Establishment of novel riboswitch -based regulatory system useful in metabolic engineering of B. methanolicus

Establishment of novel riboswitch -based regulatory system useful in metabolic engineering of B. methanolicus

Marta Irla and Sigrid Hakvåg from NTNU have characterised selected regulatory elements: promoters and lysine riboswitch in methylotrophic and thermotolerant Bacillus methanolicus. A putative lysine riboswitch in the genome of B. methanolicus was detected based on the RNA-seq data, and its functionality was confirmed through analysis of changes in sfGFP fluorescence with and without the presence of L-lysine. The response of the lysine riboswitch to changes in intra- and extracellular concentrations of L-lysine B. methanolicus demonstrates its feasibility for potential use in metabolic engineering for metabolic flux control or as a lysine sensor. Furthermore, ten novel promoters were predicted based on RNA-seq data and here experimentally shown to be functional in B. methanolicus. The promoters differ in their strength, ranging from 0.1- to 5.9-fold of the activity of the control promoter. A system for regulation of gene expression based on the B. subtilis-derived pbuE riboswitch was created and shown to be functional in B. methanolicus. Lastly, the genetic tools were used in combination, presenting the potential for creation of novel genetic systems for control of gene expression in B. methanolicus.

 

Irla M, Hakvåg S, Brautaset T (2021) Developing a riboswitch-mediated regulatory system for metabolic flux control in thermophilic Bacillus methanolicus. Int J Mol Sci. 22(9):4686. doi: 10.3390/ijms22094686.

 

Combining crystal structure with CRISPRi analyses to study methylotrophy

Combining crystal structure with CRISPRi analyses to study methylotrophy

Kerstin Schultenkämper and Marina Gil López from the C1Pro teams of Bielefeld Unversity and NTNU characterized the fructose bisphosphate aldolases of B. methanolicus. CRISPRi gene repression revealed a genetic compensation mechanism, i.e, the plasmid gene fbaP was upregulated when the chromosomal gene fbaC was repressed and vice versa. The crystal structures of both enzymes were solved by collaborators from Freiburg and Lorraine universities. Site-directed mutagenesis of the fructose bisphoshate binding site changed the major glycolytic enzyme to a gluconeogenic enzyme. This insight into methylotrophy may prove valuable for methanol-based production.

 

Schultenkämper K, Gütle DD, Lopez MG, Keller LB, Zhang L, Einsle O, Jacquot J-P, Wendisch VF (2021) Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front. Microbiol. 12: 669220. doi: 10.3389/fmicb.2021.669220

 

Characterization of the response of B. methanolicus to 5-aminovalerate

Characterization of the response of B. methanolicus to 5-aminovalerate

Carsten Haupka and Luciana Brito from the C1Pro teams of Bielefeld Unversity and NTNU characterized the response of B. methanolicus to 5-aminovalerate. B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4-C6) amino acids, diamines and dicarboxylic acids. A combined adaptive laboratory (ALE), omics, and genetics approach helped elucidating the physiological response of B. methanolicus to 5-aminovalerate and will guide future strain development for its production from methanol.

 

Haupka, C, Brito LF, Busche T, Wibberg D, Wendisch VF (2021) Genomic and transcriptomic investigation of the physiological response of the methylotroph Bacillus methanolicus to 5-aminovalerate. Front. Microbiol. 12: 664598. doi: 10.3389/fmicb.2021.664598

 

Dissemination of C1Pro activities in Teutolab Biotechnologie Online

Dissemination of C1Pro activities in Teutolab Biotechnologie Online

January, 2021: As part of our dissemination activities, Kerstin Schultenkämper and Tatjana Walter explained their research to pupils in Teutolab-Biotechnologie-Online: The video covers ERACoBioTech projects C1Pro and INDIE.

 

Flux enforcement for fermentative production of 5-aminovalerate and glutarate by Corynebacterium glutamicum

Flux enforcement for fermentative production of 5-aminovalerate and glutarate by Corynebacterium glutamicum

Carsten Haupka, Baudoin Délepine, Marta Irla, Stéphanie Heux and Volker F. Wendisch from the C1Pro teams of INSA, NTNU and UNIBI experimentally determined the efficiencies of flux enforcement with either one or two coupling sites for production of glutarate via 5-aminovalerate. Flux enforcement may be suitable for methanol-based 5-aminovalerate production by B. methanolicus once gene deletions are possible in this bacterium.

