Methods for cloning key primary metabolic enzymes and ancillary proteins associated with the acetone-butanol fermentation of Clostridium acetobutylicum.

The unavailability of genetically defined mutants for complementation has intensified the problems inherent in cloning genes from C. acetobutylicum. The uniqueness of some of the pathways of this organism coupled with the relative inefficiency of transformation of clostridia and few characterized mutants in these pathways have made cloning these genes by traditional complementation methods impractical. Oligonucleotide hybridization techniques have been shown to circumvent many problems involved in detecting protein expression. The ease of hybridization screening of plaques allows phage libraries to be examined more readily than is generally the case with colony screening techniques. Recombinant lambda phages also contain more DNA per insert than most plasmid vectors can maintain, thus further decreasing the amount of screening necessary. Cosmid libraries, offering even greater length of individual inserts, can be screened in a similar manner, although such screening incorporates the limitations of colony screening techniques. It is true that the technique hinges on the ability to obtain an amino acid sequence from which an oligonucleotide can be designed. In the past, the ability to obtain sequences was limited because the quantity and number of purified proteins were limited or the proteins were amino-terminally blocked. However, recent technological advances in this area, such as high-resolution gel separation techniques coupled with microsequencing, have opened the door to proteins previously inaccessible. Deformylation methods have been developed to deblock amino-terminally formylated proteins, and successful internal amino acid sequence analysis by in situ protease digestion has also been reported using only picomolar quantities of proteins separated by one- or two-dimensional gel electrophoresis. Protein and DNA sequence data banks have been significantly upgraded in the past few years. A proposed oligonucleotide sequence can be evaluated to determine what other possible sequences have similar homology; moreover, protein similarity comparisons between related species might possibly supplant the need for protein isolation if regions of highly conserved amino acid sequences are found. To our knowledge, this represents the first reported use of oligonucleotide probe hybridization screening technology as a strategy for cloning solvent pathway genes of C. acetobutylicum. Despite the deleterious effects on hybridization inherent in the high A + T content of C. acetobutylicum gene specific-directed oligonucleotides, the technique has been shown to function with few modifications to previously recorded systems.