Debottlenecking recombinant protein production in Bacillus megaterium under large‐scale conditions—targeted precursor feeding designed from metabolomics

Abstract

In the present work the impact of large production scale was investigated for Bacillus megaterium expressing green fluorescent protein (GFP). Specifically designed scale-down studies, mimicking the intermittent and continuous nutrient supply of large- and small-scale processes, were carried out for this purpose. The recombinant strain revealed a 40% reduced GFP yield for the large-scale conditions. In line with extended carbon loss via formation of acetate and carbon dioxide, this indicated obvious limitations in the underlying metabolism of B. megaterium under the large-scale conditions. Quantitative analysis of intracellular amino acids via validated fast filtration protocols revealed that their level strongly differed between the two scenarios. During cultivation in large-scale set-up, the availability of most amino acids, serving as key building blocks of the recombinant protein, was substantially reduced. This was most pronounced for tryptophan, aspartate, histidine, glutamine, and lysine. In contrast alanine was increased, probably related to a bottleneck at the level of pyruvate which also triggered acetate overflow metabolism. The pre-cursor quantifications could then be exploited to verify the presumed bottlenecks and improve recombinant protein production under large-scale conditions. Addition of only 5 mM tryptophan, aspartate, histidine, glutamine, and lysine to the feed solution increased the GFP yield by 100%. This rational concept of driving the lab scale productivity of recombinant microorganisms under suboptimal feeding conditions emulating large scale can easily be extended to other processes and production hosts.

Transcriptional analysis of degenerate strain Clostridium beijerinckii DG-8052 reveals a pleiotropic response to CaCO3-associated recovery of solvent production

“…The biosynthesis of amino acid is the most energetically expensive pathway. On the other hand, reduced availability of metabolic energy might be involved in the reduced supply of the ‘expensive’ amino acids28. Therefore, DG-8052 cells metabolized glucose into pyruvate, which was in turn converted into acetyl-CoA, followed by entering acids/solvents pathway instead of TCA cycle, which may cause the up-regulation of phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine, and down-regulation of arginine, proline, alanine, aspartate and glutamate (Fig.…”

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

“…Metabolomics therefore may be used to complement current metabolic engineering strategies for optimizing biological production of chemicals within microorganisms (Oldiges et al, 2007;Putri et al, 2013). Through the detection of relevant metabolic perturbations, metabolomics can identify specific targets for strain improvement that may include detection of pathway bottlenecks, metabolite or product toxicity, imbalanced cofactor supply, or draining of metabolites by alternative pathways (Gold et al, 2015;Hasunuma et al, 2011;Korneli et al, 2012;Noguchi et al, 2016;Ohta et al, 2015;Teoh et al, 2015;Xu et al, 2016). Furthermore, metabolomics provides a comprehensive analysis of the metabolome that allows a deeper understanding of cellular metabolism and how gene modifications can be used for metabolic engineering.…”

Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst

“…The fermentation process was adapted from Korneli and coworkers [37]. A -80°C glycerol stock was used to inoculate the first pre-culture with LB medium which was used to inoculate a second pre-culture with batch medium (3.52 g/L KH 2 PO 4 ; 6.62 g/L Na 2 HPO 4 *2H 2 O; 0.3 g/L MgSO 4 *7 H 2 O; 25 g/L (NH 4 )2SO 4 ; 1 g/L yeast extract and trace elements as described [37]).…”

“…A -80°C glycerol stock was used to inoculate the first pre-culture with LB medium which was used to inoculate a second pre-culture with batch medium (3.52 g/L KH 2 PO 4 ; 6.62 g/L Na 2 HPO 4 *2H 2 O; 0.3 g/L MgSO 4 *7 H 2 O; 25 g/L (NH 4 )2SO 4 ; 1 g/L yeast extract and trace elements as described [37]). The first pre-culture was incubated for 8 h in a 100 mL shake flask with 10 mL LB medium at 150 rpm, 37°C.…”