Bioethanol from microalgae and cyanobacteria: A review and technological outlook

Engineering cyanobacteria for direct biofuel production from CO2

Cyanobacteria: Promising biocatalysts for sustainable chemical production

In this chapter, we mainly focus on the acclimation of cyanobacteria to the changing ambient CO2 and discuss mechanisms of inorganic carbon (Ci) uptake, photorespiration, and the regulation among the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth. The structural components for several of the transport and uptake mechanisms are described and the progress towards elucidating their regulation is discussed in the context of studies, which have documented metabolomic changes in response to changes in Ci availability. Genes for several of the transport and uptake mechanisms are regulated by transcriptional regulators that are in the LysR-transcriptional regulator family and are known to act in concert with small molecule effectors, which appear to be well-known metabolites. Signals that trigger changes in gene expression and enzyme activity correspond to specific "regulatory metabolites" whose concentrations depend on the ambient Ci availability. Finally, emerging evidence for an additional layer of regulatory complexity involving small non-coding RNAs is discussed.

https://www.mdpi.com/2075-1729/5/1/348/pdf

Regulation of CO2 Concentrating Mechanism in Cyanobacteria.

Cyanobacteria are promising microorganisms for sustainable biotechnologies, yet unlocking their potential requires radical re-engineering and application of cutting-edge synthetic biology techniques. In recent years, the available devices and strategies for modifying cyanobacteria have been increasing, including advances in the design of genetic promoters, ribosome binding sites, riboswitches, reporter proteins, modular vector systems, and markerless selection systems. Because of these new toolkits, cyanobacteria have been successfully engineered to express heterologous pathways for the production of a wide variety of valuable compounds. Cyanobacterial strains with the potential to be used in real-world applications will require the refinement of genetic circuits used to express the heterologous pathways and development of accurate models that predict how these pathways can be best integrated into the larger cellular metabolic network. Herein, we review advances that have been made to translate synthetic biology tools into cyanobacterial model organisms and summarize experimental and in silico strategies that have been employed to increase their bioproduction potential. Despite the advances in synthetic biology and metabolic engineering during the last years, it is clear that still further improvements are required if cyanobacteria are to be competitive with heterotrophic microorganisms for the bioproduction of added-value compounds.

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New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering.

Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms, which are engineered to synthesize valuable products directly from CO2 and sunlight. As cyanobacteria can be cultivated in large scale on non-arable land, these phototrophic bacteria have become attractive organisms for production of biofuels. Synechococcus sp. PCC 7002, one of the cyanobacterial model organisms, provides many attractive properties for biofuel production such as tolerance of seawater and high light intensities. Here, we performed a systems analysis of an engineered ethanol-producing strain of the cyanobacterium Synechococcus sp. PCC 7002, which was grown in artificial seawater medium over 30 days applying a 12:12 h day–night cycle. Biosynthesis of ethanol resulted in a final accumulation of 0.25% (v/v) ethanol, including ethanol lost due to evaporation. The cultivation experiment revealed three production phases. The highest production rate was observed in the initial phase when cells were actively growing. In phase II growth of the producer strain stopped, but ethanol production rate was still high. Phase III was characterized by a decrease of both ethanol production and optical density of the culture. Metabolomics revealed that the carbon drain due to ethanol diffusion from the cell resulted in the expected reduction of pyruvate-based intermediates. Carbon-saving strategies successfully compensated the decrease of central intermediates of carbon metabolism during the first phase of fermentation. However, during long-term ethanol production the producer strain showed clear indications of intracellular carbon limitation. Despite the decreased levels of glycolytic and tricarboxylic acid cycle intermediates, soluble sugars and even glycogen accumulated in the producer strain. The changes in carbon assimilation patterns are partly supported by proteome analysis, which detected decreased levels of many enzymes and also revealed the stress phenotype of ethanol-producing cells. Strategies towards improved ethanol production are discussed. Systems analysis of ethanol production in Synechococcus sp. PCC 7002 revealed initial compensation followed by increasing metabolic limitation due to excessive carbon drain from primary metabolism.

/pdf/systems-analysis-of-ethanol-production-in-the-genetically-4990maqia8.pdf

Systems analysis of ethanol production in the genetically engineered cyanobacterium Synechococcus sp. PCC 7002.

Background
The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.

/pdf/transcriptomic-response-to-prolonged-ethanol-production-in-21g319yaxw.pdf

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803.

Ethylene is a gaseous signal sensed by plants and bacteria. Heterologous expression of the ethylene-forming enzyme (EFE) from Pseudomonas syringae in cyanobacteria leads to the production of ethylene under photoautotrophic conditions. The recent characterization of an ethylene-responsive signalling pathway affecting phototaxis in the cyanobacterium Synechocystis sp. PCC 6803 implied that biotechnologically relevant ethylene synthesis may induce regulatory processes that are not related to changes in metabolism. Here, we provide data that indicate that endogenously produced ethylene accelerates the movement of cells towards light. Microarray analysis demonstrates that ethylene mainly deactivates transcription from the csiR1/lsiR promoter, which is under the control of the two-component system consisting of the ethylene- and UV-A-sensing histidine kinase UirS and the DNA-binding response regulator UirR. Surprisingly, ethylene production triggers a very specific transcriptional response and only a few other smaller transcriptional changes are detected in the microarray analysis.

Ekaterina Kuchmina

Papers

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

Systems analysis of ethanol production in the genetically engineered cyanobacterium Synechococcus sp. PCC 7002.

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803.

Ethylene production in Synechocystis sp. PCC 6803 promotes phototactic movement.

Engineering cyanobacteria for industrial products