Shu-Jen Chiang

Strain improvement for fermentation and biocatalysis processes by genetic engineering technology

Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized. Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition. Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering. How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity. Where will this emerging technology lead us in the twenty-first century?

Cloning and expression of a cytochrome P450 hydroxylase gene from Amycolatopsis orientalis: hydroxylation of epothilone B for the production of epothilone F

Degenerate PCR primers were used to amplify cytochrome P450 gene fragments from the high-GC gram-negative bacteria Amycolatopsis orientalis, which catalyzes the hydroxylation of epothilone B to produce epothilone F. The amplified fragments were used as hybridization probes to identify and clone two intact cytochrome P450 genes. The expression of one of the cloned genes in a Streptomyces lividans transformant resulted in the biotransformation of epothilone B to epothilone F. The conversion of epothilone B to epothilone F by the S. lividans transformant was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.

Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over that in the untransformed host.

Engineering enzymes for improved catalytic efficiency: a computational study of site mutagenesis in epothilone-B hydroxylase

Epothilone F, 21-hydroxyl-epothilone B, is an intermediate in the synthesis of BMS-310705, an antitumor compound that has been evaluated in Phase I clinical trials. A bioconversion process utilizing the Gram-positive bacterium Amycolatopsis orientalis was used to prepare epothilone F from epothilone B. In order to improve the yield of epothilone F, a mutagenesis program was performed with the goal of engineering the epothilone-B hydroxylase (EBH) enzyme to improve the yield of epothilone F through oxidative biotransformation. The mutations in EBH increased the yield of epothilone F from 21% in the recombinant expression system to higher than 80% utilizing the best EBH mutants. The studies described here show how a homology model of EBH was used to obtain an understanding of the possible mechanism that led to improved yield of epothilone F in the mutated enzymes. A novel aspect of this study is that it provides some insight into how mutations distant from the binding site can affect enzyme activity.

Expression of a cephalosporin C esterase gene in Acremonium chrysogenum for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.