Abstract
Zero-growth processes are a promising strategy for the production of reduced molecules and depict a steady transition from aerobic to anaerobic conditions. To investigate the adaptation of Corynebacterium glutamicum to altering oxygen availabilities, we conceived a triple-phase fermentation process that describes a gradual reduction of dissolved oxygen with a shift from aerobiosis via microaerobiosis to anaerobiosis. The distinct process phases were clearly bordered by the bacteria’s physiologic response such as reduced growth rate, biomass substrate yield and altered yield of fermentation products. During the process, sequential samples were drawn at six points and analyzed via RNA-sequencing, for metabolite concentrations and for enzyme activities. We found transcriptional alterations of almost 50% (1421 genes) of the entire protein coding genes and observed an upregulation of fermentative pathways, a rearrangement of respiration, and mitigation of the basic cellular mechanisms such as transcription, translation and replication as a transient response related to the installed oxygen dependent process phases. To investigate the regulatory regime, 18 transcriptionally altered (putative) transcriptional regulators were deleted, but none of the deletion strains showed noticeable growth kinetics under an oxygen restricted environment. However, the described transcriptional adaptation of C. glutamicum resolved to varying oxygen availabilities provides a useful basis for future process and strain engineering.
Highlights
Corynebacterium glutamicum is an established workhorse in industrial biotechnology and is used for the production of amino acids such as monosodium glutamate (MSG) and L-lysine with a market size of 3.1 and 2.2 million tons per year in 2015 [1]
Zero-growth production processes often start with an aerobic phase for biomass formation, which is accompanied by an anaerobic production phase with resting cells either in separated vessels
C. glutamicum ATCC 13032 was purchased from the American Type Culture Collection and was cultivated in 2× yeast extract tryptone (YT) complex medium [60] and modified CGXII minimal medium based on literature [61,62]
Summary
Corynebacterium glutamicum is an established workhorse in industrial biotechnology and is used for the production of amino acids such as monosodium glutamate (MSG) and L-lysine with a market size of 3.1 and 2.2 million tons per year in 2015 [1]. Applying triple-phase processes, which provide an oxygen-limited interface, led to a (partial) restoration of the performance in the successive anaerobic production phase [11,12,13,14] It was shown with a lactate dehydrogenase-deficient strain of C. glutamicum that a progressive deoxygenation enhanced succinate and acetate titers by up to 640% [15]. Microaerobiosis is discussed to negatively impact cell viability in large-scale bioreactors, where bacteria face changing oxygen availabilities due to insufficient power input and mixing [16,17,18] Such fluctuations might go hand in hand with reduced productivities and product yields [18,19,20,21]
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