探討細菌血紅蛋白對Serratia marcescens C3生產天然抗癌藥物-靈菌紅素之影響

A study on the production of prodigiosin using Vitreoscilla hemoglobin by Serratia marcescens C3

林吟臻(Yin-Chen Lin)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2009年)

摘要

靈菌紅素（Prodigiosin）是由 Serratia marcescens 及其他革蘭氏陰性菌所代謝之紅色色素(red pigment)，其中以S. marcescens為靈菌紅素主要生產菌株。近年來已有多篇文獻證實，靈菌紅素具有抗真菌、免疫抑制及拮抗腫瘤細胞增生之能力，並能誘導特定腫瘤細胞進行細胞凋亡 (apoptosis)。基於前述特質，靈菌紅素在臨床醫療應用上，可作為一抗癌藥物。S. marcescens代謝靈菌紅素為一極端好氧之發酵過程，因此優越之氧氣傳送效率，將有助於靈菌紅素的生產。本研究主要目的為將具有協助菌體於貧氧環境中有效利用氧氣之細菌血紅蛋白基因(Vitreoscilla hemoglobin gene, vgb)送進S. marcescens C3中，以克服靈菌紅素發酵後期溶氧不足的問題。首先，分別建構帶有vgb之持續性表現質體(pTC3)與誘導式質體(pU5K)，以電穿孔(Electroporation)的方式將其轉型至菌體中，之後以Kanamycin 篩選轉型成功的菌株。藉由西方墨點法及一氧化碳光譜差分析，確認S. marcescens(pTC3和pU5K)成功表現細菌血紅蛋白(Vitreoscilla hemoglobin,VHb)；接著以本實驗室所開發的最適化培養基SMP培養轉型株(CT)，所得靈菌紅素產量為11.01 g/L，與控制組(CB)之產能(11.37 g/L)相差無幾，推測可能與VHb之濃度不足有關，故針對可誘導之轉型株(U5K)進行測試，藉由添加IPTG來獲得較高的VHb濃度，結果所得靈菌紅素產量為15.80 g/L，高於控制組11.96 g/L，顯示VHb可提升菌體代謝靈菌紅素的能力。

關鍵字

靈菌紅素,細菌血紅蛋白,Serratia marcescens C3

並列摘要

Prodigiosin is a red pigment, which produced by Serratia marcescens and other gram-native bacteria. Its well known for anti-fungal, immunosuppressive and anti-cancer activities, besides, has possession of potential for applications in pharmaceutical industrial. Some literatures show that the prodigiosin production is limited under the condition of poor oxygen. Hence, this study will focus on construction one mutant carrying Vitreoscilla hemoglobin gene (vgb) to overcome the question of oxygen uptake. Many studies show that vgb can assist host with catching oxygen in poor environment. First, we construct two plasmids pTC3 (constitutive) and pU5K (inducible), and transfer them into S. marcescens C3 by electroporation. Then, we demonstrated Vitreoscilla hemoglobin (VHb) is successful expression in our host by western blotting and carbon monoxide difference spectra analysis. Finally, we have tested the ability for the production of prodigiosin using the mutant strain. In S. marcescens(pTC3), the concentration of prodigiosin is 11.01 g/L, lower than control strain(11.37 g/L). From these results, the VHb seem to not promote the prodigiosin production. We presume that concentration of VHb is insufficient, so we test the other one, S. marcescens(pU5K). By add 1mM IPTG in medium to produce more VHb. The concentration of prodigiosin is 15.80 g/L, higher than control strain(11.96 g/L). This result indicate that VHb could promote the prodigiosin production by the mutant strain S. marcescens pU5K .

並列關鍵字

Prodigiosin, Serratia marcescens C3, Vitreoscilla Hemoglobin Gene

參考文獻

Wakabayashi, S., H. Matsubara, and D. A. Webster. 1986. Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature 322:481-483.

Wei, M. L., D. A. Webster, and B. C. Stark. 1998. Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size. Biotechnol. Bioeng. 57:477-483.

Wei, M. L., D. A. Webster, and B. C. Stark. 1998. Metabolic engineering of Serratia marcescens with the bacterial hemoglobin gene: alterations in fermentation pathways. Biotechnol. Bioeng. 59:640-646.