Metabolic engineering of Escherichia coli into a versatile glycosylation platform : production of bio‐active quercetin glycosides

Background: Flavonoids are bio-active specialized plant metabolites which mainly occur as different glycosides. Due to the increasing market demand, various biotechnological approaches have been developed which use Escherichia coli as a microbial catalyst for the stereospecific glycosylation of flavonoids. Despite these efforts, most processes still display low production rates and titers, which render them unsuitable for large-scale applications. Results: In this contribution, we expanded a previously developed in vivo glucosylation platform in E. coli W, into an efficient system for selective galactosylation and rhamnosylation. The rational of the novel metabolic engineering strategy constitutes of the introduction of an alternative sucrose metabolism in the form of a sucrose phosphorylase, which cleaves sucrose into fructose and glucose 1-phosphate as precursor for UDP-glucose. To preserve these intermediates for glycosylation purposes, metabolization reactions were knocked-out. Due to the pivotal role of UDP-glucose, overexpression of the interconverting enzymes galE and MUM4 ensured the formation of both UDP-galactose and UDP-rhamnose, respectively. By additionally supplying exogenously fed quercetin and overexpressing a flavonol galactosyltransferase (F3GT) or a rhamnosyltransferase (RhaGT), 0.94 g/L hyperoside (quercetin 3-O-galactoside) and 1.12 g/L quercitrin (quercetin 3-O-rhamnoside) could be produced, respectively. In addition, both strains showed activity towards other promising dietary flavonols like kaempferol, fisetin, morin and myricetin. Conclusions: Two E. coli W mutants were engineered that could effectively produce the bio-active flavonol glycosides hyperoside and quercitrin starting from the cheap substrates sucrose and quercetin. This novel fermentation-based glycosylation strategy will allow the economically viable production of various glycosides

A sigma factor toolbox for orthogonal gene expression in Escherichia coli

Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future

Predictive design of sigma factor-specific promoters

To engineer synthetic gene circuits, molecular building blocks are developed which can modulate gene expression without interference, mutually or with the host's cell machinery. As the complexity of gene circuits increases, automated design tools and tailored building blocks to ensure perfect tuning of all components in the network are required. Despite the efforts to develop prediction tools that allow forward engineering of promoter transcription initiation frequency (TIF), such a tool is still lacking. Here, we use promoter libraries of E. coli sigma factor 70 (sigma (70))- and B. subtilis sigma (B)-, sigma (F)- and sigma (W)-dependent promoters to construct prediction models, capable of both predicting promoter TIF and orthogonality of the sigma -specific promoters. This is achieved by training a convolutional neural network with high-throughput DNA sequencing data from fluorescence-activated cell sorted promoter libraries. This model functions as the base of the online promoter design tool (ProD), providing tailored promoters for tailored genetic systems. Automated design tools and tailored subunits are beneficial in fine-tuning all components of a complex genetic circuit. Here the authors create E. coli and B. subtilis promoter libraries using FACS and HTS, from which an online promoter design tool has been developed using CNN

Orthogonal gene expression : promoter libraries linked to different sigma factors which are orthogonal between each other and towards the host

Technological advances in synthetic biology, systems biology, and metabolic engineering have boosted applications of industrial biotechnology for an increasing number of complex and high added-value molecules. In general, the transfer of multi-gene or poorly understood heterologous pathways into the production host leads to imbalances due to lack of adequate regulatory mechanisms. Hence, fine-tuning expression of synthesis pathways in specific conditions is mandatory together with the decoupling of host systems. Here we develop an orthogonal expression system in Escherichia coli with three heterologous sigma factors that function orthogonal between each other and towards the host organism. Furthermore, for each of these sigma factors, a promoter library will be created that retains this orthogonal feature. First, several sigma factors originating from Bacillus subtilis were tested for their orthogonality in E. coli on the level of promoter recognition, by using a red-fluorescent reporter system. Secondly, the potential of sigma factors, and mutants thereof, from B. subtilis to work together with the E. coli core RNA polymerase was tested. This was accomplished by expressing these proteins together with their promoters; three distinct promoters for each factor. Subsequently, cross talk between the heterologous sigma factors and the promoters was evaluated by examining all combinations. Based on the results three heterologous sigma factors with one of their respective promoters were chosen for further work. For each of these factors a promoter library will be generated that remains orthogonal towards the host and towards the alternative sigma factors. The promoter libraries will be attained by randomising part of the promoter, one strategy will be to randomise regions of 5 nucleotides throughout the promoter while the second strategy will be randomisation of the complete linker sequence. Subsequently the promoter strengths will be measured through sequential rounds of fluorescent-activated cell sorting (FACS) to result in a set of orthogonal promoters for each sigma factor, covering a range of different strengths. The use of these different orthogonal sigma factors combined with a range of promoter strengths for each factor offers the opportunity to fine-tune expression of different genes in heterologous pathways