ACS Synthetic Biology最新文献

Bacteria in the gastrointestinal tract play a crucial role in intestinal motility, homeostasis, and dysfunction. Unraveling the mechanisms by which microbes impact the host poses many challenges due to the extensive array of metabolites produced or metabolized by bacteria in the gut. Here, we describe the engineering of a gut commensal bacterium, Escherichia coli Nissle 1917, to biosynthesize the human metabolite serotonin for examining the effects of microbially produced biogenic amines on host physiology. Upon oral administration to mice, our engineered bacteria reach the large intestine, where they produce serotonin. Mice treated with serotonin-producing bacteria exhibited biological changes in the gut at transcriptional and physiological levels. This work establishes a novel framework employing engineered bacteria to modulate luminal serotonin levels and suggests potential clinical applications of modified microbial therapeutics to address gut disorders in humans.

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.

Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.

Benzylisoquinoline alkaloids (BIAs) are a class of natural compounds found in plants of the Ranunculaceae family, known for their diverse pharmacological activities. However, the extraction yields of BIAs from plants are limited, and the cost of chemical synthesis is prohibitively high. Recent advancements in systems metabolic engineering and genomics have made it feasible to use microbes as bioreactors for BIAs production. This review explores recent progress in enhancing the production and yields of BIAs in two microbial systems: Escherichia coli and Saccharomyces cerevisiae. It covers various BIAs, including (S)-reticuline, morphinane, protoberberine, and aporphine alkaloids. The review provides strategies and technologies for BIAs synthesis, analyzes current challenges in BIAs research, and offers recommendations for future research directions.

Ribosome arrest peptides (RAPs) such as the SecM arrest peptide (SecM AP: FSTPVWISQAQGIRAGP) and WPPP with consecutive Pro residues are known to induce translational stalling in Escherichia coli. We demonstrate that the translation-enhancing SKIK peptide tag, which consists of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviates translational arrest caused by WPPP. Moreover, the proximity between SKIK and WPPP significantly influences the extent of this alleviation, observed in both PURE cell-free protein synthesis and in vivo protein production systems, resulting in a substantial increase in the yield of proteins containing such RAPs. Furthermore, we unveil that nascent SKIK peptide tag and translation elongation factor P (EF-P) alleviate ribosome stalling in consecutive-Pro-rich protein to synergistically promote translation. A kinetic analysis based on the generation of superfolder green fluorescent protein under in vitro translation reaction reveals that the ribosome turnover is enhanced by more than 10-fold when the SKIK peptide tag is positioned immediately upstream of the SecM AP sequence. Our findings provide valuable insights into optimizing protein production processes, which are essential for advancing synthetic biology applications.

The study of ecosystems, both natural and artificial, has historically been mediated by population dynamics theories. In this framework, quantifying population numbers and related variables (associated with metabolism or biological-environmental interactions) plays a central role in measuring and predicting system-level properties. As we move toward advanced technological engineering of cells and organisms, the possibility of bioengineering ecosystems (from the gut microbiome to wildlands) opens several questions that will require quantitative models to find answers. Here, we present a comprehensive survey of quantitative modeling approaches for managing three kinds of synthetic ecosystems, sharing the presence of engineered strains. These include test tube examples of ecosystems hosting a relatively low number of interacting species, mesoscale closed ecosystems (or ecospheres), and macro-scale, engineered ecosystems. The potential outcomes of synthetic ecosystem designs and their limits will be relevant to different disciplines, including biomedical engineering, astrobiology, space exploration and fighting climate change impacts on endangered ecosystems. We propose a space of possible ecosystems that captures this broad range of scenarios and a tentative roadmap for open problems and further exploration.

