Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.

Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes.

D-glucaric acid can be used as a building block for biopolymers as well as in the formulation of detergents and corrosion inhibitors. A biosynthetic route for production in E. coli has been developed (Moon et al., 2009), but previous work with the glucaric acid pathway has indicated that competition with endogenous metabolism may limit carbon flux into the pathway. Our group has recently developed an E. coli strain where phosphofructokinase (Pfk) activity can be dynamically controlled and demonstrated its use for improving yields and titers of the glucaric acid precursor myo-inositol on glucose minimal medium. In this work, we have explored the further applicability of this strain for glucaric acid production in a supplemented medium more relevant for scale-up studies, both under batch conditions and with glucose feeding via in situ enzymatic starch hydrolysis. It was found that glucaric acid titers could be improved by up to 42% with appropriately timed knockdown of Pfk activity during glucose feeding. The glucose feeding protocol could also be used for reduction of acetate production in the wild type and modified E. coli strains.