Trends in Biotechnology
Volume 39, Issue 9, September 2021, Pages 875-889
Journal home page for Trends in Biotechnology

Review
Biocatalysis in Green and Blue: Cyanobacteria

https://doi.org/10.1016/j.tibtech.2020.12.009Get rights and content

Highlights

  • Cyanobacteria use photosynthesis to harvest the Sun’s energy to bind atmospheric CO2 and accumulate biomass. This ability renders cyanobacteria interesting hosts for sustainable industrial processes.

  • The photoautotrophic metabolism allows a constant supply with oxygen and NADPH, and so overcoming limitations, often faced in biotransformation with heterotrophic hosts.

  • Compared to established biocatalysis hosts, cyanobacteria lag several years of development. Exploitation of their metabolism, especially of the photosynthesis apparatus, still shows room for improvement.

  • Many aspects for whole-cell catalysis have not been investigated carefully enough, including mass transfer of substrate and product or tolerance towards stressors, such as solvents or ROS.

  • Novel photo-bioreactor concepts and also new, and more efficient strains must be introduced to achieve economically feasible production.

Recently, several studies have proven the potential of cyanobacteria as whole-cell biocatalysts for biotransformation. Compared to heterotrophic hosts, cyanobacteria show unique advantages thanks to their photoautotrophic metabolism. Their ability to use light as energy and CO2 as carbon source promises a truly sustainable production platform. Their photoautotrophic metabolism offers an encouraging source of reducing power, which makes them attractive for redox-based biotechnological purposes. To exploit the full potential of these whole-cell biocatalysts, cyanobacterial cells must be considered in their entirety. With this emphasis, this review summarizes the latest developments in cyanobacteria research with a strong focus on the benefits associated with their unique metabolism. Remaining bottlenecks and recent strategies to overcome them are evaluated for their potential in future applications.

Section snippets

CO2 Capture and Valorization with Cyanobacteria

Global emissions increased from 2 billion tons of CO2 per year to over 36 billion tons within the last 120 years (https://ourworldindata.org/co2-and-other-greenhouse-gas-emissions). This continuous increase of CO2 leads to a steady rise in global temperature. Catastrophic weather phenomena such as droughts, glacier retreats, and sea level rises are only some of the associated effects [1]. These are already noticeable, and the related consequences will be the greatest threat to humankind and the

Enzyme Synthesis and Genetic Engineering

Biotransformations with wild-type cyanobacterial strains quickly reach their limits regarding the range of applicable enzymes and expression levels. Optimized production strains contribute significantly for a realization on industrial scale. Genetic engineering has become an integral part of biotechnology and has its share when it comes to biocatalysis.

A key step in microbial engineering is the expression of one or more enzymes, catalyzing the desired reactions. Early attempts on expressing

Biocatalysis and Photoautotrophic Metabolism

Once the enzyme of interest is successfully expressed in the cyanobacterial host, its activity is often dependent on various cofactors or cosubstrates. Shortages here can lead to reduced yields or even the absence of any enzymatic transformation. In this regard, whole-cell mediated biotransformations have a clear advantage: (i) essential cofactors are provided by the cell and are continuously recycled; (ii) enzyme production is performed by the host; and (iii) valuable building blocks of the

Provision of Cofactors

Many enzymes are cofactor/substrate dependent. Their availability is crucial for their catalytic performance. Compared to heterotrophic hosts, cyanobacteria differ significantly in the availability and the ratio of the cofactors.

In cyanobacteria, the absolute NADPH concentration is reported to be approximately 6.5-fold higher than that of NADH [36]. In comparison, common other hosts, such as E. coli, or Pseudomonas, show 4–5 times more NADH than NADPH [37]. This large NADPH pool in

Provision of Electrons

Photosynthesis generates a surplus of redox power, besides NADPH, which promises a usable source for biotechnological exploitation. This reducing power can be used directly by other electron acceptors, including recombinant enzymes (Figure 2). Coupling of recombinant enzymes to photosynthesis is a relatively new idea and was proven only for a few enzymes, such as hydrogenases, nitrogenases or cytochrome P450s [48]. Potent coupling partners are small electron carrier proteins (Figure 2,

Provision of Oxygen

Molecular oxygen is a predominant side product of photosynthesis and plays an important role as oxidant in biocatalysis [55]. Efficient gas–liquid mass transfer of oxygen represents a major problem, in particular for large-scale cultivations, that limits production yields substantially [56]. Oxygen availability in heterotrophic hosts suffers from a high demand of oxygen for the cell’s respiration, which competes with the target reaction [4,57]. A novel concept shows that by using photosynthetic