Biological systems can directly upgrade carbon dioxide (CO₂) into chemicals. The CO₂ fixation rate of autotrophic organisms, however, is too slow for industrial utility, and the breadth of engineered metabolic pathways for the synthesis of value-added chemicals is too limited. Biotechnology workhorse organisms with extensively engineered metabolic pathways have recently been engineered for CO₂ fixation. Yet, their low carbon fixation rate, compounded by the fact that living organisms split their carbon between cell growth and chemical synthesis, has led to only cell growth with no chemical synthesis achieved to date. Here, we engineer a lysate-based cell-free expression (CFE)-based multienzyme biocatalyst for the carbon negative synthesis of the industrially relevant amino acids glycine and serine from CO₂ equivalents─formate and bicarbonate─and ammonia. The formate-to-serine biocatalyst leverages tetrahydrofolate (THF)-dependent formate fixation, reductive glycine synthesis, serine synthesis, and phosphite dehydrogenase-dependent NAD(P)H regeneration to convert 30% of formate into serine and glycine, surpassing the previous 22% conversion using a purified enzyme system. We find that (1) the CFE-based biocatalyst is active even after 200-fold dilution, enabling higher substrate loading and product synthesis without incurring additional cell lysate cost, (2) NAD(P)H regeneration is pivotal to driving forward reactions close to thermodynamic equilibrium, (3) balancing the ratio of the formate-to-serine pathway genes added to the CFE is key to improving amino acid synthesis, and (4) efficient THF recycling enables lowering the loading of this cofactor, reducing the cost of the CFE-based biocatalyst. To our knowledge, this is the first synthesis of amino acids that can capture CO₂ equivalents for the carbon negative synthesis of amino acids using a CFE-based biocatalyst. Looking ahead, the CFE-based biocatalyst process could be extended beyond serine to pyruvate, a key intermediate, to access a variety of chemicals from aromatics and terpenes to alcohols and polymers.

Cell-free transcription–translation (TX–TL) systems have been used for diverse applications, but their performance and scope are limited by variability and poor predictability. To understand the drivers of this variability, we explored the effects of metabolic perturbations to anEscherichia coli (E. coli) Rosetta2 TX–TL system. We targeted three classes of molecules: energy molecules, in the form of nucleotide triphosphates (NTPs); central carbon “fuel” molecules, which regenerate NTPs; and magnesium ions (Mg²⁺). Using malachite green mRNA aptamer (MG aptamer) and destabilized enhanced green fluorescent protein (deGFP) as transcriptional and translational readouts, respectively, we report the presence of a trade-off between optimizing total protein yield and optimizing total mRNA yield, as measured by integrating the area under the curve for mRNA time-course dynamics. We found that a system’s position along the trade-off curve is strongly determined by Mg²⁺ concentration, fuel type and concentration, and cell lysate preparation and that variability can be reduced by modulating these components. Our results further suggest that the trade-off arises from limitations in translation regulation and inefficient energy regeneration. This work advances our understanding of the effects of fuel and energy metabolism on TX–TL in cell-free systems and lays a foundation for improving TX–TL performance, lifetime, standardization, and prediction.

Typical dammarane-type ginsenosides are well-known tetracyclic triterpenoids with significant pharmacological effects including antitumor, cardiovascular protection, and neuroprotection. Polyene-type ginsenosides exhibit stronger biological activities than common ginsenosides; however, their contents are low, and most are converted from ginsenosides through a series of processing steps, resulting in higher preparation costs. In this study, a dammaradienol synthase, AarOSC20433, was identified for the first time from Artemisia argyi H. Lév. & Vaniot (A. argyi). The high-yielding squalene strain constructed in this study was used as the chassis strain. Yeast heterologous biosynthesis of the polyene-type ginsenoside precursor dammaradienol was achieved via metabolic engineering strategies, including optimization of the terpene supply, increase in copy number of AarOSC20433, and rational enzyme design. Eventually, through replenishment and batch fermentation, the titer of dammaradienol reached 1.037 g/L (4.3 mg/L/OD), laying a solid foundation for the construction of a polyene-type ginsenoside cell factory.

Efficient bioassimilation of one-carbon (C1) feedstocks is often hindered by the toxicity of C1 substrates and/or intermediates. We compared the toxicity of several common C1 substrates/intermediates and found that formaldehyde imposes the highest toxicity on the representative bacterium Escherichia coli. Besides causing chromosomal DNA and protein damage effects, here, we revealed that formaldehyde greatly impairs cell membranes. To this end, here, we sought to remodel the cell membrane of E. coli by introducing a non-native, eukaryote-featured membrane phospholipid composition, phosphatidylcholine (PC). This engineered E. coli strain exhibited significantly increased membrane integrity, resulting in enhanced formaldehyde tolerance. When applied to C1 assimilation, the PC-harboring E. coli consumed up to 4.7 g/L methanol, which is 23-fold higher than that of the control strain (0.2 g/L). In summary, the present study highlights the detrimental impact of formaldehyde-induced membrane damage and thus underscores the significance of membrane remodeling in enhancing formaldehyde tolerance and facilitating the assimilation of C1 substrates.