Characterizing a benzoic acid inducible promoter system from the fungus Aspergillus niger
Abstract
Functional studies of proteins in plants can be significantly facilitated by controlling the timing of expression of their genes. In addition, commercial transgenic crops could also benefit from a controlled expression of transferred genes. This control can be accomplished through the use of chemically inducible promoter systems. Currently available systems have many disadvantages, the main one being the high toxicity of the inducer compounds. Therefore, there is a need for chemically inducible promoters that utilize more suitable inducers that are non-toxic and inexpensive, to allow this technology to be transferred to commercial crops. In this context, the bphA gene from the fungus Aspergillus niger has the potential to provide all the necessary components for the engineering of such a system. This gene encodes the enzyme benzoate-para-hydroxylase (BPH), and its expression is tightly regulated by benzoic acid. Expression of bphA is rapidly upregulated when benzoic acid is added to the media. Concentrations as low as 0.8 mM of benzoic acid are able to induce expression of the bphA gene. Other aromatic compounds with structure similar to benzoic acid also act as inducers of this gene. However, the components of the regulatory mechanism that determines bphA expression are not known. The present work presents evidence that a 200 by region in the promoter of the bphA gene is necessary for benzoic acid inducibility of this promoter. Additionally, a 51-bp fragment contained within this region shows consistent and specific binding by factors present in a total protein extract from A. niger cells. This fragment contains a pair of direct hexameric repeats, with the sequence 5′ -TAGTCA-3′, flanking an almost perfect palindrome. Base-pair mutagenesis results show that the second hexameric repeat is involved in this factor binding. Also, to identify the factors that interact with this fragment, a yeast one-hybrid screen yielded seventeen positive clones. Two of these cDNAs encoded polypeptides that bound to the 51-bp fragment, and thus, are candidate factors that may be involved in modulating the activity of the promoter. Finally, I propose the design of a synthetic benzoic acid inducible promoter system to be tested in plants.
Degree
Ph.D.
Advisors
Carpita, Purdue University.
Subject Area
Molecular biology
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