Judul: The Enhancement of Thiamine Accumulation in Escherichia coli Culture by Enhancing Oxygenation Level and Deletion of rpoS
Abstrak: Thiamine is one of the essential compounds for microorganisms in the metabolic process of their growth. Thiamine plays a role as a precursor in the pathway for thiamine diphosphate formation. Thiamine diphosphate is a coenzyme for essential enzymes involved in the metabolic pathway of Escherichia coli. Escherichia coli can synthesize thiamine by de novo. Several things that affect the synthesis of thiamine in Escherichia coli such as nutritional deficiencies, oxidative stress cause of exposure to ROS (Reactive Oxygen Species) and the process of respiration in cells to produce ATP. In stress environmental conditions, thiamine plays a protective role in maintaining metabolic processes. In addition, Escherichia coli also has the global regulator rpoS which controls regulatory and structural genes when cells are under stress conditions, including oxidative stress. Enhancing accumulation of thiamine in Escherichia coli cells can be accomplished through inducing oxygenation and deleting global regulator rpoS. The first stage in this study was investigated the effect of inducing oxygenation on thiamine accumulation in Escherichia coli BW25113 culture (parent strain) by giving rotation speed variation treatment in 150 rpm and 200 rpm shaking culture as physical oxidative stress cultivated on Luria Bertani (LB) media enriched high glucose concentration (40 g/L) for 24 hours at 37℃ . A further enhancing thiamine accumulation, confirmed by cultivation of Escherichia coli BW25113-∆rpoS (rpoS mutant) compared to the parent strain at high rotation speed at shaking culture (200 rpm). The parameters investigated were the concentration of thiamine formed and the direction of carbon central metabolism were analyzed based on dry cell weight, substrate consumption, and acetate formation. The results showed that inducing oxygenation at rotation speed at shaking culture from 150 rpm to 200 rpm could enhance thiamine accumulation in Escherichia coli BW25113 culture (parent strain) from 11.87 g/g DCW to 16.81 g/g DCW. Enhancing accumulation of thiamine is affected by oxidative stress physically provided in this case oxygen can induce thiamine synthesis and it is thought an indication of the protective role of thiamine to maintain its metabolism. However, under these conditions there is competition with the presence of rpoS which controls various types of stress conditions and active under oxidative stress. The presence of rpoS at conditions of high oxygen and high glucose causes the direction of cell metabolism tend to formation pathway of by-products, namely acetate. Thus, further confirmation of enhancing thiamine through the deleting of rpoS. The results showed that there was an enhancing thiamine accumulation in rpoS mutant compared to the parent strain by 9.5%. This can be occured because rpoS has a negative regulation of purine metabolism and the thiI gene which is a metabolic pathway and a gene that contributes to the thiamine formation pathway., Tiamin merupakan salah satu senyawa esensial bagi mikroorganisme dalam proses metabolisme pertumbuhannya. Tiamin berperan sebagai prekursor pada jalur pembentukan tiamin difosfat. Tiamin difosfat merupakan koenzim bagi enzim-enzim esensial yang terlibat pada jalur metabolisme Escherichia coli. Escherichia coli dapat mensintesis tiamin secara de novo. Beberapa hal yang mempengaruhi sintesis tiamin pada Escherichia coli di antaranya kekurangan nutrisi, stres oksidatif akibat dari paparan ROS (Reactive Oxygen Species) serta
Keyword: Thiamine accumulation, Escherichia coli, rpoS mutant, oxidative stress