Physiological Response of Corynebacterium glutamicum to Increasingly Nutrient-Rich Growth Conditions

Abstract

To ensure economic competitiveness, bioprocesses should achieve maximum productivities enabled by high growth rates (μ) and equally high substrate consumption rates (qS) as a prerequisite of sufficient carbon-to-product conversion. Both traits were investigated and improved via bioprocess engineering approaches studying the industrial work horse Corynebacterium glutamicum. Standard minimal medium CGXII with glucose as sole carbon source was supplemented with complex brain-heart-infusion (BHI) or amino acid (AA) cocktails. Maximum μ of 0.67 h-1 was exclusively observed in 37 g BHI L-1 whereas only minor growth stimulation was found after AA supplementation (μ = 0.468 h-1). Increasing glucose consumption rates (qGlc) were solely observed in certain dosages of BHI (1–10 g L-1), while 37 g BHI L-1 and AA addition revealed qGlc below the reference experiments. Moreover, BHI supplementation revealed Monod-type saturation kinetics of μ (KBHI = 2.73 g BHI L-1) referring to the preference of non-AAs as key boosting nutrients. ATP-demands under reference, 1 g BHI L-1, and AA conditions were nearly constant but halved in BHI concentrations above 5 g L-1 reflecting the energetic advantage of consuming complex nutrient components in addition to “simple” building blocks such as AAs. Furthermore, C. glutamicum revealed maximum biomass per carbon yields of about 18 gCDW C-mol-1 irrespective of the medium. In AA supplementation experiments, simultaneous uptake of 17 AAs was observed, maximum individual consumption rates determined, and L-asparagine and L-glutamine were distinguished as compounds with the highest consumption rates. Employment of the expanded stoichiometric model iMG481 successfully reproduced experimental results and revealed the importance of C. glutamicum’s transaminase network to compensate needs of limiting AA supply. Model-based sensitivity studies attributed the highest impact on μ to AAs with high ATP and NADPH demands such as L-tryptophan or L-phenylalanine.

Microbial single-cell growth response at defined carbon limiting conditions

“…Among the most commonly used devices are table-top fermenters, baffled or non-baffled cultivation asks and miniature bioreactor systems (MBRs) in micro-well plate formats. 3,4 Apart from their sizes, the most striking differences between these macrouidic devices are the throughput capacities, costs of maintenance and operating principles. Cultivation asks and MBRs are mainly operated in batch mode.…”

Revisiting the Growth Modulon of Corynebacterium glutamicum Under Glucose Limited Chemostat Conditions

“…The metabolic flux map of Figure 8 mirrors cellular efforts to provide sufficient metabolic precursors, reduction equivalents, and ATP at a growth rate of 0.4 h –1 that represents growth conditions typically observed under non-limited batch conditions ( Blombach et al, 2013 ; Grünberger et al, 2013 ; Graf et al, 2018 ; Haas et al, 2019 ). Apparently, the central metabolism of C. glutamicum serves the pivotal need of precursor supply for biomass formation which is mirrored by the strong activity of glycolysis fueling the TCA cycle.…”

“…However, a putative drawback lies in the somewhat reduced growth rates compared to other hosts such as Escherichia coli . With about 0.4 h –1 wild-type (WT) C. glutamicum shows only moderate growth in synthetic media in classical batch cultivations ( Blombach et al, 2013 ; Grünberger et al, 2013 ; Graf et al, 2018 ; Haas et al, 2019 ) compared to about 0.7–0.8 h –1 maximum growth rate of other prokaryotes such as E. coli , 1.7 h –1 for the putative future competitors Vibrio natriegens ( Hoffart et al, 2017 ) and Geobacillus LC300 with 2.15 h –1 ( Long et al, 2017 ). Relatively low space-time yields compared to other hosts may be the consequence.…”

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities

“…Strain SA-7 carrying an empty plasmid produced 10.9 ± 0.92 g/L of shikimic acid (Figure 4, orange bar) with a yield of 0.22 g shikimate/g-glucose, about two-fold higher than SA-7 cultivated in CGXII minimal medium. Graf et al reported that ATP and NADPH generation was enhanced in nutrient-rich medium (Graf et al, 2018), contributing to increased shikimic acid production. SA-7 overexpressing aroG and aroB strains produced 10.9 ± 0.38 g/L and 10.6 ± 0.36 g/L of shikimic acid in CGXIIY medium (Figure 4, orange bars), respectively, almost the same as SA-7 carrying an empty vector.…”