Expanding the synthetic biology repertoire of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801.

Synechococcus elongatus PCC 11801 is a fast-growing cyanobacterium, exhibiting high tolerance to environmental stresses. We have earlier characterized its genome and analysed its transcriptome and proteome. However, to deploy it as a potential cell factory, it is necessary to expand its synthetic biology toolbox, including promoter elements and ribosome binding sites (RBSs). Here, based on the global transcriptome analysis, 48 native promoters of the genes with high transcript count were characterized using a fluorescent reporter system. The promoters PcpcB, PpsbA1, and P11770 exhibited consistently high fluorescence under all the cultivation conditions. Similarly, from the genome data and proteome analysis, 534 operons were identified. Fifteen intergenic regions exhibiting higher protein expression from the downstream gene were systematically characterized for identifying RBSs, using an operon construct comprising fluorescent protein genes eyfp and mTurq under PcpcB (PcpcB:eyfp:RBS:mTurq:TrrnB). Overall, the work presents promoter and RBS sequence libraries, with varying strengths, to expedite bioengineering of PCC 11801.

Global Transcriptome-Guided Identification of Neutral Sites for Engineering Synechococcus elongatus PCC 11801.

Engineered cyanobacteria are attractive hosts for the phototrophic conversion of CO2 to chemicals. Synechococcus elongatus PCC11801, a novel, fast-growing, and stress-tolerant cyanobacterium, has the potential to be a platform cell factory, and hence, it necessitates the development of a synthetic biology toolbox. Considering the widely followed cyanobacterial engineering strategy of chromosomal integration of heterologous DNA, it is of interest to discover and validate new chromosomal neutral sites (NSs) in this strain. To that end, global transcriptome analysis was performed using RNA Seq under the conditions of high temperature (HT), carbon (HC), and salt (HS) and ambient growth conditions. We found upregulation of 445, 138, and 87 genes and downregulation of 333, 125, and 132 genes, under HC, HT, and HS, respectively. Following nonhierarchical clustering, gene enrichment, and bioinformatics analysis, 27 putative NSs were predicted. Six of them were experimentally tested, and five showed confirmed neutrality, based on unaltered cell growth. Thus, global transcriptomic analysis was effectively exploited for NS annotation and would be advantageous for multiplexed genome editing.

High cell density cultivation of E. coli in shake flasks for the production of recombinant proteins.

Batch cultivation of recombinant bacteria in shake flasks typically results in low cell density due to nutrient depletion. Previous studies on high cell density cultivation in shake flasks have relied mainly on controlled release mechanisms. Here, we report a true fed-batch strategy to achieve high cell density of recombinant E. coli in shake flasks in 24 h by feeding a mixture of glycerol and yeast extract with a syringe pump. Feed composition and feed rate were obtained by cybernetic model-based, multi-objective optimization. Model parameters were estimated from time-course measurement of substrate, biomass, and dissolved oxygen levels. The optimized process yielded 20.7 g dry cell weight/L, in agreement with the model prediction. Volumetric protein productivity improved by 10-34-fold compared to batch cultivation with 2.8-fold further improvement when the fed-batch process was replicated in a 3 L bioreactor. The process has significance in the routine laboratory cultivations and in scaleup studies.

Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance.

Cyanobacteria hold promise as cell factories for the photoautotrophic conversion of carbon dioxide to useful chemicals. For the eventual commercial viability of such processes, cyanobacteria need to be engineered for (i) efficient channeling of carbon flux toward the product of interest and (ii) improved product tolerance, the latter being the focus of this study. We chose the recently reported, fast-growing, high light and CO2 tolerant cyanobacterium Synechococcus elongatus PCC 11801 for adaptive laboratory evolution. In two parallel experiments that lasted over 8400 h of culturing and 100 serial passages, S. elongatus PCC 11801 was evolved to tolerate 5 g/L n-butanol or 30 g/L 2,3-butanediol representing a 100% improvement in concentrations tolerated. The evolved strains retained alcohol tolerance even after being passaged several times without the alcohol stress suggesting that the changes were permanent. Whole genome sequencing of the n-butanol evolved strains revealed mutations in a number of stress responsive genes encoding translation initiation factors, RpoB and an ABC transporter. In 2,3-butanediol evolved strains, genes for ClpC, a different ABC transporter, glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase were found to be mutated. Furthermore, the evolved strains showed significant improvement in tolerance toward several other alcohols. Notably, the n-butanol evolved strain could tolerate up to 32 g/L ethanol, thereby making it a promising host for photosynthetic production of biofuels via metabolic engineering.

