Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Abstract

Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017.

Scale-Up of Capsular Polysaccharide Production Process by Haemophilus influenzae Type b Using kLa Criterion

“…It was confirmed that PRP synthesis is totally associated with the cell growth, according to the model described by Luedeking-Piret and reported by Takagi et al [ 19 ] for H. influenzae type b. The controlling glucose feed rate was started in order to avoid the accumulation of metabolites such as acetate formation [ 12 , 49 ]. Acetate in the protonated form (acetic acid) can diffuse freely across cell membranes, resulting an accumulation of acetate anions and protons in the cell cytoplasm and a reduction in intracellular pH of 4.76.…”