Itaconic acid production by co-feeding of Ustilago maydis: A combined approach of experimental data, design of experiments, and metabolic modeling.

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 g L - 1 $\,\text{g L}{}^{-1}$ glucose and 12 g L - 1 $\,\text{g L}{}^{-1}$ acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide.

Adaptive laboratory evolution in a novel parallel shaken pH-auxostat.

Adaptive laboratory evolution (ALE) is a widely used microbial strain development and optimization method. ALE experiments, to select for faster-growing strains, are commonly performed as serial batch cultivations in shake flasks, serum bottles, or microtiter plates or as continuous cultivations in bioreactors on a laboratory scale. To combine the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting ALE experiments, a new Continuous parallel shaken pH-auxostat (CPA) was developed. The CPA consists of six autonomous parallel shaken cylindrical reactors, equipped with real-time pH control of the culture medium. The noninvasive pH measurement and control are realized by biocompatible pH sensor spots and a programmable pump module, to adjust the dilution rate of fresh medium for each reactor separately. Two different strains of the methylotrophic yeast Ogataea polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. The pH control was optimized by conducting a step-response experiment and obtaining tuned PI controller parameters by the Chien-Hrones-Reswick (CHR) PID tuning method. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, up to 4.8-fold faster-growing strains were selected. The increased specific maximum growth rates of the selected strains were confirmed in subsequent batch cultivations.

A genetic toolbox to empower Paracoccus pantotrophus DSM 2944 as a metabolically versatile SynBio chassis.

BACKGROUND: To contribute to the discovery of new microbial strains with metabolic and physiological robustness and develop them into successful chasses, Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium from the phylum Alphaproteobacteria and the family Rhodobacteraceae, was chosen. The strain possesses an innate ability to tolerate high salt concentrations. It utilizes diverse substrates, including cheap and renewable feedstocks, such as C1 and C2 compounds. Also, it can consume short-chain alkanes, predominately found in hydrocarbon-rich environments, making it a potential bioremediation agent. The demonstrated metabolic versatility, coupled with the synthesis of the biodegradable polymer polyhydroxyalkanoate, positions this microbial strain as a noteworthy candidate for advancing the principles of a circular bioeconomy. RESULTS: The study aims to follow the chassis roadmap, as depicted by Calero and Nikel, and de Lorenzo, to transform wild-type P. pantotrophus DSM 2944 into a proficient SynBio (Synthetic Biology) chassis. The initial findings highlight the antibiotic resistance profile of this prospective SynBio chassis. Subsequently, the best origin of replication (ori) was identified as RK2. In contrast, the non-replicative ori R6K was selected for the development of a suicide plasmid necessary for genome integration or gene deletion. Moreover, when assessing the most effective method for gene transfer, it was observed that conjugation had superior efficiency compared to electroporation, while transformation by heat shock was ineffective. Robust host fitness was demonstrated by stable plasmid maintenance, while standardized gene expression using an array of synthetic promoters could be shown. pEMG-based scarless gene deletion was successfully adapted, allowing gene deletion and integration. The successful integration of a gene cassette for terephthalic acid degradation is showcased. The resulting strain can grow on both monomers of polyethylene terephthalate (PET), with an increased growth rate achieved through adaptive laboratory evolution. CONCLUSION: The chassis roadmap for the development of P. pantotrophus DSM 2944 into a proficient SynBio chassis was implemented. The presented genetic toolkit allows genome editing and therewith the possibility to exploit Paracoccus for a myriad of applications.

Biotechnological production of polyphosphate from industrial wash water.

Phosphate is mined from phosphate rock, which is a limited resource on a human time scale. For a sustainable phosphate supply, strategies for efficient use and recycling of phosphate must be developed. A German chemical company produces annually wash water containing phosphate and other inorganic substances (e.g., sodium, potassium, sulfate, and chloride) at a ton scale. Chemical precipitation is mostly used for phosphate removal. In this study, a biotechnological process utilizing Saccharomyces cerevisiae to upcycle phosphate-containing wastewater into pure sodium polyphosphate in powder form was developed. The process comprises fermentation and downstream processing (polyphosphate yields: 25% and 36%, respectively). The polyphosphate quality was independent of the wash water composition. Polyphosphate with a purity of 23% molar ratio Na to Na, K, and Mg of > 90%, and with an average chain length of 12.5 phosphate subunits was produced. The upcycled polyphosphate can be reused compared to phosphate fertilizer in many different applications. Overall, the here developed process can contribute to truly slowing down phosphate mining and finally enable a sustainable utilization of phosphate. Thereby, the benefit of the process is the cascade use of phosphate, reducing the need for phosphate rock before the phosphate ends up in the soil and ultimately in the sea.

