The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts by employing CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome.
Abstract
The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.
Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4′-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl2. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.
Efficient genetic engineering of lactic acid bacteria remains technically challenging due to their thick peptidoglycan cell wall, low transformation efficiency, strain-specific restriction–modification systems, and sensitivity to Cas9-induced double-strand breaks. In this study, we adapted an established CRISPR/Cas9 approach for the targeted disruption of plnD, a key negative regulatory gene within the plantaricin quorum-sensing network of Lactiplantibacillus plantarum 8P-A3 through extensive optimization of transformation and genome-editing conditions. The genetically modified strain exhibited upregulation of plnA, plnE, and plnF, accompanied by elevated antimicrobial activity. These findings underscore the feasibility of rationally reconfiguring a quorum-sensing-associated regulatory circuit and provide a practical strategy for successful genetic engineering in L. plantarum for elevated bacteriocin production.
Rajat Anand, Rudolf Lütticken, Laura De Laporte et al.· Journal of Biological Engine...· 0 citations
2'-Fucosyllactose (2'-FL), the most abundant human milk oligosaccharide (HMO), has attracted considerable interest for its prebiotic and immunomodulatory functions, with broad applications in infant nutrition. In this study, we report the development of a high-yield, genome-integrated 2'-FL-producing strain based on Escherichia coli MG1655 through systematic modular optimization. Starting from a single-copy BKHT strain (MGC06), we first optimized the copy number of the α-1,2-fucosyltransferase (α-1,2-FT) gene BKHT. Subsequently, the GDP-L-fucose supply was enhanced through coordinated genomic integration of the gene clusters cpsG-cpsB and gmd-fcl, while the multidrug efflux transporter gene mdfA was integrated to improve product export and strain robustness. BKHT copy number was then re-evaluated in the optimized background, with four copies yielding the highest production. The final engineered strain, harboring all genetic modifications stably integrated into the chromosome, produced 17.18 g/L 2'-FL in shake-flask culture. In fed-batch fermentation using a 5-L bioreactor, this strain achieved a titer of 154.12 g/L after 60 h, with a productivity of 2.57 g/L/h. Notably, throughout the entire fermentation process, no antibiotics or inducers were supplemented, underscoring the genetic stability and regulatory compliance of this plasmid-free system. To our knowledge, this represents the highest 2'-FL titer reported to date, positioning our engineered strain as a promising candidate for commercial 2'-FL production.
Roulin Chen, Longhao Yang, Hao Wang et al.· Enzyme and Microbial Technol...· 0 citations
Tandem Interspaced Guide RNA (TIGR)–TIGR-associated (Tas) systems are a newly discovered family of ultracompact, modular RNA-guided DNA-targeting proteins that function without a protospacer adjacent motif (PAM) requirement. Their utility as genome engineering tools in microbes remains unexplored. Here, we report the first functional implementation of TIGR-Tas in Saccharomyces cerevisiae for genome engineering. We show that TasR from Parcubacteria (ParTasR) can be programmed by user-defined tigRNAs to generate targeted DNA double-strand breaks at yeast endogenous loci. By co-delivering ParTasR with customized tigRNAs and donor templates, we achieved precise gene fragment deletion and targeted codon substitutions at multiple genomic loci. The multiplex genome engineering capability of this TIGR-Tas system was demonstrated through high-efficiency multiplex gene disruption and chromosomal assembly of a lycopene biosynthesis pathway while inactivating an endogenous gene. This work establishes TIGR-Tas as a valuable addition to the yeast genome engineering toolbox, particularly for applications requiring PAM-independent targeting or compact delivery.
MORTISE is introduced, a compact Cas6f-based RNA system to repress target bacterial genes without relying on host RNA factors, enables multiplex control across bacteria, and supports pathway balancing when genetic knockouts are unsuitable.
5-Hydroxytryptophan (5-HTP) is a functional precursor of serotonin; however, achieving sustained 5-HTP production in vivo by engineered microbes remains challenging. To address this, we developed a genome-integrated engineered probiotic system enabling gut-local biosynthesis of 5-HTP. Escherichia coli Nissle 1917 (EcN), a clinically validated probiotic with an established safety profile for intestinal application, was selected as the chassis. Constitutive expression was employed to eliminate inducer dependence, while genomic integration was used to enhance genetic stability and avoid reliance on antibiotic selection for pathway maintenance. In addition, BH4 biosynthesis and regeneration modules were incorporated to address the limited endogenous BH4 availability in the gut environment. Different administration strategies were systematically evaluated to characterize in vivo performance. Notably, our results showed that the hTPH2 hydroxylation module, together with the BH4 biosynthesis and regeneration system, supported 5-HTP biosynthesis in EcN. The engineered EcN strain achieved a 5-HTP titer of 863.2 mg/L, the highest level reported in EcN-based systems. Under non-selective conditions, EcNPconst−5−HTP carrying the chromosomally integrated pathway maintained stable 5-HTP production in vitro and retained 5-HTP-producing capacity after intestinal passage, whereas plasmid loss in non-integrated constructs resulted in a pronounced decrease in production. In vivo analysis showed that single-dose administration induced a transient elevation in plasma 5-HTP, whereas repeated dosing generated reproducible, time-dependent fluctuations in fecal bacterial abundance and gut-local biosynthetic activity. Treatment with the engineered probiotic further improved behavioral phenotypes and modulated brain 5-HT-related metabolites in a reserpine-induced mouse model. Together, these findings provide a translational framework linking in vitro 5-HTP production by engineered probiotics to their in vivo functional performance.
Jing Wu, Yifan Yang, Taiyu Li et al.· Synthetic and Systems Biotec...· 0 citations