Metabolic Engineering Of Cyanobacteria Research Articles

Development of a base editor for convenient and multiplex genome editing in cyanobacteria

Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria.

Tailoring regulatory components for metabolic engineering in cyanobacteria.

The looming climate crisis has prompted an ever-growing interest in cyanobacteria due to their potential as sustainable production platforms for the synthesis of energy carriers and value-added chemicals from CO2 and sunlight. Nonetheless, cyanobacteria are yet to compete with heterotrophic systems in terms of space-time yields and consequently production costs. One major drawback leading to the low production performance observed in cyanobacteria is the limited ability to utilize the full capacity of the photosynthetic apparatus and its associated systems, i.e. CO2 fixation and the directly connected metabolism. In this review, novel insights into various levels of metabolic regulation of cyanobacteria are discussed, including the potential of targeting these regulatory mechanisms to create a chassis with a phenotype favorable for photoautotrophic production. Compared to conventional metabolic engineering approaches, minor perturbations of regulatory mechanisms can have wide-ranging effects.

Photosynthetic Conversion of CO2 Into Pinene Using Engineered Synechococcus sp. PCC 7002

Metabolic engineering of cyanobacteria has received much attention as a sustainable strategy to convert CO2 to various longer carbon chain fuels. Pinene has become increasingly attractive since pinene dimers contain high volumetric energy and have been proposed to act as potential aircraft fuels. However, cyanobacteria cannot directly convert geranyl pyrophosphate into pinene due to the lack of endogenous pinene synthase. Herein, we integrated the gene encoding Abies grandis pinene synthase into the model cyanobacterium Synechococcus sp. PCC 7002 through homologous recombination. The genetically modified cyanobacteria achieved a pinene titer of 1.525 ± 0.l45 mg L−1 in the lab-scale tube photobioreactor with CO2 aeration. Specifically, the results showed a mixture of α- and β-pinene (∼33:67 ratio). The ratio of β-pinene in the product was significantly increased compared with that previously reported in the engineered Escherichia coli. Furthermore, we investigated the photoautotrophic growth performances of Synechococcus overlaid with different concentrations of dodecane. The work demonstrates that the engineered Synechococcus is a suitable potential platform for β-pinene production.

Escherichia coli AraJ boosts utilization of arabinose in metabolically engineered cyanobacterium Synechocystis sp. PCC 6803

Lignocellulosic biomass can serve as an inexpensive and renewable source of carbon for the biosynthesis of commercially important compounds. L-arabinose is the second most abundant pentose sugar present in the plant materials. Model cyanobacterium Synechocystis sp. PCC 6803 is incapable of catabolism of L-arabinose as a source of carbon and energy. In this study, all the heterologous genes expressed in Synechocystis were derived from Escherichia coli K-12. Initially we constructed four Synechocystis strains that expressed AraBAD enzymes involved in L-arabinose catabolism, either in combination with or without one of the three arabinose transporters, AraE, AraFGH or AraJ. Among the recombinants, the strain possessing AraJ transporter was observed to be the most efficient in terms of dry biomass production and L-arabinose consumption. Later, an additional strain was generated by the expression of AraJ in the AraE-possessing strain. The resultant strain was shown to be advantageous over its parent. This study demonstrates that AraJ, a protein with hitherto unknown function plays a role in the uptake of L-arabinose to boost its catabolism in the transgenic Synechocystis strains. The work also contributes to the current knowledge regarding metabolic engineering of cyanobacteria for the utilization of pentose sugars.

Designing and Constructing Artificial Small RNAs for Gene Regulation and Carbon Flux Redirection in Photosynthetic Cyanobacteria.

Photosynthetic cyanobacteria are not only model organisms for studying photosynthesis and biological cycling of carbon in biosphere but also potential "green microbial factories" to produce renewable fuels and chemicals, due to their capability to utilizing solar energy and CO2. Therefore, strategies for gene regulation and carbon flux redirection are important for both fundamental research and metabolic engineering of cyanobacteria. To address the challenges, regulatory tools based on artificial small RNAs have been developed with satisfactory effects for single or multiple gene(s) regulation in various cyanobacterial species. When combined with the promoters of varying gradient strength and the inducible switches developed in recent years, it is now feasible to realize precise gene regulation in photosynthetic cyanobacteria for producing fuels and chemicals. Here in this chapter, we provide a detailed introduction of the design principles and constructing methods of the artificial sRNA tools to achieve accurate inducible regulation of cyanobacterial gene(s).

Sigma Factor Modulation for Cyanobacterial Metabolic Engineering.

