GLOBAL REGULATORS AS NON-TRADITIONAL TARGETS FOR ENHANCING PRODUCTION OF COMPOUNDS OF BIOTECHNOLOGICAL INTEREST IN Escherichia coli

M. JULIA PETTINARI; DIAZ PEÑA ROCIO; DIEGO EGOBURO

San Miguel de Tucuman

Congreso; XII Congreso de Microbiologia General; 2017

SAMIGE

Transcriptional regulation in Escherichia coli comprise a network of specific and global regulators. Some of the latter control operons related to central metabolism affecting carbon flow and reducing power. Mutations in these genes may be advantageous for the production of compounds of biotechnological interest. Among different global regulators that affect central metabolism we studied ArcA and CreC, members of two components systems, Cra and Rob. ArcA is known to be one of the main regulators that affects C metabolism in response to O2 availability, while CreC is known to respond to both C source and aeration. The majority of Cra targets (over 180 genes) are genes coding for the enzymes involved in central carbon metabolism. Rob is involved in antibiotic resistance, solvent tolerance and affects some genes of glucose metabolism and TCA cycle. To investigate the effect of deleting these regulators on the production of industrial compounds, cultures of mutants in each regulator carrying the genes for synthesis of 1,3-propanediol from Klebsiella pneumoniae were carried on in M9 or LB medium supplemented with glycerol. Different conditions of oxygen availability were assayed: (I) low aeration (125 rpm and 1:2 flask-volumen:medium-volume ratio) and (ii) aerobiosis (200 rpm and 1:10 ratio). Glycerol 10 g·liter-1 and 30 g·liter-1 were used, respectively. Production of 1,3-PDO, glycerol consumption and yield production were measured. All regulators affected the synthesis of this diol in both aeration conditions and both culture medium.ΔarcA mutants grown in low aeration conditions and M9 showed a slightly increase in the production of 1,3-PDO and yield production were increased for ΔarcA and ΔcreC mutants. Cultures in LB revealed different effects compared to M9. Microaerobic assays showed a significant decrease of 1,3-PDO titer for ΔarcA mutants in contrast to those in minimal medium. Additionally, a slightly increase of 1,3-PDO production for Δcra mutants was achieved in aerobic condition. Interestingly, a 2-fold higher production of this solvent was observed for ΔarcA strain in aerobiosis in this culture medium. Furthermore, all parameters measured were remarkably increased for this mutant. Global regulators mutants carrying the genes for polyhydroxibutirate synthesis from Ralstonia eutropha were also evaluated for the production of this polymer in M9 glucose 30 g·liter-1 in low aerobic conditions described above. Although no significant differences were seen, slightly increases of PHB accumulation for Δrob and ΔcreC mutants were observed. Finally, endogenous ethanol synthesis was measured for each mutant in low aerobic conditions in M9 glucose 5 g·liter-1. ΔcreC strain showed higher ethanol production per unit weight biomass after 24 h culture. In conclusion, all global regulators seemed to affect carbon distribution and to behave differently in the aeration and culture medium conditions tested. Results obtained indicates that Δcra and ΔarcA mutant could be potentially interesting for 1,3-PDO production. As for PHB, even slightly effects were seen in the microaerobic conditions, more experiments must be done to evaluate the roles of these regulators on polymer production. In the case of ethanol, results suggest that ΔcreC mutant could be suitable for alcohol production. Furthermore, results showed that culture conditions could be optimized to design more efficient and sustainable processes for biocompounds production.