A novel membrane stirrer system enables foam-free biosurfactant production.

Bioreactors are the operative backbone, for example, for the production of biopharmaceuticals, biomaterials in tissue engineering, and sustainable substitutes for chemicals. Still, the Achilles' heel of bioreactors nowadays is the aeration which is based on intense stirring and gas sparging, yielding inherent drawbacks such as shear stress, foaming, and sterility concerns. We present the synergistic combination of simulations and experiments toward a membrane stirrer for the efficient bubble-free aeration of bioreactors. A digital twin of the bioreactor with an integrated membrane-module stirrer (MemStir) was developed with computational fluid dynamics (CFD) studies addressing the determination of fluid mixing, shear rates, and local oxygen concentration. Usability of the MemStir is shown in a foam-free recombinant production process of biosurfactants (rhamnolipids) from glucose with different strains of Pseudomonas putida KT2440 in a 3-L vessel and benchmarked against a regular aerated process. The MemStir delivered a maximal oxygen transfer rate (OTRmax ) of 175 mmol L-1 h-1 in completely foam-free cultivations. With a high space-time yield (STY) of 118 mgRL L-1 h-1 during a fed-batch fermentation, the effectiveness of the novel MemStir is demonstrated. Simulations show the generic value of the MemStir beyond biosurfactant production, for example, for animal cell cultivation.

Self-Assembly of Rhamnolipid Bioamphiphiles: Understanding the Structure-Property Relationship Using Small-Angle X-ray Scattering.

The structure-property relationship of rhamnolipids, RLs, well-known microbial bioamphiphiles (biosurfactants), is explored in detail by coupling cryogenic transmission electron microscopy (cryo-TEM) and both ex situ and in situ small-angle X-ray scattering (SAXS). The self-assembly of three RLs with reasoned variation of their molecular structure (RhaC10, RhaC10C10, and RhaRhaC10C10) and a rhamnose-free C10C10 fatty acid is studied in water as a function of pH. It is found that RhaC10 and RhaRhaC10C10 form micelles in a broad pH range and RhaC10C10 undergoes a micelle-to-vesicle transition from basic to acid pH occurring at pH 6.5. Modeling coupled to fitting SAXS data allows a good estimation of the hydrophobic core radius (or length), the hydrophilic shell thickness, the aggregation number, and the surface area per RL. The essentially micellar morphology found for RhaC10 and RhaRhaC10C10 and the micelle-to-vesicle transition found for RhaC10C10 are reasonably well explained by employing the packing parameter (PP) model, provided a good estimation of the surface area per RL. On the contrary, the PP model fails to explain the lamellar phase found for the protonated RhaRhaC10C10 at acidic pH. The lamellar phase can only be explained by values of the surface area per RL being counterintuitively small for a di-rhamnose group and folding of the C10C10 chain. These structural features are only possible for a change in the conformation of the di-rhamnose group between the alkaline and acidic pH.

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

RATIONALE: Methyl ketones are of interest for the application as biofuels. The fatty acid metabolism of different microbes has been rearranged to achieve a sustainable production of methyl ketones. The biofuel properties and possible further chemical modifications of these methyl ketones are influenced by their chain length, as well as their degree of saturation and the corresponding double bond position. METHODS: A method based on gas chromatography-electron ionization; mass spectrometry (GC-EI-MS) was used to determine the double bond position of methyl ketones. Derivatization using dimethyl disulfide (DMDS) and an iodine catalyst enabled the activation of the double bonds for selective fragmentation during electron ionization. The cleavage led to key fragments in the Orbitrap high-resolution mass spectrum and allowed the unequivocal localization of the double bond position of the respective monounsaturated methyl ketone. RESULTS: The double bond position of several medium chain length methyl ketones originating from the gram-negative bacterium Pseudomonas taiwanensis (P. taiwanensis) VLB120 was determined. The DMDS derivatives of methyl ketones can yield isobaric fragment ions for different possible double bond positions, which can be distinguished only using high-resolution MS. The double bond position of all methyl ketones deriving from P. taiwanensis VLB120 was the same, counting from the end of the aliphatic chain, and was determined as ω-7. CONCLUSIONS: The derivatization of medium chain length monounsaturated methyl ketones with DMDS allowed the determination of the corresponding double bond position via GC-EI-MS. High-resolution MS is needed to differentiate possible double bond positions that yield isobaric fragment ions.

