Trends in Biotechnology
ReviewBiocatalysis in Green and Blue: Cyanobacteria
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
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These authors contributed equally