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.

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.

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.

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.

Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO 2 -fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO) 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 H 2 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 H 2 production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated. P urple nonsulfur bacteria (PNSB) are renowned for their ability to employ versatile metabolic modules to thrive under different growth conditions. PNSB can grow photoautotrophically using light for energy, inorganic compounds other than water (e.g., thiosulfate, Fe 2ϩ ) for electrons, and CO 2 for carbon. The Calvin cycle is well-known for permitting autotrophic growth by converting CO 2 into organic precursors for biosynthesis (Fig. 1). In this pathway phosphoribulokinase (PRK) expends ATP to generate ribulose 1,5-bisphosphate (RuBP). RuBP is then combined with CO 2 via ribulose 1,5-bisphosphate carboxylase (RubisCO), resulting in two molecules of 3-phosphoglycerate. CO 2 fixation generates relatively oxidized metabolites that accept electrons from NAD(P)H via glyceraldehyde-3-phosphate dehydrogenase.,PNSB can also grow photoheterotrophically using light for energy and organic compounds for carbon and electrons. Unlike the process in a respiring heterotroph, reducing power from oxidative pathways [e.g., NAD(P)H] is not used to reduce a terminal electron acceptor and generate ATP by oxidative phosphorylation. Rather, ATP generation is largely decoupled from the oxidative pathways of central metabolism as photoheterotrophs repeatedly energize electrons and shuttle them through a H ϩ -pumping electron transfer chain to generate ATP by cyclic photophosphorylation (Fig. 1). Nevertheless, photoheterotrophs generate ample reducing power that must be oxidized to replenish pools of oxidized electron carriers [e.g., NAD(P) ϩ ] and maintain metabolic flow. CO 2 fixation was first hypothesized to fulfill this essential role of maintaining oxidized electron carriers during photoheterotrophic growth in 1933 by Muller (1), prior to the elucidation of the Calvin cycle itself (2) (Fig. 1, model 1). Muller devised this hypothesis to explain why there was net CO 2 fixation when PNSB grew photoheterotrophically on compounds, like butyrate, that are more electron rich than the average carbon in biomass (1).Supporting this hypothesis, it was later shown that PNSB could be grown photoheterotrophically on butyrate without added CO 2 if an electron acceptor like dimethyl sulfoxide (DMSO) was provided (3) or i...