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Microbial production of value-added chemicals from biomass is a sustainable alternative to chemical synthesis. To improve product titer, yield, and selectivity, the pathways engineered into microbes must be optimized. One strategy for optimization is dynamic pathway regulation, which modulates expression of pathway-relevant enzymes over the course of fermentation. Metabolic engineers have used dynamic regulation to redirect endogenous flux toward product formation, balance the production and consumption rates of key intermediates, and suppress production of toxic intermediates until later in the fermentation. Most cases, however, have utilized a single strategy for dynamically regulating pathway fluxes. Here we layer two orthogonal, autonomous, and tunable dynamic regulation strategies to independently modulate expression of two different enzymes to improve production of D-glucaric acid from a heterologous pathway. The first strategy uses a previously described pathway-independent quorum sensing system to dynamically knock down glycolytic flux and redirect carbon into production of glucaric acid, thereby switching cells from “growth” to “production” mode. The second strategy, developed in this work, uses a biosensor for myo-inositol (MI), an intermediate in the glucaric acid production pathway, to induce expression of a downstream enzyme upon sufficient buildup of MI. The latter, pathway-dependent strategy leads to a 2.5-fold increase in titer when used in isolation and a fourfold increase when added to a strain employing the former, pathway-independent regulatory system. The dual-regulation strain produces nearly 2 g/L glucaric acid, representing the highest glucaric acid titer reported to date in Escherichia coli K-12 strains.

Establishment of a highly efficient cell factory is imperative for 5-aminolevulinic acid (5-ALA) biomanufacturing.A streamlined workflow is described that enables highly efficient 5-ALA synthase mining.Genome-scale model-guided identification and combination of multiplex targets are reported.An artificial homeostasis was designed for dynamically responding to, and fine-tuning, redox status.Final collaborative optimization resulted in the highest 5-ALA biomanufacturing performance achieved to date. The versatile applications of 5-aminolevulinic acid (5-ALA) across the fields of agriculture, livestock, and medicine necessitate a cost-efficient biomanufacturing process. In this study, we achieved the economic viability of biomanufacturing this compound through a systematic engineering framework. First, we obtained a 5-ALA synthase (ALAS) with superior performance by exploring its natural diversity with divergent evolution. Subsequently, using a genome-scale model, we identified and modified four key targets from distinct pathways in Escherichia coli, resulting in a final enhancement of 5-ALA titers up to 21.82 g/l in a 5-l bioreactor. Furthermore, recognizing that an imbalance of redox equivalents hindered further titer improvement, we developed a dynamic control system that effectively balances redox status and carbon flux. Ultimately, we collaboratively optimized the artificial redox homeostasis system at the transcription level with other cofactors at the feeding level, demonstrating the highest recorded performance to date with a titer of 63.39 g/l for the biomanufacturing of 5-ALA. Graphical abstract [Display omitted] The economic viability of biomanufacturing 5-aminolevulinic acid (5-ALA) was successfully demonstrated in this study, showcasing excellent production performance. The titer reached a record-breaking 63.39 g/l at 44 h, representing the highest reported value to date, with the productivity of 1.44 g/l/h. Although the yield (0.384 mol/mol glucose) was lower than theoretically expected, the significant value of 5-ALA positions our developed cell factory competitively for efficient industrial-scale biomanufacturing. Therefore, no challenges unique to this compound can be identified for full-scale fermentation, particularly considering that our 5-ALA cell factory was derived from a widely utilized Escherichia coli strain. Currently, two primary approaches are used to produce 5-ALA: chemical synthesis and biomanufacturing. Microbial biosynthesis of 5-ALA presents a more facile, environmentally benign, and low-cost alternative. Given that 5-ALA is a non-protein amino acid, we anticipated that the entire biomanufacturing process would be analogous to the biomanufacturing of canonical amino acids. Therefore, although a separation process was not implemented in this study, we believe that our developed 5-ALA cell factory exhibits excellent parameters and represents a highly cost-efficient biomanufacturing process. Within NASA’s Technology Readiness Level (TRL) system, we propose that this 5-ALA cell factory has reached TRL 6, indicating a fully functional prototype suitable for demonstration in real production scenarios. A systematic engineering framework was demonstrated to construct a 5-aminolevulinic acid (5-ALA) cell factory, achieving the economic viability of biomanufacturing this compound with an unprecedented production performance (63.39 g/l). This comprehensive framework encompasses enzyme mining, multitarget engineering, artificial homeostasis design, and collaborative optimization.

