Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002.

RNase III is a ribonuclease that recognizes and cleaves double-stranded RNA. Across bacteria, RNase III is involved in rRNA maturation, CRISPR RNA maturation, controlling gene expression, and turnover of messenger RNAs. Many organisms have only one RNase III while others have both a full-length RNase III and another version that lacks a double-stranded RNA binding domain (mini-III). The genome of the cyanobacterium Synechococcus sp. strain PCC 7002 (PCC 7002) encodes three homologs of RNase III, two full-length and one mini-III, that are not essential even when deleted in combination. To discern if each enzyme had distinct responsibilities, we collected and sequenced global RNA samples from the wild type strain, the single, double, and triple RNase III mutants. Approximately 20% of genes were differentially expressed in various mutants with some operons and regulons showing complex changes in expression levels between mutants. Two RNase III's had a role in 23S rRNA maturation and the third was involved in copy number regulation one of six native plasmids. In vitro, purified RNase III enzymes were capable of cleaving some of the known Escherichia coli RNase III target sequences, highlighting the remarkably conserved substrate specificity between organisms yet complex regulation of gene expression.

Regulatory Tools for Controlling Gene Expression in Cyanobacteria.

Cyanobacteria are attractive hosts for converting carbon dioxide and sunlight into desirable chemical products. To engineer these organisms and manipulate their metabolic pathways, the biotechnology community has developed genetic tools to control gene expression. Many native cyanobacterial promoters and related sequence elements have been used to regulate genes of interest, and heterologous tools that use non-native small molecules to induce gene expression have been demonstrated. Overall, IPTG-based induction systems seem to be leaky and initially demonstrate small dynamic ranges in cyanobacteria. Consequently, a variety of other induction systems have been optimized to enable tighter control of gene expression. Tools require significant optimization because they function quite differently in cyanobacteria when compared to analogous use in model heterotrophs. We hypothesize that these differences are due to fundamental differences in physiology between organisms. This review is not intended to summarize all known products made in cyanobacteria nor the performance (titer, rate, yield) of individual strains, but instead will focus on the genetic tools and the inherent aspects of cellular physiology that influence gene expression in cyanobacteria.

CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002.

Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.

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.

Calvin cycle mutants of photoheterotrophic purple nonsulfur bacteria fail to grow due to an electron imbalance rather than toxic metabolite accumulation.

Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO2-fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO), prevents photoheterotrophic growth unless an electron acceptor is provided or if cells can dispose of electrons as H2. Such observations led to the long-standing model wherein the Calvin cycle is necessary during photoheterotrophic growth to maintain a pool of oxidized electron carriers. This model was recently challenged with an alternative model wherein disrupting RubisCO activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate (RuBP) (D. Wang, Y. Zhang, E. L. Pohlmann, J. Li, and G. P. Roberts, J. Bacteriol. 193:3293-3303, 2011, http://dx.doi.org/10.1128/JB.00265-11). Here, we confirm that RuBP accumulation can impede the growth of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) RubisCO-deficient (ΔRubisCO) mutants under conditions where electron carrier oxidation is coupled to H2 production. However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H2 production is permitted. Thus, the Calvin cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H2 production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated.

Genome-Wide Analysis of RNA Decay in the Cyanobacterium Synechococcus sp. Strain PCC 7002.

RNA degradation is an important process that influences the ultimate concentration of individual proteins inside cells. While the main enzymes that facilitate this process have been identified, global maps of RNA turnover are available for only a few species. Even in these cases, there are few sequence elements that are known to enhance or destabilize a native transcript; even fewer confer the same effect when added to a heterologous transcript. To address this knowledge gap, we assayed genome-wide RNA degradation in the cyanobacterium Synechococcus sp. strain PCC 7002 by collecting total RNA samples after stopping nascent transcription with rifampin. We quantified the abundance of each position in the transcriptome as a function of time using RNA-sequencing data and later analyzed the global mRNA decay map using machine learning principles. Half-lives, calculated on a per-ORF (open reading frame) basis, were extremely short, with a median half-life of only 0.97 min. Despite extremely rapid turnover of most mRNA, transcripts encoding proteins involved in photosynthesis were both highly expressed and highly stable. Upon inspection of these stable transcripts, we identified an enriched motif in the 3' untranslated region (UTR) that had similarity to Rho-independent terminators. We built statistical models for half-life prediction and used them to systematically identify sequence motifs in both 5' and 3' UTRs that correlate with stabilized transcripts. We found that transcripts linked to a terminator containing a poly(U) tract had a longer half-life than both those without a poly(U) tract and those without a terminator.IMPORTANCE RNA degradation is an important process that affects the final concentration of individual mRNAs, affecting protein expression and cellular physiology. Studies of how RNA is degraded increase our knowledge of this fundamental process as well as enable the creation of genetic tools to manipulate RNA stability. By studying global transcript turnover, we searched for sequence elements that correlated with transcript (in)stability and used these sequences to guide tool design. This study probes global RNA turnover in a cyanobacterium, Synechococcus sp. strain PCC 7002, that both has a unique array of RNases that facilitate RNA degradation and is an industrially relevant strain that could be used to convert CO2 and sunlight into useful products.

High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria.

As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO2-concentrating mechanism (CCM) to impose a high-CO2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. We showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 108 CFU.

RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA.

Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK, and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism.IMPORTANCE The concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. With this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.

Genome sequence and analysis of Escherichia coli production strain LS5218.

Escherichia coli strain LS5218 is a useful host for the production of fatty acid derived products, but the genetics underlying this utility have not been fully investigated. Here, we report the genome sequence of LS5218 and a list of large mutations and single nucleotide permutations (SNPs) relative to E. coli K-12 strain MG1655. We discuss how genetic differences may affect the physiological differences between LS5218 and MG1655. We find that LS5218 is more closely related to E. coli strain NCM3722 and suspect that small genetic differences between K-12 derived strains may have a significant impact on metabolic engineering efforts.

Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases.

Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.

Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002.

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system had a 48-fold dynamic range and was shown to out-perform Ptrc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.