Harnessing transcription for bioproduction in cyanobacteria

Frontiers in bioengineering and biotechnology, 2014

Cyanobacteria can function as solar-driven biofactories thanks to their ability to perform photosynthesis and the ease with which they are genetically modified. In this review, we discuss transcriptional parts and promoters available for engineering cyanobacteria. First, we go through special cyanobacterial characteristics that may impact engineering, including the unusual cyanobacterial RNA polymerase, sigma factors and promoter types, mRNA stability, circadian rhythm, and gene dosage effects. Then, we continue with discussing component characteristics that are desirable for synthetic biology approaches, including decoupling, modularity, and orthogonality. We then summarize and discuss the latest promoters for use in cyanobacteria regarding characteristics such as regulation, strength, and dynamic range and suggest potential uses. Finally, we provide an outlook and suggest future developments that would advance the field and accelerate the use of cyanobacteria for renewable biotech...

Life, 2019

In recent years, metabolic engineering of microorganisms has attained much research interest to produce biofuels and industrially pertinent chemicals. Owing to the relatively fast growth rate, genetic malleability, and carbon neutral production process, cyanobacteria has been recognized as a specialized microorganism with a significant biotechnological perspective. Metabolically engineering cyanobacterial strains have shown great potential for the photosynthetic production of an array of valuable native or non-native chemicals and metabolites with profound agricultural and pharmaceutical significance using CO 2 as a building block. In recent years, substantial improvements in developing and introducing novel and efficient genetic tools such as genome-scale modeling, high throughput omics analyses, synthetic/system biology tools, metabolic flux analysis and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (CRISPR/cas) systems have been made for engineering cyanobacterial strains. Use of these tools and technologies has led to a greater understanding of the host metabolism, as well as endogenous and heterologous carbon regulation mechanisms which consequently results in the expansion of maximum productive ability and biochemical diversity. This review summarizes recent advances in engineering cyanobacteria to produce biofuel and industrially relevant fine chemicals of high interest. Moreover, the development and applications of cutting-edge toolboxes such as the CRISPR-cas9 system, synthetic biology, high-throughput "omics", and metabolic flux analysis to engineer cyanobacteria for large-scale cultivation are also discussed.

Scientific reports, 2014

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 cyanoba...

Photosynthetic organisms, and especially cyanobacteria, hold great promise as sources of renewably-produced fuels, bulk and specialty chemicals, and nutritional products. Synthetic biology tools can help unlock cyanobacteria's potential for these functions, but unfortunately tool development for these organisms has lagged behind that for S. cerevisiae and E. coli. While these organisms may in many cases be more difficult to work with as "chassis" strains for synthetic biology than certain heterotrophs, the unique advantages of autotrophs in biotechnology applications as well as the scientific importance of improved understanding of photosynthesis warrant the development of these systems into something akin to a "green E. coli." In this review, we highlight unique challenges and opportunities for development of synthetic biology approaches in cyanobacteria. We review classical and recently developed methods for constructing targeted mutants in various cyanobacterial strains, and offer perspective on what genetic tools might most greatly expand the ability to engineer new functions in such strains. Similarly, we review what genetic parts are most needed for the development of cyanobacterial synthetic biology. Finally, we highlight recent methods to construct genome-scale models of cyanobacterial metabolism and to use those models to measure properties of autotrophic metabolism. Throughout this paper, we discuss some of the unique challenges of a diurnal, autotrophic lifestyle along with how the development of synthetic biology and biotechnology in cyanobacteria must fit within those constraints.

Renewable and Sustainable Energy Reviews, 2016

Higher oil prices and the necessity for long-term energy security have increased the public and scientific attention on the production of biofuels. Bioenergy is much cleaner, safer, and more economical source of energy than fossil-based fuels. Of several organisms, cyanobacteria are attractive source of biofuels because of their genetic tractability, photosynthetic capability and lack of dependency on fertile land. Synthetic biology and metabolic engineering approaches have been successfully used towards the production of biofuels including ethanol, butanol, biodiesel and hydrogen. This review highlights the recent advances of pathway engineering and uses of synthetic biology tools in cyanobacteria for the production of economical and ecologically biofuels.

ACS Synthetic Biology

Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gltA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a non-repression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gltA gene, we could modulate GltA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These

Metabolites, 2015

Engineering cyanobacteria into photosynthetic microbial cell factories for the production of biochemicals and biofuels is a promising approach toward sustainability. Cyanobacteria naturally grow on light and carbon dioxide, bypassing the need of fermentable plant biomass and arable land. By tapping into the central metabolism and rerouting carbon flux towards desirable compound production, cyanobacteria are engineered to directly convert CO₂ into various chemicals. This review discusses the diversity of bioproducts synthesized by engineered cyanobacteria, the metabolic pathways used, and the current engineering strategies used for increasing their titers.

Frontiers in Bioengineering and Biotechnology, 2022

Biological chemical production has gained traction in recent years as a promising renewable alternative to traditional petrochemical based synthesis. Of particular interest in the field of metabolic engineering are photosynthetic microorganisms capable of sequestering atmospheric carbon dioxide. CO2 levels have continued to rise at alarming rates leading to an increasingly uncertain climate. CO2 can be sequestered by engineered photosynthetic microorganisms and used for chemical production, representing a renewable production method for valuable chemical commodities such as biofuels, plastics, and food additives. The main challenges in using photosynthetic microorganisms for chemical production stem from the seemingly inherent limitations of carbon fixation and photosynthesis resulting in slower growth and lower average product titers compared to heterotrophic organisms. Recently, there has been an increase in research around improving photosynthetic microorganisms as renewable chem...

2016

Engineering cyanobacteria into photosynthetic microbial cell factories for the production of biochemicals and biofuels is a promising approach toward sustainability. Cyanobacteria naturally grow on light and carbon dioxide, bypassing the need of fermentable plant biomass and arable land. By tapping into the central metabolism and rerouting carbon flux towards desirable compound production, cyanobacteria are engineered to directly convert CO2 into various chemicals. This review discusses the diversity of bioproducts synthesized by engineered cyanobacteria, the metabolic pathways used, and the current engineering strategies used for increasing their titers.

The development of renewable energy is rapidly being embraced by our society and industry to meet the energy growth and emission reduction goals. Ethanol has recently surged to the forefront of biofuels technology. Most of the current ethanol production is from the biomass fermentation of agricultural crops and residues. It remains a challenge for ethanol distilleries to be environmentally friendly and economically competitive using biomass as the raw materials. The technology reported in this paper involves genetic engineering of cyanobacterial strains for direct conversion of sunlight and the greenhouse gas CO2 into ethanol. It will integrate photosynthesis, emission reduction and biofuels production in one single process. Another important advantage for our approach is that by directly using CO2 and solar energy for cyanobacteria growth and ethanol synthesis, agricultural space can be preserved for food production. This novel ethanol production approach thus has great potential for industrial applications for many interrelated sociological/political/environmental/economical reasons.