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Regulation and Metabolic Engineering of Secondary Metabolite Biosynthesis

A large number of industrially important pharmaceuticals, including antibiotics, anticancer agents, and immunosuppressors, are products of secondary metabolism in microbial or plant systems. Regulation of secondary metabolite biosynthesis includes at least four layers of cellular control. At the first level of the metabolic scheme is the influence of global and specific regulators of secondary biosynthetic gene expression. These genetic elements although poorly understood are capable of influencing biosynthetic rates significantly in microorganisms that produce natural products. At the second level of the biosynthetic scheme are the structural genes and protein products that specify biosynthesis of a particular secondary metabolite. At this level, individual enzymes that represent rate-limiting steps can influence the rate of microbial metabolite production. Similarly, enzyme co-factor and precursor supply represents a third level of cellular control of biosynthesis in multistep biosynthetic pathways. At the fourth level of the metabolic scheme, the degree to which an individual cell can effectively transport (excrete) complex metabolites represents yet another influence on production efficiency of natural products. The long-term goal of our work in this area is to unveil the regulatory hierarchy of secondary metabolism and to develop a rational approach for metabolic engineering of secondary metabolite production using microbial genomic technologies. The model system used is the b-lactam antibiotic biosynthesis in Streptomyces clavuligerus. The project involves both Prof. Hu's research group and Prof. Sherman's at the Department of Microbiology at University of Minnesota.

Relaxing precursor flux control by genetic engineering

Using known enzyme kinetic data and physiological information we constructed a kinetic model for biosynthesis of cephamycin C. By performing control strength analysis we identified ACV synthetase and lysine e-aminotransferase (LAT), the first enzymes in the biosynthetic pathway of cephamycin C, as the rate-limiting steps in cephamycin production. Increasing LAT levels by introducing an additional copy of the gene encoding for LAT in S. clavuligerus chromosome (LHM100) increased cephamycin production by two to five fold. To investigate the temporal profile of LAT activity a fusion protein of LAT and green fluorescent protein (GFP) was constructed and expressed in S. clavuligerus. The transformants exhibited the characteristic green fluorescence of GFP. Laser scanning confocal microscopy was used to examine the spatial distribution of LAT in the mycelia. Unique spatial patterns of green fluorescence were observed in mycelia during various stages of cultivation. The results suggest that the genes for cephamycin C production be regulated both spatially and temporally. The potential for use of GFP as a physiological and metabolic probe in Streptomyces species was verified.

Precursor and cofactor as a check valve for cephamycin biosynthesis

The biosynthesis of secondary metabolites is closely linked to primary metabolism via the supply of precursors, cofactors, and cellular energy. A combined experimental and kinetic modeling approach was used to examine the regulation of flux in the cephamycin biosynthetic pathway in S. clavuligerus. The Km values for lysine and a-ketoglutarate were substantially higher than their intracellular concentrations. Furthermore, their Km values are much higher than that tRNAlys acyl transferase and for other enzymes in primary and secondary metabolism which utilize as a-ketoglutarate a substrate. This suggests that lysine and a-ketoglutarate play a key role in regulating the flux of cephamycin biosynthesis ensuring that the flux through LAT will not be a diversion of resources needed for primary metabolism.

Analysis of the spatio-temporal gene expressions using GFP as a reporter

Secondary metabolite synthesis is related to cellular differentiation. The expression of the machinery for secondary metabolite biosynthesis occurs only in late exponential growth and may be heterogeneous in mycelia. We investigated the temporal and spatial distribution of the rate limiting biosynthetic enzyme Lysine 6 aminotransferase (LAT) by constructing green fluorescent protein (GFP) and LAT fusion protein (LAT-GFP) in S. clavuligerus. Using confocal microscopy we demonstrated that the expression of LAT quickly diminishes after inoculation from seed culture into main culture. It reappears only in some mycelia in early exponential phase. All mycelia became fluorescent in late exponential phase and green fluorescence diminishes again in stationary phase.

