Over-expression of an electron transport protein OmcS provides sufficient NADH for D-lactate production in cyanobacterium.

BACKGROUND: An efficient supply of reducing equivalent is essential for chemicals production by engineered microbes. In phototrophic microbes, the NADPH generated from photosynthesis is the dominant form of reducing equivalent. However, most dehydrogenases prefer to utilize NADH as a cofactor. Thus, sufficient NADH supply is crucial to produce dehydrogenase-derived chemicals in cyanobacteria. Photosynthetic electron is the sole energy source and excess electrons are wasted in the light reactions of photosynthesis. RESULTS: Here we propose a novel strategy to direct the electrons to generate more ATP from light reactions to provide sufficient NADH for lactate production. To this end, we introduced an electron transport protein-encoding gene omcS into cyanobacterium Synechococcus elongatus UTEX 2973 and demonstrated that the introduced OmcS directs excess electrons from plastoquinone (PQ) to photosystem I (PSI) to stimulate cyclic electron transfer (CET). As a result, an approximately 30% increased intracellular ATP, 60% increased intracellular NADH concentrations and up to 60% increased biomass production with fourfold increased D-lactate production were achieved. Comparative transcriptome analysis showed upregulation of proteins involved in linear electron transfer (LET), CET, and downregulation of proteins involved in respiratory electron transfer (RET), giving hints to understand the increased levels of ATP and NADH. CONCLUSIONS: This strategy provides a novel orthologous way to improve photosynthesis via enhancing CET and supply sufficient NADH for the photosynthetic production of chemicals.

Impairing photorespiration increases photosynthetic conversion of CO2 to isoprene in engineered cyanobacteria.

Photorespiration consumes fixed carbon and energy generated from photosynthesis to recycle glycolate and dissipate excess energy. The aim of this study was to investigate whether we can use the energy that is otherwise consumed by photorespiration to improve the production of chemicals which requires energy input. To this end, we designed and introduced an isoprene synthetic pathway, which requires ATP and NADPH input, into the cyanobacterium Synechocystis sp. 6803. We then deleted the glcD1 and glcD2 genes which encode glycolate dehydrogenase to impair photorespiration in isoprene-producing strain of Synechocystis. Production of isoprene in glcD1/glcD2 disrupted strain doubled, and stoichiometric analysis indicated that the energy saved from the impaired photorespiration was redirected to increase production of isoprene. Thus, we demonstrate we can use the energy consumed by photorespiration of cyanobacteria to increase the energy-dependent production of chemicals from CO2.

Development of a longevous two-species biophotovoltaics with constrained electron flow.

Microbial biophotovoltaics (BPV) offers a biological solution for renewable energy production by using photosynthetic microorganisms as light absorbers. Although abiotic engineering approaches, e.g., electrode modification and device optimization, can enhance the electrochemical communication between living cells and electrodes, the power densities of BPV are still low due to the weak exoelectrogenic activity of photosynthetic microorganisms. Here, we develop a BPV based on a D-lactate mediated microbial consortium consisting of photosynthetic cyanobacteria and exoelectrogenic Shewanella. By directing solar energy from photons to D-lactate, then to electricity, this BPV generates a power density of over 150 mW·m-2 in a temporal separation setup. Furthermore, a spatial-temporal separation setup with medium replenishment enables stable operation for over 40 days with an average power density of 135 mW·m-2. These results demonstrate the electron flow constrained microbial consortium can facilitate electron export from photosynthetic cells and achieve an efficient and durable power output.

Production of Industrial Chemicals from CO2 by Engineering Cyanobacteria.

As photosynthetic prokaryotes, cyanobacteria can directly convert CO2 to organic compounds and grow rapidly using sunlight as the sole source of energy. The direct biosynthesis of chemicals from CO2 and sunlight in cyanobacteria is therefore theoretically more attractive than using glucose as carbon source in heterotrophic bacteria. To date, more than 20 different target chemicals have been synthesized from CO2 in cyanobacteria. However, the yield and productivity of the constructed strains is about 100-fold lower than what can be obtained using heterotrophic bacteria, and only a few products reached the gram level. The main bottleneck in optimizing cyanobacterial cell factories is the relative complexity of the metabolism of photoautotrophic bacteria. In heterotrophic bacteria, energy metabolism is integrated with the carbon metabolism, so that glucose can provide both energy and carbon for the synthesis of target chemicals. By contrast, the energy and carbon metabolism of cyanobacteria are separated. First, solar energy is converted into chemical energy and reducing power via the light reactions of photosynthesis. Subsequently, CO2 is reduced to organic compounds using this chemical energy and reducing power. Finally, the reduced CO2 provides the carbon source and chemical energy for the synthesis of target chemicals and cell growth. Consequently, the unique nature of the cyanobacterial energy and carbon metabolism determines the specific metabolic engineering strategies required for these organisms. In this chapter, we will describe the specific characteristics of cyanobacteria regarding their metabolism of carbon and energy, summarize and analyze the specific strategies for the production of chemicals in cyanobacteria, and propose metabolic engineering strategies which may be most suitable for cyanobacteria.

Introducing extra NADPH consumption ability significantly increases the photosynthetic efficiency and biomass production of cyanobacteria.

Increasing photosynthetic efficiency is crucial to increasing biomass production to meet the growing demands for food and energy. Previous theoretical arithmetic analysis suggests that the light reactions and dark reactions are imperfectly coupled due to shortage of ATP supply, or accumulation of NADPH. Here we hypothesized that solely increasing NADPH consumption might improve the coupling of light reactions and dark reactions, thereby increasing the photosynthetic efficiency and biomass production. To test this hypothesis, an NADPH consumption pathway was constructed in cyanobacterium Synechocystis sp. PCC 6803. The resulting extra NADPH-consuming mutant grew much faster and achieved a higher biomass concentration. Analyses of photosynthesis characteristics showed the activities of photosystem II and photosystem I and the light saturation point of the NADPH-consuming mutant all significantly increased. Thus, we demonstrated that introducing extra NADPH consumption ability is a promising strategy to increase photosynthetic efficiency and to enable utilization of high-intensity lights.

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment.

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

Cyanobacteria are oxygenic photosynthetic prokaryotes that play important roles in the global carbon cycle. Recently, engineered cyanobacteria capable of producing various small molecules from CO2 have been developed. However, cyanobacteria are seldom considered as factories for producing proteins, mainly because of the lack of efficient strong promoters. Here, we report the discovery and verification of a super-strong promoter P(cpc560), which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Using P(cpc560), functional proteins were produced at a level of up to 15% of total soluble protein in the cyanobacterium Synechocystis sp. 6803, a level comparable to that produced in Escherichia coli. We demonstrated that the presence of multiple TFBSs in P(cpc560) is crucial for its promoter strength. Genetically transformable cyanobacteria neither have endotoxins nor form inclusion bodies; therefore, P(cpc560) opens the possibility to use cyanobacteria as alternative hosts for producing heterogeneous proteins from CO2 and inorganic nutrients.