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 ß-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.

A Novel Cyanobacterium Synechococcus elongatus PCC 11802 has Distinct Genomic and Metabolomic Characteristics Compared to its Neighbor PCC 11801.

Cyanobacteria, a group of photosynthetic prokaryotes, are attractive hosts for biotechnological applications. It is envisaged that future biorefineries will deploy engineered cyanobacteria for the conversion of carbon dioxide to useful chemicals via light-driven, endergonic reactions. Fast-growing, genetically amenable, and stress-tolerant cyanobacteria are desirable as chassis for such applications. The recently reported strains such as Synechococcus elongatus UTEX 2973 and PCC 11801 hold promise, but additional strains may be needed for the ongoing efforts of metabolic engineering. Here, we report a novel, fast-growing, and naturally transformable cyanobacterium, S. elongatus PCC 11802, that shares 97% genome identity with its closest neighbor S. elongatus PCC 11801. The new isolate has a doubling time of 2.8 h at 1% CO2, 1000 µmole photons.m-2.s-1 and grows faster under high CO2 and temperature compared to PCC 11801 thus making it an attractive host for outdoor cultivations and eventual applications in the biorefinery. Furthermore, S. elongatus PCC 11802 shows higher levels of key intermediate metabolites suggesting that this strain might be better suited for achieving high metabolic flux in engineered pathways. Importantly, metabolite profiles suggest that the key enzymes of the Calvin cycle are not repressed under elevated CO2 in the new isolate, unlike its closest neighbor.

The role of systems biology in developing non-model cyanobacteria as hosts for chemical production.

Cyanobacteria, a group of photosynthetic prokaryotes, can be engineered for direct conversion of carbon dioxide to value added products. Despite two decades of progress in the metabolic engineering of these photoautotrophs, the reported productivities are below those needed for commercialization. While much of this work has been performed with a handful of model cyanobacteria, new fast-growing and robust cyanobacterial strains may afford significant improvements in productivity. The focus now is on isolating and developing cyanobacteria that can be deployed as industrial strains for the production of a wide range of chemicals. Systems-level characterization, development of synthetic biology tools and improvement in photosynthetic efficiency would be an integral part of host engineering efforts. The knowledge base that exists for model cyanobacteria together with the systems biology data from newer strains will provide the foundation for the development of new hosts.

Ex vivo Caprine Model to Study Virulence Factors in Keratitis.

PURPOSE: To develop an infectious keratitis model using caprine (goat) corneas and to investigate the expression of virulence factors during infection. METHODS: Goat eyes were surface-sterilized and dissected, and the corneas were placed on an agarose-gelatin solid support (0.5% in phosphate-buffered saline) in a 12-well culture plate containing 10% fetal bovine serum-supplemented culture medium for 3 weeks. Cell viability tests (trypan blue and MTT) were performed on the cultured corneas. Corneas were infected with Pseudomonas aeruginosa and Fusarium solani separately. Infection progression was observed via histological analysis and hematoxylin and eosin (H-E) staining. For Pseudomonas-infected corneas, expression of eight virulence genes (exoS, exoT, exoY, alpR, prpL, lasA, lasB, and algD) was determined via quantitative real-time PCR (qRT-PCR) at 48-h and 72-h time-points. For Fusarium-infected corneas, expression of five proteases (C7Z0E6, C7ZFW9, C7Z7U2, C7ZNV5, and C7YY94) was quantified via qRT-PCR at 2, 4, and 8 days after infection. Protease from infected corneas was detected via gelatin zymography. RESULTS: Goat corneas with a viable epithelium could be maintained for 15 days. Pseudomonas infection progressed rapidly, and complete corneal degradation was observed on day 4 after infection. Fusarium infection progressed more slowly. Histological analysis and H-E staining of Fusarium-infected cornea revealed mycelia penetrating all layers of the cornea. qRT-PCR revealed expression of all eight virulence factors, and statistically significant difference in expression of prpL and alpR in Pseudomonas-infected corneas. Expression of C7ZNV5 was highest in Fusarium-infected corneas. CONCLUSION: Goat corneas can be used to evaluate the expression of virulence factors involved in Pseudomonas and Fusarium infection.