Development of a system for genetic exchange and studies of genome structure and fermentation pathways in Clostridium papyrosolvens C7

• Main Author: He, Jiancai
• Language: ENG
• Published: ScholarWorks@UMass Amherst 1999
• Subjects: Microbiology|Genetics|Food science
• Online Access: https://scholarworks.umass.edu/dissertations/AAI9932320

Mesophilic cellulolytic Clostridium papyrosolvens C7 potentially could be used by industry for ethanol production from cellulose fermentation. The first goal of this study was to determine the pathways utilized by C. papyrosolvens C7 for the fermentation of cellulose and cellobiose, the soluble disaccharide product of cellulose hydrolysis. High-performance-liquid chromatography and gas chromatography analyses showed that acetate, ethanol, formate, lactate, malate, CO2, and H 2 were the end products of cellobiose fermentation by C. papyrosolvens C7. These products were quantified and a fermentation balance was calculated. Based on these analyses and the results of enzyme assays, a biochemical model for the fermentation pathways of C. papyrosolvens C7 is presented. It is suggested that formation of malate, a very uncommon fermentation product, served as an electron consuming reaction used to regenerate electron carriers (e.g., NAD+) during cell growth. In order to facilitate the metabolic engineering of C. papyrosolvens C7 for increased ethanol production and other properties desirable for industrial applications, a genetic exchange system involving the conjugative transposon Tn916 was developed. Tn916 was transferred from Enterococcus faecalis to C. papyrosolvens C7 in filter mating experiments. The tetM marker, conferring resistance to tetracycline, was used in the primary selection of transconjugants. Tn916 transfer frequencies ranged from 10 −7 to 10−5 per recipient. Highest transfer frequencies were obtained when recipient cells received a heat shock treatment, and both the donor and recipient cells were in the logarithmic phase of growth. A tetM gene probe hybridized to chromosomal DNA extracted from randomly selected C. papyrosolvens C7 transconjugants. These experiments indicated that Tn916 inserted into different sites on the chromosome of C. papyrosolvens C7. Tn 916 transposon mutagenesis was used to select low-acid-producing mutants. Fourteen mutant strains were selected by using the proton suicide method. Most of the mutant strains selected showed higher ethanol production. The third goal of this project was to determine the genome size of C. papyrosolvens C7. The restriction enzyme NotI was used to cleave intact C. papyrosolvens C7 DNA in agarose plugs. The resulting five DNA fragments were resolved by using pulse-field gel electrophoresis. The minimum size of the C. papyrosolvens C7 genome was determined to be 4.88 Mb.