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Decoupling growth from product synthesis is a promising strategy to increase carbon partitioning and maximize productivity in cell factories. However, reduction in both substrate uptake rate and metabolic activity in the production phase are an underlying problem for upscaling. Here, we used CRISPR interference to repress growth in lactate-producing Synechocystis sp. PCC 6803. Carbon partitioning to lactate in the production phase exceeded 90%, but CO2 uptake was severely reduced compared to uptake during the growth phase. We characterized strains during the onset of growth arrest using transcriptomics and proteomics. Multiple genes involved in ATP homeostasis were regulated once growth was inhibited, which suggests an alteration of energy charge that may lead to reduced substrate uptake. In order to overcome the reduced metabolic activity and take advantage of increased carbon partitioning, we tested a novel production strategy that involved alternating growth arrest and recovery by periodic addition of an inducer molecule to activate CRISPRi. Using this strategy, we maintained lactate biosynthesis in Synechocystis for 30 days in a constant light turbidostat cultivation. Cumulative lactate titers were also increased by 100% compared to a constant growth-arrest regime, and reached 1 g/L. Further, the cultivation produced lactate for 30 days, compared to 20 days for the non-growth arrest cultivation. Periodic growth arrest could be applicable for other products, and in cyanobacteria, could be linked to internal circadian rhythms that persist in constant light., QC 20211101

To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (Delta PSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the Delta PSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of Delta PSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (Delta PSI/Delta SynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing Delta PSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP(+)-reduction.

The approach of providing an oxygenic photosynthetic organism with a cyclic electron transfer system, i.e., a far-red light-driven proton pump, is widely proposed to maximize photosynthetic efficiency via expanding the absorption spectrum of photosynthetically active radiation. As a first step in this approach, Gloeobacter rhodopsin was expressed in a PSI-deletion strain of Synechocystis sp. PCC6803. Functional expression of Gloeobacter rhodopsin, in contrast to Proteorhodopsin, did not stimulate the rate of photoheterotrophic growth of this Synechocystis strain, analyzed with growth rate measurements and competition experiments. Nevertheless, analysis of oxygen uptake and-production rates of the Gloeobacter rhodopsin-expressing strains, relative to the 1 PSI control strain, confirm that the proton-pumping Gloeobacter rhodopsin provides the cells with additional capacity to generate proton motive force. Significantly, expression of the Gloeobacter rhodopsin did modulate levels of pigment formation in the transgenic strain.

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis. Despite widespread vaccination, its incidence has been rising alarmingly, and yet, the physiology of B. pertussis remains poorly understood. We combined genome-scale metabolic reconstruction, a novel optimization algorithm, and experimental data to probe the full metabolic potential of this pathogen, using B. pertussis strain Tohama I as a reference. Experimental validation showed that B. pertussis secretes a significant proportion of nitrogen as arginine and purine nucleosides, which may contribute to modulation of the host response. We also found that B. pertussis can be unexpectedly versatile, being able to metabolize many compounds while displaying minimal nutrient requirements. It can grow without cysteine, using inorganic sulfur sources, such as thiosulfate, and it can grow on organic acids, such as citrate or lactate, as sole carbon sources, providing in vivo demonstration that its tricarboxylic acid (TCA) cycle is functional. Although the metabolic reconstruction of eight additional strains indicates that the structural genes underlying this metabolic flexibility are widespread, experimental validation suggests a role of strain-specific regulatory mechanisms in shaping metabolic capabilities. Among five alternative strains tested, three strains were shown to grow on substrate combinations requiring a functional TCA cycle, but only one strain could use thiosulfate. Finally, the metabolic model was used to rationally design growth media with > 2-fold improvements in pertussis toxin production. This study thus provides novel insights into B. pertussis physiology and highlights the potential, but also the limitations, of models based solely on metabolic gene content., SCOPUS: ar.j, info:eu-repo/semantics/published