Sigma (σ) factors are key regulatory proteins that control the transcription initiation in prokaryotes. In response to environmental or developmental cues, σ factors initiate the transcription of necessary genes responsible for maintaining a life-sustaining metabolic balance. Due to the significant role of σ factors in bacterial metabolism, their rational engineering for commercial metabolite production in photoautotrophic, cyanobacterial cells is a desirable venture. As cyanobacterial genomes typically encode multiple σ factors, effective execution of metabolic engineering efforts largely relies on uncovering the complicated gene regulatory network and further characterization of the members of σ factor regulatory circuits. This review outlines the prospects of σ factor in metabolic engineering of cyanobacteria, summarizes the challenges in the path towards an efficient strain construction and highlights the genomic context of putative regulators of cyanobacterial σ factors.

A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria.

Cyanobacteria are emerging as hosts for various biotechnological applications. The ability to engineer these photosynthetic prokaryotes greatly depends on the availability of well-characterized promoters. Inducer-free promoters of a range of activities may be desirable for the eventual large-scale, outdoor cultivations. Further, several native promoters of cyanobacteria are repressed by high carbon dioxide or light, and it would be of interest to alter this property. We started with PrbcL and PcpcB, the well-characterized native promoters of the model cyanobacterium Synechococcus elongatus PCC 7942, found upstream of the two abundantly expressed genes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and phycocyanin β-1 subunit, respectively. The library of 48 promoters created via error-prone PCR of these 300-bp-long native promoters showed 2 orders of magnitude dynamic range with activities that were both lower and higher than those of the wild-type promoters. A few mutants of the PrbcL showed greater strength than PcpcB, which is widely considered a superstrong promoter. A number of mutant promoters did not show repression by high CO2 or light, typically found for PrbcL and PcpcB, respectively. Further, the wild-type and mutant promoters showed comparable activities in the fast-growing and stress-tolerant strains S. elongatus PCC 11801 and PCC 11802, suggesting that the library can be used in different cyanobacteria. Interestingly, the majority of the promoters showed strong expression in E. coli, thus adding to the repertoire of inducer-free promoters for this heterotrophic workhorse. Our results have implications in the metabolic engineering of cyanobacteria and E. coli.

CRISPRi-dCas12a: A dCas12a-Mediated CRISPR Interference for Repression of Multiple Genes and Metabolic Engineering in Cyanobacteria.

In cyanobacteria, metabolic engineering using synthetic biology tools is limited to build a biosolar cell factory that converts CO2 to value-added chemicals, as repression of essential genes has not been achieved. In this study, we developed a dCas12a-mediated CRISPR interference system (CRISPRi-dCas12a) in cyanobacteria that effectively blocked the transcriptional initiation by means of a CRISPR-RNA (crRNA) and 19-nt direct repeat, resulting in 53-94% gene repression. The repression of multiple genes in a single crRNA array was also successfully achieved without a loss in repression strength. In addition, as a demonstration of the dCas12a-mediated CRISPRi for metabolic engineering, photosynthetic squalene production was improved by repressing the essential genes of either acnB encoding for aconitase or cpcB2 encoding for phycocyanin β-subunit in Synechococcus elongatus PCC 7942. The ability to regulate gene repression will promote the construction of biosolar cell factories to produce value-added chemicals.

A Heterocyst-Specific Antisense RNA Contributes to Metabolic Reprogramming in Nostoc sp. PCC 7120.

Upon nitrogen deficiency, some filamentous cyanobacteria differentiate specialized cells, called heterocysts, devoted to N2 fixation. Heterocysts appear regularly spaced along the filaments and exhibit structural and metabolic adaptations, such as loss of photosynthetic CO2 fixation or increased respiration, to provide a proper microaerobic environment for its specialized function. Heterocyst development is under transcriptional control of the global nitrogen regulator NtcA and the specific regulator HetR. Transcription of a large number of genes is induced or repressed upon nitrogen deficiency specifically in cells undergoing differentiation. In recent years, the HetR regulon has been described to include heterocyst-specific trans-acting small RNAs and antisense RNAs (asRNAs), suggesting that there is an additional layer of post-transcriptional regulation involved in heterocyst development. Here, we characterize in the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 an asRNA, that we call as_glpX, transcribed within the glpX gene encoding the Calvin cycle bifunctional enzyme sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase). Transcription of as_glpX is restricted to heterocysts and is induced very early during the process of differentiation. Expression of as_glpX RNA promotes the cleavage of the glpX mRNA by RNase III, resulting in a reduced amount of SBPase. Therefore, the early expression of this asRNA could contribute to the quick shut-down of CO2 fixation in those cells in the filament that are undergoing differentiation into heterocysts. In summary, as_glpX is the first naturally occurring asRNA shown to rapidly and dynamically regulate metabolic transformation in Nostoc heterocysts. The use of antisense transcripts to manipulate gene expression specifically in heterocysts could became a useful tool for metabolic engineering in cyanobacteria.

