Synthetic Promoter for Expressing Corynebacteria

본 발명은 코리네박테리아 발현용 합성프로모터에 관한 것으로, 더욱 자세하게는 코리네박테리아에서 높은 발현효율을 나타낼 수 있는 합성프로모터의 스크리닝 방법 및 상기 방법으로 제조된 코리네박테리아 발현용 합성프로모터에 관한 것이다.본 발명의 프로모터를 사용하면, 기존의 코리네박테리움 속 균주에서 재조합 단백질 대량 생산에 사용되던 프로모터들에 비하여 훨씬 강한 유전자 발현을 나타내어, 코리네박테리움 속에서 생산되는 재조합 단백질을 보다 높은 수율로 생산할 수 있다

헤미셀룰로오스를 탄소원으로 이용하는 코리네박테리움 글루타미쿰 균주의 개량

학위논문(석사) - 한국과학기술원 : 생명화학공학과, 2013.2, [ v, 57 p. ]Xylan is a major component of hemicellulose which is considered to be the second most abundant natural polymers in nature and it is regarded as one of the most important renewable resources. By enzymatic reaction, xylan can be degraded into xylose, its main building block, and then the resulting sugar (xylose) can be used as a major carbon source in the growth of microorganisms and further production of useful things. In this study, I engi-neered Corynenacterium glutamicum for the direct utilization of xylan as a carbon source. C. glutamicum is a Gram positive, non-sporulating bacteria and is also one of the most important bacteria in the industrial production of various chemicals and biomolecules including amino acids, lactate, and recombinant proteins. Overall scheme of this research was two-step process comprising (i) enzymatic hydrolysis of hemicellulose and (ii) xylose utilization as a carbon source by introducing heterologous xylose metabolic pathway. For the hydrolysis of xylan, two heterologous enzymes (endoxylanase and xylosidase) were expressed in C. glutamicum. The xylan can be decomposed into xylobiose or xylotriose by secreted endoxy-lanase in the media and then further hydrolyzed into xylose in the cytoplasm of cells. Due to the lack of xylose metabolic pathway, wild type of C. glutamicum cannot utilize the xylose as a carbon source. Next, to give the ability of xylose metabolism, heterologous cytoplasmic expression of the xylose isomerase (xylA) and xylulokinase (xylB) genes were introduced in C. glutamicum, mediating the conversion of xylose into xylulose 5-phosphate (an intermediate of the pentose phosphate pathway). As a result, xylose metabolism could be successfully introduced and I confirmed the cell growth by utilization of xylose as a carbon source in minimal media. Finally, two steps (xylan hydrolysis and xylose metabolism) were successfully combined as a whole cell system in which xylan can be directly utilized for growth of C. glutamicum.한국과학기술원 : 생명화학공학과

Engineering outer membrane anchoring motifs of Escherichi coli OmpX for bacterial surface display systems

Bacterial cell surface display is one of the most powerful tools which have various applications including protein engineering, whole cell biocatalist, biotherapy with antibody or vaccine, biosensor etc. In the bacterial cell surface display system, it is the most important step to choose the appropriate anchoring motif which is linked to target molecules. To discover new anchoring motif which allows more stable and efficient protein display on bacterial cell surface, we first analyzed the outer membrane proteome of E. coli. Based on proteome analysis, we chose outer membrane protein X (OmpX) which has a small (16.3 kDa), monomeric and β-barrel structure with high expression level. The usefulness of OmpX as an anchoring motif for cell surface was demonstrated with two important industrial enzymes, lipase and endoxylanase. By fusion with truncated OmpX, both lipase and endoxylanase showed the efficient display and also showed higher activities than those in free-state. These results revealed that use of OmpX as an anchoring motif provide an efficient way for displaying functional enzymes on the surface of E. coli host and can be used for many other applications of E. coli cell surface display such as high throughput screening method in development of antibody