Abstract
Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.
Highlights
Global market values involving biotechnology have been forecasted to reach US$2–4 trillion by 2030–2040
The concentrations of biomass, penicillin G (PenG), phenylacetic acid (PAA), oxopiperidine-2-carboxylic acid (OPC), o-OH-PAA, 6-APA and their biomass-specific consumption/production rates are shown in Figure 1 for the chemostat process with PAA as the precursor and continuous supplementation with other media components for the synthesis of PenG
It is of vital importance to gain quantitative insight into the cellular regulation mechanism in response to relevant dissolved oxygen (DO) perturbations typically occurring in large-scale settings
Summary
Global market values involving biotechnology have been forecasted to reach US$2–4 trillion by 2030–2040. As industry has grown to rely heavily on microbes, only one in 5000–10,000 biotechnology innovations survives the long route from initial findings, through academia to product commercialization at the industrial level [2]. This valley of death in industrial biotechnology is often encountered when strains and/or processes, once optimized at the laboratory scale, are directly transferred to large-scale bioreactors for actual industrial production. This so-called scale-up effect often leads to reduced production performance in terms of titer, rate and yield
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