Understanding the interplay of carbon and nitrogen supply for ectoines production and metabolic overflow in high density cultures of Chromohalobacter salexigens.
BACKGROUND
The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines.


RESULTS
Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium.13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect-mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L-1) and volumetric ectoine yields (up to 4.21 g L-1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes.


CONCLUSIONS
Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.
Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", 30100, Murcia, Spain.|Departamento de Ingeniería Química y Ambiental, Universidad Politécnica de Cartagena, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", Campus Muralla del MarCalle Doctor Fleming S/N, 30202, Cartagena, Spain.
María J
MJ
Salar-García
Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", 30100, Murcia, Spain. vicente.bernal@gmail.com.|Área de Biología, Dirección de Tecnología Química y Nuevas Energías, Centro de Tecnología de Repsol S.A., Ctra. de Extremadura A-5, Km. 18, 28375, Móstoles, Spain. vicente.bernal@gmail.com.
Vicente
V
Bernal
Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", 30100, Murcia, Spain.
José M
JM
Pastor
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012, Seville, Spain.
Manuel
M
Salvador
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012, Seville, Spain.
Montserrat
M
Argandoña
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012, Seville, Spain.
Joaquín J
JJ
Nieto
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012, Seville, Spain.
Carmen
C
Vargas
Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", 30100, Murcia, Spain. mcanovas@um.es.
Manuel
M
Cánovas
D000599:Amino Acids, Diamino / Q000096:biosynthesis*; D000641:Ammonia / Q000494:pharmacology; D061249:Batch Cell Culture Techniques; D018533:Biomass; D050260:Carbohydrate Metabolism; D002244:Carbon / Q000378:metabolism*; D055353:Chromohalobacter / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D009997:Osmotic Pressure; D019289:Pyruvic Acid / Q000032:analysis; D054712:Salinity
D000599:Amino Acids, Diamino; D003470:Culture Media; D002244:Carbon; D000641:Ammonia; C045628:ectoine; D019289:Pyruvic Acid; D005947:Glucose; D009584:Nitrogen
C. salexigens; Carbon overflow; Ectoines; Fed-batch; Halophilism; Nitrogen assimilation