Accurate scaleable microfermentation screening for microbial cell line development of therapeutic proteins

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Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology

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Engineering ionic liquid tolerant enzymes

As media for biocatalysis, imidazolium based ionic liquids (ILs) have many applications, including improving enzyme refolding, selectivity, and replacing organic solvents as either the bulk media or cosolvent for biocatalysis. Despite their potential as media for biocatalysis, however, ILs have received underwhelming results due to the broad inactivation of enzymes in this media. Activity profiles of enzymes in increasing concentrations of the ionic liquid 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) show increasing deactivation over time. The deactivation appears unrelated to protein solubility losses but correlated with unfolding based on ultra-violet spectroscopy and fluorescence measurements, respectively. Additionally, equilibrium dialysis measurements show increased IL-enzyme binding at higher concentrations where the enzyme is thought to be unfolded. Increased binding to the denatured state will favor unfolding. Crystallographic studies further shed light on enzyme-IL binding by finding the binding modes of [BMIM][Cl] with enzymes tend to be based on cation-π stacking and hydrophobic interactions. Aromatic and hydrophobic groups are typically in the core of proteins. Unfolding will therefore expose these binding sites allowing for increased binding, as seen in the equilibrium dialysis results. Our approach to stabilize enzymes in ILs was to mediate preferential IL-enzyme binding. There is a non-specific and site-specific mechanism by which enzymes are proposed to be stabilized. The non-specific approach is based on mutating residues to negatively charged amino acids. The negative charge is thought to repel the anion, [Cl], more than it attracts the cation, [BMIM], resulting in a net preferential exclusion of the IL. This non-specific preferential exclusion will, hypothetically, drive native state formation based on surface tension effects around the negative charge much like trehalose or other non-specifically excluded osmolytes. Alternatively, specific high-affinity sites where [BMIM][Cl] binds may be targeted such as tyrosines. If a tyrosine is exposed in the native state, it can likely still only bind one [BMIM] molecule from the exposed side. In the unfolded state, the tyrosine can now bind two [BMIM] molecules (one on either side). In theory, mutation of this high affinity residue will result in stabilization by eliminating one binding site in the folded protein and two binding sites in the unfolded protein. Non-specific chemical modification approaches to model enzymes found that, in four cases, removing positively charged residues for a neutral moiety improved enzyme activity retention. Meanwhile, in all four cases, removing negatively charged residues for a neutral moiety was deleterious for enzyme stability. Certainly, this observation is a result of the total stability of the enzyme, which is the sum of its intrinsic stability (i.e. in buffer alone) and its ability to favorably mediate IL-solvent interactions. However, it was found that the intrinsic stability, measured via urea unfolding midpoint in buffer, was lower for all variants than the wild-type for two enzymes (the other two were not measured). While enzyme stabilization in ionic liquids may not be achieved via this chemical modification with every enzyme, it provides support for surface charge as a tool to non-specifically alter enzyme stability in ILs. A site-specific approach to mediating binding interactions was employed using 2D HSQC NMR. [BMIM][Cl] was titrated into the enzyme sample, and perturbations in the resonance values of various peaks were observed. These peaks were interpreted as binding sites. In support of this, soaking enzyme crystals in [BMIM][Cl] resulted in resolution of [BMIM] and [Cl] molecules surrounding the residues with the largest resonance perturbations. Mutation to a glutamic acid at the location with the largest perturbations resulted in decreased chemical shift perturbations of the amino acids surrounding the mutation upon titration of the mutant enzyme with [BMIM][Cl]. Mutagenesis also resulted in an enhanced activity retention profile in [BMIM][Cl]. Moreover, two highly exposed positive charges on the enzyme were mutated to a glutamic acid in attempt to non-specifically exclude [BMIM][Cl]. Both mutations resulted in enhanced activity retention of the enzyme in [BMIM][Cl], although to varying degrees. Combination of stabilizing mutations around the surface of the enzyme provided an additive effect on stability. The mutations did not, however, improve the melting temperature of the enzyme in the absence of IL, suggesting the intrinsic stability of the enzyme in buffer was not enhanced. Moreover, activity retention profiles in [BMIM][Cl] show an equilibrium being achieved which is higher for the mutant enzyme than the wild-type, suggesting thermodynamic stabilization, and not purely a kinetic effect based on altering the unfolding barrier. Crystallographic studies of the mutant and wild-type enzymes showed a [BMIM] molecule resolved at tyrosine 49 for the wild-type enzyme, but not at glutamic acid 49 in the mutant. Also, equilibrium dialysis suggested decreased binding of [BMIM][Cl] to the mutant relative to the wild-type enzyme, consistent with the proposed mechanism of stabilization via reducing IL-enzyme binding, which would be stronger (and therefore more reduced by mutation) in the denatured state

Towards understanding directed evolution: more than half of all amino acid positions contribute to ionic liquid resistance of Bacillus subtilis lipase A

Ionic liquids (ILs) are attractive (co-)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50–69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6–13 % of substitutions led to improved resistance. Among these, 66–95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance