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.

Mutations Suppressing the Lack of Prepilin Peptidase Provide Insights Into the Maturation of the Major Pilin Protein in Cyanobacteria

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

Evaluation of the Growth Performance of Microalgae Based on Fine pH Changes

Microalgae are photosynthetic microorganisms with many potential applications in the food, cosmetics, pharmaceutical and environmental industries. Currently, commercial microalgae production remains limited. Therefore, improving the growth and the culture density of the microalgae cultivation is one of the key enablers to open the way to mass production and commercialisation of these microorganisms. The effect of culture pH on the photoautotrophic growth of C. vulgaris over a large range of values has been investigated in pH-regulated cultures. For each microalgae culture, the specific growth rate, the cell density, the chlorophyll content, the intracellular carbon content and the nitrogen source consumption were monitored. Optimal growth and carbon incorporation have been observed at pH of 7.0. The fastest growth rate and highest biomass production of C. vulgaris were 0.074 h-1 and 0.896 g/L respectively. Under these conditions, a maximum carbon content of cells was 49 % (w/w).

Correction of Frameshift Mutations in the atpB Gene by Translational Recoding in Chloroplasts of Oenothera and Tobacco

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of mutation that otherwise would be lethal.

Comparing Biochemical and Raman Microscopy Analyses of Starch, Lipids, Polyphosphate, and Guanine Pools during the Cell Cycle of Desmodesmus quadricauda

Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods—as well as conventional fluorescence microscopy—were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.

The essential role of sodium bioenergetics and ATP homeostasis in the developmental transitions of a cyanobacterium

The ability to resume growth after a dormant period is an important strategy for the survival and spreading of bacterial populations. Energy homeostasis is critical in the transition into and out of a quiescent state. Synechocystis sp. PCC 6803, a non-diazotrophic cyanobacterium, enters metabolic dormancy as a response to nitrogen starvation. We used Synechocystis as a model to investigate the regulation of ATP homeostasis during dormancy and unraveled a critical role for sodium bioenergetics in dormant cells. During nitrogen starvation, cells reduce their ATP levels and engage sodium bioenergetics to maintain the minimum ATP content required for viability. When nitrogen becomes available, energy requirements rise, and cells immediately increase ATP levels employing sodium bioenergetics and glycogen catabolism. These processes allow them to restore the photosynthetic machinery and resume photoautotrophic growth. Our work reveals a precise regulation of the energy metabolism essential for bacterial survival during periods of nutrient deprivation.

Photosynthesis without β-carotene

Carotenoids are essential in oxygenic photosynthesis: they stabilize the pigment–protein complexes, are active in harvesting sunlight and in photoprotection. In plants, they are present as carotenes and their oxygenated derivatives, xanthophylls. While mutant plants lacking xanthophylls are capable of photoautotrophic growth, no plants without carotenes in their photosystems have been reported so far, which has led to the common opinion that carotenes are essential for photosynthesis. Here, we report the first plant that grows photoautotrophically in the absence of carotenes: a tobacco plant containing only the xanthophyll astaxanthin. Surprisingly, both photosystems are fully functional despite their carotenoid-binding sites being occupied by astaxanthin instead of β-carotene or remaining empty (i.e. are not occupied by carotenoids). These plants display non-photochemical quenching, despite the absence of both zeaxanthin and lutein and show that tobacco can regulate the ratio between the two photosystems in a very large dynamic range to optimize electron transport.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding, is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch of atpB (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, transplastomic tobacco lines were generated, in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We found that insertion of two adenines was more efficiently compensated than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding. Plants carrying a disrupted oligoA stretch have an albino-phenotype, indicating absence of indel correction. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in mutants that otherwise would be lethal.

Engineered Terpenoid Production in Synechocystis sp. PCC 6803 Under Different Growth Conditions

ABSTRACTTerpenoids are the largest class of natural products and have applications in a wide variety of industries. Many terpenoids can be chemically synthesized or extracted from plants, but this is often uneconomical or unsustainable. An alternative production method relies on the heterologous expression of terpene synthase enzymes in cyanobacteria, producing the desired compounds directly from carbon dioxide. In this work, a patchoulol synthase enzyme from Pogostemon cablin (patchouli) was expressed in the cyanobacterium Synechocystis sp. PCC 6803 under four different growth conditions. Final yields of patchoulol from each growth condition were as follows: 249 μg L−1, photoautotrophic growth; 6.5 μg L−1, mixotrophic growth; 27.6 μg L−1, bicarbonate low light; 31.7 μg L−1, bicarbonate high light. By comparing patchoulol production across growth conditions, we identified a significant positive correlation between the production of photopigments (chlorophyll and carotenoids) and the production of patchoulol. Importantly, this relationship was found to be stronger than the correlation between cell density and patchoulol production across growth conditions, which was not statistically significant. The relationship between photopigments and patchoulol should be generalizable to the production of other terpenoids that rely on expression of the endogenous methylerythritol phosphate (MEP) pathway in cyanobacteria. Based on the results of this work, we propose a strategy for maximizing terpenoid production in cyanobacteria by optimizing growth conditions for photopigment production, resulting in increased flux through the MEP pathway. This strategy has the advantage of facile photopigment quantification using simple spectroscopic methods, and optimized growth conditions can be utilized in partnership with conventional terpenoid production strategies to further improve yields.