Insights from novel mechanistic paradigms in gene expression control have led to the development of new gene expression systems for bioproduction, control, and sensing applications. Coupled with a greater understanding of synthetic burden and modern creative biodesign approaches, contemporary bacterial gene expression tools and systems are emerging that permit fine-tuning of expression, enabling greater predictability and maximisation of specific productivity, while minimising deleterious effects upon cell viability. These advances have been achieved by using a plethora of regulatory tools, operating at all levels of the so-called ‘central dogma’ of molecular biology. In this review, we discuss these gene regulation tools in the context of their design, prototyping, integration into expression systems, and biotechnological application. A huge array of genetic regulatory tools are now available that permit gene expression control at all layers of the central dogma.Matching production demand with cellular capacity can reduce burden and allow stable production over longer timescales.Modern in silico design tools allow rapid design and optimisation of complex genetic circuitry.Cell-free prototyping of genetic parts and devices is an emerging, yet powerful tool to improve the design–build–test–learn cycle.The use of feedback loops to facilitate dynamic regulation of gene expression allows researchers to construct responsive pathways to minimise cellular metabolic burden.Stress-linked expression regulation enables coupling of heterologous production with endogenous stress response pathways.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most popular flavors with wide applications in food, fragrance, and pharmaceutical industries. However, the high cost and limited yield of plant extraction failed to meet the vast market demand of natural vanillin. Vanillin biotechnology has emerged as a sustainable and cost-effective alternative to supply vanillin. In this review, we explored recent advances in vanillin biosynthesis and highlighted the potential of vanillin biotechnology. In particular, we addressed key challenges in using microorganisms and provided promising approaches for improving vanillin production with a special focus on chassis development, pathway construction and process optimization. Future directions of vanillin biosynthesis using inexpensive precursors are also thoroughly discussed.

The sustainable, bio‐based production of industrially valuable chemicals and materials from renewable, non‐edible biomass through biorefineries has emerged as a vital strategy for tackling urgent global challenges, including climate change, and for realizing the “net zero carbon” commitments recently pledged by nations worldwide. Metabolic engineering has played a central role in enabling the development of microbial strains capable of efficiently overproducing a diverse array of target compounds. Nevertheless, engineered microbial cell factories often face inherent trade‐offs between product synthesis and cell growth, frequently resulting in diminished fitness or loss‐of‐function phenotypes. This review highlights recent advances in metabolic engineering strategies aims at reconciling this conflict, encompassing pathway optimization, dynamic regulation, orthogonal system design, microbial consortia engineering, fermentation process control, and integrative metabolic modeling. It also explores the remaining challenges and future directions for reprogramming microbial metabolism to harmonize growth with high‐level production. This review presents state‐of‐the‐art metabolic engineering strategies to balance microbial cell growth and product synthesis in biorefineries. It surveys pathway engineering, dynamic genetic circuits, orthogonal control systems, synthetic microbial consortia, and fermentation optimization, alongside integrative modeling approaches. Current challenges and future directions for reprogramming cellular metabolism to harmonize high‐level production with robust growth are also explored.

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.

