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

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). The ease of cultivation, dependency on light as an energy source, CO2 as a carbon source, and water as electron donor paved the way for a rapid interest in these biological platforms (Knoot et al., 2018). Cyanobacteria harvest (sun)light energy converting it to reducing power that can be redirected for the production of valuable compounds and/or for the activity of biocatalysts. This is a particularly interesting approach for redox transformations, one of the most important reactions in organic synthesis which requires reduced electron donors such as nicotinamide cofactors (NADPH) (Hollmann et al., 2011). In cyanobacteria coupling whole-cell redox reactions to photosynthetic electron flow will allow for the substitution of sacrificial co-substrates by using water as the electron donor (Köninger et al., 2016).

In the last years, the progress of synthetic biology of cyanobacteria provided the tools for heterologous expression of oxidoreductive enzymes benefiting from the steady photosynthetic production of cofactors (Schmermund et al., 2019), with several examples ranging from ene-reductases (Assil-Companioni et al., 2020, Köninger et al., 2016), Baeyer–Villiger monooxygenase (Böhmer et al., 2017, Erdem et al., 2021), imine reductase (Büchsenschütz et al., 2020), AlkBGT hydroxylation system (Hoschek et al., 2019a), hydroxyisocaproate dehydrogenases (Jurkaš et al., 2021) and cytochrome P450 monooxygenase (Hoschek et al., 2019b), underline the versatility of this approach. Previously reported rates for whole-cell biotransformations with Synechocystis sp. PCC 6803 cells expression heterologous enzymes are in the range of 5.5 U gDCW−1 for the monooxygenase AlkBGT (Hoschek et al., 2019a), 5.7 U gDCW−1 for the monooxygenase CHMO (Böhmer et al., 2017), 20 U gDCW−1 for an imine reductase (Büchsenschütz et al., 2020), 39 U gDCW−1 for a P450 monooxygenase (Hoschek et al., 2019b) and above 100 U gDCW−1 for the YqjM ene-reductase (Assil-Companioni et al., 2020, Köninger et al., 2016). Rates above 100 U gDCW−1 are of particular interest for the establishment of industrial applications towards the synthesis of fine chemicals requiring a productivity of 1–10 g L−1 h−1 (Hoschek et al., 2018). Engineering cyanobacterial hosts/chassis by directing more reducing power to fuel the activities of heterologous enzymes represents an attractive approach to improve the reaction rates. Since one of the limitations for high reaction rates could be the competition from native electron transfer pathways, along with the inherent regulation of the photosynthetic electron transport that dissipates the excess electrons (Russo et al., 2019), the rewiring of photosynthesis offers the possibility of redirecting excess reducing potential to catalyze the formation of high-value compounds, enabling the tapping of electrons that otherwise would be wasted. Despite the vast knowledge regarding regulatory mechanisms around the cyanobacterial photosynthetic machinery (Lea-Smith et al., 2016, Mullineaux, 2014, Nikkanen et al., 2021), cyanobacterial chassis customization for enhancing the availability of reductive power to drive biocatalytic reactions within the cell is yet to be realized.

To maintain redox balance and prevent dangerous over-reduction of the electron transport chain, cyanobacteria have evolved a remarkable number of mechanisms to adjust the balance of photosynthetic outputs. They do so by mainly removing electrons from the photosynthetic electron transport chain through the activity of switches known as “electron valves” or by balancing cyclic vs. linear electron transport (Mullineaux, 2014, Murata et al., 2012). Flavodiiron proteins function as efficient release valves, reducing O2 to H2O, in a Mehler-like reaction (Allahverdiyeva et al., 2013). Recently, it was demonstrated that in Synechocystis sp. PCC 6803 the activity of a heterologous ene-reductase and a Baeyer–Villiger monooxygenase could be enhanced by the deletion of flavodiiron-protein encoding genes (Assil-Companioni et al., 2020, Erdem et al., 2021). The bidirectional hydrogenase was suggested to act as a valve for low-potential electrons (Appel et al., 2000, Tamagnini et al., 2007), being insensitive to light, reversibly inactivated by O2, and quickly reactivated by NADH or NADPH (Cournac et al., 2004). A markerless deletion mutant (ΔhoxYH) lacking the functional bidirectional hydrogenase was previously constructed and characterized as a robust photoautotrophic chassis (Pinto et al., 2015), and used for the expression of heterologous hydroxyisocaproate dehydrogenases (Jurkaš et al., 2021). Regarding the cyclic electron transport, one of the routes involves electron transfer from ferredoxin to plastoquinone via NAD(P)H dehydrogenase-like complex 1 (NDH-1), particularly subunit NdhD2 (Battchikova et al., 2011). It was previously shown that deletion of the gene coding for NdhD2 subunit led to increased activity of a heterologous cytochrome P450 expressed in Synechococcus sp. PCC 7002 (Berepiki et al., 2018).

To meet the goal of engineering the photosynthetic electron flow to create a customized Synechocystis sp. PCC 6803 based chassis optimized for redox-dependent enzymatic reactions, we focused on deleting several natural electron switches, dispensable under controlled laboratory conditions. The strategy consisted of inactivating genes coding for Flv3 (sll0550) or NdhD2 (slr1291) in Synechocystis sp. PCC 6803 wild-type (from now on wt) or by using the previously generated Synechocystis ΔhoxYH mutant and a novel double ΔhoxYHΔflv3 mutant (Pinto et al., 2012). To evaluate the effects of these deletions on the functionality and productivity of the chassis, we chose to express the well-characterized heterologous ene-reductase YqjM from Bacillus subtilis. This enzyme displays high conversion and enhanced enantioselectivity of fast Cdouble bondC double-bond reduction without side reactions constituting, therefore, a promising approach to evaluate rational design (Köninger et al., 2016). Our results demonstrate that engineering cyanobacterial chassis customized for biotransformations leads to improved activities and reaction rates of the YqjM-catalyzed ene-reduction.

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