Integrated bioprocessing for the pH-dependent production of 4-valerolactone from levulinate in Pseudomonas putida KT2440.

In nature, metabolism is not restricted to the cytosol. Metabolic activity can occur in the periplasm, on cell surfaces, or even extracellularly in the environment. When extracytosolic enzyme expression occurs, it is often to convey an advantage to the cell that is not possible with the enzyme in the cytosol. For instance, Escherichia coli alkaline phosphatase is expressed in the periplasm to detoxify compounds before they can enter the cell and to allow the enzyme better access to the extracellular environment for scavenging of phosphate (8). Penicillium decumbens secretes cellulases, presumably to break down extracellular substrates into a form amenable to uptake by the cell for further metabolism (18). Each of these enzymes exists as a component of larger metabolic pathways (phosphate and cellulosic material metabolism), and each has evolved for noncytosolic expression to facilitate the physiological goals of the respective pathways. In microbial biotechnology, the objective is typically not physiological but commercial in nature: to increase the titer of a small-molecule metabolic product. Though the goal has changed, the lessons we can learn from natural systems remain. The cytoplasm is not always the best choice for enzyme expression because cytoplasmic conditions are not necessarily optimal for enzyme productivity. One such system is the intramolecular lactonization of hydroxyacids, such as 4-hydroxyvalerate (4HV), to lactones, such as 4-valerolactone (4VL). This reaction, catalyzed in this work by the G3C9 variant of human paraoxonase I (PON1) (1), is known to be highly pH dependent. Because lactonization is acid catalyzed and because hydroxyacids and lactones exist in pH-dependent equilibrium with each other, control of the pH at which lactonization occurs is critical to achieving high titers of lactones (19). The cytoplasmic pH, typically about 7.5, is too high to achieve good titers of lactones at equilibrium (20). This limitation in the lactone titer is thermodynamic in nature, meaning that overexpressing the lactonase or most other traditional metabolic engineering techniques aimed at increasing flux toward the product would be largely ineffective at improving lactone production. However, having the lactonase perform catalysis outside the cytosol, where the pH can be lowered, would alter the equilibrium in favor of lactone production, thereby increasing the product titer. To implement such a system, we used extracytosolically expressed PON1 to catalyze the lactonization reaction in acidic medium (pH ∼6). Recently we found that Pseudomonas putida was capable of producing high titers of 4HV from the renewable carbon source levulinic acid (15). This process is achieved through coenzyme A (CoA) carriers, and the secretion of 4HV into the extracellular medium was enhanced by the expression of an intracellular thioesterase. Thus, P. putida can serve as an intracellular source of 4HV. This 4HV can then be lactonized by extracytosolically expressed PON1 in acidic medium to yield 4VL (Fig. ​(Fig.1).1). This general strategy is termed “integrated bioprocessing” for its integration of cytosolic and extracytosolic biocatalysis to enhance production. FIG. 1. Integrated bioprocessing system for the production of 4VL from levulinate. Production of 4HV from levulinate occurs intracellularly, while the lactonization reaction takes place extracytosolically in acidic medium. The enzyme(s) responsible reaction step(s) ... 4VL has been reported to be an ideal compound for use as a fuel and in the production of carbon-based chemicals (9). It has also seen extensive use as a component of block copolymers for drug delivery (2, 6) and as a precursor for acrylic compounds (13) and can be used as a precursor for the production of “bio-nylon” polymers (10). Current synthetic methods for 4VL also utilize levulinate as the starting material but require supercritical solvents (14) or carbon dioxide (3) with hydrogen gas under harsh conditions (∼10 to 40 MPa and ∼200°C) and ruthenium-based catalysts. In contrast, our biological method of production is done under mild conditions without the need for harsh solvents, hydrogen, rare metal catalysts, or supercritical fluids. This integrated bioprocessing system to produce 4VL from levulinate (Fig. ​(Fig.1)1) was compared to an entirely intracellular 4VL production pathway using cytosolically expressed PON1 to demonstrate the effectiveness of integrated bioprocessing to improve product titers at different pH values. The system was tested in a 2.0-liter bioreactor to further increase product titers and take advantage of automated pH control. To our knowledge, this work represents the first report of 4VL synthesis in a biological system.