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Naringenin, a flavanone and a precursor for a variety of flavonoids, has potential applications in the health and pharmaceutical sectors. The biological production of naringenin using genetically engineered microbes is considered as a promising strategy. The naringenin synthesis pathway involving chalcone synthase (CHS) and chalcone isomerase (CHI) relies on the efficient supply of key substrates, malonyl-CoA and p-coumaroyl-CoA. In this research, we utilized a soil bacterium, Acinetobacter baylyi ADP1, which exhibits several characteristics that make it a suitable candidate for naringenin biosynthesis; the strain naturally tolerates and can uptake and metabolize p-coumaric acid, a primary compound in alkaline-pretreated lignin and a precursor for naringenin production. A. baylyi ADP1 also produces intracellular lipids, such as wax esters, thereby being able to provide malonyl-CoA for naringenin biosynthesis. Moreover, the genomic engineering of this strain is notably straightforward. In the course of the construction of a naringenin-producing strain, the p-coumarate catabolism was eliminated by a single gene knockout (ΔhcaA) and various combinations of plant-derived CHS and CHI were evaluated. The best performance was obtained by a novel combination of genes encoding for a CHS from Hypericum androsaemum and a CHI from Medicago sativa, that enabled the production of 17.9 mg/L naringenin in batch cultivations from p-coumarate. Furthermore, the implementation of a fed-batch system led to a 3.7-fold increase (66.4 mg/L) in naringenin production. These findings underscore the potential of A. baylyi ADP1 as a host for naringenin biosynthesis as well as advancement of lignin-based bioproduction.

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 ​g/g glucose) and 3.15-fold improvement in titer (1.82 ​g/L) and productivity (0.038 ​g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

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. Keywords: Lipid biosynthesis, Wax esters, Acetate, Dynamic control, Decoupling, Autonomous circuit