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Cited by 17Microbial Metabolic Engineering and BioproductionBiochemical and biochemical processesEnzyme Catalysis and Immobilization

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Exploitation of Hetero- and Phototrophic Metabolic Modules for Redox-Intensive Whole-Cell Biocatalysis

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Theodosiou, Eleni, Tüllinghoff, Adrian, Toepel, Jörg, Bühler, Bruno·2022·Frontiers in Bioengineering and Biotechnology
DOI 10.3389/fbioe.2022.855715PMC PMC9043136
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Abstract

The successful realization of a sustainable manufacturing bioprocess and the maximization of its production potential and capacity are the main concerns of a bioprocess engineer. A main step towards this endeavor is the development of an efficient biocatalyst. Isolated enzyme(s), microbial cells, or (immobilized) formulations thereof can serve as biocatalysts. Living cells feature, beside active enzymes, metabolic modules that can be exploited to support energy-dependent and multi-step enzyme-catalyzed reactions. Metabolism can sustainably supply necessary cofactors or cosubstrates at the expense of readily available and cheap resources, rendering external addition of costly cosubstrates unnecessary. However, for the development of an efficient whole-cell biocatalyst, in depth comprehension of metabolic modules and their interconnection with cell growth, maintenance, and product formation is indispensable. In order to maximize the flux through biosynthetic reactions and pathways to an industrially relevant product and respective key performance indices (i.e., titer, yield, and productivity), existing metabolic modules can be redesigned and/or novel artificial ones established. This review focuses on whole-cell bioconversions that are coupled to heterotrophic or phototrophic metabolism and discusses metabolic engineering efforts aiming at 1) increasing regeneration and supply of redox equivalents, such as NAD(P/H), 2) blocking competing fluxes, and 3) increasing the availability of metabolites serving as (co)substrates of desired biosynthetic routes.