In this paper we explore further how energy network regulation might better be adapted to the uncertainty challenges that are raised by net zero climate policy. We do this with specific reference to energy regulation in the UK. We discuss the drivers of change and the nature of the uncertainty that is faced by energy regulators. Next, we examine theories of dynamic/responsive/adaptive regulation for lessons that regulators can learn in the light of net zero. We look for regulatory learning from water regulators in Scotland and England and Wales and airport regulation in London. Drawing on evidence from a recent consultation with 41 stakeholder responses, we explore how energy regulation might need to change in the areas of planning, uncertainty mechanisms, regulatory incentives, financing arrangements, stakeholder engagement, innovation processes, and industry governance.

An alcohol dehydrogenase SACE_1905 that converts NADH to NAD+ is identified in Saccharopolyspora erythraea. It is an effective target for manipulating cellular redox status by altering the NADH/NAD+ ratio.A dynamic control strategy, named diNAD, that fine-tunes SACE_1905 expression with the inducible clustered regularly interspaced short palindromic repeat interference (CRISPRi) circuit is developed for precisely adjusting cellular NADH/NAD+ ratio in a timely and appropriate manner.diNAD enables the trade-off between cell growth and antibiotic formation via the optimization of carbon metabolic flux.A paradigm for flexibly modulating cellular redox status to increase antibiotic titers is established, filling the gap in NAD-based dynamic engineering strategies in actinomycetes. Nicotinamide adenine dinucleotide (NAD) homeostasis is crucial for secondary metabolism in antibiotic-producing actinomycetes. However, NAD-based dynamic control strategies for boosting antibiotic titers have not been reported. We identified SACE_1905, an alcohol dehydrogenase in Saccharopolyspora erythraea, which converts NADH to NAD+. Overexpressing SACE_1905 lowered the cellular NADH/NAD+ ratio, facilitating carbon source utilization and erythromycin biosynthetic precursor supply, concurrently improving cell growth and erythromycin yield. To balance primary and secondary metabolism, we developed a strategy that fine-tunes SACE_1905 expression with inducible clustered regularly interspaced short palindromic repeat interference (CRISPRi) to dynamically modulate the NADH/NAD+ ratio, which we named diNAD. Optimized diNAD redirected carbon flux, maximizing erythromycin biosynthesis at a moderate NADH/NAD+ ratio during the stationary phase. Based on its utility in actinorhodin and avermectin overproduction in Streptomyces coelicolor and Streptomyces avermitilis, we show that diNAD is effective in augmenting antibiotic titers in actinomycetes. [Display omitted] The diNAD strategy for programmable control of redox status provides a straightforward and effective platform to boost secondary metabolite titers in actinomycetes. By fine-tuning the expression of a new alcohol dehydrogenase gene with an inducible clustered regularly interspaced short palindromic repeat interference (CRISPRi) circuit, we exploited the diNAD strategy to enable dynamic adjustment of the cellular NADH/NAD+ ratio in a timely and appropriate manner. The proof-of-concept has been established through successful laboratory-scale experiments, demonstrating enhanced erythromycin, actinorhodin, and avermectin production. In particular, this strategy was applied to the industrial Saccharopolyspora erythraea strain, achieving the high-level production of erythromycin in a 15-l bioreactor. Therefore, we propose that the technology has reached Technology Readiness Level (TRL) 5, as simulations have been run in environments that are as close to realistic as possible. However, challenges remain for full-scale industrial implementation. Optimizing diNAD for robust and consistent performance in large-scale fermentations is crucial. Overcoming this challenge will require further engineering and process development, including optimization of inducer delivery and real-time monitoring of cellular NADH/NAD+ level. If successful, diNAD holds significant potential to revolutionize antibiotic production in actinomycetes, impacting pharmaceutical and industrial sectors. By mining an alcohol dehydrogenase that can convert NADH to NAD+, we developed a strategy that dynamically adjusts the cellular NADH/NAD+ ratio to balance cell growth and antibiotic biosynthesis. This work establishes a NAD-based dynamic engineering strategy and provides an effective platform to boost secondary metabolite titers in actinomycetes.

