High titer methyl ketone production with tailored Pseudomonas taiwanensis VLB120

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.

Determination of double bond positions in methyl ketones by gas chromatography–mass spectrometry using dimethyl disulfide derivatives

“…Pseudomonas taiwanensis Δ6 pProd 4 was propagated in Lysogeny Broth (LB) containing 10 g L −1 peptone, 5 g L −1 sodium chloride, and 5 g L −1 yeast extract. Solid LB was prepared by adding 1.5% (w/v) agar to the liquid medium.…”

“…The industrial production of these compounds is based on hydrocarbons derived from petroleum. To achieve a sustainable production of methyl ketones, intense research has been carried out to rearrange the fatty acid metabolism of different microbes 1–8 . In this regard, fatty acid‐derived methyl ketones are of interest for the application as biofuels 1–4 .…”

“…taiwanensis ) VLB120 for the first time. The latter is of particular interest in biofuel production 4,9 . Pseudomonads are of an increasing interest in biotechnological applications 47,48 on account of their fast growth, solvent tolerance, and versatile metabolism 4,49,50 .…”

High‐throughput colorimetric assays optimized for detection of ketones and aldehydes produced by microbial cell factories

“…Promising bioproduction of acetone from acetate, for example, resulted in titers up to 122 mM acetone in 48‐h Escherichia coli cultures (Yang et al ., 2019). The highest production yield of methyl ketones reported to date has been achieved through rational strain engineering of the non‐conventional host Pseudomonas taiwanensis , leading to titers of 9.8 g l −1 and highlighting the potential for high titre production of these compounds (Nies et al ., 2020). While initial strain development is often done in a rational, human‐designed manner, novel approaches relying on computer‐aided strain design and randomized strain engineering allow tapping into the space of non‐understood metabolism through a systems metabolic engineering approach, pushing yields and productivities further (Choi et al ., 2019).…”

Synthetic metabolism for in vitro acetone biosynthesis driven by ATP regeneration

“…Supported by dedicated strain engineering approaches, previous studies shed light on the molecular mechanisms underlying the regulation of the acetone biosynthetic pathway. 6,[19][20][21][22][23] Enhanced ketones and alcohols production has been demonstrated by using engineered bacteria, adapted to consume different feedstocks under optimized fermentation setups. 24 However, limited knowledge on the biocatalysts involved often resulted in relatively low yields, which remain to be a major hurdle for the development of economically-feasible ABE fermentation and related bioprocesses.…”