• Energy Research

AbstractVolatile organic compounds play an essential role in every domain of life, with diverse functions. In this study, we use novel secondary electrospray ionisation high-resolution Orbitrap mass spectrometry (SESI-Orbitrap MS) to monitor the complete yeast volatilome every 2.3 s. Over 200 metabolites were identified during growth in shake flasks and bioreactor cultivations, all with their unique intensity profile. Special attention was paid to ethanol as biotech largest product and to acetaldehyde as an example of a low-abundance but highly-volatile metabolite. While HPLC and Orbitrap measurements show a high agreement for ethanol, acetaldehyde could be measured five hours earlier in the SESI-Orbitrap MS. Volatilome shifts are visible, e.g. after glucose depletion, fatty acids are converted to ethyl esters in a detoxification mechanism after stopped fatty acid biosynthesis. This work showcases the SESI-Orbitrap MS system for tracking microbial physiology without the need for sampling and for time-resolved discoveries during metabolic transitions.

3-Hydroxy decanoic acid (3-HDA), derivable from glucose or xylose waste-streams, was successfully upgraded electrochemically into a drop-in oxygenate with promising fuel characteristics.

Frontiers in Bioengineering and Biotechnology 8, 596414 (2020). doi:10.3389/fbioe.2020.596414 Published by Frontiers Media, Lausanne

Frontiers in Bioengineering and Biotechnology 8, 899 (2020). doi:10.3389/fbioe.2020.00899 Published by Frontiers Media, Lausanne

Autotrophic cultivation offers a path to carbon-neutral bioproduction, which is increasingly valuable in the context of climate change mitigation. In this study, the production of isopropanol by Cupriavidus necator is used as an example for CO2 valorisation, and a simple shake bottle system is introduced to facilitate the development of aerobic autotrophic cultivation processes and strain screening. Applying 1.5 bar overpressure in the bottle's headspace enhances gas transfer while pressure decrease was shown to be correlated to biomass and product formation, allowing to follow metabolic activity without sampling. After optimizing cultivation parameters and nickel feeding strategy, the system was applied to compare three different isopropanol-producing strains. The highest autotrophically obtained isopropanol concentration was 2.2 ± 0.5 g L-1 with a specific yield of 0.9 ± 0.2 g gCDW-1 and a minimal by-product concentration of 0.05 ± 0.01 g L-1 acetone. Heterotrophic cultivations were carried out for comparison, obtaining up to 3.4 ± 0.2 g L-1 final isopropanol concentration with a specific yield of 1.4 ± 0.1 g gCDW-1. Although the use of CO2 instead of fructose resulted in a slower process, the overall isopropanol production is promising. This study provides valuable insights into strain behaviour while demonstrating the utility of the presented shake bottle system for advancing autotrophic process development.

AbstractMethyl 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 engineeredPseudomonas taiwanensisVLB120 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-1methyl 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-1methyl ketones (corresponding to 69.3 g Lorg-1in thein situextraction 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 ofP. taiwanensisVLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.

Microbes harbor many traits that are dispensable or even unfavorable under industrial and laboratory settings. The elimination of such traits could improve the host's efficiency, genetic stability, and robustness, thereby increasing the predictability and boosting its performance as a microbial cell factory. We engineered solvent-tolerant Pseudomonas taiwanensis VLB120 to yield streamlined chassis strains with higher growth rates and biomass yields, enhanced solvent tolerance, and improved process performance. In total, the genome was reduced by up to 10%. This was achieved by the elimination of genes that enable the cell to swim and form biofilms and by the deletion of the megaplasmid pSTY and large proviral segments. The resulting strain GRC1 had a 15% higher growth rate and biomass yield than the wildtype. However, this strain lacks the pSTY-encoded efflux pump TtgGHI, rendering it solvent-sensitive. Through reintegration of ttgGHI by chromosomal insertion without (GRC2) and with (GRC3) the corresponding regulator genes, the solvent-tolerant phenotype was enhanced. The generated P. taiwanensis GRC strains enlarge the repertoire of streamlined chassis with enhanced key performance indicators, making them attractive hosts for biotechnological applications. The different solvent tolerance levels of GRC1, GRC2, and GRC3 enable the selection of a fitting host platform in relation to the desired process requirements in a chassis à la carte principle. This was demonstrated in a metabolic engineering approach for the production of phenol from glycerol. The streamlined producer GRC1Δ5-TPL38 outperformed the equivalent nonstreamlined producer VLB120Δ5-TPL38 concerning phenol titer, rate, and yield, thereby highlighting the added value of the streamlined chassis.