Lysate of Engineered Escherichia coli Supports Conversion of Glucose to 2,3-Butanediol with Near-Theoretical Yields and Ultrahigh Productivity

Traditionally, metabolic engineering has been applied for biochemical production in one of two ways; cell-free systems of purified enzymes and in vivo systems using engineered organisms. In vitro, the high cost of purified enzymes and cofactors has limited industrial applications, particularly when considering enzyme ensembles. In vivo, over-production of naturally high-flux nodes or byproducts in a host’s metabolism is often successful and has resulted in many industrial processes. Conversely, generating high flux through a novel pathway can be extremely difficult and development for many desirable products remains costly and slow. A key limitation is the conflict between cellular growth and adaptation objectives and engineering goals. In this work, we begin to explore a hybrid model in which metabolic engineering techniques are used to tailor a crude cell lysate for high rates, yields, and titers.

As a model pathway, we selected conversion of glucose to 2,3-butanediol (23BDO). 23BDO is a medium level commodity chemical with many industrial applications. First, a three-enzyme pathway for production of 23BDO from pyruvate was expressed in BL21(DE3) cells. Second, lysates were prepared by high-pressure homogenization and clarification of these cells. Third, lysates were combined with glutamate salts, catalytic (1 mM) cofactors NAD and ATP, and glucose to produce 23BDO. Importantly, most soluble native enzymes are present and active in the lysate, allowing the endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for 23BDO synthesis. We observed a maximal synthesis rate of meso-2,3-butanediol of 4.2 g/L/h with a theoretical yield of 80% (0.4 g m23BDO / g glucose). Titers reached 66 g/L m23BDO in a 30 hour batch reaction. Simply by removing the cellular structure and avoiding genomic regulation, productivity almost as high as any native producer and titers several fold above any previous studies in E. coli in vivo have been achieved. Our results highlight the ability for co-factor regeneration in cell-free lysates.  Further, they suggest exciting opportunities for use of lysate-based systems to (i) rapidly prototype metabolic pathways and (ii) carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation.