpressing 714 transgenes individually d Nearly all genes, including heterochromatin genes, are perturbed by these transgenes d Transcription factors are grouped for hESC differentiation into specific lineages

Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 ( FANCD2 ) and RAD51 recombinase ( RAD51 ) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies.