Since the Industrial Revolution, the annual emissions of highly endothermic greenhouse gases like carbon dioxide by humans into the atmosphere have steadily risen. This has led to the intensification of the greenhouse effect in the atmosphere, setting off a sequence of increasingly severe climate crises. Furthermore, the combustion of fossil fuels in thermal power plants stands as the primary contributor to carbon dioxide emissions, accounting for as much as one-third of the total.

Therefore, we have designed an electricity generation system for co-culture of Synechocystis sp. PCC 6803 and Shewanella oneidensis MR-1. The Synechocystis sp. PCC 6803 use solar energy to capture carbon dioxide and produce lactic acid. At the same time, Shewanella oneidensis MR-1 takes lactic acid transported by Synechocystis sp. PCC 6803 as its carbon source and oxidizes it completely to generate the power.

Meanwhile, we have learned that thermal power plants generate a significant amount of waste heat. If this waste heat can be utilized to cultivate our strains, we can not only reduce the greenhouse effect but also enhance energy efficiency.

To enhance the carbon sequestration and electricity production capabilities of our co-culture system, we concentrate on modifying the two strains, with a particular emphasis on increasing their intracellcular NADH concentration.

Chassis strains: Synechocystis sp. PCC 6803 and Shewanella oneidensis MR-1

Synechocystis sp. PCC 6803 are a group of organisms characterized by aerobic photosynthesis. The carbon dioxide fixed by Synechocystis sp. PCC 6803 is stored as a polysaccharide and is metabolized into various compounds. Synechocystis sp. PCC 6803 is the earliest model cyanobacteria to be sequenced completely, which is also a commonly used substrate cell for photosynthetic carbon sequestration and production of [1]. Lactic acid produced by Synechocystis sp. PCC 6803 for electricity production of S. oneidensis MR-1.

Fig 1. The carbon metabolic pathway in Synechocystis sp. PCC 6803.

Shewanella oneidensis MR-1 is one of the most widely studied model microorganisms in the field of bioelectrochemistry. It is also an electricity producing strain that uses electrodes as electron mediators for extracellular electron transport. The capacity of S. oneidensis MR-1’s ability to utilize a wide range of electron receptors for growth respiration has received extensive attention. In particular, in terms of electricity generation, S. oneidensis MR-1 can use lactic acid as its respiratory substrate[2]. Furthermore, S. oneidensis MR-1 is capable of utilizing lactic acid as its respiratory substrate and completely oxidizing lactic acid under anaerobic conditions. Additionally, S. oneidensis MR-1 preferentially utilizes D-lactate, and the maximum current density is positively correlated with the concentration of D-lactate. In addition, S. oneidensis MR-1 enable to use self-secreted quinone derivatives or flavin electron mediators to indirectly transport electrons to the electrode as well[3].

Strategy 1: Photosynthetic carbon sequestration in Synechocystis sp. PCC 6803

To address the greenhouse effect, we have enhanced the carbon fixation capability of Synechocystis sp. PCC 6803. Through this improvement, Synechocystis sp. PCC 6803 can continually capture carbon dioxide, engage in photosynthesis for carbon fixation, leading to lactic acid production, and then transport the lactic acid to the extracellular space.

Synechocystis sp. PCC 6803 itself lacks a pathway to produce lactic acid. Moreover, as a photoautotrophic microorganism, Synechocystis sp. PCC 6803 also lacks a lactate transporter to facilitate the export of lactic acid[4]. To address these limitations, we have introduced lactate dehydrogenase and a lactate transporter protein into Synechocystis sp. PCC 6803. This modification enables the production of lactate and the transport of the produced lactate to the extracellular layer of the organism.

We have selected ldhA as the lactate dehydrogenase gene, which enables Synechocystis sp. PCC 6803 to produce lactic acid. Lactate dehydrogenase A is a glycolytic enzyme responsible for catalyzing the reverse conversion of pyruvate into lactic acid.

NAD+ and NADP+ mainly act as not only coenzymes of dehydrogenase but also the hydrogen transmitters in enzymatic reactions. In Synechocystis sp. PCC 6803, due to the fact that the NADPH molecules produced by photosynthesis far exceed the NADH molecules produced by catabolism, NADPH is the major form of reducing equivalent in Synechocystis sp. PCC 6803.

However, the activity of NADPH is not as high as that of NADH, for this reason most dehydrogenase enzymes tend to use NADH as a cofactor. Therefore, sufficient NADH supply is significant for the production of dehydrogenase derived chemicals in Synechocystis sp. PCC 6803.

Nevertheless, the physiological environment with low NADH/NADPH ratio restricts the application of NADH dependent dehydrogenase in Synechocystis sp. PCC 6803. Therefore, we are faced with two pressing issues that require immediate attention: first, how to raise the concentration of NADH to ensure an adequate supply for lactic acid production, leading to increased lactic acid production; and second, how to facilitate the subsequent enhancement of electricity production in S. oneidensis MR-1.

At the same time, we have introduced the lldP gene to express lactate transporters, which are used to transport lactate out of cells.The LldP protein has 12 transmembrane alpha-helical segments, which can effectively transport lactic acid. Furthermore,this lactate transport process is dependent on [5], which means it also requires us to increase the photosynthetic efficiency of Synechocystis sp. PCC 6803.

As a consequence, we have brought in the gene omcS encoding electron transfer proteins into Synechocystis sp. PCC 6803 to change the direction of intracellular electron [6]. The appearance of Omcs can conduct the electrons to produce more ATP from photoreaction, increase their proton kinetic potential, and raise the concentration of NADH concurrently.

The pathways of photosynthetic electron transfer are mainly divided into linear electron transfer (LET) and cyclic electron transfer (CET). In the process of LET, electrons are ultimately transferred to NADP+ through photosystem II (PSII), cytochrome b6f (Cytb6f), and photosystem I (PSI) to produce NADPH, and also a proton gradient across the thylakoid membrane is formed to drive ATP synthase to produce ATP. Besides the LET, as auxiliary, CET cycles electrons from the PSI acceptor side back to plastoquinone (PQ), cytochrome b6f (cytb6f) complex and plastocyanin (PC), generating ATP without net NADPH production to meet metabolic demand. Synechocystis sp. PCC 6803 also have respiratory electron transfer (RET), where the NADH generated from the catabolism of organic carbon donates electrons to generate ATP for sustaining cell growth or cellular activities, resulting in low intracellular NADH concentration and no additional NADH available for chemical productions involving NADH dependent dehydrogenase. Therefore, we are able to decline the concentration of ATP produced by RET by increasing the ATP produced by photosynthetic electrons, thus saving NADH consumed by RET.

To reach the goal, we have introduced a redox protein called C-type outer membrane cytochromes which is encoded by gene omcS from Geobacter sp. into Synechocystis sp. PCC 6803. Regulating LET and CET is crucial for efficient photosynthesis. Additionally, enhance CET can drive LET as well as photosynthesis. As the most abundant mobile small electron carrier, PQ is shared by LET, CET, and RET in Synechocystis sp. PCC 6803[7]. The introduced OmcS directs excess electrons from PQ to PSI to stimulate CET and drive LET to regulate the ratio of ATP and NADPH, leading to three effects:

(1) Improves CET, which increases ATP production.

(2) Improve LET, which increases both ATP and NADPH production during photoreaction to meet metabolic needs.

(3) Represses RET, which enables NADH to accumulate that otherwise will be respired to generate ATP, thus leading to an increase NADH level to improve the activity of lactate dehydrogenase.

Fig 2. Efects of OmcS on the expression of central metabolism genes.

Strategy 2: Electricity generation of Shewanella oneidensis MR-1

Shewanella is a microorganism endowed with the capability of extracellular electron transfer (EET). This unique ability allows it to decompose organic matter and convert the stored chemical energy into electrical energy. Shewanella oneidensis MR-1, one of the most well-studied metal-reducing exoelectrogens, is capable of conducting EET to enable metal reduction or power generation in microbial fuel cells (MFCs).

However, the slow extracellular electron transfer (EET) rate of electroactive microorganisms is still the main bottleneck limiting the practical application of bioelectrochemistry. Intracellular NAD(H/+) (i.e., the total level of NADH and NAD+) is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways. An increase in intracellular NAD(H/+) results in the transfer of more electrons from the increased oxidation of the electron donor to the EET pathways of S. oneidensis MR-1, thereby enhancing intracellular electron flux and the EET rate. Accordingly, the synthetic biological strategy to improve the intracellular NAD (H/+) level is of great value to promote the efficiency of EET.

S. oneidensis MR-1 harbors heterogeneously introduced genes (ycel, pncB, nadM, nadD*, and nadE*) to enhance NAD+ biosynthesis. The network architecture of NAD+ biosynthesis in S. oneidensis MR-1 can [8]:

Module 1 involves de novo biosynthesis by assimilating L-aspartate (LAsp).

Module 2 involves salvage biosynthesis from the precursors Na (nicotinic acid) and Nm (nicotinamide). S. oneidensis MR-1 can assimilate Nm as a precursor, but cannot use Na as a precursor for lacking the enzyme that converts Na to nicotinic acid mononucleotide (NaMN). Therefore, in Module 2, in order to be able to assimilate and metabolize Na and Nm simultaneously, we have introduced the gene encoding the bifunctional transporter niaP of Na and Nm from Bacillus subtiles and the gene pncB encoding nicotinic acid phosphoribosyltransferase ycel from Salmonella typhimurium , so that we can synergistically drive the conversion of Nm and Na to NAD+.

Module 3 involves universal biosynthesis from a common portion of the de novo and salvage biosynthesis pathways. In module 3, S. oneidensis MR-1 cannot synthesize NAD+ directly from nicotinamide mononucleotide (NMN), but can only synthesize NAD+ through a circuitous pathway from NMN to NaMN and then to nicotinate adenine dinucleotide (NaAD), which leads to low efficiency of NAD+ biosynthesis. Therefore, we introduced nadM, a gene encoding NMN adenylyltransferase from Francisella tularensis , to establish a bridge between the salvage biosynthesis and universal biosynthesis to directly convert NMN to NAD+ to enhance the EET rate.

In addition, we have introduced the gene nadE* encoding NAD+ synthase and the gene nadD* encoding nicotinic acid mononucleotide adenylyltransferase from Escherichia coli to increase the original rate of NAD+ synthesis from salvage and universal biosynthetic pathways. In the end, we are able to further promote the EET rate in S. oneidensis MR-1.

Another EET mechanism of S. oneidensis MR-1 is indirect electron transfer mediated by endogenously secreted soluble electron shuttling flavin, including flavin mononucleotide (FMN) and riboflavin (RF), to enhance the electricity production capacity of S. oneidensis MR-1. In order to increase the diffusion of riboflavin from S. oneidensis MR-1, we introduced porin Oprf from Pseudomonas aeruginosa to increase the transport of riboflavin across the cell membrane to further improve the power generation capacity.

Fig 3. Schematic of modular design to enhance NAD+ biosynthesis and EET rate in S. oneidensis MR-1.

Strategy 3:Oxidative-stress detoxification and DNA protection

When our microbial fuel cell system is applied to thermal power plants, we need to take into account the impact of harsh environmental factors such as strong oxidizing exhaust gas - environmental stress will cause the accumulation of a large number of reactive oxygen radicals in cells, resulting in serious damage to proteins, membrane lipids, DNA and other cellular components.

To settle these problems, we have overexpressed GshA and GshB in Synechocystis sp. PCC 6803 to produce elevate glutathione level, which has cellular detoxification effects. Glutathione is a tripeptide compound. It exists under two forms: GSH and GSSG. The reduced form (GSH) can undergo oxidation to maintain the intracellular cell environment in a reduced state. The resulting glutathione disulfide—the oxidized form (GSSG) can be recycled into GSH by glutathione reductase. GSH is synthesized by the continuous interaction of γ-glutamyl cysteine synthetase (GshA) and glutathione synthetase (GshB). GshA synthesizes the intermediate product γ-glutamyl cysteine using glutamic acid and cysteine as substrates, and then GshB adds [9]. Therefore, the overexpressed GshA and GshB can produce more GSH in Synechocystis sp. PCC 6803, which as an electron donor of antioxidant glutathione (Grx), can catalyze the reduction of disulfide (protein-s-s-protein) or glutathione mixed disulfide (protein-s-sg), thereby maintaining the activity and stability of intracellular proteins, increasing the intracellular antioxidant capacity of Synechocystis sp. PCC 6803.

Fig 4. Schematic of GSH metabolic pathways in Synechocystis sp. PCC 6803.

Besides, we also need to protect S. oneidensis MR-1 from the damage of strong oxidizing substances in exhaust gas and enhance the intracellular antioxidant capacity. We have introduced SODA of superoxide dismutase (SOD)[10]. SOD is a widely existing antioxidant metalloenzyme, which is known to directly scavenge free radicals as well. Specifically, SOD disproportionates active superoxide radicals through the cyclic redox mechanism, with high specificity and efficiency. Moreover, it plays an important role in the[11]. Meanwhile, we have known that SOD containing Fe(II)(FeSOD) and SOD containing Mn(II) (MnSOD) are considered to be the oldest forms of SOD, which are still one of the most common forms of SOD in various modern aerobic and anaerobic bacterial strains so far.

However, we found that S. oneidensis MR-1 has high Fe(II) content but low intracellular Mn(II) concentration, which is not resistant to strong oxidizing environment, while Deinococcus radiodurans has strong resistance to this environment, which accumulates very high Mn(II) concentration and low Fe(II) concentration in the cell, and has a Mn(II)- dependent response. Then we have introduced the MnSOD of Deinococcus radiodurans , because of its better antioxidant effect . The accumulation of Mn (II) helps to improve the antioxidant capacity, can directly scavenge free radicals in its body, and better protect our S. oneidensis MR-1.

Considering that thermal power plants have other harsh environmental factors in addition to oxidative waste gas, we expect to enhance the stress resistance of Synechocystis sp. PCC 6803 and S. oneidensis MR-1, so that they can also have strong viability in harsh environments, and ensure the normal operation of our microbial fuel cells. Additionally, we have introduced damage suppressor (Dsup), a specific protein with damage inhibitory activity from tardigrades, into Synechocystis sp. PCC 6803 and S. oneidensis MR-1 to protect DNA from radiation and free radical damage. The protein is disordered in nature, which enables Dsup to adjust its structure to fit the DNA shape and facilitate complementary binding with DNA. What’s more, there is a large number of charged residues with dominance of positive (59 Lysine+12 Arginine) over negative (29 Aspartate+19 Glutamate) amino acids, which together account for 26.7% of total and yield a net charge+23[12]. There is strong electrostatic attraction between Dsup with positive net charges and DNA with negative net charges, which can better form protein-DNA aggregates to protect DNA and reduce radiation and free radical damage received by DNA damage.

Fig 5. The mechanism of Dsup protecting DNA.

Pathway construction

In the end, we have designed a total of four plasmids, which were respectively in Synechocystis sp. PCC 6803 and Shewanella oneidensis MR-1.

In Synechocystis sp. PCC 6803:

(1) pLactate: Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc

For ldhA and lldp, the promoter we selected is pcpcpc560 which is a super strong promoter suitable for Synechocystis sp. PCC 6803[13]. The ppsba2 promoter carrying omcs can prevent the disorder of electron transport[14]. Therefore, both of them can ensure the considerable expression of heterogeneously genes in Synechocystis sp. PCC 6803 which improve the photosynthetic carbon fixation efficiency of Synechocystis sp. PCC 6803 to increase lactate production.

Fig 6. The schematic of pLactate.

(2) pResistance6803: PrbcL-dsup-Tpsbc-PrbcL-gshA-gshB-Tpsbc pResistance6803 γ-Glutamylcysteine synthetase gene (gshA), glutathione synthetase gene (gshB) and damage suppressor gene dsup are expressed in combination driven by light intensity promoter prbcl to cope with the harsh environment of strong oxidative substances and teratogenicity.

Fig 7. The schematic of pResistance6803.

In Shewanella oneidensis MR-1:

In order to control the expression of heterogeneously genes, we applied the promoter PTAC in the S. oneidensis MR-1 expression system - the promoter tac is constitutive, and we have added the lactose operon to make it an inducible promoter to construct the following two plasmids.

(3) pElectricty: Ptac-ycel-pncB-TrrnB T1-Ptac-nadE*-nadD*-nadM-TrrnB T1

This plasmid is composed of following genes, including ycel, pncb, nadM, nadD*, nadE*, so as to enhance the intracellular electron flux and EET rate, thus improving the efficiency of electricity production.

Fig 8. The schematic of pElectricty

(4) pResistance MR-1: Ptac-dsup-sodA-oprf-TrrnB T1

Considering the plasmid size, we combined the nuclear perforin gene oprf, which can increase the electricity production efficiency of S. oneidensis MR-1 as well, with superoxide dismutase gene (sodA) and damage inhibitory activity gene (dsup), in order to enhance the electron transport rate of S. oneidensis MR-1 and its stress resistance.

Fig 9. The schematic of pResistance MR-1


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