Johannes Kabisch

A two‐compartment bioreactor system made of commercial parts for bioprocess scale‐down studies: Impact of oscillations on Bacillus subtilis fed‐batch cultivations

This study describes an advanced version of a two-compartment scale-down bioreactor that simulates inhomogeneities present in large-scale industrial bioreactors on the laboratory scale. The system is made of commercially available parts and is suitable for sterilization with steam. The scale-down bioreactor consists of a usual stirred tank bioreactor (STR) and a plug flow reactor (PFR) equipped with static mixer modules. The PFR module with a working volume of 1.2 L is equipped with five sample ports, and pH and dissolved oxygen (DO) sensors. The concept was applied using the non-sporulating Bacillus subtilis mutant strain AS3, characterized by a SpoIIGA gene knockout. In a fed-batch process with a constant feed rate, it is found that oscillating substrate and DO concentration led to diminished glucose uptake, ethanol formation and an altered amino acid synthesis. Sampling at the PFR module allowed the detection of dynamics at different concentrations of intermediates, such as pyruvic acid, lactic acid and amino acids. Results indicate that the carbon flux at excess glucose and low DO concentrations is shifted towards ethanol formation. As a result, the reduced carbon flux entering the tricarboxylic acid cycle is not sufficient to support amino acid synthesis following the oxaloacetic acid branch point.

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments.

Characterization and optimization of Bacillus subtilis ATCC 6051 as an expression host.

The genome sequence of Bacillus subtilis ATCC 6051 and its suitability as an expression host for recombinant protein production was determined. The comparison of this undomesticated wild type with the widely used laboratory strain B. subtilis 168 reveals a high degree of congruency between the two strains. Differences could only be detected on the level of point mutations or small insertions. B. subtilis ATCC 6051 shows none of the auxotrophies known for B. subtilis 168 and is able to produce polyketides. It exhibits better use of complex media and higher genomic stability through reduced natural competence. Consequently, B. subtilis ATCC 6051 was genetically modified to yield an optimized strain for the production of heterologously expressed proteins under control of an acetoin-inducible promoter.

Metabolic engineering of Bacillus subtilis for growth on overflow metabolites

BackgroundThe genome of the important industrial host Bacillus subtilis does not encode the glyoxylate shunt, which is necessary to utilize overflow metabolites, like acetate or acetoin, as carbon source. In this study, the operon encoding the isocitrate lyase (aceB) and malate synthase (aceA) from Bacillus licheniformis was transferred into the chromosome of B. subtilis. The resulting strain was examined in respect to growth characteristics and qualities as an expression host.ResultsOur results show that the modified B. subtilis strain is able to grow on the C2 compound acetate. A combined transcript, protein and metabolite analysis indicated a functional expression of the native glyoxylate shunt of B. lichenifomis in B. subtilis. This metabolically engineered strain revealed better growth behavior and an improved activity of an acetoin-controlled expression system.ConclusionsThe glyoxylate shunt of B. licheniformis can be functionally transferred to B. subtilis. This novel strain offers improved properties for industrial applications, such as growth on additional carbon sources and a greater robustness towards excess glucose feeding.

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.

Fueling the future with biomass: processes and pathways for a sustainable supply of hydrocarbon fuels and biogas

Global economic growth, wealth and security rely upon the availability of cheap, mostly fossil‐derived energy and chemical compounds. The replacement by sustainable resources is widely discussed. However, the current state of biotechnological processes usually restricts them to be used as a true alternative in terms of economic feasibility and even sustainability. Among the rare examples of bioprocesses applied for the energetic use of biomass are biogas and bioethanol production. Usually, these processes lack in efficiency and they cannot be operated without the support of legislation. Although they represent a first step towards a greater share of bio‐based processes for energy provision, there is no doubt that tremendous improvements in strain and process development, feedstock and process flexibility as well as in the integration of these processes into broader supply and production networks, in this review called smart bioproduction grids, are required to make them economically attractive, robust enough, and wider acceptance by society. All this requires an interdisciplinary approach, which includes the use of residues in closed carbon cycles and issues concerning the process safety. This short review aims to depict some of the promising strategies to achieve an improved process performance as a basis for future application.

An optimized technique for rapid genome modifications of Bacillus subtilis

The transformation efficiency of naturally competent Bacillus subtilis cells can be significantly increased using β recombinase binding sequences, as revealed by the results of this study. Plasmids containing different variations of these so called six-site-marker-cassettes were investigated. Furthermore, an optimized protocol for knock-out or knock-in mutations combining the Cre-lox-system and the six-sites is presented, which can be used for multiple genome modifications of B. subtilis.

Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria

Summary Mobile genomic islands distribute functional traits between microbes and habitats, yet it remains unclear how their proteins adapt to new environments. Here we used a comparative phylogenomic and proteomic approach to show that the marine bacterium Pseudoalteromonas haloplanktis ANT/505 acquired a genomic island with a functional pathway for pectin catabolism. Bioinformatics and biochemical experiments revealed that this pathway encodes a series of carbohydrate‐active enzymes including two multi‐modular pectate lyases, PelA and PelB. PelA is a large enzyme with a polysaccharide lyase family 1 (PL1) domain and a carbohydrate esterase family 8 domain, and PelB contains a PL1 domain and two carbohydrate‐binding domains of family 13. Comparative phylogenomic analyses indicate that the pathway was most likely acquired from terrestrial microbes, yet we observed multi‐modular orthologues only in marine bacteria. Proteomic experiments showed that P. haloplanktis ANT/505 secretes both pectate lyases into the environment in the presence of pectin. These multi‐modular enzymes may therefore represent a marine innovation that enhances physical interaction with pectins to reduce loss of substrate and enzymes by diffusion. Our results revealed that marine bacteria can catabolize pectin, and highlight enzyme fusion as a potential adaptation that may facilitate microbial consumption of polymeric substrates in aquatic environments.

Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor

The introduction of a three-enzyme cascade (comprising a cyclohexanone monooxygenase (CHMO), an alcohol dehydrogenase (ADH) and a lipase (CAL-A)) for the production of oligo-ε-caprolactone provided self-sufficiency with respect to NADPH-cofactor regeneration and reduced inhibiting effects on the central CHMO enzyme. For further optimization of cofactor regeneration, now a co-expression of CHMO and ADH in E. coli using a Duet™ vector was performed. This led to higher conversion values of the substrate cyclohexanol in whole-cell biocatalysis compared to an expression of both enzymes from two separate plasmids. Furthermore, a more advantageous balance of expression levels between the partial cascade enzymes was achieved via engineering of the ribosome binding site. This contributed to an even faster cofactor regeneration rate.

Draft Genome Sequence of Cutaneotrichosporon curvatus DSM 101032 (Formerly Cryptococcus curvatus), an Oleaginous Yeast Producing Polyunsaturated Fatty Acids

ABSTRACT Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species.