Flux controlling technology for central carbon metabolism for efficient microbial bio-production
Graphical abstract
Introduction
To achieve a sustainable society, bio-based production of various commodities using microorganisms from renewable biomass resources has been attracting attention [1,2]. However, the yield and production rate are still low in many bio-processes, and improving the productivity is one of the challenges for industrialization. Although various studies have focused on deletion of undesirable genes or overexpression of important genes to enhance productivity [3,4], room for further improvement still exists in many cases. To obtain ideal production of a target compound, not only is the deletion or overexpression of genes required, but fine-tuning of the flux distribution by optimizing the enzyme expression level appropriately also plays an important role. There are many genetic engineering tools used to adjust the expression level, such as inducible promoters, antisense RNA, 5′-untranslated regions (5′-UTR), and riboswitches [5, 6, 7, 8]. Since the intracellular and extracellular conditions change over time in the fermentation processes, dynamic control of the metabolic system for maintaining the cellular state appropriately is important [9]. To accomplish such a robust process, on-line monitoring of the metabolic state in the cultured cells, and the control of the carbon flux based on the obtained information would be beneficial. In this paper, we reviewed effects of the carbon flux rewiring of the central carbon metabolism in Escherichia coli and Saccharomyces cerevisiae on bio-productions, as well as techniques for detecting the intracellular metabolic state and for controlling the flux of the central carbon metabolism. Recently, various fluorescent sensors have been developed for detecting a concentration of key metabolites, such as co-factors. Furthermore, optogenetics is a promising way for developing an ON–OFF switchable system with controlling a gene expression. We present a prospect for future bio-production processes using these techniques.
Section snippets
Effect of flux regulation of the central carbon metabolism on useful compound production
Cells decompose carbon sources into small molecules through central carbon metabolism and obtain energy (ATP) and redox cofactors (NADPH). Various useful compounds are synthesized from intermediates using ATP and NADPH as driving forces. However, the original metabolic system’s primary role is generating energy and biosynthetic precursors for growth, but allows for waste and redundancy in target production. Therefore, to enhance desirable target production, it is necessary to adjust the carbon
Techniques for evaluating the metabolic state with fluorescent sensors
As described above, flux distribution in the central carbon metabolism affects the supply of precursors and cofactors and should be rewired to enhance the target production. To fine-tune the metabolic flux during the culture period, a strategy to monitor the current metabolic state and adjust the flux toward the ideal state would be effective. Although metabolome analysis with mass spectrometry and 13C-metabolic flux analysis are reliable methods, application to on-line measurements seems still
Techniques for dynamically controlling the metabolic flux
Studies that attempt to control the flux of the central carbon metabolism by using switches are summarized in this section. It has been reported that the growth and isopropanol production modes are switched by redirecting the carbon flux at the acetyl-CoA node between the tricarboxylic acid (TCA) cycle and isopropanol synthesis using a metabolic toggle switch [29]. The gltA expression of the competitive pathway which encodes the first enzyme in the TCA cycle and isopropanol synthesis genes,
Conclusions and future outlook
In this paper, the current status of the techniques used for detecting the metabolic state and for controlling carbon flux was reviewed. Furthermore, we described a future outlook on bio-processing using fluorescent metabolic sensors and light-inducible metabolic switches. The productivity of microbial production would be enhanced by optimization of the flux distribution of the central carbon metabolism. However, since the culture environment changes over time, external control of the metabolic
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
CRediT authorship contribution statement
Yoshihiro Toya: Writing - original draft, Writing - review & editing. Hiroshi Shimizu: Writing - original draft, Writing - review & editing.
Acknowledgements
This work was supported by a JST-Mirai Program Grant Number JPMJMI17EJ, and a Grant-in-Aid for Scientific Research (S) No.19H05626.
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