Armin Ehrenreich

Prp operon usually comprises prpB, prpC, prpD and prpE genes, encoding 2-methylisocitrate (2-MIC) lyase, 2-methylcitrate (2-MC) synthase, 2-MC dehydratase

Mucic acid (MA) is used as a chelating agent or as a building block for bio-based polymers. MA can be produced by regioselective oxidation of D-galacturonic acid (GA). Gluconobacter oxydans is known for the partial oxidation of various substrates via membrane-bound dehydrogenases. As the wild-type strain shows only low oxidation activity towards GA, the engineered multideletion strain G. oxydans BP9.1 pta-mGDH, overexpressing a membrane-bound glucose dehydrogenase from Pseudomonas taetrolens, was used in buffered whole-cell batch biotransformations with GA as the sole substrate. Initial cell-specific MA formation rates elevated with rising educt concentrations up to 63 g L⁻¹. At pH 4, full GA conversion was only achieved with an initial GA concentration of 10 g L⁻¹. Complete conversion of 94 g L⁻¹ of GA was achieved at pH 5 with 3.4 g L⁻¹ of G. oxydans BP9.1 pta-mGDH within 48 h, resulting in >100 g L⁻¹ of MA, corresponding to a yield of >99% (mol/mol). Isolation of MA (purity > 90%) was achieved after cell separation, followed by cooling crystallization and drying, with a yield of 94%. Complete, full-yield GA conversion using non-growing cells of engineered G. oxydans in simple phosphate buffer yielded high product concentrations and enabled simple, high-yield product isolation, thus resulting in cost-effective and sustainable bioproduction of MA.

Solventogenic Clostridium species can efficiently produce n -butanol and other valuable chemicals via acetone–butanol–ethanol (ABE) fermentation from plant-based feedstocks. For economic and ecological sustainability, cheap and abundant substrates such as lignocellulosic and hemicellulosic residues from agricultural or forestry side streams are preferable. Cereal brans, rich in hemicellulose, represent a promising substrate. However, for direct fermentation of this material, only low product titers are reported. In this study, we characterized the utilization of arabinoxylan, the main polysaccharide component of cereal bran, by the industrial ABE producer Clostridium saccharobutylicum DSM 13864 ^T and report inefficient degradation of the substrate. Supplementation with hemicellulolytic enzyme mixtures derived from the thermophilic organism Thermoclostridium stercorarium subsp. stercorarium DSM 8532 ^T significantly enhanced substrate utilization. The best improvement was achieved by the addition of the arabinofuranosidase Axh43A, which reduced the residual sugar content in the fermentation broth from 48.2 to 17.8%. Analysis of the remaining oligosaccharides after growth on arabinoxylan showed that C. saccharobutylicum cannot remove O -2 and O -3 α-L-arabinofuranosyl groups from double-substituted xyloses, creating a key bottleneck in arabinoxylan degradation that is overcome by Axh43A addition. Plasmid-based expression of Axh43A in C. saccharobutylicum DSM 13864 ^T replicated the enzymatic supplementation effects, confirming the enzyme’s role in overcoming this limitation. This underscores the potential of genetic engineering to enhance the valorization of lignocellulosic biomass in biotechnological fermentation processes. Key Points: C. saccharobutylicum DSM 13864 ^T insufficiently utilizes cereal arabinoxylan. Inability to cleave double-arabinosylated xylose moieties limits degradation. Heterologous expression of Axh43A strongly increases arabinoxylan utilization. Graphical

This study demonstrates the impact of a Structure Maintenance of Chromosome (SMC)-like Wadjet system on the horizontal gene transfer of plasmids by conjugation to a recipient that naturally containing such a system for the first time. A Clostridium cellulovorans mutant with dramatically improved efficiency to receive plasmid DNA by conjugation was isolated and sequenced. Three spontaneous chromosomal deletions included a type II restriction-modification system, a putative CRISPR system, and a cluster of ORFs named jetABCD encoding a putative Wadjet system. Since nearly nothing is known about the role of naturally occurring Wadjet systems in their native host bacteria, markerless chromosomal deletion of jetABCD in the C. cellulovorans wildtype strain 743B was achieved and the effect on conjugative plasmid uptake was studied. The transconjugation frequency of the jetABCD mutant was increased by about five orders of magnitude compared to wildtype C. cellulovorans recipient cells. Bioinformatic analysis of genome sequences of the Bacillota phylum revealed near-complete mutually exclusive possession of either plasmids < 40 kb or jetABCD genes, indicating high efficiency of Wadjet systems in small plasmid prevention in bacteria. Importantly, the implications of this study go beyond the case of C. cellulovorans . Our study demonstrates that the eradication of Wadjet systems can dramatically improve the uptake of recombinant plasmids and thereby enhance genetic engineering of bacterial strains of interest for biotechnological applications. Key points: • Native Wadjet system inhibits plasmid transfer by conjugation in C. cellulovorans • Deleting jetABCD increased plasmid uptake by about five orders of magnitude • Possession of Wadjet systems efficiently block plasmid maintenance in Bacillota Graphical

The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans . The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans . Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans . In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans -specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. Key points: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.

Cellobionic acid (CBA) can be obtained through the oxidation of cellobiose, the monomer of cellulose. CBA serves as a plant-based alternative to its stereoisomer lactobionic acid, which is used in the pharmaceutical, cosmetic, and food industries. Gluconobacter oxydans is a well-established whole-cell biocatalyst with membrane-bound dehydrogenases (mDH) for regio-specific oxidations. As G. oxydans wildtype cells show low cellobiose oxidation activities, the glucose mDH from Pseudomonas taetrolens was overexpressed in G. oxydans BP9, a multi mDH deletion strain. Whole-cell biotransformation studies were performed with resting cells of the engineered G. oxydans in stirred tank bioreactors. Initial biomass specific cellobionate formation rates increased with increasing cellobiose concentrations up to 190 g L⁻¹, and were constant until the solubility limit. The maximal volumetric CBA formation rates and the oxygen uptake rates increased linearly with the concentration of engineered G. oxydans. This enables the estimation of the maximum biocatalyst concentration limited by the maximum oxygen transfer rate of any bioreactor. Thus, 5.2 g L⁻¹ G. oxydans was sufficient to produce 502 g L⁻¹ CBA with >99% yield in a simple aerobic batch process. The highly concentrated CBA will reduce downstream processing costs considerably after cell separation.

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5–9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9–5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures.