Professor
Ph.D., Physical Biochemistry

Protein Engineering

Research Interest: 

      Enzymes are biological catalysts which determine the pattern of chemical transformation in a living system. The metabolic activities of living cells are accomplished by a regulated, highly coupled network of over 1000 enzyme-catalyzed reactions and selective membrane transport systems. Since the success of gene cloning about two decades age, there has been growing interest in increase or redirect metabolic flux studies using recombinant DNA technology.  

However, the complexity of metabolic regulation necessitates studies of the regulatory behaviour of enzymes for developing an understanding of the overall metabolism. The work of this laboratory is concerned with regulation of gene expression and metabolic control. Two projects currently under study are: (1) structure, expression and regulation of cellulase and xylanase genes in Trichoderma koningii G39 and (2) metabolic studies of genes involved in the biosynthesis of L-lysine in Escherichia coli.

Lignocelluloses are the most abundant renewable natural resources on earth. They are insoluble complexes composed of cellulose, xylan, and lignin in varying proportion. For efficient natural recycling, a large number of different enzymes acting apparently in synergy are required. Another feature is that these enzymes are highly adaptive in fungi. The inducing substrate, such as cellulose is insoluble. The fact that cellulose cannot be transported directly across the cell wall and membrane suggests that it must be first converted to soluble inducer(s). However, the true inducer(s) of cellulases is not yet unequivocally identified and the nature of enzyme induction is also largely unknown. We have now cloned three of these enzyme genes from T Koningii G39. Analysis of approximately 1.4kb of the 5' flanking region of cellobiohydrolase I gene shows that there are five consensus 5'-(G/C) PyGGGG-3' sequences identified as the carbon catabolite repressor binding sites of Aspergillus nidulans CREA and Saccharomyces cerevisiae MIG1 repressors. The presence of these five CREA/MIG1 repressor binding consensus sequences in the region suggests the wide-domain carbon catabolite repression regulatory system that controls the A. nidulans ethanol regulon and yeast GAL genes transcription might also be operative and responsible for regulation of T. Koningii cbh1 transcription. We are now sequencing the other two genes, a bifunctional b-xylosidase and an endoxylanase, and employing RT-PCR to study the problem of enzyme induction. The primary objective of the study will be to understand the mechanism of regulation and control of celluloase biosynthesis in Trichoderma spp. We are also interested in the structure and function relationships of these enzymes. They will be expressed heterologously in other hosts like E coli and S. cerevisiae and site-directed mutagenesis will be employed for the study mechanism of action of these enzymes.

Metabolic pathways in microbial cells consist of networks of enzyme-catalyzed reactions that transform substrates into products. In a metabolic pathway there are certain key enzymes that control and regulate the fluxes through different branches and concentrations of various metabolites. Generally, a particular metabolite can be overproduced by deregulating the pathway directly associated with the synthesis of that metabolite or applying recombinant DNA methods to alter pathway distributions and rates.

However, cloning and overexpression of particular enzyme gene may have other kinetic consequences which are difficult to foresee and may lead to undesirable effects on the rest of metabolism. In order to study the effects of genetic modifications of specific enzymes in the primary metabolism for enhancing metabolite yield and productivity, we have chosen overproduction of lysine in E coli as a model.

In E. coli, aspartic acid is a precursor of the essential metabolites, diaminopimelic acid, lysine, methionine, threonine and isoleucine. It is derived from a product of tricarboxylic acid cycle and thus extends into glycolysis. The lysine biosynthetic pathway in E coli commonly designates the sequential reactions that begin at aspartate and end at lysine and is composed of nine different steps. The expression of most of the corresponding genes in the lysine pathway is repressed by lysine and these genes are scattered along the chromosome. We have cloned E. coli ppc, lysC and dapA genes. ppc encodes phosphoenopyruvate carboxylase which is apparently the first committed enzyme in the biosynthesis of amino acids of the aspartate family and the glutamate family; lysC encodes lysine-sensitive aspartokinase III which catalyzes the first step in the synthesis of lysine, methionine, threonine and isoleucine from aspartate; dapA gene, encoding dihydrodipicolinic acid synthase, is the first gene of the lysine branched pathway. The main goal of the project is to study their effects on cell metabolism as well as lysine biosynthesis.

Selected Publications: 

1. Chen HJH, Chen TF, Huang SRS, Gong J, Li JC, Chen WC, Hseu TH, Hsu IC A Novel Micro-well Array Chip for Liquid Phase Biomaterial Processing and Detection. Sensors and Actuators A: Physical 108, 193-200 (2003).
   
   
2. Chen MY, Huang SY, Lin EC, Hseu TH, Lee WC Association of a single nucleotide polymorphism in the 5'-flanking region of porcine HSP70.2 with backfat thickness in Duroc breed. Asia-Aust. J. Anim. Sci. 16, 100-103 (2003).
   
   
3. Lee CF, Hseu TH.. Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches. Can J Microbiol. 48, 831-40 (2002).
   
   
4. Chiu CC, Ting JW, Hseu TH., Chang CY. Characterization and transactivation domain and developmental expression of pituitary specific transcritional factor, Pit-1 of ayu (Plecoglossus altivelis) Gen. Comp. Endocrinal. 127, 307-13 (2002).
   
   
5. Chiu CC, John JA, Hseu TH., Chang CY. Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody. Protein Purif. Expr. 24, 292-301 (2002).
   
   
6. Ou WC, Hseu TH., Wang M, Chang H, Chang D. Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation. J. Med Virol. 64, 366-373 (2001).
   
   
7. Ou WC, Chen LH, Wang M, Hseu TH., Chang D. Analysis of minimal sequences on JC virus VP1 required for capsid assembly. J. NeuroVirol. 7, 298-301. (2001).
   
   
8. Li YC, Lee C, Sanoudou D, Hseu TH., Li SY, Lin CC, Hsu TH. Interstitial colocalization of two cervid satellite DNA s involved in the genesis of the Indian muntjac karyotype. Chromosome Res. 8, 363-373 (2000).
   
   
9. Li YC, Lee C, Hseu TH, Li SY, Lin CC, Hsu TH.. Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families. Cytogenet. Cell Genet. 89, 192-198 (2000).
   
   
10. Y. C. Chou, C. C Chou, Y. K. Chen, S. Tsai, F. M. J. Hsieh, H. J. Liu and T. H. Hseu (1999) Structure and genomic organization of porcine RACK1 gene. Biochim. Biophys. Acta 1489:315-322.
    
    
11. W. C. Ou, M. L. Wang, C. Y. Fung, R. T. Tsai, P. C. Chao, T. H. Hseu and D. Chang (1999) The major capsid protein VP1 of human JCV expressed in E. coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells. J. Gen Virol. 80:39-46.