Crossing the Thauer limit: rewiring cyanobacterial metabolism to maximize fermentative H2production

Metabolic engineering of cyanobacteria with concomitant electrochemical elimination of H2uptake increases H2yield beyond the Thauer limit.

Riboregulator elements as tools to engineer gene expression in cyanobacteria.

Cyanobacteria are an ideal host for biofuel production. Although efforts have been made to genetically engineer cyanobacteria for efficient production of biofuels and other important chemicals, the tools that can be applied to cyanobacteria are still limited. A new gene regulation tool, riboregulator, has been examined for application in cyanobacteria. A riboregulator is a nature-inspired RNA tool, which is composed of two artificially designed RNA fragments. Owing to its high specificity and efficacy, it is suitable for metabolic engineering in cyanobacteria, and several studies have been done to optimize and improve the function of the riboregulator. In this review, we focus on the recent improvements made to riboregulators and compare them with other RNA-mediated gene regulation tools developed in cyanobacteria to investigate future applications of riboregulators.

Toward solar biodiesel production from CO2 using engineered cyanobacteria.

Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to various biochemicals including fatty acid-derived biodiesel. Recently, Synechococcus elongatus PCC 7942, a model cyanobacterium, has been engineered to convert CO2 to fatty acid ethyl esters (FAEEs) as biodiesel. Modular pathway has been constructed for FAEE production. Several metabolic engineering strategies were discussed to improve the production levels of FAEEs, including host engineering by improving CO2 fixation rate and photosynthetic efficiency. In addition, protein engineering of key enzyme in S. elongatus PCC 7942 was implemented to address issues on FAEE secretions toward sustainable FAEE production from CO2. Finally, advanced metabolic engineering will promote developing biosolar cell factories to convert CO2 to feasible amount of FAEEs toward solar biodiesel.

Development of SyneBrick Vectors As a Synthetic Biology Platform for Gene Expression in Synechococcus elongatus PCC 7942.

Cyanobacteria are oxygenic photosynthetic prokaryotes that are able to assimilate CO2 using solar energy and water. Metabolic engineering of cyanobacteria has suggested the possibility of direct CO2 conversion to value-added chemicals. However, engineering of cyanobacteria has been limited due to the lack of various genetic tools for expression and control of multiple genes to reconstruct metabolic pathways for biochemicals from CO2. Thus, we developed SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942 as a model cyanobacterium. The SyneBrick chromosomal integration vectors provide three inducible expression systems to control gene expression and three neutral sites for chromosomal integrations. Using a SyneBrick vector, LacI-regulated gene expression led to 24-fold induction of the eYFP reporter gene with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer in S. elongatus PCC 7942 under 5% (v/v) CO2. TetR-regulated gene expression led to 19-fold induction of the GFP gene when 100 nM anhydrotetracycline (aTc) inducer was used. Gene expression decreased after 48 h due to degradation of aTc under light. T7 RNA polymerase-based gene expression resulted in efficient expression with a lower IPTG concentration than a previously developed pTrc promoter. A library of T7 promoters can be used for tunable gene expression. In summary, SyneBrick vectors were developed as a synthetic biology platform for gene expression in S. elongatus PCC 7942. These results will accelerate metabolic engineering of biosolar cell factories through expressing and controlling multiple genes of interest.

Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs) Using Engineered Cyanobacteria.

Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to fatty acid-derived chemicals that are widely used in the food and chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium, was engineered for the first time to produce fatty acid ethyl esters (FAEEs) from CO2. Due to the lack of an endogenous ethanol production pathway and wax ester synthase (AftA) activity in the wild-type cyanobacterium, we metabolically engineered S.elongatus PCC 7942 by expressing heterologous AftA and introducing the ethanol pathway, resulting in detectable peaks of FAEEs. To enhance FAEE production, a heterologous phosphoketolase pathway was introduced in the FAEE-producing strain to supply acetyl-CoA. Subsequent optimization of the cyanobacterial culture with a hexadecane overlay resulted in engineered S.elongatus PCC 7942 that produced photosynthetic FAEEs (10.0 ± 0.7 mg/L/OD730) from CO2. This paper is the first report of photosynthetic production of FAEEs from CO2 in cyanobacteria.

Metabolic engineering of Cyanobacteria and microalgae for enhanced production of biofuels and high-value products.

