Relative abundance of enzymes in a pathway offer clues to rate-limiting step of the pathway

Identification of the rate-limiting step of a pathway is key to optimizing pathway performance and improving product titer and yield. Specifically, knowledge of the rate-limiting step affords us candidate for over-expression, which would help remove the bottleneck in the pathway for product formation. Typically, activity of enzyme is the primary yardstick for identification of rate limiting step of a pathway given that concentration of intermediates of a pathway could not be easily controlled. However, activity data of many enzymes are not widely available, and its measurement requires a tedious and laborious process of first heterologous expression, purification and biochemical characterization. One approach for circumventing the problem may to compare the relative abundance of enzymes in an endogenous pathway. Data supporting this endeavor could come from RNA-seq transcriptome of the microorganism under typical growth conditions. Expression counts of different enzymes in an endogenous pathway such as tricarboxylic acid (TCA) cycle could serve to calculate relative abundance of enzymes in the pathway. The smallest ratio could serve as indicator of the rate-limiting step. But, this remains an estimate given the lack of enzyme activity data. Overall, expression counts from RNA-seq transcriptome could serve as barometer for the relative abundance of enzymes in a pathway, and thus, help inform on the rate-limiting step of a pathway. In a cyclic pathway such as TCA cycle, such information would serve as first-pass estimate for the functional status of the pathway as well as inform efforts to further engineer gene over-expression strategies in metabolic engineering.