Recent functional genomics and genome-scale modeling approaches indicated that B(12) production in Lactobacillus reuteri could be improved by optimization of the medium. Here we show that a series of systematic single-amino-acid omissions could significantly modulate the production of B(12) from nearly undetectable levels (with omission of isoleucine) to levels 20-fold higher than the levels previously reported (with omission of cysteine). Using cDNA microarray experiments, we analyzed the transcriptional response of L. reuteri to medium lacking cysteine. The results supported the observed high level of B(12) production and provided new avenues for future improvement of production of vitamin B(12).