Construction of simultaneous saccharification and fermentation escherichia coli mutant

• Main Authors: Chun-chi,Tsai, 蔡棕麒
• Other Authors: Yuan-Tay Shyu
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/29130840386638234852

碩士 === 國立臺灣大學 === 園藝學研究所 === 84 === By the use of homologous recombinant technique, we successfully integrated the genes of the ethanol fermentation key enzymes into Escherichia coli chromosome. Integration improved the stability of the genes expression. An a-amylase expression plasmid was also constructed to achieve the goal of simultaneous saccharification and fermentation. The DNA fragment, pfl, for homology to the chromosome gene was obtained from 15224 by PCR. With the pfl fragment flanked , the ethanol fermentation genes, pdc and adhII , from pSSE217 were cloned into the PvuII site of the temperature sensitive plasmid, pSC101. The integrative vector (pST415) was transformed to 15224. By the use of selective medium, temperature control and the formation of red colonies on the aldehyde indicator plate, we got 5 transformants, SE004:1~5. We furtheridentified by PCR that SE004:2 was the strain with pdc and adhII genes integrated into the chromosome within the pfl gene. An a-amylase gene obtained from YEpSSA14 was cloned into PCRII vector. The a-amylase expression plasmid (pST617) was transformed to SE004:2. Transformants were stained by iodine vapor on the LB+2% starch plate. The halo forming colonies, SE004(pst617):1-25, indicated the expression of the a-amylase. In 30℃, 100rpm fermentation condition, the ethanol production of strain 15224(pSSE217),SE004:2, SE004 (pST617):5 were 6.1%、4.2%、3.5% in 10% glucose + LB broth and 1.7%、1.3%、 2.7% in 5% starch + LB broth, respectively. Among these, acid production and hydrolysis of macro-carbohydrate molecules were the growth limitation factors, respectively.