Chromopyrrolic acid (CPA) and its congeners are important intermediates for the biosynthesis and synthesis of various dimeric tryptophan natural products. We have constructed two E. coli strains (CPA001/CPA002) harboring a single plasmid carrying genes coding for a combination of two enzymes (LaStaO/LzrO and VioB) that are able to convert L-tryptophan (L-Trp)/5-chloro-L-tryotophan (5-Cl-L-Trp) to chromopyrrolic acid (CPA)/5,5'-dichloro-chromopyrrolic acid (5,5'-diCl-CPA). Effect on the production of CPA were evaluated by varying the parameters of strain cultivation and biotransformation process. Under the optimized conditions, up to 325 mg/L of CPA and 275 mg/L of 5,5'-diCl-CPA could be obtained by supplementing L-Trp and 5-Cl-L-Trp, respectively, to a working culture of CPA001, or to a phosphate buffer-resuspended culture of CPA002. The practicability of this whole-cell biotransformation system could also be served as a potential platform for the preparation of CPA congeners.