Most Streptomyces spp. undergoes greater morphological change during the growth cycle in solid culture as opposed to liquid culture. Time-lapsed experiments using confocal microscopy were carried out to monitor expression of the lat gene in solid media (agar plate). In solid phase analysis, abundant LAT expression was evident in the substrate mycelia but was completely absent in aerial hyphae.

It has been shown that a positive regulator, an ccaR-encoded control element located within the cephamycin gene cluster, regulates LAT expression. We have constructed a recombinant clone carrying the positive regulator ccaR fused to gfp coding for green fluorescent protein and another recombinant clone carrying lat::gfp and ccaR::cfp (coding for cyan fluorescent protein) in the chromosome. This either strain of S. clavuligerus allows simultaneous detection of CcaR and LAT in situ. Their behavior is similar to LAT-GFP. These findings may have implications for process design as well as metabolic engineering of secondary metabolite biosynthesis in Streptomyces.

Genome-wide probing of the regulation of secondary metabolism

Antibiotic productions are precisely controlled by sets of regulators in Streptomyces but most previous studies have been focused on several individual regulatory genes. To develop a rational approach for metabolic engineering of secondary metabolite production, both subset and genome-wide microarray methods are being used to analyze the regulation of secondary metabolism. Quantitative physiological and mathematical modeling approaches will be combined to obtain information on temporal and conditional expression of global and pathway-specific regulatory factors, and rate-limiting enzymes for the natural product pathways. Our research objectives encompass the following aspect.

	Genome-wide and subset microarray analysis to monitor expression of two-component regulatory genes and pathway-specific regulators involved in secondary metabolite biosynthesis in S. coelicolor.
	Microarray analysis of metabolic regulation in wild type and isogenic mutants of S. coelicolor involving two-component and secondary metabolite pathway specific regulators.
	Development of recombinant S. coelicolor strains with engineered regulatory gene::gfp fusions to study temporal and spatial expression patterns for secondary metabolism.
	Development of recombinant S. coelicolor strains with disrupted regulatory genes to study the regulatory circuits of secondary metabolism.
	To establish and refine a mathematical modeling framework for regulation of secondary metabolism in S. coelicolor as well as other model secondary metabolite biosynthetic systems.

Selected Recent Publications:

	Kyung, Y. S., W.-S. Hu and D. H. Sherman. 2000. Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein. Submitted.
	Han, L., A. Khetan, and W. -S. Hu. 1999. Time-lapsed confocal microscopy reveals temporal and spatial expression of the lysine e-aminotransferase gene in Streptomyces clavuligerus. Mol. Microbiol. 34:878-886
	Khetan, A., L.-H. Malmberg, Y. S. Kyung, D. H. Sherman and W.-S. Hu. 1999. Precursor and cofactor as a check valve for cephamycin biosynthesis in Streptomyces clavuligerus. Biotechnol. Prog. 15:1020-1027
	Khetan, A. and W. -S. Hu. 1999. Metabolic engineering of antibiotic biosynthesis for process improvement. In: Metabolic Engineering, Eds: S. Y. Lee and E. T. Papoutsakis. Marcel Dekker, New York.
	Khetan, A., L.-H. Malmberg, D. H. Sherman and W.-S. Hu. 1996. Metabolic engineering of cephalosporin biosynthesis in Streptomyces clavuligerus. Ann. N.Y. Acad. Sci. 782:17-24.
	Malmberg, L.-H., W.-S. Hu and D.H. Sherman. 1995. Effects of enhanced lysine e-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus. Appl. Microbiol. Biotechnol. 44:198-205.
	Malmberg, L.-H., W.-S. Hu and D.H. Sherman. 1993. Precursor flux control through targeted chromosomal insertion of the lysine e-aminotransferase (lat) gene in cephamycin C biosynthesis. J. Bacteriol. 175:6916-6924.
	Malmberg, L.-H. and W.-S. Hu. 1991. Kinetic analysis of cephalosporin biosynthesis in Streptomyces clavuligerus. Biotechnol. Bioeng. 38:941-947.