ACS Synthetic Biology最新文献

Lacticin 481, a ribosomally synthesized and post-translationally modified peptide (RiPP), exhibits antimicrobial activity, for which its characteristic lanthionine and methyllanthionine ring structures are essential. The post-translational introduction of (methyl)lanthionines in lacticin 481 is catalyzed by the enzyme LctM. In addition to macrocycle formation, various other post-translational modifications can enhance and modulate the chemical and functional diversity of antimicrobial peptides. The incorporation of noncanonical amino acids, occurring in many nonribosomal peptides (NRPs), is a valuable strategy to improve the properties of antimicrobial peptides. Ornithine, a noncanonical amino acid, can be integrated into RiPPs through the conversion of arginine residues by the newly characterized peptide arginase OspR. Recently, a flexible expression system was described for engineering lanthipeptides using the post-translational modification enzyme SyncM, which has a relaxed substrate specificity. This study demonstrates that SyncM is able to catalyze the production of active lacticin 481 by recognition of a designed hybrid leader peptide, which enables the incorporation of both ornithine and (methyl)lanthionine. Utilizing this hybrid leader peptide, the functional order was established for the production of active ornithine-containing lacticin 481 analogues at positions 8 and 12 in vivo. Furthermore, this study demonstrates that prior lanthionine (Lan) and methyllanthionine (MeLan) formation may preclude ornithine incorporation at specific sites of lacticin 481. The antibacterial activity of ornithine-containing lacticin 481 analogues was evaluated using Bacillus subtilis as the indicator strain. Overall, the synthetic biology pathway constructed here helped to elucidate aspects of the substrate preferences of OspR and SyncM, offering practical guidance to combine these modifications for further lantibiotic bioengineering.

Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in Escherichia coli. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system's sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP's robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.

In vitro reconstructed minimal respiratory chains are powerful tools to investigate molecular interactions between the different enzyme components and how they are influenced by their environment. One such system is the coreconstitution of the terminal cytochrome bo₃ oxidase and the ATP synthase from Escherichia coli into liposomes, where the ATP synthase activity is driven through a proton motive force (pmf) created by the bo₃ oxidase. The proton pumping activity of the bo₃ oxidase is initiated using the artificial electron mediator short-chain ubiquinone and electron source DTT. Here, we extend this system and use either complex II or NDH-2 and succinate or NADH, respectively, as electron entry points employing the natural long-chain ubiquinone Q₈ or Q₁₀. By testing different lipid compositions, we identify that negatively charged lipids are a prerequisite to allow effective NDH-2 activity. Simultaneously, negatively charged lipids decrease the overall pmf formation and ATP synthesis rates. We find that orientation of the bo₃ oxidase in liposomal membranes is governed by electrostatic interactions between enzyme and membrane surface, where positively charged lipids yield the desired bo₃ oxidase orientation but hinder reduction of the quinone pool by NDH-2. To overcome this conundrum, we exploit ionizable lipids, which are either neutral or positively charged depending on the pH value. We first coreconstituted bo₃ oxidase and ATP synthase into temporarily positively charged liposomes, followed by fusion with negatively charged empty liposomes at low pH. An increase of the pH to physiological values renders these proteoliposomes overall negatively charged, making them compatible with quinone reduction via NDH-2. Using this strategy, we not only succeeded in orienting the bo₃ oxidase essentially unidirectionally into liposomes but also found up to 3-fold increased ATP synthesis rates through the usage of natural, long-chain quinones in combination with the substrate NADH compared to the synthetic electron donor/mediator pair.

In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT7⁴) can increase the mutant frequency of the target gene by 1.12 × 10⁴-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT7⁴ system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT7⁴ to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.

Genetic functions have evolved over long timescales and can be encoded by multiple genes dispersed in different locations in genomes, and although contemporary molecular biology enables control over single genes, more complex genetic functions remain challenging. Here, we study the restructuring and mobilization of a complex genetic function encoded by 10 genes, originally expressed from four operons and two loci on the Escherichia coli genome. We observe subtle phenotypic differences and reduced fitness when expressed from episomal DNA and demonstrate that mutations in the transcriptional machinery are necessary for successful implementation in different bacteria. The work provides new approaches for advanced genome editing and constitutes a first step toward modularization and genome-level engineering of complex genetic functions.

