McCluskey, J. B. | bioRxiv Science

bioRxiv2024

The deletion of aldehyde:ferredoxin oxidoreductase-encoding genes in Clostridium ljungdahlii results in changes in the product spectrum with various carbon sources

Biofuels, such as ethanol, can be produced by the microbial fermentation of waste gases that contain carbon dioxide (CO2) and carbon monoxide (CO). The acetogenic model microbe Clostridium ljungdahlii converts those substrates into acetyl-CoA with the Wood-Ljungdahl pathway. During autotrophic conditions, acetyl-CoA can be reduced further to ethanol via acetic acid by the enzymes aldehyde:ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase. Here, the genes encoding both tungsten-dependent AORs (aor1, CLJU_c20110 and aor2, CLJU_c20210) were deleted from the genome of C. ljungdahlii. Ethanol formation was enhanced for C. ljungdahlii {Delta}aor1 with different carbon sources, that is, fructose, a mixture of hydrogen (H2) and CO2, and CO. The highest and lowest ethanol:acetic acid ratio was detected during growth with H2/CO2 and CO, respectively. Oscillating patterns were observed during growth with CO, underpinning the importance of a balanced redox metabolism.

microbiology↗

bioRxiv2022

Single-step purification of functionalized protein nanostructures using multimodal chromatography

Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. Here, we developed an efficient approach to purify functionalized protein filament assemblies directly from bacterial lysates in a single step through a type of multimodal chromatography that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. In this approach, the ultrastable filamentous protein gamma-prefoldin was employed as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using multimodal chromatography was optimized across a wide range of salt concentrations and pH. Subsequently, functionalized protein assemblies were purified from bacterial lysates using multimodal chromatography in a single step and shown to befree of unincorporated subunits. The assembly and purification of protein nanostructures with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Forster resonance energy transfer, and transmission electron microscopy. We envision that the use of multimodal chromatography will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.

bioengineering↗