Rational design of an improved transglucosylase for production of the rare sugar nigerose

The sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) can be used as a transglucosylase for the production of rare sugars. We designed variants of BaSP for the efficient synthesis of nigerose from sucrose and glucose, thereby adding to the inventory of rare sugars that can conveniently be produced from bulk sugars

Comprehensive study on Escherichia coli genomic expression : does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings

Comprehensive study on Escherichia coli genomic expression : does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively for the production of metabolites with commercial interest. Moreover, promoter and terminator databases are readily available as well as a wide amount of expression vectors and numerous gene editing technologies. Together with the ever-reducing cost of synthetic DNA, the range of possibilities in metabolic engineering is expanding even more. Recent advances have secured the possibility of integrating whole synthetic pathways with ease and high efficiency onto the bacterial genome, hereby overcoming the need for plasmid expression and their associated instability. However, the question of where and how to incorporate your novel optimized pathway remains unanswered. To address this issue, we first improved the integration methodology of genes and pathways into the chromosome. To this end we created a serine integrase recombinational engineering (SIRE) system that allows fast and highly efficient integration in the genome, in single and in multiple copies. We were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette (PMID: 30345503). This methodology allowed us to comprehensively investigate multiple factors that can potentially influence gene expression in an organisms’ chromosome. The tested factors are: 1) chromosomal locations, spread over the E. coli macrodomains, 2) the influence of carbon and nitrogen source on gene expression, 3) the effect of supercoiling on gene expression, 4) the effect of gene direction in relation to the DNA replication fork and 5) the effect of induced burden on chromosomal expression. To enable these experiments, we have designed fluorescent expression cassettes that eliminate specific local effects on gene expression originating from the surrounding genes, transcriptional read through and influence from transcription factors. Overall, we can conclude from this study that characterising multiple effects on genomic expression is crucial in the development of production hosts, in order to maximize the range in expression of pathway genes. The availability of libraries of strains with different expression levels can only augment the possibilities for the development of new production hosts

Comprehensive study on Escherichia coli genomic expression : does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively for the production of metabolites with commercial interest. Moreover, promoter and terminator databases are readily available as well as a wide amount of expression vectors and numerous gene editing technologies. Together with the ever-reducing cost of synthetic DNA, the range of possibilities in metabolic engineering is expanding even more. Recent advances have secured the possibility of integrating whole synthetic pathways with ease and high efficiency onto the bacterial genome, hereby overcoming the need for plasmid expression and their associated instability. However, the question of where and how to incorporate your novel optimized pathway remains unanswered. To address this issue, we first improved the integration methodology of genes and pathways into the chromosome. To this end we created a serine integrase recombinational engineering (SIRE) system that allows fast and highly efficient integration in the genome, in single and in multiple copies. We were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette (PMID: 30345503). This methodology allowed us to comprehensively investigate multiple factors that can potentially influence gene expression in an organisms’ chromosome. The tested factors are: 1) chromosomal locations, spread over the E. coli macrodomains, 2) the influence of carbon and nitrogen source on gene expression, 3) the effect of supercoiling on gene expression, 4) the effect of gene direction in relation to the DNA replication fork and 5) the effect of induced burden on chromosomal expression. To enable these experiments, we have designed fluorescent expression cassettes that eliminate specific local effects on gene expression originating from the surrounding genes, transcriptional read through and influence from transcription factors. Overall, we can conclude from this study that characterising multiple effects on genomic expression is crucial in the development of production hosts, in order to maximize the range in expression of pathway genes. The availability of libraries of strains with different expression levels can only augment the possibilities for the development of new production hosts