Identification and quantification of biosurfactants produced by the marine bacterium Alcanivorax borkumensis by hyphenated techniques.

A novel biosurfactant was discovered to be synthesized by the marine bacterium Alcanivorax borkumensis in 1992. This bacterium is abundant in marine environments affected by oil spills, where it helps to degrade alkanes and, under such conditions, produces a glycine-glucolipid biosurfactant. The biosurfactant enhances the bacterium's attachment to oil droplets and facilitates the uptake of hydrocarbons. Due to its useful properties expected, there is interest in the biotechnological production of this biosurfactant. To support this effort analytically, a method combining reversed-phase high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) was developed, allowing the separation and identification of glycine-glucolipid congeners. Accurate mass, retention time, and characteristic fragmentation pattern were utilized for species assignment. In addition, charged-aerosol detection (CAD) was employed to enable absolute quantification without authentic standards. The methodology was used to investigate the glycine-glucolipid production by A. borkumensis SK2 using different carbon sources. Mass spectrometry allowed us to identify congeners with varying chain lengths (C6-C12) and degrees of unsaturation (0-1 double bonds) in the incorporated 3-hydroxy-alkanoic acids, some previously unknown. Quantification using CAD revealed that the titer was approximately twice as high when grown with hexadecane as with pyruvate (49 mg/L versus 22 mg/L). The main congener for both carbon sources was glc-40:0-gly, accounting for 64% with pyruvate and 85% with hexadecane as sole carbon source. With the here presented analytical suit, complex and varying glycolipids can be identified, characterized, and quantified, as here exemplarily shown for the interesting glycine-glucolipid of A. borkumensis.

Co-feeding enhances the yield of methyl ketones.

The biotechnological production of methyl ketones is a sustainable alternative to fossil-derived chemical production. To date, the best host for microbial production of methyl ketones is a genetically engineered Pseudomonas taiwanensis VLB120 ∆6 pProd strain, achieving yields of 101 mgg-1 on glucose in batch cultivations. For competitiveness with the petrochemical production pathway, however, higher yields are necessary. Co-feeding can improve the yield by fitting the carbon-to-energy ratio to the organism and the target product. In this work, we developed co-feeding strategies for P. taiwanensis VLB120 ∆6 pProd by combined metabolic modeling and experimental work. In a first step, we conducted flux balance analysis with an expanded genome-scale metabolic model of iJN1463 and found ethanol as the most promising among five cosubstrates. Next, we performed cultivations with ethanol and found the highest reported yield in batch production of methyl ketones with P. taiwanensis VLB120 to date, namely, 154 mg g-1 methyl ketones. However, ethanol is toxic to the cell, which reflects in a lower substrate consumption and lower product concentrations when compared to production on glucose. Hence, we propose cofeeding ethanol with glucose and find that, indeed, higher concentrations than in ethanol-fed cultivation (0.84 g Laq-1 with glucose and ethanol as opposed to 0.48 g Laq-1 with only ethanol) were achieved, with a yield of 85 mg g-1. In a last step, comparing experimental with computational results suggested the potential for improving the methyl ketone yield by fed-batch cultivation, in which cell growth and methyl ketone production are separated into two phases employing optimal ethanol to glucose ratios. ONE-SENTENCE SUMMARY: By combining computational and experimental work, we demonstrate that feeding ethanol in addition to glucose improves the yield of biotechnologically produced methyl ketones.

Enzymes for microplastic-free agricultural soils.

Plastic mulch films and biofertilizers (processed sewage sludge, compost or manure) have helped to increase crop yields. However, there is increasing evidence that these practices significantly contribute to microplastic contamination in agricultural soils, affecting biodiversity and soil health. Here, we draw attention to the use of hydrolase enzymes that depolymerize polyester-based plastics as a bioremediation technique for agricultural soils (in situ), biofertilizers and irrigation water (ex situ), and discuss the need for fully biodegradable plastic mulches. We also highlight the need for ecotoxicological assessment of the proposed approach and its effects on different soil organisms. Enzymes should be optimized to work effectively and efficiently under the conditions found in natural soils (typically, moist solids at an ambient temperature with low salinity). Such optimization is also necessary to ensure that already distressed ecosystems are not disrupted any further.

The metabolic potential of plastics as biotechnological carbon sources - Review and targets for the future.