 

Haupka C, Délepine B, Irla M, Heux S, Wendisch VF (2020) Flux enforcement for fermentative production of 5-aminovalerate and glutarate by Corynebacterium glutamicum. Catalysts 10: 1065. doi: https://doi.org/10.3390/catal10091065

 

Methanol-dependent growth in non-methylotrophic bacterium

Methanol-dependent growth in non-methylotrophic bacterium

Based on their fruitful collaboration on methylotrophic Bacillus methanolicus the C1Pro teams from UNIBI,  NTNU and INSA constructed and characterized a methanol-essential Corynebacterium glutamicum strain. Adaptive laboratory evolution (ALE) selected for faster methanol-dependent growth. Of the mutations identified by genome sequencing, three were causal. They (1) increased expression of methylotrophic transgenes, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. The results may help to further our cross-species understanding of using methanol for growth and production.

 

Hennig G, Haupka C, Brito LF, Rücker C, Cahorea E, Heux S and Wendisch VF (2020) Methanol-essential growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution overcomes limitation due to methanethiol assimilation pathway. Int. J. Mol. Sci. 21:3617. doi: 10.1186/s12866-020-01750-6

 

Elucidation of metabolic landscape of B. methanolicus

Elucidation of metabolic landscape of B. methanolicus

The C1Pro teams of INSA and Bielefeld University performed a 13C metabolic flux analysis of B. methanolicus growing on methanol, mannitol, and arabitol to compare the associated metabolic states. With the exception of the pentose phosphate pathway, our studies demonstrated that non-methylotrophic growth on mannitol and on arabitol share the same features despite their associated different growth rates. Notably, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Importantly, a clearly different metabolic state was found for growth on methanol. In particular, we provided new insights related to the utilization of cyclic ribulose monophosphate (RuMP) versus linear dissimilation pathways and between the RuMP cycle variants.

Further dissemination: CeBiTec Quarterly  

Delépine B, López MG, Carnicer M, Vicente CM, Wendisch VF, Heux S (2020) Charting the metabolic landscape of the facultative methylotroph Bacillus methanolicus. mSystems 5:e00745-20. https://doi.org/10.1128/mSystems.00745-20

 

Dissemination of our research in CeBiTec-Quaterly

Dissemination of our research in CeBiTec-Quaterly

The information about joint publication of NTNU and UNIBI research groups was recently announced in the Spring 2020 edition of CeBiTec-Quaterly, a newsletter that reports about recent developments, talks, conferences, further events, and news at the Center for Biotechnology at Bielefeld University. The Drejer et al. paper describes establishment of methanol-based acetoin production by metabolically engineered Bacillus methanolicus and was published in Green Chemistry doi: 10.1039/C9GC03950C

 

Transaldolase characterization

Transaldolase characterization

The C1Pro teams of Bielefeld University and NTNU have characterized transaldolase from Bacillus methanolicus. While it is well established that B. methanolicus can use the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle, this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.

 

Pfeifenschneider J, Markert B, Stolzenberger J, Brauatset T, Wendisch VF (2020) Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle. BMC Microbiology 20: 63. doi: 10.1186/s12866-020-01750-6

 

Review on biotechnological production of amino acid derivatives

Review on biotechnological production of amino acid derivatives

Volker F. Wendisch from the C1Pro team of Bielefeld University together with Melanie Mindt, Tatjana Walter and Pierre Kugler reviewed microbial production of compounds that can be derived from amines and amino acids by N-functionalization. Currently, these fermentative processes, for example N-hydroxylation of L-pipecolic acid, rely on sugars as carbon sources. However, it is conceivable to transfer these processes to methanol-based production with B. methanolicus.

 

Mindt M, Walter T, Kugler P and Wendisch VF (2020) Microbial engineering for production of N-functionalized amino acids and amines. Biotechnol. J. in press. doi: https://doi.org/10.1002/biot.201900451

 

Review on metabolic engineering with CRISPR interference

Review on metabolic engineering with CRISPR interference

Kerstin Schultenkämper, Luciana F. Brito and Volker F. Wendisch from the C1Pro teams of Bielefeld University and NTNU reviewed how the latest developments of CRISPR interference (CRISPRi) improved metabolic engineering. They provide an up-to-date overview of CRISPRi applications used to improve white biotech processes and forecast the vast potential of this genetic perturbation technique for enabling sought after bioprocesses such as methanol-based production with B. methanolicus.