A lot of research has been performed on Cyanobacteria and microalgae with the aim to produce numerous biotechnological products. However, native strains have a few shortcomings, like limitations in cultivation, harvesting and product extraction, which prevents reaching optimal production value at lowest costs. Such limitations require the intervention of genetic engineering to produce strains with superior properties. Promising advancements in the cultivation of Cyanobacteria and microalgae have been achieved by improving photosynthetic efficiency through increasing RuBisCO activity and truncation of light-harvesting antennae. Genetic engineering has also contributed to final product extraction by inducing autolysis and product secretory systems, to enable direct product recovery without going through costly extraction steps. In this review, we summarize the different enzymes and pathways that have been targeted thus far for improving cultivation aspects, harvesting and product extraction in Cyanobacteria and microalgae. With synthetic biology advancements, genetically engineered strains can be generated to resolve demanding process issues and achieve economic practicality. This comprehensive overview of gene modifications will be useful to researchers in the field to employ on their strains to increase their yields and improve the economic feasibility of the production process.

Diel light:dark cycles significantly reduce FFA accumulation in FFA producing mutants of Synechocystis sp. PCC 6803 compared to continuous light

We examine the effect of diel light:dark cycles (12:12 LD) on the production of free fatty acids (FFAs) in Synechocystis sp. PCC 6803. Our results show that FFA titers in FFA producing strains of Synechocystis were significantly reduced in 12:12 LD compared to continuous light. On the transcriptional level, we saw no significant changes of the fatty acid biosynthesis pathway as a result of increased FFA production. In addition, we describe our efforts in increasing FFA production by introducing 'tesA from Escherichia coli. Despite evidence of 'tesA mRNA transcript abundance, no measurable TesA enzymatic activity was observed in our Synechocystis mutants. Overall, our work demonstrates the importance of considering the effects of diel light:dark cycles in the metabolic engineering of cyanobacteria.

Metabolic engineering of Synechococcus sp. PCC 7002 to produce poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-4-hydroxybutyrate

Cyanobacteria are an important group of photoautotrophic bacteria that have been engineered and used to produce a wide range of biomaterials and biofuels, which are usually derived from important intermediates of the central metabolic pathways. In this study, the production of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-4-hydroxybutyrate in cyanobacteria was studied, and metabolic engineering strategies to improve the yields were also investigated. The genes involved in the biosynthetic pathway for poly-3-hydroxybutyrate from Chlorogloeopsis fritschii PCC 9212 were introduced into Synechococcus sp. PCC 7002, and the resulting strain was able to accumulate 2.77% of total cell dry weight as poly-3-hydroxybutyrate. When the ccmR gene was deleted in this strain, the yield of poly-3-hydroxybutyrate increased to 3.97% of total cell dry weight. A biosynthetic pathway for the production of 4-hydroxybutyryl-CoA was also constructed and introduced into the poly-3-hydroxybutyrate-producing strain. The resulting strain was able to produce ~4.5% of total cell dry weight as poly-3-hydroxybutyrate-co-4-hydroxybutyrate, in which 4-hydroxybutyrate accounted for ~12% of the co-polymer. These results demonstrate that poly-3-hydroxybutyrate-co-4-hydroxybutyrate can be produced in cyanobacteria and confirm that succinic semialdehyde is an important TCA cycle metabolite in cyanobacteria. This study also demonstrates the potential for future metabolic engineering in cyanobacteria that is based on recently discovered metabolites.

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the Pcpc560 or the Ptrc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.

Genetic and genomic analysis of RNases in model cyanobacteria.

Cyanobacteria are diverse photosynthetic microbes with the ability to convert CO2 into useful products. However, metabolic engineering of cyanobacteria remains challenging because of the limited resources for modifying the expression of endogenous and exogenous biochemical pathways. Fine-tuned control of protein production will be critical to optimize the biological conversion of CO2 into desirable molecules. Messenger RNAs (mRNAs) are labile intermediates that play critical roles in determining the translation rate and steady-state protein concentrations in the cell. The majority of studies on mRNA turnover have focused on the model heterotrophic bacteria Escherichia coli and Bacillus subtilis. These studies have elucidated many RNA modifying and processing enzymes and have highlighted the differences between these Gram-negative and Gram-positive bacteria, respectively. In contrast, much less is known about mRNA turnover in cyanobacteria. We generated a compendium of the major ribonucleases (RNases) and provide an in-depth analysis of RNase III-like enzymes in commonly studied and diverse cyanobacteria. Furthermore, using targeted gene deletion, we genetically dissected the RNases in Synechococcus sp. PCC 7002, one of the fastest growing and industrially attractive cyanobacterial strains. We found that all three cyanobacterial homologs of RNase III and a member of the RNase II/R family are not essential under standard laboratory conditions, while homologs of RNase E/G, RNase J1/J2, PNPase, and a different member of the RNase II/R family appear to be essential for growth. This work will enhance our understanding of native control of gene expression and will facilitate the development of an RNA-based toolkit for metabolic engineering in cyanobacteria.