In vitro biosynthesis of 3-hydroxypropionic acid from glucose

• Main Authors: LIOU,TING-YIN, 劉庭穎
• Other Authors: CHENG,YU-SHEN
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/u5hpr6

碩士 === 國立雲林科技大學 === 化學工程與材料工程系 === 105 === Value-added chemicals and fuels derived from renewable biomass have gained considerable attentions in the past decades because of the environmental problems caused by over exploitation of fossil resources. 3-Hydroxypropionic acid (3-HP), suggested by U.S Department of Energy, is one of the top twelve value-added planform chemicals from biomass that has great potential to derive a wide range of commodity chemicals. Considerable research effort has been put in the microbial fermentation-based processes for 3-HP production, because fermentation has been utilized by humankind's for thousands of years and the technological knowledge of the processes is well developed. However, there are also constraints in the production of non-natural products such as 3-HP through traditional fermentation of native microorganisms. In vitro biosynthesis, also call in vitro metabolic engineering, has emerged as a potential platform for bioproductions without worrying the limits associated with living cells. In this study, a novel pathway for in vitro biosynthesis of 3-HP was designed and constructed by assembling ten enzymes. This innovative pathway is vitamin B12 independent and designed to have the balances between the consumption and generation of ATP/ADP and NAD+/NADH. The expression plasmids of required enzymes were obtained from other research institutes or by self-construction and the enzymes were produced by using E. coli expression system. The expressed enzymes were purified before using or applied directly as crude cell lysate. The 3-HP production was conducted in batch systems with designated concentration of proteins or cell lysate. The yields of 3-hydroxypropionic acid were 75.6 ± 4.6% and 24.7 ± 0.3% respectively in purified enzyme batch and crude cell lysate batch. Although the experimental yields were still less than therotical value (100%), especially in the crude cell lysate batch. The new design pathway is capable to generate 3-HP from glucose; with further optimizations, the developed process can be useful for 3-HP production.