In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness.

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named "LAbrini", exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain "LAbrini" to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

Phylogenomics and genetic analysis of solvent-producing Clostridium species.

The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii, 57 C. saccharobutylicum, 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production.

Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale.

Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

Carbon recovery by fermentation of CO-rich off gases - Turning steel mills into biorefineries.

Technological solutions to reduce greenhouse gas (GHG) emissions from anthropogenic sources are required. Heavy industrial processes, such as steel making, contribute considerably to GHG emissions. Fermentation of carbon monoxide (CO)-rich off gases with wild-type acetogenic bacteria can be used to produce ethanol, acetate, and 2,3-butanediol, thereby, reducing the carbon footprint of heavy industries. Here, the processes for the production of ethanol from CO-rich off gases are discussed and a perspective on further routes towards an integrated biorefinery at a steel mill is given. Recent achievements in genetic engineering as well as integration of other biotechnology platforms to increase the product portfolio are summarized. Already, yields have been increased and the portfolio of products broadened. To develop a commercially viable process, however, the extraction from dilute product streams is a critical step and alternatives to distillation are discussed. Finally, another critical step is waste(water) treatment with the possibility to recover resources.

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.