Biology subjects

Ballerstedt, H.

Publications and source records attributed to Ballerstedt, H..

5 recordsLinked to original sources

bioRxiv2026

Engineering Pseudomonas putida KT2440 for open-loop upcycling of mixed plastics

Current mechanical and chemical recycling strategies address less than 10% of global plastic waste, necessitating alternative valorization routes. Biological upcycling via enzymatic depolymerization combined with microbial conversion of the resulting monomers offers a promising pathway to transform mixed plastic waste into valuable alternatives. Here, we employed a single engineered Pseudomonas putida KT2440 for simultaneous co-utilization of five plastic monomers including ethylene glycol, terephthalic acid, adipic acid, 1,4-butanediol, and L-lactic acid, which can be derived from enzymatic hydrolysis of polyethylene terephthalate (PET), polybutylene adipate-co-terephthalate (PBAT), polyester-polyurethanes (PUs), and polylactic acid (PLA). Continuous fermentation over 21 days with alternating mixed-monomer feeds achieved steady state growth and complete substrate depletion, yielding adaptive mutations that informed iterative strain improvement. Further engineering enabled the biosynthesis of (R)-3-hydroxybutyrate (R-3HB), and 0.70 g L-1 R-3HB was produced directly from enzymatic hydrolysates of blended PET, PBAT, and TPU. These results establish a viable bio-based approach for upcycling realistic mixed plastics into value-added bioproducts.

microbiology↗

bioRxiv2025

Optimization of a T7 RNA polymerase expression system for high-yield protein production in Cupriavidus necator H16

Many chemical manufacturing routes are being replaced with enzymatic processes to improve sustainability. In biocatalysis, enzyme production is often the main bottleneck. Bacterial proteins are frequently produced using the workhorse E. coli BL21(DE3) and its derivatives. However, other bacteria with beneficial characteristics can also be engineered for this purpose. Cupriavidus necator H16 (C. necator), for example, is a Gram-negative bacterium well-known for its chemolithoautotrophic metabolism and high polyhydroxybutyrate (PHB) accumulation. Previous studies have demonstrated protein production without inclusion body formation, which is one of the main challenges when producing enzymes in E. coli. Nevertheless, high-yield protein production in C. necator remains an understudied field. Here, we investigated the bottlenecks limiting protein production in C. necator. We optimized a T7 RNA polymerase genetic system and quantified the impact of several genetic elements such as T7 promoter, RBS strength and codon usage towards GFP production. Codon usage was the main factor limiting protein production in C. necator. Tuning the RBS strength and selecting a different T7 promoter strongly influenced expression leakiness. We then produced the ene-reductase YqjM from Bacillus subtilis in C. necator, analyzing the performance of the engineered C. necator strain and our previously developed pMVRha expression plasmid. C. necator produced high amounts of soluble protein and outperformed the gold standard, E. coli BL21(DE3) in producing FMN-loaded enzyme. This result highlights the potential of non-model bacteria to achieve high-yield enzyme production and promote the transition to biocatalysis-driven chemical synthesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/688765v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1a6584corg.highwire.dtl.DTLVardef@1ef094dorg.highwire.dtl.DTLVardef@4bb4c3org.highwire.dtl.DTLVardef@489c85_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Using Cupriavidus necator H16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria

Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting non-model bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in non-model bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO2 and hydrogen. However, its low electroporation efficiency hinders the high-throughput genetic modifications required to develop C. necator into an industrially relevant host organism. First, we propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of non-model bacteria. TABLE OF CONTENT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/596136v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@dfc76aorg.highwire.dtl.DTLVardef@19d130forg.highwire.dtl.DTLVardef@14e6ed3org.highwire.dtl.DTLVardef@4e0024_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Synthetic bacterial consortium for degradation of plastic pyrolysis oil waste

The plasic crisis is ominipresent, from littering macroplastic to reports that document plastic in every niche of this planet, including the human body. In order to achieve higher recycling quotas, especially of mixed plastic waste, pyrolysis seems to be a viable option. However, depending on the process parameters, plastic pyrolysis oil waste is encountered, which is difficult to valorize, due to the enormous spread of the molecules included. To reduce the molecular heterogeneity, we here artificially compounded, monitored, and optimized the performance of a bacterial consortium, which has the ability to tolerate organic pollutants and use them as energy and carbon sources for their own metabolic activity. The primary constituents of the here used plastic pyrolysis oil waste (PPOW) were alkanes and {varepsilon}-caprolactam. The bacterial community exhibited noteworthy efficacy in eliminating alkanes of diverse chain lengths ranging from 71% to 100%. Additionally, within 7-days, the microbial community demonstrated a removal efficiency surpassing 50% for various aromatic hydrocarbons, along with complete eradication of {varepsilon}-caprolactam and naphthalene. Besides, a back-propagation (BP) neural network method is applied to evaluate O2 consumption as a measure of microbial activity. The insights gained were used to build a model, which is able to predict O2 depletion in long-time experiments and other experimental conditions. The results are discussed in the context of a developing (open) circular plastic economy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/590079v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@628f68org.highwire.dtl.DTLVardef@b5274eorg.highwire.dtl.DTLVardef@1278ceaorg.highwire.dtl.DTLVardef@194887a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightO_LISynthetic bacterial communities are used to remove plastic hydrolysis oil waste C_LIO_LIThe optimized biphase reaction system can remove the majority of pollutants C_LIO_LIThe biodegradation process can be monitored in a real-time bioprocess software C_LIO_LINeural network techniques are used to model and predict the removal process C_LI

bioengineering↗

bioRxiv2019

Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficient Pseudomonas putida KT2440

Pseudomonas putida KT2440 is known to metabolise glycerol via glycerol-3-phosphate using glycerol kinase an enzyme previously described as critical for glycerol metabolism (1). However, when glycerol kinase was knocked out in P. putida KT2440 it retained the ability to use glycerol as the sole carbon source, albeit with a much-extended lag period and 2 fold lower final biomass compared to the wild type strain. A metabolomic study identified glycerate as a major and the most abundant intermediate in glycerol metabolism in this mutated strain with levels 21-fold higher than wild type. Erythrose-4-phosphate was detected in the mutant strain, but not in the wild type strain. Glyceraldehyde and glycraldehyde-3-phosphate were detected at similar levels in the mutant strain and the wild type. Transcriptomic studies identified 191 genes that were more than 2-fold upregulated in the mutant compared to the wild type and 175 that were down regulated. The genes involved in short chain length fatty acid metabolism were highly upregulated in the mutant strain. The genes encoding 3-hydroxybutyrate dehydrogenase were 5.8-fold upregulated and thus the gene was cloned, expressed and purified to reveal it can act on glyceraldehyde but not glycerol as a substrate.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC=\"FIGDIR/small/567230_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@1b5298eorg.highwire.dtl.DTLVardef@bb4841org.highwire.dtl.DTLVardef@184de3forg.highwire.dtl.DTLVardef@1b2e2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