An industrial perspective on in vivo kinetics of central metabolite pools and dynamic flux responses in Penicillium chrysogenum during periodic dissolved oxygen feast-famine cycles in a scale-down system
Authors: Wang Guan , Wang Xueting , yang Qi , Haringa Cees , Wang Zejian , Chu Ju , Zhuang Ying-Ping 
Published: August 2023

Limitations in mixing and mass transfer coupled with high hydrostatic
pressures lead to significant spatial variations in dissolved oxygen
(DO) concentrations in large-scale bioreactors. While traveling through
different zones in the bioreactor, microbes are subjected to fluctuating
DO conditions at the timescales of global circulation time. In this
study, to mimic industrial-scale spatial DOT gradients, we present a
scale-down model based on dynamic feast/famine regime (150 s) that leads
to repetitive cycles with rapid changes in DO availability in
glucose-limited chemostat cultures of Penicillium chrysogenum.
The results revealed that the exposure time to the low DO level (less
than 10%) imposed a significant impact on the biomass growth and
penicillin productivity was considerably reduced by a factor of two,
while the averaged substrate consumption rates were comparable under the
DO oscillation condition compared to that of 60% DO steady-state
condition. Quantitative metabolomics data showed that the DO
feast/famine induced a stable and repetitive pattern with a reproducible
metabolic response in time. The dynamic response of intracellular
metabolites under such DO oscillating conditions showed specific
differences in comparison to repetitive substrate pulse experiments. Due
to invariable the specific glucose uptake rate ( q s ) during a cycle,
the variation in the intracellular pools size of amino acids, sugar
phosphates and organic acids was less pronounced in terms of both
coverage and magnitude under DO fluctuations than under repetitive
substrate pulses featured with a marked variation in the q s .
Remarkably, intracellular sugar polyols were considerably increased as
the hallmark metabolites to reserve carbon source and reducing
equivalent, which likely provide short-term benefits in such changing
environments. Furthermore, the calculated cytosolic NADH/NAD
ratio under the DO oscillating condition indicated a
dynamic and higher redox state of the cytosol, which has been reported
to negatively affect the maintenance of penicillin productivity. Despite
the increased availability of NADPH for penicillin production under the
oscillatory DO conditions, this positive effect may be counteracted by
the decreased ATP supply. From an economical point of view, it is
interesting to note that not only the penicillin productivity was
reduced under such oscillating DO conditions, but also that of the
unrecyclable byproduct ortho-hydroxyphenyl acetic acid (ο-OH-PAA) and
degeneration of penicillin productivity induced by low extracellular
glucose sensing. Furthermore, dynamic metabolic flux analysis based on
constraining time-resolved metabolite data into genome-scale metabolic
model showed that Penicillium chrysogenum metabolism shifted from
penicillin production to maintaining biomass growth upon a reduction of
oxygen supply. The relative decreasing fluxes of amino acid metabolic
pathways and fatty acid biosynthetic pathways were assumed to relieve
the energy demand for balanced cellular metabolism. Taken together, the
metabolic responses of Penicillium chrysogenum to DOT gradients
reported here are important for elucidating metabolic regulation
mechanisms, improving bioreactor design and scale-up procedures as well
as for constructing robust cell strains to cope with heterogenous
industrial culture conditions.