Genetic Engineering, Synthetic Biology and the Light Reactions of Photosynthesis

Abstract

One-sentence summary: Applications of synthetic biology to photosynthesis currently range Oxygenic photosynthesis is imperfect and the evolutionarily conditioned patchwork nature of 32 the light reactions in plants provides ample scope for their improvement (Leister, 2012; 33 Blankenship and Chen, 2013). In fact, only around 40% of the incident solar energy is used 34 for photosynthesis. Two obvious ways of reducing energy loss are to expand the spectral 35 band used for photosynthesis and to shift saturation of the process to higher light intensities. 36Indeed, even minor enhancements to the efficiency or stress resistance of the light reactions 37 of photosynthesis should have a positive impact on biomass production and yield (Leister, 38 2012; Blankenship and Chen, 2013; Long et al., 2015). 3in successful outcomes and is limited to a small subset of regulatory proteins (Pribil et al., 50 2010; Kromdijk et al., 2016; Glowacka et al., 2018). 51In addition to enhancing the light reactions for improved biomass production and 52 yield, concepts have been developed for the direct coupling of photosynthesis to other 53 important pathways that are indirectly connected to photosynthesis in natural systems, for 54 instance, because they reside in different subcellular compartments (Lassen et al., 2014a). 55Direct coupling would be expected to boost the production of rare compounds in cells and 56 contribute to the biotechnological production of high-value compounds in vivo. 57In vitro, it is possible to functionally link components of photosynthesis with entirely 58 unrelated biotic or abiotic catalysts, or with abiotic electrode materials. In fact, Photosystem I 59 (PSI) is naturally adapted for highly efficient light harvesting and charge separation (Kargul 60 et al., 2012;Nguyen and Bruce, 2014; Martin and Frymier, 2017), and has been described as 61 the most efficient natural nano-photochemical machine (Nelson, 2009). Upon light excitation, 62PSI produces the most powerful naturally occurring reducing agent -P700* -which, 63together with an exceptionally long-lived charge-separated state, provides sufficient driving 64 force to reduce protons to H 2 at neutral pH. PSI operates with a quantum yield close to 1.0, 65 and currently no synthetic system has approached its remarkable efficiency. Moreover, PSI 66 preparations are generally robust, especially those obtained from extremophilic microalgae 67 (Kubota et al., 2010; Haniewicz et al., 2018 introduced into photosynthetic species that lack them. In the subsequent two sections, the 106 combination of non-photosynthetic (Bio-bio hybrids, using photosynthesis as an electron 107 source for unrelated biological processes) and non-biological building blocks (Bio-nano 108 hybrids, using photosynthesis as an electron source for non-biological processes) with 109 components of the photosynthetic machinery are described. An overview of these approaches 110 is provided in Figure 2. (Carpenter et al., 1993; Vermaas et al., 1996) that lack the corre...

Improved recombinant expression and purification of functional plant Rubisco

“…As the pET28 and pET11a plasmids (for expression of Rubisco and chaperonins, respectively) share the pBR322 origin of replication, the long‐term coexistence of these plasmids may result in variation of copy number and thus fluctuations in expression levels . Thus, applications sensitive to fluctuations of Rubisco expression, such as directed evolution screening or metabolic engineering , may require the use of compatible plasmids.…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Their unique combination of characteristics, including relatively fast photosynthetic growth, availability of redox power, a plethora of internal membranes, and subcellular microcompartments, opens up a completely new palette of synthetic biology strategies that is not possible in other well-studied heterotrophic host organisms. The photosynthetic machinery is full of interesting targets for synthetic biology strategies (Leister, 2019), such as enhancing photosynthesis for increased biomass production and yield (Zhu et al, 2010), direct coupling of metabolic pathways to photosynthetic reducing power (Mellor et al, 2017), and creation of bio-nano hybrids where photosynthetic modules are used as a source of electrons for nonbiological processes by linking them to abiotic catalysts or electrode nanomaterials (Saar et al, 2018) or by derivatization of photosynthetic electrons by redox mediators (Longatte et al, 2015;Fu et al, 2017). Although in principle such applications can be hosted in plants, photosynthetic microorganisms offer distinct advantages: the combination of photosynthesis with simple unicellular organization, facile genetic manipulation strategies, quick growth in liquid cultures, relative ease of scale-up, and, if grown in contained facilities, potentially fewer regulatory challenges.…”

Shedding Light on the Power of Light

“…In this special issue on synthetic biology, Leister (2018) sheds light on the synthetic biology strategies that have applied the light reactions of photosynthesis for not only increasing biomass and crop yield, but also for coupling evolutionarily unconnected pathways to produce high-value compounds in vivo and for the generation of hydrogen or electricity in vitro. The evolutionarily conserved photosynthetic modules in plants, algae, and cyanobacteria have encouraged many groups to study the effect of swapping homologous proteins between species.…”

“…However, these simple genetic engineering feats have resulted in reduced photosynthetic efficiency or a total loss of photoautotrophy, highlighting the incompatibility of such approaches, despite the high similarity between core subunit proteins of photosystems. As discussed in Leister (2018), this inability to exchange conserved modules is a prime example of how evolution has optimized individual components of multicomponent systems to adapt to their intrinsic environments for optimal interactions and thus optimum function. As a result, exchanging single proteins disturbs the evolved optimal state and causes suboptimal photoautotrophy, a phenomenon that was initially coined as the "frozen metabolic accident" (Shi et al, 2005) and then generalized to the "frozen metabolic state" (Gimpel et al, 2016).…”

“…It is also interesting to note that the reintroduction of photosynthetic proteins lost in the evolutionary transition from cyanobacteria to chloroplasts in algae and plants, has demonstrated enhanced stress tolerance and an advantage to growth and photosynthesis in the transgenic plant, which contrasts with the unsuccessful expression of plant proteins in cyanobacteria. Leister (2018) also extensively discusses the various synthetic biology applications of exploiting the photosynthetic machinery for driving unrelated biological and nonbiological processes. The light reactions have an inherent capacity to generate reducing power (NADPH), which can be coupled to reactions that have a high demand for electrons.…”