Flux controlling technology for central carbon metabolism for efficient microbial bio-production

doi:10.1016/j.copbio.2020.04.003

Current Opinion in Biotechnology

Volume 64, August 2020, Pages 169-174

https://doi.org/10.1016/j.copbio.2020.04.003 Get rights and content

Highlights

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Flux fine-tuning is important to optimize cofactors supplies for target synthesis.
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Biosensors for detecting accumulation of various intermediates have been developed.
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Various metabolic switches have been developed for rewiring a flux of the pathways.
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Optogenetic switch is an ON–OFF switchable system for controlling a gene expression.
•
Metabolic sensors and switches may provide a novel process for flux fine-tuning.

Syntheses of many commodities that are produced using microorganisms require cofactors such as ATP and NAD(P)H. Thus, optimization of the flux distribution in central carbon metabolism, which plays a key role in cofactor regeneration, is critical for enhancing the production of the target compounds. Since the intracellular and extracellular conditions change over time in the fermentation process, dynamic control of the metabolic system for maintaining the cellular state appropriately is necessary. Here, we review techniques for detecting the intracellular metabolic state with fluorescent sensors and controlling the flux of central carbon metabolism with optogenetic tools, as well as present a prospect of bio-production processes for fine-tuning the flux distribution.

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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View full text

Flux controlling technology for central carbon metabolism for efficient microbial bio-production

Highlights

Graphical abstract

Introduction

Section snippets

Effect of flux regulation of the central carbon metabolism on useful compound production

Techniques for evaluating the metabolic state with fluorescent sensors

Techniques for dynamically controlling the metabolic flux

Conclusions and future outlook

Conflict of interest statement

References and recommended reading

CRediT authorship contribution statement

Acknowledgements

Curr Opin Biotechnol

Cell

Synth Syst Biotechnol

Metab Eng

Bioresour Technol

Appl Microbiol Biotechnol

Metab Eng

Sci Rep

Appl Microbiol Biotechnol

Metab Eng

Metab Eng

Appl Microbiol Biotechnol

Nucleic Acids Res

Proc Natl Acad Sci U S A

Metabolic engineering of strains: from industrial-scale to lab-scale chemical production

J Ind Microbiol Biotechnol

Synthetic and systems biology for microbial production of commodity chemicals

NPJ Syst Biol Appl

Engineered promoters enable constant gene expression at any copy number in bacteria

Nat Biotechnol

Automated design of synthetic ribosome binding sites to control protein expression

Nat Biotechnol

A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli

Appl Environ Microbiol

Molecular parts and genetic circuits for metabolic engineering of microorganisms

FEMS Microbiol Lett

Effect of precise control of flux ratio between the glycolytic pathways on mevalonate production in Escherichia coli

Biotechnol Bioeng

Redirection of the glycolytic flux enhances isoprenoid production in Saccharomyces cerevisiae

Biotechnol J