Abstract
For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strictly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost and sustainable substrates.
Highlights
Metabolic engineering and synthetic biology provide powerful means for the bio-based production of a variety of chemicals and other commodities by engineered microbes
We observed that when grown on glucose, the cells grow more slowly, but produce wax esters (WE) titers comparable to those of the wild type strain; after 48 h of cultivation, wt had produced 470 ± 150 mg/l WEs compared to 460 ± 40 mg/l WEs produced by the knock-out strain
As acetyl-CoA represents the key precursor in both the biomass production through the glyoxylate shunt and the WE biosynthesis, we hypothesized that by dynamically regulating the isocitrate lyase, the state of the cells could be switched between biomass and lipid synthesis modes (Fig. 1)
Summary
Metabolic engineering and synthetic biology provide powerful means for the bio-based production of a variety of chemicals and other commodities by engineered microbes. While growthassociated genes responsible for central carbon metabolism cannot be directly deleted, various strategies for decoupling growth and product synthesis have been introduced; Soma et al constructed a metabolic toggle switch in Escherichia coli for conditional knockout of citrate synthase gltA, an enzyme required for functional tricarboxylic acid (TCA) cycle (Soma et al, 2014). Solomon et al introduced a dynamic approach to controlling the glycolytic flux; antisense RNA technology and an inverting gene circuit were employed for inhibiting the activity of glucokinase (Glk), resulting in a controlled growth rate and a reduced production of acetate in E. coli (Solomon et al, 2012). Brockman and Prather introduced another example of a dynamic regulation system, where they developed a circuit for dynamic knockdown of phosphofructokinase-1 (Pfk-1), the enzyme responsible for the key step in the glycolytic pathway regulating glucose-6-phosphate flux (Brockman and Prather, 2015). By the temporal control of Pfk-1 degradation, glucose6-phosphate could be efficiently directed to a heterologous myo-
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