The goal of biomanufacturing is to create cost-effective, sustainable and renewable routes for production of biologics, pharmaceuticals, biofuels, commodity chemicals, and materials. Central to these efforts is the endowment of cells with new enzymatic machinery and altering of cellular metabolism to support carbon flux to the desired products. These tasks create a large array of unsolved challenges, from the determination and heterologous expression of enzymes that can perform requisite chemical transformations to the toxicity caused to the host by enzymes, pathway intermediate metabolites, and products. Furthermore, the task of re-directing carbon flux from natural pathways to the desired product is often non-intuitive since cells can drastically shift their metabolism in response to engineering manipulations, necessitating technologies that can rapidly produce and evaluate many manipulations of cellular metabolism in high throughput. Altogether, these issues create a need for new tools and approaches that resolve long-standing challenges in the engineering of cells for biomanufacturing. In this work, we explore the challenges associated with this goal as well as develop and introduce new synthetic biology technologies, particularly involving new advances in RNA gene expression regulation, that will speed the pace of cellular engineering and enhance the production capabilities of engineered cells. We begin by applying classical metabolic engineering tools to a pathway for eukaryotic protein glycosylation in Escherichia coli that lays the groundwork for efficient production of designer therapeutic glycoproteins. Building off this work, we explored the application of synthetic RNA regulators for high-throughput screening of endogenous gene knockdown in the context of the protein glycosylation pathway and propose a general approach for high-throughput strain engineering with RNA regulators. Finally, we developed and applied a new technology for implementing dynamic feedback control of gene expression in response to toxicity and burden imposed on cells by heterologous pathways and toxic intermediates. In addition to other examples, we applied this technology to a pathway for Taxol precursor production to improve its titer and productivity. Altogether, this work elucidates contemporary obstacles in engineering cells for biomanufacturing and provides new tools and approaches to address these obstacles.