In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.

As one of the three important branched-chain amino acids, l-isoleucine has a wide range of applications in the fields of medicine, food, and feed. Currently, the production of l-isoleucine is well-studied by Corynebacterium glutamicum, while the autonomous and efficient production of l-isoleucine in Escherichia coli has not been reported. Here, we developed a production strategy that combined metabolic engineering with bacterial quorum sensing to achieve the efficient production of l-isoleucine. First, we enhanced the l-isoleucine synthesis pathway by overexpressing the genes ilvIH1, CgilvA1, and ygaZH. Second, the precursor supply was increased by knocking out the gene rhtC, while deletion of the gene livJ was implemented to maximize the accumulation of l-isoleucine. Finally, the artificial quorum sensing system was applied to the efficient production of l-isoleucine, and self-induced protein expression in E. coli was realized through self-regulation during fermentation. In this study, an l-threonine high-yielding strain of E. coli TWF106 was used as the starting strain, and the final strain TWF127/pST1011, pST1042-IH1ZHA1 obtained 49.3 g/L l-isoleucine with a yield of 0.32 g/g glucose and a productivity of 1.03 g/(L·h). This autonomous production strategy without the addition of inducers can also be used in other biosynthetic pathways to increase yields while also providing the possibility for various natural products to be applied to industrial production.

Production of chemicals via metabolic engineering of microbes is becoming highly important for sustainable bioeconomy. Conventional metabolic engineering methodologies typically involve labor-intensive and time-consuming processes of iterative genetic modifications, which are inefficient in identifying new genetic targets for the construction of robust industrial strains on a large scale. To accelerate the creation of efficient microbial cell factories and enhance our insights into cellular metabolism, diverse large-scale genome libraries are emerging as powerful tools, which can be established through multiplex or parallel genome editing, gene expression regulation, and incorporation of evolutionary strategies. In this review, we discuss the latest advancements in the construction of genome-scale libraries as well as their applications within the domain of metabolic engineering. We also address the limitations of various techniques and provide insights into future prospects for the field.

The domesticated silkworm Bombyx mori, an essential industrial animal for silk production, has attracted attention as a host for protein production due to its remarkable protein synthesis capability. Here, we applied genetic code expansion (GCE) using a versatile pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl pair from Methanosarcina mazei to B. mori; GCE enables synthetic amino acid incorporation into proteins to give them non-natural functions. Transgenic B. mori lines expressing M. mazei PylRS and its cognate tRNA^Pyl were generated and cross-mated to obtain their F₁ hybrid. Orally administering a click-compatible synthetic amino acid, trans-cyclooctene-lysine (TCO-Lys), to the F₁ hybrid has led to the production of silk fiber incorporated with TCO-Lys. TCO-Lys incorporation in silk fiber was verified by selective labeling of the TCO group by click chemistry. The developed system is available for large-scale protein production with a wide variety of synthetic amino acids.

Galactose oxidase (GOase) is a versatile biocatalyst with a wide range of potential applications, ranging from synthetic chemistry to bioelectrochemical devices. Previous GOase engineering by directed evolution generated the M-RQW mutant, with unprecedented new-to-nature oxidation activity at the C6-OH group of glucose, and a mutational backbone that helped to unlock its promiscuity toward other molecules, including secondary alcohols. In the current study, we have used the M-RQW mutant as a starting point to engineer a set of GOases that are very thermostable and that are easily produced at high titers in yeast, enzymes with latent activities applicable to sustainable chemistry. To boost the generation of sequence and functional diversity, the directed evolution workflow incorporated one-shot computational mutagenesis by the PROSS algorithm and ancestral sequence reconstruction. This synergetic approach helped produce a rapid rise in functional expression by Pichia pastoris, achieving g/L production in a fed-batch bioreactor while the different GOases designed were resistant to pH and high temperature, with T₅₀ enhancements up to 27 °C over the parental M-RQW. These designs displayed latent activity against glucose and an array of secondary aromatic alcohols with different degrees of bulkiness, becoming a suitable point of departure for the future engineering of industrial GOases.