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FIGURE 1 Interconversion and regeneration of redox equivalents via reactions that are not directly coupled to the central carbon metabolism: NAD+ kinase (EC 2.7.1.23); NADH kinase (EC 2.7.1.86), NADP+ phosphatase (EC 2.7.1.23); NAD+-dependent dehydrogenase (EC:1.12.1.2); NADP(H)+-dependent dehydrogenases (EC 1.12.1.3); energy-independent soluble pyridine nucleotide transhydrogenase (UdhA, EC 1.6.1.1); energy-dependent proton translocating membrane-bound pyridine nucleotide transhydrogenase (PntAB, EC 1.6.1.2); ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2).
FIGURE 1 Interconversion and regeneration of redox equivalents via reactions that are not directly coupled to the central carbon metabolism: NAD+ kinase (EC 2.7.1.23); NADH kinase (EC 2.7.1.86), NADP+ phosphatase (EC 2.7.1.23); NAD+-dependent dehydrogenase (EC:1.12.1.2); NADP(H)+-dependent dehydrogenases (EC 1.12.1.3); energy-independent soluble pyridine nucleotide transhydrogenase (UdhA, EC 1.6.1.1); energy-dependent proton translocating membrane-bound pyridine nucleotide transhydrogenase (PntAB, EC 1.6.1.2); ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2).CC BY · PubMed Central
FIGURE 2 Overview of the NAD(P)+/NAD(P)H dependent reactions in central carbon metabolic modules (glycolysis, pentose phosphate pathway, TCA cycle, and Entner-Doudoroff pathway) and heterologous redox cofactor regeneration systems commonly used to fuel redox-intensive production processes. Green arrows and red arrows indicate reactions, which have been upregulated and downregulated/deleted, respectively, to promote/improve redox intensive production schemes. Enzyme names are accompanied by their abbreviations as cited in the text. Respective genes are: the PPP genes zwf (glucose 6-phosphate dehydrogenase, G6PDH) and gnd (6-phosphogluconate dehydrogenase, 6PGD) both encoding NADPH forming enzymes (Lim et al., 2002; Poulsen et al., 2005; Zhang et al., 2016a); the glycolytic genes pgi (glucose-6-phosphate isomerase, GPI), pfkA/B (phosphofructokinase I/II, PFK), and gapA (G3P dehydrogenase, GAPDH); the TCA cycle genes maeA/B (malic enzyme, ME) and icd (Isocitrate dehydrogenase, IDH). The symbol ( * ) denotes enzymes that can be exchanged with functionally equivalent isozymes but with different cofactor preference (i.e., NADP+-dependent variants of aldehyde dehydrogenase and GAPDH). The symbol ( ** ) denotes reactions that have been either activated or deactivated depending on the envisaged redox related metabolic engineering target, as in the case of isocitrate lyase and succinyl-coA synthetase. With activated glyoxylate shunt, more malate is formed which in turn can be converted to pyruvate by the ME involving NADPH formation. In order to promote α-KG dependent redox biotransformations, the glyoxylate shunt has been deactivated forcing metabolic flux through the introduced enzyme (e.g., proline or isoleucine dioxygenases). Succinyl-CoA synthetase activity significantly promotes NADPH allocation (via IDH) for NADPH-dependent syntheses (e.g., xylitol production). On the other hand, deletion or reduction of this activity promotes α-KG-dependent redox biotransformations.
FIGURE 2 Overview of the NAD(P)+/NAD(P)H dependent reactions in central carbon metabolic modules (glycolysis, pentose phosphate pathway, TCA cycle, and Entner-Doudoroff pathway) and heterologous redox cofactor regeneration systems commonly used to fuel redox-intensive production processes. Green arrows and red arrows indicate reactions, which have been upregulated and downregulated/deleted, respectively, to promote/improve redox intensive production schemes. Enzyme names are accompanied by their abbreviations as cited in the text. Respective genes are: the PPP genes zwf (glucose 6-phosphate dehydrogenase, G6PDH) and gnd (6-phosphogluconate dehydrogenase, 6PGD) both encoding NADPH forming enzymes (Lim et al., 2002; Poulsen et al., 2005; Zhang et al., 2016a); the glycolytic genes pgi (glucose-6-phosphate isomerase, GPI), pfkA/B (phosphofructokinase I/II, PFK), and gapA (G3P dehydrogenase, GAPDH); the TCA cycle genes maeA/B (malic enzyme, ME) and icd (Isocitrate dehydrogenase, IDH). The symbol ( * ) denotes enzymes that can be exchanged with functionally equivalent isozymes but with different cofactor preference (i.e., NADP+-dependent variants of aldehyde dehydrogenase and GAPDH). The symbol ( ** ) denotes reactions that have been either activated or deactivated depending on the envisaged redox related metabolic engineering target, as in the case of isocitrate lyase and succinyl-coA synthetase. With activated glyoxylate shunt, more malate is formed which in turn can be converted to pyruvate by the ME involving NADPH formation. In order to promote α-KG dependent redox biotransformations, the glyoxylate shunt has been deactivated forcing metabolic flux through the introduced enzyme (e.g., proline or isoleucine dioxygenases). Succinyl-CoA synthetase activity significantly promotes NADPH allocation (via IDH) for NADPH-dependent syntheses (e.g., xylitol production). On the other hand, deletion or reduction of this activity promotes α-KG-dependent redox biotransformations.CC BY · PubMed Central
FIGURE 3 Coupling of α-ketoglutarate dependent oxygenases to phototrophic and heterotrophic metabolism. These enzymes catalyze a remarkable range of reactions, such as hydroxylation, desaturation, and epoxidation. They utilize iron (II) as the metallo-cofactor and α-ketoglutarate as the co-substrate that is oxidatively decarboxylated to succinate and CO2.
