Have you sequenced the construct? One possibility is the presence of a mutation introducing a frameshift or a premature stop codon in one of the CDS.

If the sequence is OK, next check whether there are differences in the efficiency of translational initiation between both genes, using something like the RBS calculator at https://www.denovodna.com/software/

Next, compare both genes for codon usage regarding highly expressed E. coli genes. Any of the codon adaptation index (CAI) calculators available online will do. Fixing that (if there are problems) will require changing the sequence of the affected gene or expression in Rosetta strains.

Hope it helps!

thanks for your reply. But i have checked the sequence no frameshift mutation is there. Also both the genes are under separate T7 promoters. Both genes are codon optimized for E.coli strain.

It does not matter that both genes are under separate T7 promoters, it's translational, not transcriptional inititation that I was talking about. Do check the two genes in RBS calculator, you never know.

By the way, have you checked expression of the 'missing' protein by Western blot? There's a difference regarding troubleshooting between 'no expression' and 'low expression'.

Thanks Alejandro Martin for your suggestions. I checked the biotransformation activity using those cells its giving me good conversions. Probably one gene is showing very good expression as seen in SDS PAGE and the other gene is not having as good expression and is minutely showing on the gel.