High titer methyl ketone production with tailoredPseudomonas taiwanensisVLB120

Abstract

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineered Pseudomonas taiwanensis VLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g Laq -1 methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g Laq -1 methyl ketones (corresponding to 69.3 g Lorg -1 in the in situ extraction phase) at 53 % of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential of P. taiwanensis VLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.

Co-feeding enhances the yield of methyl ketones

“…The network i JN1463 contains 2 153 metabolites, 2 927 reactions, and 1 463 genes. Herein, the network i JN1463 was adjusted mainly according to the modifications introduced by Nies et al ( 2020 ). In their GEM for P. taiwanensis VLB120, named i JN1411_MK, they took the metabolic network reconstruction i JN1411 for P. putida KT2440 (Nogales et al, 2017 ) as a basis.…”

“…In the model i JN1411_MK, Nies et al ( 2020 ) added a methyl ketone pooling reaction for methyl ketone congeners. Contrary to their suggestion, in this work, we accounted for the C17-congener 10-heptadecen-2-one.…”

“…where e indicates that the metabolites are extracellular and where the stoichiometry corresponds to the molar ratio of the methyl ketone congeners that was determined experimentally by Nies et al ( 2020 ). Note that, as the reaction is a so-called exchange reaction, only reactants and no products are assigned to this reaction.…”

“…Additionally, P. taiwanensis VLB120 shows a high tolerance to lignin-derived aromatic compounds that have an inhibitory effect on many other microorganisms (Lenzen et al, 2019 ; Wordofa & Kristensen, 2018 ). Nies et al ( 2020 ) engineered P. taiwanensis VLB120 for the production of medium-chain-length methyl ketones. They established the truncated β -oxidation pathway from Goh et al ( 2012 ) to Goh et al ( 2014 ).…”

“…In a fed-batch bioprocess with glucose as a carbon source, a product titer of 9.8 g L aq −1 at 53% of the theoretical maximum yield was achieved. This is the highest recombinantly produced methyl ketone yield to date (Nies et al, 2020 ).…”