RESUMEN
The salt-tolerant marine microalgae Dunaliella tertiolecta is reported to generate significant amounts of intracellular glycerol as an osmoprotectant under high salt conditions. This study highlights the phylogenetic distribution and comparative glycerol biosynthesis of seven new Dunaliella isolates compared to a D. tertiolecta reference strain. Phylogenetic analysis indicates that all Dunaliella isolates are newly discovered and do not relate to the D. tertiolecta reference. Several studies have identified light color and intensity and salt concentration alone as the most inducing factors impacting glycerol productivity. This study aims to optimize glycerol production by investigating these described factors singularly and in combination to improve the glycerol product titer. Glycerol production data indicate that cultivation with white light of an intensity between 500 and 2000 µmol m-2 s-1 as opposed to 100 µmol m-2 s-1 achieves higher biomass and thereby higher glycerol titers for all our tested Dunaliella strains. Moreover, applying higher light intensity in a cultivation of 1.5 M NaCl and an increase to 3 M NaCl resulted in hyperosmotic stress conditions, providing the highest glycerol titer. Under these optimal light intensity and salt conditions, the glycerol titer of D. tertiolecta could be doubled to 0.79 mg mL-1 in comparison to 100 µmol m-2 s-1 and salt stress to 2 M NaCl, and was higher compared to singularly optimized conditions. Furthermore, under the same conditions, glycerol extracts from new Dunaliella isolates did provide up to 0.94 mg mL-1. This highly pure algae-glycerol obtained under optimal production conditions can find widespread applications, e.g., in the pharmaceutical industry or the production of sustainable carbon fibers.
RESUMEN
BACKGROUND: L-cysteine is an essential chemical building block in the pharmaceutical-, cosmetic-, food and agricultural sector. Conventionally, L-cysteine production relies on the conversion of keratinous biomass mediated by hydrochloric acid. Today, fermentative production based on recombinant E. coli, where L-cysteine production is streamlined and facilitated by synthetic plasmid constructs, is an alternative process at industrial scale. However, metabolic stress and the resulting production escape mechanisms in evolving populations are severely limiting factors during industrial biomanufacturing. We emulate high generation numbers typically reached in industrial fermentation processes with Escherichia coli harbouring L-cysteine production plasmid constructs. So far no genotypic and phenotypic alterations in early and late L-cysteine producing E. coli populations have been studied. RESULTS: In a comparative experimental design, the E. coli K12 production strain W3110 and the reduced genome strain MDS42, almost free of insertion sequences, were used as hosts. Data indicates that W3110 populations acquire growth fitness at the expense of L-cysteine productivity within 60 generations, while production in MDS42 populations remains stable. For the first time, the negative impact of predominantly insertion sequence family 3 and 5 transposases on L-cysteine production is reported, by combining differential transcriptome analysis with NGS based deep plasmid sequencing. Furthermore, metabolic clustering of differentially expressed genes supports the hypothesis, that metabolic stress induces rapid propagation of plasmid rearrangements, leading to reduced L-cysteine yields in evolving populations over industrial fermentation time scales. CONCLUSION: The results of this study implicate how selective deletion of insertion sequence families could be a new route for improving industrial L-cysteine or even general amino acid production using recombinant E. coli hosts. Instead of using minimal genome strains, a selective deletion of certain IS families could offer the benefits of adaptive laboratory evolution (ALE) while maintaining enhanced L-cysteine production stability.
