Reconstruction of the Violacein Biosynthetic Pathway From Chromobacterium violaceum in Escherichia coli.

Introduction: Violacein, a bis-indole produced by various bacteria is known to display extensive pharmaceutical properties. Unfortunately, low productivities in natural violacein producers have led to inconsistent violacein supply, limiting its application as future therapeutics. Heterologous expression systems such as Escherichia coli and Pichia pastoris offers an alternative means of producing these high-valued secondary metabolites. This work describes the development of a genetic system for violacein heterologous production in E. coli. Methods: A violacein producer, C. violaceum, MTH01, was isolated from the limnological grounds of Universiti Sains Malaysia, Penang. Using gene specific primers, the entire 7.3 kb violacein gene cluster was successfully amplified from C. violaceum MTH01 DNA, cloned into pUC19 vector (pVio19) and then subcloned into pET-3a and pET-11b, yielding recombinant vectors, pVio3a and pVio11b, respectively. For heterologous expression, parameters such as carbon sources, temperature, inducer (IPTG) and L-tryptophan were optimised. Violacein extracted from purple-pigmented E. coli transformants were analysed using TLC and FTIR. Results: After a few days, E. coli transformants containing either pVio3a or pVio11b developed purple colonies, indicating that violacein was successfully expressed in E. coli. TLC analysis of the pigment extract revealed Rf values comparable to deoxyviolacein, an intermediary metabolite in violacein biosynthesis, while FTIR spectra revealed the presence of amine and carbonyl groups, both of which are characteristics of an indole. Conclusion: The E. coli heterologous system described here could utilize either glucose or glycerol as carbon source. Addition of L-tryptophan to growth medium was necessary for successful expression of the violacein pathway. [ABSTRACT FROM AUTHOR]