- Email: [email protected]
Metabolic design of a platform Escherichia coli strain for the production of chorismate derivatives
Abstracts / New Biotechnology 33S (2016) S1–S213
Acknowledgments: This work was supported by the National Science Council of Taiwan (MOST 104-2221-E-218-030-). http://dx.doi.org/10.1016/j.nbt.2016.06.1386
P32-13 Production of polysaccharides with a biotechnological potential by the Rhizobium leguminosarum bv. trifolii rosR mutant Rt2472 2, ´ Monika Janczarek 1,∗ , Kamila Rachwał 1 , Magdalena Kopycinska Teresa Urbanik-Sypniewska 2 1 2
Maria Curie-Sklodowska University, Poland M. Curie-Sklodowska University, Poland
The production of industrially applicable microbial polysaccharides (PSs) has recently gained much interest since these types of biopolymers often possess unique physical and chemical properties. Moreover, they are renewable, biocompatible, biodegradable, non-toxic and can be obtained on a regular basis. Bacterial polymers can be successfully employed in areas such as medicine and food, petroleum, cosmetic and textile industries. Rhizobium leguminosarum bv. trifolii is a promising source of various PSs. It is a gram-negative bacterium, which exists in soil in a free-living form or as nitrogen-fixing bacteroids inside clover root nodules. This microorganism is able to produce various PSs, such as exopolysaccharide (EPS), which is secreted into the environment, and -glucan, lipopolysaccharide, glucomannan, capsular polysaccharide (CPS), and gel-forming polysaccharide (GPS), which are tightly associated with the cells. In this bacterium, rosR encodes a global transcriptional regulator positively affecting EPS production but negatively influencing synthesis of remaining PSs. In the present study, sugar polymers produced by the wild-type strain Rt24.2 and the rosR mutant Rt2472 were isolated and the efficiency of their synthesis was estimated. The results obtained indicate that the rosR mutant synthesizes significantly increased amounts of GPS, CPS, glucomannan, and -glucan in relation to the wild-type. These PSs were heterogeneous in respect to their molecular weight. Among them, GPS as a neutral polymer with gel-forming capability, which consists of galactose, mannose, and glucose, could be used in various industrial branches including the cosmetic industry. This work was supported by the grant of the National Science Centre no 2012/07/B/NZ1/00099. http://dx.doi.org/10.1016/j.nbt.2016.06.1387
P32-14 Metabolic design of a platform Escherichia coli strain for the production of chorismate derivatives Shuhei Noda ∗ , Tomokazu Shirai, Akihiko Kondo RIKEN, Japan Recently, chemicals and fuel production from renewable resources, such as biomass resources, have attracted attention due to global warming and limited amounts of fossil fuels. The aromatic compounds include a huge number of industrially important materials, and production of them using microorganisms is an active research area, as well as production of biofuel and other buildingblock compounds. In the present study, a synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An l-phenylalanine-overproducing
S193
E. coli strain was engineered to increase the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E. coli as a base strain, we tested our system by carrying out the production of salicylate, a valuable aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. To demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1–3 g/L in test tube-scale culture. http://dx.doi.org/10.1016/j.nbt.2016.06.1388
P32-15 Implementation of a synthetic transcription termination signal limiting T7 RNA polymerase read-through Johanna Jarmer 1,∗ , Ruth Eytner 2 , Simon Stammen 2 , Reingard Grabherr 1 1 2
University of Natural Resources and Life Sciences, Austria Boehringer Ingelheim RCV, Austria
T7 RNA polymerase based expression systems are characterized by high expression rates and are thus valuable and widely used tools for manufacturing biopharmaceuticals in Escherichia coli. The high processivity of the T7 RNA polymerase, however, is also associated with termination signal read-through. The standard native T7 terminator for example exerts only a termination efficiency of 50–80% depending on its DNA sequence context. Elevated transcriptional read-through leads to undesired phenomena like increased metabolic load and bioprocess instability. Mairhofer et al developed a new synthetic transcription terminator with increased efficiency of 99%. It combines an array of three different termination signals, a modified T7 terminator, the wellknown rrnBT1 and the native T7 terminator [Mairhofer et al., 2014, ACS Synthetic Biology]. At Boehringer Ingelheim, this synthetic terminator was implemented into the proprietary modular vector system for E. coli and the termination efficiency was determined by in vitro transcription and qPCR assays. For several therapeutically relevant proteins of interest (Fab and VHH antibody fragments) the high efficiency of the new synthetic terminator was confirmed in the context of the vector system independent of the gene of interest. In fed-batch cultivations improved bacterial growth could be shown while no negative impact on plasmid stability was revealed. A patent application (WO 2012/164100) for this highly efficient transcription terminator has been submitted by Boehringer Ingelheim and expression plasmids with this improved transcription terminator module are now available to customers. http://dx.doi.org/10.1016/j.nbt.2016.06.1389
