Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate

Abstract

Mycoplasma hyopneumoniae is cultured on large‐scale to produce antigen for inactivated whole‐cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint‐based genome‐scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time‐series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non‐growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model‐driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3–4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. Biotechnol. Bioeng. 2017;114: 2339–2347. © 2017 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

Combined Transcriptome Sequencing of Mycoplasma hyopneumoniae and Infected Pig Lung Tissue Reveals Up-Regulation of Bacterial F1-Like ATPase and Down-Regulation of the P102 Cilium Adhesin in vivo

“…There was also down-regulation in vivo of the operons related to myo-inositol and ribose transport and catabolism ( Figure 4 ). As the in vitro medium does not contain high levels of unbound myo-inositol ( Kamminga et al, 2017b ), up-regulation of this pathway under in vitro conditions can be expected. Another down-regulated operon is involved in oligopeptide transport.…”

“…Sequencing of the library was done using the Illumina HiSeq 2500: single-end reads, 50 cycles. Quality assessment of reads was done as previously described ( Kamminga et al, 2017b ). Reads were aligned to the strain 98 genome using TopHat v2.1.0 ( Kim et al, 2013 ) with the standard settings for strand-specific alignment enabled (fr-firststrand).…”

“…There was down-regulation under in vivo conditions of the glycerol uptake facilitator and the glycerol kinase (Figure 4, homologs of MHP_RS01925 and MHP_RS01930). Glycerol is a possible energy source for M. hyopneumoniae and is used for production of cardiolipin (Kamminga et al, 2017b). Glycerol consumption by mycoplasma species has been associated with virulence because hydrogen peroxide is released when glycerol-3-phosphate is converted to di-hydroxyacetone phosphate via the glycerol oxidase (Vilei and Frey, 2001).…”

Genomic Variability and Post-translational Protein Processing Enhance the Immune Evasion of Mycoplasma hyopneumoniae and Its Interaction With the Porcine Immune System

“…In order to improve the immunogenicity and biological characteristics of Mhp strains, the self-replicating plasmids with the origin of replication ( oriC ) sequences from Mhp could be useful genetic engineering vectors for strain improvement, which had expressed EGFP in Mhp cells successfully ( 85 , 101 ). On the other hand, genome-scale metabolic engineering techniques provided more information for the optimization of fermentation process, studies had reported that the addition of pyruvate could increase Mhp growth rate in special fermentation experiment ( 102 ).…”

Risk-Based Bioengineering Strategies for Reliable Bacterial Vaccine Production

“…Design strategies vary based on the type of antigen that is needed ( Figure 1A). GEMs can be applied in this stage to predict the effect of using gene knockout or knock-in mutant strains with respect to optimal growth rates or antigen production [14]. Although not all bacterial pathogenic strains are considered to be genetically amenable, recent advances in gene editing techniques have considerably expanded the toolbox enabling researchers to make targeted gene deletions even in bacteria that were previously thought to be inaccessible [15,16].…”