11 research outputs found
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
Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides
Metabolic Engineering @ CSB : platform tools and technologies for the production of complex metabolites
Fast and combinatorial construction and optimization of synthetic pathways
The advancements in the field of metabolic engineering and synthetic biology have allowed the rapid de novo construction of multi-enzyme heterologous pathways. However, in order to obtain an optimal flux through a pathway, the various regulatory elements (e.g. promoters, ribosome binding sites) need to be optimized. For example, the massive over-expression of a gene may result in metabolic burden, due to the withdrawal of NADH, ATP, and amino acids from the central metabolism required for the synthesis of the corresponding protein and the concurrent depletion of intermediates required for biomass synthesis or may lead to the accumulation of toxic intermediates due to an unbalanced pathway.
The current lack of in-depth knowledge on the various regulatory control levels renders combinatorial approaches popular for pathway optimization. In this context, methods to rapidly and efficiently create variability are crucial. To generate this variability, the promoter and the ribosome binding sites are typically randomized to modulate gene expression and to create gene expression libraries.
In this research Gibson assembly was used to introduce promoter and RBS variability into a construct. The biggest advantages are the speed of assembly and the standardized procedure which allow for high-throughput assembly lines.
Several libraries were tested: promoter and RBS libraries, but also combinations of both. Furthermore, this developed system was applied for the introduction of multiple libraries at once. Multiple genes could be differentially expressed using a promoter, RBS or promoter-RBS combination library.
The research was successfully used for the fast and scarless introduction of various promoter and RBS libraries. This methodology was validated using several fluorescent proteins, and can thus be applied for the combinatorial building of new and synthetic pathways
Expanding the bio-production palette: how to speed up the construction and optimization of novel pathways
In the last couple of years, metabolic engineering is undergoing a transition in which the challenge of microbial synthesis of more complex molecules is tackled by merging metabolic engineering with other disciplines such as systems biology, synthetic biology and protein engineering. As the synthetic pathway of these complex molecules involves multiple, heavily controlled and poorly expressed reactions steps, fine tuning of the pathway is mandatory.
To find the optimum flux through the pathway of the multivariate, non-lineair solution space, a combinatorial / semi-rational approach is being developed. The solution space is explored by introducing promotor, ribosome binding site or protein libraries, or combinations hereof. Optimal pathways can be traced back using a high throughput screen.
A novel method, called Single Stranded Assembly (SSA), allows for a fast and reliable introduction of different libraries like e.g. promoter, RBS and protein libraries. This SSA method was optimized and validated for different oligonucleotide concentrations and setups. Furthermore, this developed system was applied for the introduction of multiple libraries at once: promoter – RBS as well as two promoter libraries in front of two different reporter genes.
In view of the current evolutions in the field of metabolic engineering and systems biology, which increasingly focus on the biotechnological production of highly complex secondary molecules and the concomitant need to independently fine tune the expression of the multiple genes involved in the pathway, due to, e.g., metabolic burden and the accumulation of toxic intermediates, the SSA method described here is a valuable addition to the state of the art tools since it can be used successfully for the modulation of transcription, translation and enzyme activity in order to make an optimal production strain
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.status: publishe
Development of microbial cell factories for the production of chito-oligosaccharides
Chito-oligosaccharides have garnered ample interest for promising applications in various domains ranging from health care to agriculture. However, the considerable product dispersity obtained with todays’ production technologies severely hampers the exploitation of their application potential, as these applications likely rely on stringent structure/function relationships. To this day, the developed biotechnological alternatives, which have the potential to yield such high purity products by employing highly specific chitin synthases and deacetylases, neither achieved the desired product characteristics.
In response, we developed an efficient Escherichia coli-based platform for the production of a portfolio of pure and well-defined chito-oligosaccharides (Figure 1). To this end, E. coli was massively tuned by integrating metabolic engineering and synthetic biology with a view to i) increasing product yield and productivity, ii) improving product purity, and iii) expanding the library of chito-oligosaccharides produced in vivo
Splitting the E. coli metabolism for the production of fructose-6-P derived chemicals
In the past, coupling product formation with growth has proven to be a highly successful metabolic engineering strategy for the biotechnological production of numerous molecules, e.g., succinate (Hong, 2002). The guaranteed (high) product yield, the fast recuperation of cofactors and abundance of intermediates are some of the main advantages.
Such a strategy was developed for the production of fructose-6-phosphate derived molecules in E. coli, as many high added value molecules, such as for example chito-oligomers, are derived from fructose-6-P. However, compared to previous examples of this strategy, fructose-6-phosphate is a key metabolite in central metabolism, that intervenes in the glycolysis, the pentose phosphate shunt, etc. which renders such a strategy intrinsically more challenging.
The coupling between fructose-6-phosphate production and growth was achieved by using the heteromeric substrate sucrose, which is cleaved by a heterologously expressed sucrose phosphorylase into glucose-1-phosphate and fructose. A knock-out strategy was developed using metabolic models and C13 labeled flux analysis to preserve the fructose moiety for the production of fructose-6-phosphate derived molecules, while growth is fueled by metabolizing glucose-1-phosphate, hence, resulting in guaranteed product yields and a reasonable productivity.
To achieve this split metabolism, since fructose-6-P is a key intermediate in the central metabolism, the fluxes of the central carbon metabolism have to be redirected severely, e.g., the glycolysis has to be split by knocking out the genes pgi and pfkAB, which prevents the flow from fructose-6-phosphate through the glycolysis to the Krebs cycle. Different additional knockouts were evaluated in order to achieve a complete split metabolism. C13 labeled flux analysis, using OpenFlux, was performed to evaluate the fluxome of the various knock-out mutants, which ultimately showed that the fructose moiety of sucrose can be completely preserved for the formation of fructose-6-P derived products, rendering such a strain the ideal base strain for the development of a wide variety of production hosts