Engineering polyester monomer diversity through novel pathway design.

Polyesters composed of hydroxy acids (HAs) and diols serve many material niches and are invaluable to our daily lives. However, their traditional synthesis from petrochemicals creates many environmental concerns. Metabolically engineered microorganisms have been leveraged for the industrially competitive production of a few polyesters with properties that limit their application. Designing new metabolic pathways to polyester building blocks is essential to broadening material property diversity and improving carbon and energy usage of current bioproduction schemes. This review focuses on recently developed pathways to HAs and diols. Specifically, new pathways to 2,3- and ω-Hydroxy acids, as well as C3-C4 and medium-chain-length diols, are discussed. Pathways to the same compound are compared on the basis of criteria such as energy usage, number of pathway steps, and titer. Finally, suggestions for improvements and next steps for each pathway are also discussed.

Layered and multi-input autonomous dynamic control strategies for metabolic engineering.

Metabolic engineering seeks to reprogram cells to efficiently produce value-added chemicals. Traditionally, this is achieved by overexpressing the production pathway and/or knocking out competing endogenous pathways. However, limitations in some pathways are more effectively addressed through dynamic metabolic flux control to favor different cellular objectives over the course of the fermentation. Dynamic control circuits can autonomously actuate changes in metabolic fluxes in response to changing fermentation conditions, cell density, or metabolite concentrations. In this review, we discuss recent studies focused on multiplexed autonomous strategies which (1) combine regulatory circuits to control metabolic flux at multiple nodes or (2) respond to more than one input signal. These strategies have the potential to address challenging pathway scenarios, actuate more complex response profiles, and improve the specificity of the criteria that actuate the dynamic response.

Metabolic engineering strategies to overcome precursor limitations in isoprenoid biosynthesis.

Isoprenoid biosynthesis has been a focus of metabolic engineering due to the broad spectrum of uses of isoprenoid products and the limited capacity to source them from plants or chemically synthesize them. Microbial synthesis offers the potential to cost-effectively produce isoprenoids in a stable and scalable manner. One bottleneck in advancing microbial engineering for isoprenoid biosynthesis has been limited supply of precursor molecules to the isoprenoid pathway. This article reviews strategies that have been employed to overcome such limitations. These include methods for enhancing reactions in the native pathway and preventing substrate depletion by competing pathways, the use of heterologous enzymes and pathways, and methods that reduce the metabolic burden imposed by heterologous reactions. Additionally, this article discusses challenges for the synthesis of novel products and maps some new directions for future research.

Dynamic pathway regulation: recent advances and methods of construction.

Microbial cell factories are a renewable source for the production of biofuels and valuable chemicals. Dynamic pathway regulation has proved successful in improving production of molecules by balancing flux between growth of cells and production of metabolites. Systems for autonomous induction of pathway regulation are increasingly being developed, which include metabolite responsive promoters, biosensors, and quorum sensing systems. Since engineering such systems are dependent on the available methods for controlling protein abundance in the desired host, we review recent tools used for gene repression at the transcriptional, post-transcriptional and post-translational levels in Escherichia coli and Saccharomyces cerevisiae. These approaches may facilitate pathway engineering for biofuel and biochemical production.

Screening and modular design for metabolic pathway optimization.

Biological conversion of substrate sugars to a variety of products is an increasingly popular option for chemical transformation due to its high specificity and because of significant interest in the use of renewable feedstocks. However, pathway optimization through metabolic engineering is often needed to make such molecules economically at a relevant scale. Employing effective methods to search and narrow the immense pathway parameter space is essential to meet performance metrics such as high titer, yield and productivity with efficiency. This review focuses on two practices that increase the likelihood of finding a more advantageous pathway solution: implementing a screen to identify high producers and utilizing modular pathway design to streamline engineering efforts. While screens seek to couple product titer with a high-throughput measurement output, modular design aims to rationally construct pathways to allow parallel optimization of various units. Both of these methodologies have proven widely successful in metabolic engineering, with combinations of them resulting in synergistic enhancements to pathway optimization. This review will particularly highlight their utility for microbially derived acid and alcohol products, which are of interest as fuels and value added products.