Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol

Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized β-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as 'plug-and-play' module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.Microbial Biotechnolog

Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2.

Anaplerosis is an essential feature of metabolism that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO2-fixation pathway that features several C3-C5 biosynthetic precursors. We drafted four different anaplerotic modules that use CO2 to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C21-macrolide backbone of erythromycin. Our best design allowed the carbon-positive synthesis of 6-DEB via 54 enzymatic reactions in vitro at yields comparable to those with isolated 6-DEB polyketide synthase (DEBS). Our work showcases how new-to-nature anaplerotic modules can be designed and tailored to enhance and expand the synthetic capabilities of complex catalytic in vitro reaction networks. © 2022. The Author(s)

Methylocystis sp. Strain SC2 Acclimatizes to Increasing NH4(+) Levels by a Precise Rebalancing of Enzymes and Osmolyte Composition

A high NH4(+) load is known to inhibit bacterial methane oxidation. This is due to a competition between CH4 and NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of Methylocystis sp. strain SC2 to high NH4(+) levels. Relative to 1 mM NH4(+), a high (50 mM and 75 mM) NH4(+) load under CH4-replete conditions significantly increased the lag phase duration required for proteome adjustment. The number of differentially regulated proteins was highly significantly correlated with an increasing NH4(+) load. The cellular responses to increasing ionic and osmotic stress involved a significant upregulation of stress-responsive proteins, the K(+) "salt-in" strategy, the synthesis of compatible solutes (glutamate and proline), and the induction of the glutathione metabolism pathway. A significant increase in the apparent Km value for CH4 oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2(-) and, upon decreasing O2 tension, N2O. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH4(+) exposure, but the need to simultaneously combat both ionic-osmotic stress and the toxic effects of hydroxylamine may be the reason why its acclimatization capacity is limited to 75 mM NH4(+). IMPORTANCE In addition to reducing CH4 emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus Methylocystis contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of Methylocystis spp. is, however, sensitive to high NH4(+) concentrations. This is due to the competition of CH4 and NH3 for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH4(+) load. An understanding of the physiological and molecular response mechanisms of Methylocystis spp. is therefore of great importance. Here, we combined global proteomics with amino acid profiling and NOx measurements to disentangle the cellular mechanisms underlying the acclimatization of Methylocystis sp. strain SC2 to an increasing NH4(+) load

A Modular In Vitro Platform for the Production of Terpenes and Polyketides from CO2

A long-term goal in realizing a sustainable biocatalysis and organic synthesis is the direct use of the greenhouse gas CO2 as feedstock for the production of bulk and fine chemicals, such as pharmaceuticals, fragrances and food additives. Here we developed a modular in vitro platform for the continuous conversion of CO2 into complex multi-carbon compounds, such as monoterpenes (C10 ), sesquiterpenes (C15 ) and polyketides. Combining natural and synthetic metabolic pathway modules, we established a route from CO2 into the key intermediates acetyl- and malonyl-CoA, which can be subsequently diversified through the action of different terpene and polyketide synthases. Our proof-of-principle study demonstrates the simultaneous operation of different metabolic modules comprising of up to 29 enzymes in one pot, which paves the way for developing and optimizing synthesis routes for the generation of complex CO2 -based chemicals in the future

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H-2 to the culture headspace had the most significant positive effect on the growth yield under CH4 (6%) and O-2 (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (+/- 0.52) to 13.82 (+/- 0.69) mg cell dry weight per mmol CH4, while CH4 consumption was significantly reduced. Regardless of H-2 addition, CH4 utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O-2/CH4 mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H-2 addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H-2 (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H-2 may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH4, such as the production of high-quality feed protein