 

Schultenkämper K, Brito LF and Wendisch VF (2020) Impact of CRISPR interference on strain development in biotechnology. Biotechnol. Appl. Biochem. 67: 7-21. doi: https://doi.org/10.1002/bab.1901

Methanol-based acetoin production

Methanol-based acetoin production

Based on their fruitful collaboration on 5-aminovalerate production NTNU and UNIBI established further common projects. A study on methanol-based acetoin production by B. methanolicus led by Marta Irla from NTNU has just been accepted for publication by Green Chemistry, a high impact journal focusing on the development of alternative sustainable technologies.

 

Drejer EB, Chan D, Haupka C, Wendisch VF, Brautaset T and Irla M (2019) Methanol-based acetoin production by genetically engineered Bacillus methanolicus. Green Chem. 22:788-802. doi: 10.1039/C9GC03950C

Advances in metabolic engineering

Advances in metabolic engineering

Volker F. Wendisch from the C1Pro team of Bielefeld University reviewed recent advances of metabolic engineering for amino acid production. Prospects driven by technological push and market pull altering this field are exemplified for specialty proteinogenic amino acids, cyclic amino acids, omega-amino acids, and amino acids functionalized by hydroxylation, halogenation and N-methylation. Alternative feedstocks including methanol are covered as well.


Wendisch VF (2020) Metabolic engineering advances and prospects for amino acid production. Metab. Eng. 58:17-34. doi: 10.1016/j.ymben.2019.03.008 

 

Review on synthetic microbial consortia

Review on synthetic microbial consortia

Volker F. Wendisch from the C1Pro team of Bielefeld University together with Elvira Sgobba reviewed how synthetic microbial consortia extend the scope of metabolic engineering by division of labor between subpopulations e.g. to enable access to more complex feedstocks and biosynthetic routes. Although strain and process control strategies for specifically designed synthetic consortia still are in their infancy, this concept may hold promise also for methanol-based production of value-added compounds.


Sgobba E and Wendisch VF (2020) Synthetic microbial consortia for small molecule production. Curr. Opin. Biotechnol. 62:72-79. doi: https://doi.org/10.1016/j.copbio.2019.09.011

 

Characterization of arabitol utilization

Characterization of arabitol utilization

The C1Pro teams of Bielefeld University and NTNU have characterized an operon for D-arabitol utilization of B. methanolicus. The operon was identified by RNAseq and qRT-PCR analysis and functional insight was gained from complementation experiments of an arabitol negative Corynebacterium glutamicum strain. Enzyme activity measurements showed that B. methanolicus possesses arabitol phosphate dehydrogenase, which may constitute an D-arabitol catabolic pathway together with the putative PTS AtlABC. D-arabitol is one of the four carbon sources supporting growth of the facultative methylotroph B. methanolicus.


López MG, Irla M, Brito LF and Wendisch VF (2019) Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus. Front. Microbiol. 10:1725. doi: 10.3389/fmicb.2019.01725 

 

Development of CRISPR interference system

Development of CRISPR interference system

A CRISPR interference system has been developed for B. methanolicus by the C1Pro teams of Bielefeld University and NTNU. For the first time, it is possible to perform gene knockdown experiments with this bacterium. The versatility of this tool to study the importance of individual genes experimentally has been showcased with respect to sporulation, hydrogen peroxide detoxification, and mannitol catabolism. Gene repression of spo0A confirmed its role in sporulation and biofilm formation. Furthermore it was shown that hydrogen peroxide detoxification required katA and that growth with mannitol as carbon source depended on mtlD. Kerstin Schultenkämper won the best poster award at the conference "BACELL 2019" (link=http://subtiwiki.uni-goettingen.de/uploads/BACELL_2019.pdf) held Apr 09-10, 2019, in Ljubljana, Slovenia.


Schultenkämper K, Brito LF, López MG, Brautaset T, Wendisch VF (2019) Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus. Appl. Microbiol. Biotechnol. 103:5879-5889. doi: 10.1007/s00253-019-09907-8