Enzymes revolutionize the bioproduction of value-added compounds: From enzyme discovery to special applications.

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

CbbR and RegA regulate cbb operon transcription in Ralstonia eutropha H16.

The biotechnologically important Gram-negative ß-proteobacterium Ralstonia eutropha H16 is able to grow lithoautotrophically by utilizing CO2 and H2 as sole carbon and energy sources, respectively. CO2 is fixed by the CBB cycle, which is encoded in duplicate on the genome of R. eutropha H16. The transcription of both cbb operons is controlled by the transcription regulator CbbR dependent on intracellular PEP levels as a response to the carbon-state of the cell. As demonstrated in this study transcription control of both cbb operons appears to be more complex and additionally involves, next to CbbR, the transcription regulator RegA as part of the global transcription regulation system RegA/RegB. The identification of a highly conserved RegA/RegB homologue in R. eutropha H16 and experimental evidence gathered in this study reveal that RegA plays a crucial role in the transcription control of both cbb promoters. RegA is able to induce cbb promoter activity and controls transcription in combination with CbbR dependent on cellular PEP concentrations. These results clearly demonstrate that RegA plays an important role in cbb operon transcription regulation and may also be relevant for the control of other energy-utilizing and energy-generating pathways of R. eutropha H16. In addition to promoting a more complete understanding of the CO2 fixation mechanism of R. eutropha H16 these findings also provide crucial insights for the utilization of this bacterium in biotechnological applications with respect to CO2 fixation.

Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strains.

Ralstonia eutropha H16 (Cupriavidus necator H16) is a Gram-negative, facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole energy and carbon sources in the absence of organic substrates. The biotechnological use of R. eutropha H16 on an industrial scale has already been established; however, only a small number of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the number of suitable inducible expression systems for the production of metabolites and proteins with R. eutropha H16.

Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16.

The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements.

Reprint of "versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16".

The Gram-negative ß-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16.

The Gram-negative ß-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.