FIGURE 3 Coupling of α-ketoglutarate dependent oxygenases to phototrophic and heterotrophic metabolism. These enzymes catalyze a remarkable range of reactions, such as hydroxylation, desaturation, and epoxidation. They utilize iron (II) as the metallo-cofactor and α-ketoglutarate as the co-substrate that is oxidatively decarboxylated to succinate and CO2.CC BY · PubMed Central
FIGURE 4 Tapping the reductive potential of photosynthesis. Green: components of the photosynthetic electron transfer chain (PETC); yellow: internal electron sinks; blue: (native) hydrogenase (Hox); red: (recombinant) redox enzymes. Electrons derived from water splitting enter the photosynthetic electron transport chain at photosystem II, where energy from light quanta promotes them to a higher excitation state (a more negative potential). This excited state can be used for photochemistry, i.e., the reduction of the primary acceptor QA/QB, followed by electron transport over plastoquinone (PQ) and cytochromes (Cytb/d and Cytc) along a gradient of redox potentials. Photosystem I again absorbs light energy and constitutes a reducing agent strong enough to transfer electrons to Ferredoxin (Fd). The minimal redox potentials in photosystems are not drawn to scale. Ferredoxin-NADPH-reductase (FNR) allocates a big share of these electrons to NADPH, which either can be used for C-fixation or can be converted to NADH by transhydrogenases (TH). Alternatively, electrons can be distributed to N- and S-assimilation (Fd-nitrate-reductase NarB, Fd-nitrite-reductase NirA, and Fd-sulfite-reductase SiR), regulatory elements (e.g., Fd-thioredoxin-reductase FTR), or back to photosystem I, referred to as cyclic electron transfer (CET). At different points of the PETC, reductive stress can be released by transferring electrons to acceptors like the flavodiiron proteins Flv2/4 and Flv1/3 and respiratory oxidases like the PQ-terminal oxidase PTOX, Cytb/d- oxidase Cyd, and Cytc-oxidase Cox. Redox potentials of electron sinks are not drawn to scale. So far, the reductive potential of photosynthesis was tapped at or downstream of PS I, by fusion constructs (dotted line), at the level of Fd (Hox and CYP450), and at the level of NADPH (Baeyer-Villiger monooxygenase BVMO and ene-reductase Yqjm).
FIGURE 4 Tapping the reductive potential of photosynthesis. Green: components of the photosynthetic electron transfer chain (PETC); yellow: internal electron sinks; blue: (native) hydrogenase (Hox); red: (recombinant) redox enzymes. Electrons derived from water splitting enter the photosynthetic electron transport chain at photosystem II, where energy from light quanta promotes them to a higher excitation state (a more negative potential). This excited state can be used for photochemistry, i.e., the reduction of the primary acceptor QA/QB, followed by electron transport over plastoquinone (PQ) and cytochromes (Cytb/d and Cytc) along a gradient of redox potentials. Photosystem I again absorbs light energy and constitutes a reducing agent strong enough to transfer electrons to Ferredoxin (Fd). The minimal redox potentials in photosystems are not drawn to scale. Ferredoxin-NADPH-reductase (FNR) allocates a big share of these electrons to NADPH, which either can be used for C-fixation or can be converted to NADH by transhydrogenases (TH). Alternatively, electrons can be distributed to N- and S-assimilation (Fd-nitrate-reductase NarB, Fd-nitrite-reductase NirA, and Fd-sulfite-reductase SiR), regulatory elements (e.g., Fd-thioredoxin-reductase FTR), or back to photosystem I, referred to as cyclic electron transfer (CET). At different points of the PETC, reductive stress can be released by transferring electrons to acceptors like the flavodiiron proteins Flv2/4 and Flv1/3 and respiratory oxidases like the PQ-terminal oxidase PTOX, Cytb/d- oxidase Cyd, and Cytc-oxidase Cox. Redox potentials of electron sinks are not drawn to scale. So far, the reductive potential of photosynthesis was tapped at or downstream of PS I, by fusion constructs (dotted line), at the level of Fd (Hox and CYP450), and at the level of NADPH (Baeyer-Villiger monooxygenase BVMO and ene-reductase Yqjm).CC BY · PubMed Central

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Metadata

Authors
Theodosiou, E., Tüllinghoff, A., Toepel, J., & Bühler, B.
Affiliations
  • Centre for Research and Technology Hellas (GR)
  • Helmholtz Centre for Environmental Research (DE)
Corresponding author
Bühler, Bruno
ORCID
Venue
Frontiers in Bioengineering and Biotechnology
Publisher
Frontiers Media SA
Published
2022-04-13
Volume
10
Pages
855715-855715
ISSN
2296-4185 (ISSN-L)
DOI
10.3389/fbioe.2022.855715
PMC
PMC9043136
OpenAlex
W4224125904
Semantic Scholar
e00eaee1b714c8d312839e3acb7eed6543ad4153
Cited by
17
References
239
Citations by year
2026: 6 · 2025: 4 · 2024: 1 · 2023: 5 · 2022: 1
FWCI
0.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Bioengineering and Biotechnology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Helmholtz-Zentrum für Umweltforschung
  • European Regional Development Fund
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Biochemical and biochemical processes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
whole-cell biocatalysis, metabolic modules, heterotrophic metabolism, redox metabolism, NAD(P)H regeneration, metabolic engineering, flux control, bioprocess intensification

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