Acknowledgments: This work was supported by the National Science Council of Taiwan (MOST 104-2221-E-218-030-). http://dx.doi.org/10.1016/j.nbt.2016.06.1386
P32-13 Production of polysaccharides with a biotechnological potential by the Rhizobium leguminosarum bv. trifolii rosR mutant Rt2472 2, ´ Monika Janczarek 1,∗ , Kamila Rachwał 1 , Magdalena Kopycinska Teresa Urbanik-Sypniewska 2 1 2
Maria Curie-Sklodowska University, Poland M. Curie-Sklodowska University, Poland
The production of industrially applicable microbial polysaccharides (PSs) has recently gained much interest since these types of biopolymers often possess unique physical and chemical properties. Moreover, they are renewable, biocompatible, biodegradable, non-toxic and can be obtained on a regular basis. Bacterial polymers can be successfully employed in areas such as medicine and food, petroleum, cosmetic and textile industries. Rhizobium leguminosarum bv. trifolii is a promising source of various PSs. It is a gram-negative bacterium, which exists in soil in a free-living form or as nitrogen-fixing bacteroids inside clover root nodules. This microorganism is able to produce various PSs, such as exopolysaccharide (EPS), which is secreted into the environment, and -glucan, lipopolysaccharide, glucomannan, capsular polysaccharide (CPS), and gel-forming polysaccharide (GPS), which are tightly associated with the cells. In this bacterium, rosR encodes a global transcriptional regulator positively affecting EPS production but negatively influencing synthesis of remaining PSs. In the present study, sugar polymers produced by the wild-type strain Rt24.2 and the rosR mutant Rt2472 were isolated and the efficiency of their synthesis was estimated. The results obtained indicate that the rosR mutant synthesizes significantly increased amounts of GPS, CPS, glucomannan, and -glucan in relation to the wild-type. These PSs were heterogeneous in respect to their molecular weight. Among them, GPS as a neutral polymer with gel-forming capability, which consists of galactose, mannose, and glucose, could be used in various industrial branches including the cosmetic industry. This work was supported by the grant of the National Science Centre no 2012/07/B/NZ1/00099. http://dx.doi.org/10.1016/j.nbt.2016.06.1387
P32-14 Metabolic design of a platform Escherichia coli strain for the production of chorismate derivatives Shuhei Noda ∗ , Tomokazu Shirai, Akihiko Kondo RIKEN, Japan Recently, chemicals and fuel production from renewable resources, such as biomass resources, have attracted attention due to global warming and limited amounts of fossil fuels. The aromatic compounds include a huge number of industrially important materials, and production of them using microorganisms is an active research area, as well as production of biofuel and other buildingblock compounds. In the present study, a synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An l-phenylalanine-overproducing
S193
E. coli strain was engineered to increase the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E. coli as a base strain, we tested our system by carrying out the production of salicylate, a valuable aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. To demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1–3 g/L in test tube-scale culture. http://dx.doi.org/10.1016/j.nbt.2016.06.1388
P32-15 Implementation of a synthetic transcription termination signal limiting T7 RNA polymerase read-through Johanna Jarmer 1,∗ , Ruth Eytner 2 , Simon Stammen 2 , Reingard Grabherr 1 1 2
University of Natural Resources and Life Sciences, Austria Boehringer Ingelheim RCV, Austria
T7 RNA polymerase based expression systems are characterized by high expression rates and are thus valuable and widely used tools for manufacturing biopharmaceuticals in Escherichia coli. The high processivity of the T7 RNA polymerase, however, is also associated with termination signal read-through. The standard native T7 terminator for example exerts only a termination efficiency of 50–80% depending on its DNA sequence context. Elevated transcriptional read-through leads to undesired phenomena like increased metabolic load and bioprocess instability. Mairhofer et al developed a new synthetic transcription terminator with increased efficiency of 99%. It combines an array of three different termination signals, a modified T7 terminator, the wellknown rrnBT1 and the native T7 terminator [Mairhofer et al., 2014, ACS Synthetic Biology]. At Boehringer Ingelheim, this synthetic terminator was implemented into the proprietary modular vector system for E. coli and the termination efficiency was determined by in vitro transcription and qPCR assays. For several therapeutically relevant proteins of interest (Fab and VHH antibody fragments) the high efficiency of the new synthetic terminator was confirmed in the context of the vector system independent of the gene of interest. In fed-batch cultivations improved bacterial growth could be shown while no negative impact on plasmid stability was revealed. A patent application (WO 2012/164100) for this highly efficient transcription terminator has been submitted by Boehringer Ingelheim and expression plasmids with this improved transcription terminator module are now available to customers. http://dx.doi.org/10.1016/j.nbt.2016.06.1389