Balancing and increasing the flux through an engineered heterologous pathway in a target organism to achieve high yield and productivity remains an overwhelming challenge in metabolic engineering. Here we report a novel strategy combining directed evolution and promoter engineering for rapid and efficient multi-gene pathway optimization. As proof of concept, this strategy was applied to optimize a cellobiose utilizing pathway in an industrial Saccharomyces cerevisiae strain for highly efficient cellulosic biofuels production. The resulting strain exhibited significantly higher cellobiose consumption rate (6.41-fold) and ethanol productivity (6.36-fold) compared to its parent strain. This study also showed that both the ratios and absolute values of the expression levels of the genes in the cellobiose utilizing pathway play an important role in cellobiose uptake, and -glucose is likely one of the key factors affecting cellobiose metabolism. Biotechnol. Biotechnol. Bioeng. 2013;110: 2874-2881. (c) 2013 Wiley Periodicals, Inc.