Mass Transfer

Mass transfer of substrate and product is an additionally crucial aspect in biocatalysis. Cell membranes separate the cell as reaction unit from the environment. For whole-cell catalysis, this ensures stable conditions, constant catalyst production, and recycling of cofactors [4]. The cellular structure can limit substrate availability and product removal, thus often reducing the overall efficiency (Table 2, entries 1, 2, and 5) [28]. The composition and thickness of the cell wall and membrane

Stress Tolerance

The impact of stress on overall productivity of biocatalytic hosts is well known. Organisms under stress have a slower metabolism, which translates into reduced biomass including lower product titers. Energy that would otherwise be put into the reproduction process or the target reaction is required by the cell to buffer the external stress source [67., 68., 69.].

The biocatalytic reaction poses a substantial stress source. Substrates, products, and solvents are often toxic and lead to reduced

Solvent Tolerance and Substrate/Product Toxicity

Organic solvents have a major impact on biocatalyst performance due to the substrate/product solubility and their toxic effects on the host cell [70]. In cyanobacteria, solvents impair the membrane integrity by intercalating into the phospholipid bilayer and disrupt energy maintenance and overall functions of the membrane that are essential for cell viability [71]. In order to use cyanobacteria as cell factories, solvent tolerance is essential to ensure profitable production processes.

Reactive Oxygen Species (ROS)

Oxygen activating enzymes – oxygenases – are an interesting class of enzymes, which are prone to be used in cyanobacteria. They require reducing equivalents and molecular oxygen to function. Both requirements are fulfilled in cyanobacteria. If the activated oxygen is not consumed efficiently, uncoupling takes place and ROS are generated [76,77]. Since excessive ROS have a negative impact on the overall performance of the cell, mitigating these effects is of interest to increase cyanobacteria’s

Scaleup

Biotransformations to produce fine chemicals become economical feasible at a productivity of at least 1–10 g/L/h [85]. Current literature suggests that cyanobacterial biotransformations are at the bottom of this range, explaining why they have not yet been used in large scale. Probable reasons are the slow growth kinetics of cyanobacteria and low cell densities, caused by self-shading [86]. Here, we briefly describe the hurdles of cyanobacterial high-density cultivation and possible solutions.

Concluding Remarks

Cyanobacteria use sunlight to power chemical reactions and thereby bind CO2 into biomass. Their efficient photosynthesis apparatus, high growth rate, and quantum efficiency, compared to land plants, and accessibility to genetic engineering make them promising candidates to drive chemical reactions on industrial scale in a highly sustainable process, capturing greenhouse gases, requiring minimal substrates in their medium and not competing for arable land.

Their exceptional photoautotrophic

Glossary

Biotransformation
transformation of an additional substrate into a value-added product by enzymatic catalysis. Conversion can occur by native enzymes, or by recombinant enzymes. The substrate itself is not used as source of energy or carbon source by the cell.
Cofactor
nonprotein molecule or metallic ion that is required for an enzyme’s activity or increases its conversion rate. These cofactors can either be simple inorganic metal ions (copper, iron, zinc, etc.) or complex organic molecule called

References (124)

  • G.T. Hanke

    A screen for potential ferredoxin electron transfer partners uncovers new, redox dependent interactions

    Biochim. Biophys. Acta - Proteins Proteomics

    (2011)
  • E.A. Peden

    Identification of global ferredoxin interaction networks in chlamydomonas reinhardtii

    J. Biol. Chem.

    (2013)
  • W. Baebprasert

    Increased H2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway

    Metab. Eng.

    (2011)
  • S.B. Mellor

    Defining optimal electron transfer partners for light-driven cytochrome P450 reactions

    Metab. Eng.

    (2019)
  • W.A. Duetz

    Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis

    Curr. Opin. Biotechnol.

    (2001)
  • S.A. Nicolaou

    A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation

    Metab. Eng.

    (2010)
  • H. Kusumawardhani

    Solvent tolerance in bacteria: fulfilling the promise of the biotech era?

    Trends Biotechnol.

    (2018)
  • Y. Nishiyama

    A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

    Biochim. Biophys. Acta Bioenerg.

    (2006)
  • Y.Y. He et al.

    UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine

    J. Photochem. Photobiol. B Biol.

    (2002)
  • A.J.J. Straathof

    The production of fine chemicals by biotransformations

    Curr. Opin. Biotechnol.

