Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria.

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Systematic analysis of intracellular mechanisms of propanol production in the engineered Thermobifida fusca B6 strain.

Thermobifida fusca is a moderately thermophilic actinobacterium naturally capable of utilizing lignocellulosic biomass. The B6 strain of T. fusca was previously engineered to produce 1-propanol directly on lignocellulosic biomass by expressing a bifunctional butyraldehyde/alcohol dehydrogenase (adhE2). To characterize the intracellular mechanisms related to the accumulation of 1-propanol, the engineered B6 and wild-type (WT) strains were systematically compared by analysis of the transcriptome and intracellular metabolome during exponential growth on glucose, cellobiose, and Avicel. Of the 18 known cellulases in T. fusca, 10 cellulase genes were transcriptionally expressed on all three substrates along with three hemicellulases. Transcriptomic analysis of cellodextrin and cellulose transport revealed that Tfu_0936 (multiple sugar transport system permease) was the key enzyme regulating the uptake of sugars in T. fusca. For both WT and B6 strains, it was found that growth in oxygen-limited conditions resulted in a blocked tricarboxylic acid (TCA) cycle caused by repressed expression of Tfu_1925 (aconitate hydratase). Further, the transcriptome suggested a pathway for synthesizing succinyl-CoA: oxaloacetate to malate (by malate dehydrogenase), malate to fumarate (by fumarate hydratase), and fumarate to succinate (by succinate dehydrogenase/fumarate reductase) which was ultimately converted to succinyl-CoA by succinyl-CoA synthetase. Both the transcriptome and the intracellular metabolome confirmed that 1-propanol was produced through succinyl-CoA, L-methylmalonyl-CoA, D-methylmalonyl-CoA, and propionyl-CoA in the B6 strain.

Ex vivo DNA Assembly.

Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid, and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

Evolutionary engineering for industrial microbiology.

Superficially, evolutionary engineering is a paradoxical field that balances competing interests. In natural settings, evolution iteratively selects and enriches subpopulations that are best adapted to a particular ecological niche using random processes such as genetic mutation. In engineering desired approaches utilize rational prospective design to address targeted problems. When considering details of evolutionary and engineering processes, more commonality can be found. Engineering relies on detailed knowledge of the problem parameters and design properties in order to predict design outcomes that would be an optimized solution. When detailed knowledge of a system is lacking, engineers often employ algorithmic search strategies to identify empirical solutions. Evolution epitomizes this iterative optimization by continuously diversifying design options from a parental design, and then selecting the progeny designs that represent satisfactory solutions. In this chapter, the technique of applying the natural principles of evolution to engineer microbes for industrial applications is discussed to highlight the challenges and principles of evolutionary engineering.