Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.     

Metabolic pathway optimization for biosynthesis of 1,2,4-butanetriol from xylose by engineered Escherichia coli

1,2,4-Butanetriol (BT) and related derivatives have been widely used in many fields, especially in the military and in medicine. In this paper, we systematically optimized the BT biosynthetic pathway. We first investigated the activities of various NADH dependent aldehyde reductases (ALRs), which catalyze the fourth reaction in the four-step pathway for BT production from xylose in E. coli, and found that a combination of multiple endogenous enzymes catalyzed aldehyde reduction in the BT production bioprocess and that YqhD in E. coli was a main ALR for BT production. In addition, ADH2 from Saccharomyces cerevisiae can effectively catalyze 3,4-dihydroxybutanal to BT. Also, YjhG was identified as the major xylonate dehydratase and was co-overexpressed with YqhD, resulting in an improvement of BT production by 30%. Moreover, we identified and eliminated the competing branch pathway by inactivating 2-keto acid reductases (yiaE). Finally, the combination of these approaches led to BT production of 5.1 g/L. In summary, our study provides insights into the biosynthetic pathway for BT production, demonstrates an effective strategy to enhance BT production, and paves the way toward in-depth research on BT biosynthesis.     

Artificial Electron Carriers for Preparative Biocatalytic Redox Reactions Forming Reversibly Carbon Hydrogen Bonds with Enzymes Present in Strict or Facultative Anaerobes

The application of various artificial electron mediators in combination with redox enzymes present in anaerobically grown cells is described. Examples include viologens of different redox potential, cobalt complexes, anthraquinones, phenoxazines, phenazines and others. The regeneration of the reduced or oxidised mediators by various methods is discussed, with hydrogen gas, carbon monoxide, formate or a cathode as electron donors, and dimethylsulphoxide, quinones, oxygen or an anode as electron acceptors. The enzymes used, usually in the form of resting cells or crude cell extracts of clostridia or Proteus species are mostly reversible. Examples for preparative reductions as well as for dehydrogenations are given.