    (2002)
  • M. Jahn

    Growth of Cyanobacteria is constrained by the abundance of light and carbon assimilation proteins

    Cell Rep.

    (2018)
  • S. Li

    Development and optimization of genetic toolboxes for a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

    Metab. Eng.

    (2018)
  • S.Y. Lee

    High cell-density culture of Escherichia coli

    Trends Biotechnol.

    (1996)
  • S.H. Schneider

    Global climate change in the human perspective

  • Gaseous Carbon Waste Streams Utilization: Status and Research Needs

    (2019)
  • T.L. Hamilton

    The role of biology in planetary evolution: Cyanobacterial primary production in low-oxygen Proterozoic oceans

    Environ. Microbiol.

    (2016)
  • M. Schrewe

    Whole-cell biocatalysis for selective and productive C–O functional group introduction and modification

    Chem. Soc. Rev.

    (2013)
  • L. Al-Haj

    Cyanobacteria as Chassis for industrial biotechnology: progress and prospects

    Life

    (2016)
  • J. Kumar

    Cyanobacteria: Applications in Biotechnology

    (2018)
  • J. Singh et al.

    Overview of carbon capture technology: microalgal biorefinery concept and state-of-the-art

    Front. Mar. Sci.

    (2019)
  • A. Zhang

    Carbon recycling by cyanobacteria: Improving CO2 fixation through chemical production

    FEMS Microbiol. Lett.

    (2017)
  • S.A. Angermayr et al.

    On the use of metabolic control analysis in the optimization of cyanobacterial biosolar cell factories

    J. Phys. Chem. B

    (2013)
  • J. Zhou

    Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria

    Sci. Rep.

    (2014)
  • A. Behle

    Comparative dose-response analysis of inducible promoters in cyanobacteria

    ACS Synth. Biol.

    (2020)
  • E. Englund

    Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

    Sci. Rep.

    (2016)
  • K. Thiel

    Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803

    Microb. Cell Factories

    (2018)
  • H.M. Salis

    Automated design of synthetic ribosome binding sites to control protein expression

    Nat. Biotechnol.

    (2009)
  • R. Li et al.

    Enhancer activity of light-responsive regulatory elements in the untranslated leader regions of cyanobacterial psbA genes

    Proc. Natl. Acad. Sci. U. S. A.

    (1993)
  • J. Mitschke

    An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803

    Proc. Natl. Acad. Sci. U. S. A.

    (2011)
  • J.C. Cameron

    Genetic and genomic analysis of RNases in model cyanobacteria

    Photosynth. Res.

    (2015)
  • G.C. Gordon et al.

    Regulatory tools for controlling gene expression in cyanobacteria

    Adv. Exp. Med. Biol.

    (2018)
  • C.M. Immethun et al.

    Synthetic gene regulation in cyanobacteria

    Adv. Exp. Med. Biol.

    (2018)
  • R. Vasudevan

    Cyanogate: a modular cloning suite for engineering cyanobacteria based on the plant moclo syntax

    Plant Physiol.

    (2019)
  • B. Wang

    Application of synthetic biology in cyanobacteria and algae

    Front. Microbiol.

    (2012)
  • G.M. Taylor

    Start-Stop Assembly : a functionally scarless DNA assembly system optimized for metabolic engineering

    Nucleic Acids Res.

    (2019)
  • N. Betterle

    Cyanobacterial production of biopharmaceutical and biotherapeutic proteins

    Front. Plant Sci.

    (2020)
  • J. Tony Pembroke

    Metabolic engineering of the model photoautotrophic cyanobacterium Synechocystis for ethanol production: optimization strategies and challenges. Fuel Ethanol Production from Sugarcane

    (2019)
  • B. Lin et al.

    Whole-cell biocatalysts by design

    Microb. Cell Factories

    (2017)
  • G.A.R. Gale

    Emerging species and genome editing tools : future prospects in cyanobacterial synthetic biology

    Microorganisms

    (2019)
  • A. Hitchcock

    Progress and challenges in engineering cyanobacteria as chassis for light-driven biotechnology

    Microb. Biotechnol.

    (2020)
  • Cited by (32)

    • Engineering cyanobacterial chassis for improved electron supply toward a heterologous ene-reductase

      2022, Journal of Biotechnology
      Citation Excerpt :

      In the light of establishing primary producers as chassis for light-driven biotechnology, cyanobacteria are remarkably gaining attention for photobiotransformations (Jodlbauer et al., 2021; Schmermund et al., 2019).

    View all citing articles on Scopus
    3

    These authors contributed equally

    View full text