The plastic crisis requires drastic measures, especially for the plastics' end-of-life. Mixed plastic fractions are currently difficult to recycle, but microbial metabolism might open new pathways. With new technologies for degradation of plastics to oligo- and monomers, these carbon sources can be used in biotechnology for the upcycling of plastic waste to valuable products, such as bioplastics and biosurfactants. We briefly summarize well-known monomer degradation pathways and computed their theoretical yields for industrially interesting products. With this information in hand, we calculated replacement scenarios of existing fossil-based synthesis routes for the same products. Thereby, we highlight fossil-based products for which plastic monomers might be attractive alternative carbon sources. Notably, not the highest yield of product on substrate of the biochemical route, but rather the (in-)efficiency of the petrochemical routes (i.e., carbon, energy use) determines the potential of biochemical plastic upcycling. Our results might serve as a guide for future metabolic engineering efforts towards a sustainable plastic economy.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida's diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators, which are of particular interest for metabolic engineering.

An integrated yeast-based process for cis,cis-muconic acid production.

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

Early prediction of decompensation (EPOD) score: Non-invasive determination of cirrhosis decompensation risk.

BACKGROUND & AIMS: Decompensation is a hallmark of disease progression in cirrhotic patients. Early detection of a phase transition from compensated cirrhosis to decompensation would enable targeted therapeutic interventions potentially extending life expectancy. This study aims to (a) identify the predictors of decompensation in a large, multicentric cohort of patients with compensated cirrhosis, (b) to build a reliable prognostic score for decompensation and (c) to evaluate the score in independent cohorts. METHODS: Decompensation was identified in electronic health records data from 6049 cirrhosis patients in the IBM Explorys database training cohort by diagnostic codes for variceal bleeding, encephalopathy, ascites, hepato-renal syndrome and/or jaundice. We identified predictors of clinical decompensation and developed a prognostic score using Cox regression analysis. The score was evaluated using the IBM Explorys database validation cohort (N = 17662), the Penn Medicine BioBank (N = 1326) and the UK Biobank (N = 317). RESULTS: The new Early Prediction of Decompensation (EPOD) score uses platelet count, albumin, and bilirubin concentration. It predicts decompensation during a 3-year follow-up in three validation cohorts with AUROCs of 0.69, 0.69 and 0.77, respectively, and outperforms the well-known MELD and Child-Pugh score in predicting decompensation. Furthermore, the EPOD score predicted the 3-year probability of decompensation. CONCLUSIONS: The EPOD score provides a prediction tool for the risk of decompensation in patients with cirrhosis that outperforms well-known cirrhosis scores. Since EPOD is based on three blood parameters, only, it provides maximal clinical feasibility at minimal costs.

Yeast-based production and in situ purification of acetaldehyde.

Acetaldehyde is a platform chemical with a production volume of more than 1 Mt/a, but is chiefly synthesized from petrochemical feedstocks. We propose the fermentative conversion of glucose towards acetaldehyde via genetically modified S. cerevisiae. This allows for ethanol-free bioactaldehyde production. Exploiting the high volatility of the product, in situ gas stripping in an aerated reactor is inevitable and crucial due to the respiratory toxicity effects of the acetaldehyde overproduction. We devise a lab-scale setup for the recovery of the product from the off-gas. Water was chosen as a suitable solvent and the Henry coefficient of acetaldehyde in water was validated experimentally. Based on an experimentally verified capture efficiency of 75%, an acetaldehyde production rate of over 100 mg/g/h was reached in 200 mL lab-scale fermentations.

Genome-scale model reconstruction of the methylotrophic yeast Ogataea polymorpha.

BACKGROUND: Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. RESULTS: To overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model. CONCLUSION: The developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.

Towards bio-upcycling of polyethylene terephthalate.

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films are hydrolyzed by a thermostable polyester hydrolase yielding highly pure terephthalate and ethylene glycol. The obtained hydrolysate is used directly as a feedstock for a terephthalate-degrading Pseudomonas umsongensis GO16, also evolved to efficiently metabolize ethylene glycol, to produce PHA. The strain is further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which are used as monomers for the chemo-catalytic synthesis of bio-PU. In short, a novel value-chain for PET upcycling is shown that circumvents the costly purification of PET monomers, adding technological flexibility to the global challenge of end-of-life management of plastics.

Engineering adipic acid metabolism in Pseudomonas putida.

Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant ß-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h-1, reaching a final biomass yield of 0.27 ± 0.00 gCDW gadipate-1. In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts.

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.