A tetracycline repressor protein (TetR)/tetO2 inducible system was constructed and optimized in Pichia pastoris.The TetR/tetO2 inducible system dynamically enhanced homologous recombination (HR) for precise editing of multiple genes.Dynamically enhancing HR did not compromise cellular growth or product biosynthesis. The methylotrophic yeast Pichia pastoris (also known as Komagataella pastoris) is an ideal host for producing proteins and natural products. Enhancing homologous recombination (HR) is helpful for improving the precision of genome editing, but results in stress to cellular fitness and is harmful for industrial applications. To overcome these challenges, we developed a tetracycline repressor protein (TetR)/tetO2 inducible system to dynamically regulate the HR-related gene RAD52 in P. pastoris. This approach significantly improved the positivity rate of single gene deletion to 81%. Furthermore, inducible overexpression of endogenous MUS81-MMS4 resulted in high-efficiency (81%) genome assembly of multiple genes. This inducible system had no adverse effect on cell growth in different media and resulted in greater fatty alcohol production from methanol compared with a strain constitutively overexpressing HR-related genes. We anticipate that this inducible regulation is applicable for enhancing HR for precise genome editing in P. pastoris and other non-conventional microbes without compromising cellular fitness. [Display omitted] An anhydrotetracycline (aTc) inducible system was developed to dynamically overexpress homologous recombination-related genes for precise genome engineering in Pichia pastoris. By using this dynamic regulation system, the positive rate of precise gene editing reached 80%, without interrupting cellular growth or fatty alcohol production. An inducible CRISPR-Cas9 gene-editing strategy was constructed in Pichia pastoris, demonstrating excellent homologous recombination (HR) efficiency in genomic integration. This inducible system promoted the positive rates of genome integration of single and multiple genes by up to 80%, without comprising either cellular growth using methanol as a carbon source or fatty alcohol production; in addition, constitutively enhancing HR significantly impaired fatty alcohol biosynthesis. Currently, CRISPR-Cas9 gene-editing tools have successful applications in unconventional yeasts, and enhancement of the HR repair pathway could be a feasible strategy to increase the gene editing efficiency. However, enhancing HR also impacts cellular metabolism and production biosynthesis, effects that would be amplified in large-scale production. Thus, there is an urgent need to coordinate gene-editing efficiency and chemical production. In our study, although the inducible HR system was only performed in P. pastoris and evaluated for fatty alcohol production under shake flask fermentation, it should be evaluated for other products in larger scale biomanufacturing systems, such as bioreactors. Therefore, the current Technology Readiness Level (TRL) of this technology lies between 4 and 5.

The coal seams mined in the Sandaogou Coal Mine are characterized by shallow burial depth, high susceptibility to spontaneous combustion, and near-horizontal occurrence. Surface air leakage not only leads to abnormal gas emissions from the goaf but also results in frequent low-oxygen conditions at the working face. Meanwhile, it increases the oxygen concentration in the composite goaf, thereby elevating the risk of spontaneous combustion of residual coal.To address the above issues, a comprehensive intelligent ventilation regulation system was established. Based on the on-site meteorological conditions, gas emission rates, and the number of personnel, the required airflow for the working face was calculated, resulting in a demand of 1122.66 m 3 /min.The ventilation route was determined, and an equal-pressure system—comprising fans, air doors, sensors, and other components—was designed. Based on ventilation resistance calculations and the functional relationships among fan frequency, rotational speed, air pressure, and airflow, the FBD-№12.5 2 × 75 kW variable-frequency equal-pressure fan was selected. An intelligent remote control system for air doors and an intelligent monitoring system for local fans were developed, integrating multiple types of sensors—including differential pressure, multi-parameter, photoelectric, and proximity sensors—to enable automated control and coordinated operation of air doors, air windows, and fans. An intelligent monitoring software platform consisting of seven functional modules was established, achieving real-time monitoring and dynamic balancing of working-face air pressure, airflow, and goaf differential pressure. This system significantly enhances the level of intelligent safety management in coal mine operations.