Biology subjects

Hilzinger, J. M.

Publications and source records attributed to Hilzinger, J. M..

4 recordsLinked to original sources

bioRxiv2022

Acetaminophen production in the edible, filamentous cyanobacterium Arthrospira platensis

Spirulina is the common name for the edible, non-heterocystous, filamentous cyanobacterium Arthrospira platensis that is grown industrially as a food supplement, animal feedstock, and pigment source. Although there are many applications for engineering this organism1-3, until recently no genetic tools or reproducible transformation methods have been published. While recent work showed the production of a diversity of proteins in A. platensis, including single domain antibodies for oral delivery, there remains a need for a modular, characterized genetic toolkit4. Here, we establish and characterize a genetic toolkit and reproducible method for the transformation of A. platensis and engineer this bacterium to produce acetaminophen as proof-of-concept for small molecule production in an edible host from CO2, H2O, and light. This work opens A. platensis to the wider scientific community for future engineering as a functional food for nutritional enhancement, modification of organoleptic traits, and production of pharmaceuticals for oral delivery.

bioengineering↗

bioRxiv2022

Charting a narrow course for direct electron uptake-facilitated electromicrobial production

Electromicrobial production (EMP) processes based on CO2-fixing microbes that directly accept electrons from a cathode have received significant attention in the past decade. However, fundamental questions about the performance limits and viability of this strategy remain unanswered. Here, we sought to determine what would be necessary for such a system to compete with alternative sustainable production technologies based on H2-mediated EMP and traditional bioprocessing with crop feedstocks. Using global warming potential as the metric for comparison, we show that each EMP process can outperform sugarcane-based sucrose production. Following a stoichiometric and energetic analysis, direct electron uptake-based EMP would need to achieve a current density >48 mA/cm2 to reach parity with the H2- mediated system. Because this is currently only practical with a gas diffusion electrode (GDE) architecture, we developed a physical model of the proposed bio-GDE and used it to determine the conditions that a microbial catalyst would experience in a reactor. Our analysis demonstrates that unavoidable inefficiencies in the reactor (e.g., kinetic overpotentials and Ohmic losses) require additional energy input, increasing the breakeven current density to [~]91 mA/cm2. At this current density, the microbial catalyst would need to withstand a pH >10.4 and a total salinity >18.8%. Because currently-known electroautotrophs are not adapted to such extreme conditions, we discuss potential improvements to reactor design that may alleviate these challenges, and consider the implications these results have on the engineerability and feasibility of direct electron uptake-based EMP.

bioengineering↗

bioRxiv2020

Systems-informed genome mining for electroautotrophic microbial production

Microbial electrosynthesis (MES) systems can store renewable energy and CO2 in many-carbon molecules inaccessible to abiotic electrochemistry. Here, we develop a multiphysics model to investigate the fundamental and practical limits of MES enabled by direct electron uptake and we identify organisms in which this biotechnological CO2-fixation strategy can be realized. Systematic model comparisons of microbial respiration and carbon fixation strategies revealed that, under aerobic conditions, the CO2 fixation rate is limited to <6 mol/cm2/hr by O2 mass transport despite efficient electron utilization. In contrast, anaerobic nitrate respiration enables CO2 fixation rates >50 mol/cm2/hr for microbes using the reductive tricarboxylic acid cycle. Phylogenetic analysis, validated by recapitulating experimental demonstrations of electroautotrophy, uncovered multiple probable electroautotrophic organisms and a significant number of genetically tractable strains that require heterologous expression of <5 proteins to gain electroautotrophic function. The model and analysis presented here will guide microbial engineering and reactor design for practical MES systems.

bioengineering↗

bioRxiv2017

dRNA-seq implicates sulfide as master regulator of S(0) metabolism in Chlorobaculum tepidum and other green sulfur bacteria

The green sulfur bacteria (Chlorobiaceae) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S(0), and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum, the model system for the Chlorobiaceae, both produces and consumes extracellular S(0) globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e. specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA-seq (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S(0), and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide mediated repression is the dominant regulatory mode in Cba. tepidum. First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the gene CT1277, encoding a putative regulatory protein, leads to constitutive over-expression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae. Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other Chlorobiaceae.\n\nImportanceElemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum both produces and consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur, and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This study identifies new genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this study paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements to control gene expression, a key element of strain engineering.

microbiology↗