Engineering of an environmental isolate of bacillus megaterium for biochemical production under supercritical CO2

Continuous processing is a mainstay for chemical production but is far less common for biochemical processes. The increase in productivity and corresponding decrease in costs make continuous processing an intriguing option for bulk chemicals where price is a major consideration. Among the various challenges of continuous bioprocessing are the risks of contamination and the toxicity of the target products. Supercritical carbon dioxide (scCO2) may provide a means to address both of these issues. scCO2 is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones and aldehydes. scCO2 is also known for its broad microbial lethality. The isolation and engineering of a microbe that is capable of growth and production in the presence of scCO2 thus represents an opportunity to create a production environment that is both resist to contamination and capable of sequestering toxic products through phase separation. Using a targeted bioprospecting approach by sampling fluid from a natural, deep-subsurface scCO2 well, a strain of Bacillus megaterium was isolated that is able to germinate and grow in the presence of scCO2. Transformation is possible using a protoplast-based method, which permitted the identification of promoters capable of inducible heterologous protein expression in both aerobic and anaerobic conditions. A xylose-inducible promoter was evaluated under scCO2 and found to have similar expression under both conditions. We engineered the B. megaterium strain to produce isobutanol from 2-ketoisovalerate by introducing a two-enzyme pathway (2- ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (Adh)). Due to the strong partition of the aldehyde to the scCO2 phase, we tested five homologous Adh enzymes and found that YqhD from E. coli resulted in greater than 85% conversion when grown aerobically. Isobutanol production was also observed when our recombinant strain was cultured under scCO2. Finally, we have developed a process model for an integrated bioprocess and have found conditions that are comparable if not better than existing in situ extraction techniques such as gas stripping. Boock, J.T., A.J.E. Freedman, G.A. Tompsett, S.K. Muse, A.J. Allen, L.A. Jackson, B. Castro-Dominguez, M.T. Timko, K.L.J. Prather (co-corresponding author), J.R. Thompson. 2019. “Engineered microbial biofuel production and recovery under supercritical carbon dioxide.” Nat. Commun. 10:587. DOI: 10.1038/s41467- 019-08486-6

An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in <i>Escherichia coli</i>

The extracellular expression of recombinant proteins using laboratory strains of <i>Escherichia coli</i> is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (<i>i.e.</i>, 10^10–12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of <i>E. coli</i> single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanismsenabling the exploration of protein secretion in pathogenesis as well as the creation of designer <i>E. coli</i> strains with greatly expanded secretomesall without the need for expensive exogenous reagents, assay instruments, or robotic automation

Recordant el Dr. Llobet, professor de geografia

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing

Engineered microbial biofuel production and recovery under supercritical carbon dioxide

End-product toxicity, culture contamination, and energy efficient product recovery are long-standing issues in bioprocessing. Here, the authors address these problems using a fermentation strategy that combines microbial production of branched alcohols with supercritical carbon dioxide extraction

Universal Genetic Assay for Engineering Extracellular Protein Expression

A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli. However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli<i>.</i> Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible <i>in situ</i> without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli. Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production

Directed Evolution of <i>Mycobacterium tuberculosis</i> β-Lactamase Reveals Gatekeeper Residue That Regulates Antibiotic Resistance and Catalytic Efficiency

<div><p>Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium <i>Mycobacterium tuberculosis,</i> which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a ‘gatekeeper’ residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.</p></div

Structural basis for enhanced BlaC-mediated resistance.

<p>(a) Active sites of wt BlaC (top), BlaC(I105F) (middle), or structural alignment of both (bottom). (b) Structural alignment of wt BlaC (yellow), BlaC(I105F) (cyan), and TEM-1 Bla (magenta). Arrow indicates aromatic residues of BlaC(I105F), and TEM-1 Bla.</p

Heterospecific expression of <i>M. tuberculosis</i> BlaC in <i>E. coli</i>.

<p>(a) Serially diluted wt or Δ<i>tatC</i> cells expressing ssTorA-Bla or ssTorA-BlaC chimeras were spotted on Amp. (b) Western blot analysis of cytoplasmic (cyt) and periplasmic (per) fractions prepared from wt cells expressing ssTorA-BlaC, full-length BlaC, or BlaC lacking a signal peptide (ΔspBlaC). Arrow indicates BlaC. Samples prepared from an equivalent number of cells were loaded in each lane. Blots were probed with anti-FLAG antibodies.</p

Ligand binding and allostery can emerge simultaneously

A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 β-lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has ∼106 M−1 affinity for Zn2+, a property that neither of the parental proteins possesses. Furthermore, Zn2+ is a negative effector that noncompetitively switches off β-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn2+-binding site does not involve a histidine or a cysteine, which is atypical of natural Zn2+-binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously