Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium.

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

Funneling modulatory peptide design with generative models: Discovery and characterization of disruptors of calcineurin protein-protein interactions.

Design of peptide binders is an attractive strategy for targeting "undruggable" protein-protein interfaces. Current design protocols rely on the extraction of an initial sequence from one known protein interactor of the target protein, followed by in-silico or in-vitro mutagenesis-based optimization of its binding affinity. Wet lab protocols can explore only a minor portion of the vast sequence space and cannot efficiently screen for other desirable properties such as high specificity and low toxicity, while in-silico design requires intensive computational resources and often relies on simplified binding models. Yet, for a multivalent protein target, dozens to hundreds of natural protein partners already exist in the cellular environment. Here, we describe a peptide design protocol that harnesses this diversity via a machine learning generative model. After identifying putative natural binding fragments by literature and homology search, a compositional Restricted Boltzmann Machine is trained and sampled to yield hundreds of diverse candidate peptides. The latter are further filtered via flexible molecular docking and an in-vitro microchip-based binding assay. We validate and test our protocol on calcineurin, a calcium-dependent protein phosphatase involved in various cellular pathways in health and disease. In a single screening round, we identified multiple 16-length peptides with up to six mutations from their closest natural sequence that successfully interfere with the binding of calcineurin to its substrates. In summary, integrating protein interaction and sequence databases, generative modeling, molecular docking and interaction assays enables the discovery of novel protein-protein interaction modulators.

Mutant allele knockout with novel CRISPR nuclease promotes myelopoiesis in ELANE neutropenia.

Severe congenital neutropenia (SCN) is a life-threatening marrow failure disorder, usually caused by heterozygous mutations in ELANE. Potential genetic treatment strategies include biallelic knockout or gene correction via homology-directed repair (HDR). Such strategies, however, involve the potential loss of the essential function of the normal allele product or limited coverage of diverse monogenic mutations within the patient population, respectively. As an alternative, we have developed a novel CRISPR-based monoallelic knockout strategy that precisely targets the heterozygous sites of single-nucleotide polymorphisms (SNPs) associated with most ELANE mutated alleles. In vitro studies demonstrate that patients' unedited hematopoietic CD34+ cells have significant abnormalities in differentiation and maturation, consistent with the hematopoietic defect in SCN patients. Selective knockout of the mutant ELANE allele alleviated these cellular abnormalities and resulted in about 50%-70% increase in normally functioning neutrophils (p < 0.0001). Genomic analysis confirmed that ELANE knockout was specific to the mutant allele and involved no off-targets. These results demonstrate the therapeutic potential of selective allele editing that may be applicable to SCN and other autosomal dominant disorders.