7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

PD Dr.

Styrene oxide Isomerase, a catalytic efficient enzyme acting as peroxidase, peroxygenase, and isomerase

Biotechnology & Synthetic Microbiology

Styrene oxide isomerase (SOI), an integral membrane protein in the upper styrene degradation pathway, which catalyses a rare chemistry of Meinwald rearrangement in converting epoxides into aldehyde by 1,2-hydride/methyl shift1. The stereo- and regio-selective conversion by SOI makes it appealing for biocatalysis. But the molecular mechanism of SOI was not studied so far. Our recent collaborative work on SOI, and the cryo-EM structure showed that the enzyme possesses heme as a cofactor and active site. Hence, the study aimed at understanding the mechanism as well as to uncover the potential of heme-based isomerase.This study included screening of eight SOIs from different classes of bacteria and fungi, where all the enzymes were purified to its homogeneity and screened for specific activity of its natural substrate (R/S)-styrene oxide. Out of all the SOIs tested, the enzyme from Zavarzinia compransoris Z-1155 (ZcSOI) stood out in protein yield and activity which was further characterized for its stability and kinetics. The enzyme showed a turnover frequency of about 250 s-1 with a catalytic efficiency of 2.5×106 s-1M-1. The mutation and reduction study revealed that both tyrosine and heme are co-ordinating the substrate and required for effective catalysis. Owing to the fact that heme is the active site and reducible, the study was conducted to utilize ZcSOI as peroxygenase. The ABTS assay proved that ZcSOI can accepts H2O2 with kcat of 2.3 s-1, hence the enzyme was used to achieve both epoxidation and isomerization of styrene to produce phenylacetaldehyde. The biotransformation of ZcSOI in presence of H2O2 showed about 10-15% conversion of styrene to phenylacetaldehyde after 2h, indicating that the enzyme functions as peroxygenases in addition to isomerase. This study opened up the new regime in the biocatalysis of industrially high valuable product production with single enzyme.Xin, R. P., See, W. W. L., Yun, H., Li, X. R. &amp; Li, Z. Enzyme-Catalyzed Meinwald Rearrangement with an Unusual Regioselective and Stereospecific 1,2-Methyl Shift. Angewandte Chemie-International Edition 61 (2022). https://doi.org/10.1002/anie.202204889

Oral Presentation

Reprogramming a protein ligase for genetic code expansion

Nature employs a limited and conservative set of amino acids to synthesize proteins. The ability to genetically encode an extended set of building blocks can be used in diverse applications, including approaches to study and control protein function as well as to design novel therapeutics. Non-natural amino acids (NAA) are co-translationally incorporated into proteins by orthogonal pairs consisting of aminoacyl-tRNA synthetase and cognate tRNA. However, the current repertoire can neither display the full natural diversity of NAAs and is especially limited for backbone modifications. Excitingly, we now succeeded in reverse engineering a protein ligase into a new-to-nature tRNA synthetase (aaRS-β) that can load tRNA with β-amino acids (βaa). Additionally, we derived a variant aarRS-α accepting α-amino acids that cannot be integrated into the genetic code by any other means including adavanced glycation endproducts (AGEs). Our discovery lays the foundation for understanding the role of AGEs in the development and pathogenesis of major diseases such as diabetes and atherosclerosis as well as in the process of aging. At the same time the co-translational incorporation of βaa into the nascent chain will allow the cost-effective production of antimicrobial peptides with high protease stability and increased activity against multidrug-resistant pathogens.

Upcycling of PBAT to 4-coumaric acid by combined enzymatic hydrolysis and monomer conversion with engineered Pseudomonas taiwanensis

Bioplastics present a very promising approach to help solving the plastic crisis. However, it is crucial to establish efficient recycling strategies for these materials to ensure circularity of the plastic economy. Microbial upcycling is an important option for the end of life of more complex polymers or blends as well as mixed plastic waste. We engineered the metabolism of the PBAT monomer 1,4-butanediol in an aromatic-producing Pseudomonas taiwanensis strain with the ultimate goal of upcycling PBAT to valuable aromatic compounds. Growth on 1,4-butanediol was achieved via adaptive laboratory evolution. Subsequent genome sequencing and reverse engineering revealed a mutation upstream of PVLB_10545 encoding a putative ethanol dehydrogenase as essential for growth on 1,4-butanediol. This dehydrogenase likely oxidizes 1,4-butanediol to 4-hydroxybutyrate thus substituting for PedE which is present in P. putida but not P. taiwanensis (1). Adding two further mutations enhanced growth on 1,4-butanediol to ALE-like growth. One of these mutations resulted in an amino acid substitution in a LysR family transcriptional regulator (PVLB_12690), the homologue of which was also mutated in P. putida evolved on 1,4-butanediol (1). After successful reverse engineering, the strain was utilized in combination with two other strains growing on the PBAT monomers adipate and terephthalate and the PBAT hydrolyzing enzyme HiC for a process intensification, in which PBAT was hydrolyzed and the monomers were upcycled to 4-coumarate within one reactor.

Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling

Vitamin D (VitD) deficiency, which is associated with several acute and chronic diseases, is prevalent in populations worldwide and has inspired researchers to develop strategies to increase serum VitD levels via VitD-fortified food and supplementation.1 In this context, the physiologically active form in the human body, 25-OH-VitD3, has been shown to be significantly more effective as a supplement.2 Enzymatic systems to produce this circulating form of VitD3 typically use oxygen-dependent P450 monooxygenases, but these enzymes often produce undesired by-products and require costly NAD(P)H.3As an alternative, we have developed a VitD3-hydroxylating whole-cell system using a water-dependent steroid C25 dehydrogenase (S25DH) from denitrifying Sterolibacterium denitrificans in the related Thauera aromatica K172 as heterologous production host. Using resting cells of the S25DH-producing whole-cell biocatalyst at high densities, potassium ferricyanide as electron acceptor, 2-hydroxypropyl-β-cyclodextrin (HPCD) as solubiliser and ethanol as co-solvent, we demonstrated the formation of up to 1.85 g L-1 within 50 h at a 100 mL scale, making this the highest published 25-OH-VitD3 titer achieved to date. In addition, no common by-products such as the di-hydroxlated 1α,25-di-OH-VitD3 were formed. We also showed that the VitD3-solubilising HPCD can be recycled and used for up to 10 consecutive conversion cycles without a significant loss of quality or quantity, making the entire process economically feasible.41 Holick, M. et al., Rev. Endocr. Metab. Disord. 18, 153-165 (2017)2 Bouillon, R. and Quesada-Gomez, J., J. Steroid Biochem. Mol. Biol. 228, 106248 (2023)3 Wang, Z. et al., Biotechnol. Biofuels 15, 109 (2022)4 Kosian, D. et al., Microb. Cell Fact. 23, 30 (2024)

Photosystem I-Powered Hydrogen Production in Phormidium lacuna: A Green Solution for Climate-Friendly Fuel

Ongoing global climate change, driven by greenhouse gas emissions and fossil resource exploitation, increases demand for renewable energy sources. Especially green H2 is an emerging emission-free alternative. Biological solar H2 is produced as a byproduct of photosynthesis by green algae and cyanobacteria via hydrogenases. H2 production is electron-dependent and limited by the electron availability. However, recent studies have shown that coupling of the hydrogenase to the Photosystem I (PSI) ensures a spatial proximity to the FeS cluster of PSI subunit PsaC so that the electron flow can be channelled towards H2 production1,2.The goal of this study is to establish efficient solar H2 production in the filamentous and biofilm forming cyanobacterium Phormidium lacuna.To achieve this, different types of hydrogenases from various organisms are coupled to a full-length or truncated version of PSI subunit PsaD from P. lacuna and genomically integrated via natural transformation3 into P. lacuna. H2 production of the mutants are then measured via gas chromatography or H2 microsensor relative to the wild type P. lacuna. Via biochemical analyses the coupling of the hydrogenases to PsaD are verified.Preliminary results from the H2 measurements of a coupled NiFe-hydrogenase to a full-length version of PsaD, show no increase in H2 production, although western blot analyses using an anti-PsaD antibody verify the presence of PsaD chimeras. Sequencing results of the mutants also show a correctly integrated hydrogenase gene.Based on the results a truncated PsaD is coupled to various hydrogenases which might ensure a closer proximity to the FeS cluster and possibly a higher H2 production yield respectively. Two possible truncated PsaD versions as well as two hydrogenases from Chlamydomonas reinhardtii and Cupriavidus necator are currently investigated.Appel, J. et al. Nat Energy 5, 458–467; DOI: 10.1038/s41560-020-0609-6 (2020).Kanygin, A. et al. Energy Environ. Sci. 13, 2903–2914; DOI: 10.1039/C9EE03859K (2020).Nies, F. et al. PLOS ONE 15, e0234440; DOI: 10.1371/journal.pone.0234440 (2020).

Jun.-Prof. Dr.

Influence of expression vector design on the simultaneous heterologous production of cytochromes and rusticyanin from Acidithiobacillus ferrooxidans in Escherichia coli and Vibrio natriegens

Heterologous protein production is often employed to accelerate protein characterization and interaction studies. This can be especially relevant for proteins from acidophiles since these microorganisms only produce low biomass. However, the production of proteins from acidophiles in neutrophilic hosts, e.g. Escherichia coli, often poses a great challenge. Correct protein folding and co-factor integration need to be achieved despite the difference in extracellular and periplasmic pH. The Gram-negative acidophile Acidithiobacillus ferrooxidans possesses the remarkable ability to transfer electrons through its outer membrane to exocellular iron. This requires an electron transfer chain that spans both membranes and the periplasm and consists of three c-type cytochromes and the copper redox protein rusticyanin. We aim to investigate the functionality of this exocellular electron transfer chain and reconstructed it in several E. coli expression strains and Vibrio natriegens Vmax X2. We previously reported that E. coli and V. natriegens were able to express the genes from a pET16b-based vector construct and translocated all proteins to the correct cell compartment. Recent experiments focused on the impact of the expression vector design on the production of our four proteins of interest. The genes were encoded on a pRSF-1b vector, significantly reducing plasmid size compared to the pET16b-based construct. Additionally, we investigated a pCOLADuet-1-based construct where the inner membrane c-type cytochrome Cyc2 is under the control of a separate T7 promoter. The use of the pCOLADuet-1-based construct improved the production of Cyc2 in V. natriegens, while the viability of the E. coli hosts was reduced. This indicates that V. natriegens might be an interesting chassis for the production of integral membrane proteins. Additionally, V. natriegens possess a distinct advantage over E. coli for the production of c-type cytochromes. It does not require the co-expression of the cytochrome c maturation genes from an additional plasmid under aerobic conditions.

Oral Session
Biotechnology & Synthetic Microbiology 2

Termin

Chair

Thema

Programm

Oral SessionBiotechnology & Synthetic Microbiology 2

Termin

Chair

Thema

Programm

Oral Session
Biotechnology & Synthetic Microbiology 2