Challenges and advances towards the rational design of microalgal synthetic promoters in Chlamydomonas reinhardtii.

Microalgae hold enormous potential to provide a safe and sustainable source of high-value compounds, acting as carbon-fixing biofactories that could help to mitigate rapidly progressing climate change. Bioengineering microalgal strains will be key to optimizing and modifying their metabolic outputs, and to render them competitive with established industrial biotechnology hosts, such as bacteria or yeast. To achieve this, precise and tuneable control over transgene expression will be essential, which would require the development and rational design of synthetic promoters as a key strategy. Among green microalgae, Chlamydomonas reinhardtii represents the reference species for bioengineering and synthetic biology; however, the repertoire of functional synthetic promoters for this species, and for microalgae generally, is limited in comparison to other commercial chassis, emphasizing the need to expand the current microalgal gene expression toolbox. Here, we discuss state-of-the-art promoter analyses, and highlight areas of research required to advance synthetic promoter development in C. reinhardtii. In particular, we exemplify high-throughput studies performed in other model systems that could be applicable to microalgae, and propose novel approaches to interrogating algal promoters. We lastly outline the major limitations hindering microalgal promoter development, while providing novel suggestions and perspectives for how to overcome them.

Surfactin Shows Relatively Low Antimicrobial Activity against Bacillus subtilis and Other Bacterial Model Organisms in the Absence of Synergistic Metabolites.

Surfactin is described as a powerful biosurfactant and is natively produced by Bacillus subtilis in notable quantities. Among other industrially relevant characteristics, antimicrobial properties have been attributed to surfactin-producing Bacillus isolates. To investigate this property, stress approaches were carried out with biotechnologically established strains of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, the highest growth-reducing effects were detectable in B. subtilis and E. coli after surfactin treatment of 100 g/L with 35 and 33%, respectively, while P. putida showed no growth-specific response. In contrast, other antimicrobial biosurfactants, like rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. Since the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B. subtilis by about 40%, the initial stress response at the protein level was analyzed by mass spectrometry, showing induction of stress proteins under control of alternative sigma factors σB and σW as well as the activation of LiaRS two-component system. Overall, although surfactin is associated with antimicrobial properties, relatively low growth-reducing effects could be demonstrated after the surfactin addition, challenging the general claim of the antimicrobial properties of surfactin.

Influence of B. subtilis 3NA mutations in spo0A and abrB on surfactin production in B. subtilis 168.

BACKGROUND: Bacillus subtilis is a well-established host for a variety of bioproduction processes, with much interest focused on the production of biosurfactants such as the cyclic lipopeptide surfactin. Surfactin production is tightly intertwined with quorum sensing and regulatory cell differentiation processes. As previous studies have shown, a non-sporulating B. subtilis strain 3NA encoding a functional sfp locus but mutations in the spo0A and abrB loci, called JABs32, exhibits noticeably increased surfactin production capabilities. In this work, the impacts of introducing JABs32 mutations in the genes spo0A, abrB and abh from 3NA into strain KM1016, a surfactin-forming derivative of B. subtilis 168, was investigated. This study aims to show these mutations are responsible for the surfactin producing performance of strain JABs32 in fed-batch bioreactor cultivations. RESULTS: Single and double mutant strains of B. subtilis KM1016 were constructed encoding gene deletions of spo0A, abrB and homologous abh. Furthermore, an elongated abrB version, called abrB*, as described for JABs32 was integrated. Single and combinatory mutant strains were analysed in respect of growth behaviour, native PsrfA promoter expression and surfactin production. Deletion of spo0A led to increased growth rates with lowered surfactin titers, while deletion or elongation of abrB resulted in lowered growth rates and high surfactin yields, compared to KM1016. The double mutant strains B. subtilis KM1036 and KM1020 encoding Δspo0A abrB* and Δspo0A ΔabrB were compared to reference strain JABs32, with KM1036 exhibiting similar production parameters and impeded cell growth and surfactin production for KM1020. Bioreactor fed-batch cultivations comparing a Δspo0A abrB* mutant of KM1016, KM681, with JABs32 showed a decrease of 32% in surfactin concentration. CONCLUSIONS: The genetic differences of B. subtilis KM1016 and JABs32 give rise to new and improved fermentation methods through high cell density processes. Deletion of the spo0A locus was shown to be the reason for higher biomass concentrations. Only in combination with an elongation of abrB was this strain able to reach high surfactin titers of 18.27 g L-1 in fed-batch cultivations. This work shows, that a B. subtilis strain can be turned into a high cell density surfactin production strain by introduction of two mutations.

Matrix-screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins.

RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs are often poor predictors of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory possibilities. To probe the potential for complex formation among RBPs, we assembled a library of 978 mammalian RBPs and used rec-Y2H matrix screening to detect direct interactions between RBPs, sampling > 600 K interactions. We discovered 1994 new interactions and demonstrate that interacting RBPs bind RNAs adjacently in vivo. We further find that the mRNA binding region and motif preferences of RBPs deviate, depending on their adjacently binding interaction partners. Finally, we reveal novel RBP interaction networks among major RNA processing steps and show that splicing impairing RBP mutations observed in cancer rewire spliceosomal interaction networks. The dataset we provide will be a valuable resource for understanding the combinatorial interactions of RBPs with RNAs and the resulting regulatory outcomes.

Bacillus subtilis High Cell Density Fermentation Using a Sporulation-Deficient Strain for the Production of Surfactin.

Bacillus subtilis 3NA is a strain capable of reaching high cell densities. A surfactin producing sfp+ variant of this strain, named JABs32, was utilized in fed-batch cultivation processes. Both a glucose and an ammonia solution were fed to set a steady growth rate µ of 0.1 h-1. In this process, a cell dry weight of up to 88 g L-1 was reached after 38 h of cultivation, and surfactin titers of up to 26.5 g L-1 were detected in this high cell density fermentation process, achieving a YP/X value of 0.23 g g-1 as well as a qP/X of 0.007 g g-1 h-1. In sum, a 21-fold increase in surfactin titer was obtained compared with cultivations in shake flasks. In contrast to fed-batch operations using Bacillus subtilis JABs24, an sfp+ variant derived from B. subtilis 168, JABs32, reached an up to fourfold increase in surfactin titers using the same fed-batch protocol. Additionally, a two-stage feed process was established utilizing strain JABs32. Using an optimized mineral salt medium in this high cell density fermentation approach, after 31 h of cultivation, surfactin titers of 23.7 g L-1 were reached with a biomass concentration of 41.3 g L-1, thus achieving an enhanced YP/X value of 0.57 g g-1 as well as a qP/X of 0.018 g g-1 h-1. The mutation of spo0A locus and an elongation of AbrB in the strain utilized in combination with a high cell density fed-batch process represents a promising new route for future enhancements on surfactin production. KEY POINTS: • Utilization of a sporulation deficient strain for fed-batch operations • High cell density process with Bacillus subtilis for lipopeptide production was established • High titer surfactin production capabilities confirm highly promising future platform strain.