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Background In industrial fermentation, pH fluctuation resulted from microbial metabolism influences the strain performance and the final production. The common way to control pH is adding acid or alkali after probe detection, which is not a fine-tuned method and often leads to increased costs and complex downstream processing. Here, we constructed an intelligent pH-sensing and controlling genetic circuits called “Genetic pH Shooting (GPS)” to realize microbial self-regulation of pH. Results In order to achieve the self-regulation of pH, GPS circuits consisting of pH-sensing promoters and acid-/alkali-producing genes were designed and constructed. Designed pH-sensing promoters in the GPS can respond to high or low pHs and generate acidic or alkaline substances, achieving endogenously self-responsive pH adjustments. Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. Furthermore, the genetic circuits including GPS, BSC and ASC were applied to lycopene production with a higher yield without an artificial pH regulation compared with the control under pH values ranging from 5.0 to 9.0. In scale-up fermentations, the lycopene titer in the engineered strain harboring GPS was increased by 137.3% and ammonia usage decreased by 35.6%. Conclusions The pH self-regulation achieved through the GPS circuits is helpful to construct intelligent microbial cell factories and reduce the production costs, which would be much useful in industrial applications.

Microorganisms have long been used as chemical plant to convert simple substrates into complex molecules. Various metabolic pathways have been optimised over the past few decades, but the progresses were limited due to our finite knowledge on metabolism. Evolution is a knowledge-free genetic randomisation approach, employed to improve the chemical production in microbial cell factories. However, evolution of large, complex pathway was a great challenge. The invention of continuous culturing systems and in vivo genetic diversification technologies have changed the way how laboratory evolution is conducted, render optimisation of large, complex pathway possible. In vivo genetic diversification, phenotypic selection, and continuous cultivation are the key elements in in vivo continuous evolution, where any human intervention in the process is prohibited. This approach is crucial in highly efficient evolution strategy of metabolic pathway evolution.

Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory.Key points• Pseudomonas putida advances to a global industrial cell factory.• Novel tools enable system-wide understanding and streamlined genomic engineering.• Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.

Genome-scale metabolic models enable the rational design of microbial cell factories by quantitatively evaluating metabolic potential and guiding engineering strategies.The increased biological fidelity of advanced models, achieved through incorporating more constraints and cellular processes, drives the need for codeveloped algorithms to fully leverage their predictive power.The design of microbial cocultures requires distinct algorithms and modeling frameworks that address community-level objectives.The integration of artificial intelligence with genome-scale models and the emergence of self-driving laboratories are becoming transformative trends for accelerating biological design cycles. Microbial cell factories are powerful platforms for the sustainable production of chemicals, foods, medicines, and energy. Enhancing production efficiency requires a deep understanding of the underlying metabolic mechanisms. Over the past 3 decades, genome-scale models have enabled quantitative metabolic simulation and driven the development of algorithms for guiding rational metabolic engineering. In this review, we provide an overview of recent algorithms for microbial cell design and the most comprehensive evolutionary roadmap to date, emphasizing their development and implementation. We also summarize advances in modeling frameworks and algorithms for coculture design, compare algorithms for microbial cells and cocultures, and discuss emerging trends shaping future model and algorithm development. Microbial cell factories are powerful platforms for the sustainable production of chemicals, foods, medicines, and energy. Enhancing production efficiency requires a deep understanding of the underlying metabolic mechanisms. Over the past 3 decades, genome-scale models have enabled quantitative metabolic simulation and driven the development of algorithms for guiding rational metabolic engineering. In this review, we provide an overview of recent algorithms for microbial cell design and the most comprehensive evolutionary roadmap to date, emphasizing their development and implementation. We also summarize advances in modeling frameworks and algorithms for coculture design, compare algorithms for microbial cells and cocultures, and discuss emerging trends shaping future model and algorithm development.

Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Salmonella cerevisiae and Bacillus subtilis . In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.

Background As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. Results First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. Conclusions High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.

The development of CRISPR base editors, CRISPR prime editors, and EvolvR has enabled more efficient and precise gene editing without relying on double-stranded DNA breaks.Transcriptional CRISPRi and CRISPRa systems have been developed with higher efficiency and tunability, enabling the design of more advanced regulation circuits.The CRISPR toolbox has been expanded to protein-level regulation by the Cas6-enabled dynamic enzyme assembly.The expanded CRISPR toolbox has comprehensively promoted the engineering of various components and cellular processes in microbes, driving the advancement of microbial cell factory construction. Microbial cell factories (MCFs) convert low-cost carbon sources into valuable compounds. The CRISPR/Cas9 system has revolutionized MCF construction as a remarkable genome editing tool with unprecedented programmability. Recently, the CRISPR toolbox has been significantly expanded through the exploration of new CRISPR systems, the engineering of Cas effectors, and the incorporation of other effectors, enabling multi-level regulation and gene editing free of double-strand breaks. This expanded CRISPR toolbox powerfully promotes MCF construction by facilitating pathway construction, enzyme engineering, flux redistribution, and metabolic burden control. In this article, we summarize different CRISPR tool designs and their applications in MCF construction for gene editing, transcriptional regulation, and enzyme modulation. Finally, we also discuss future perspectives for the development and application of the CRISPR toolbox. Microbial cell factories (MCFs) convert low-cost carbon sources into valuable compounds. The CRISPR/Cas9 system has revolutionized MCF construction as a remarkable genome editing tool with unprecedented programmability. Recently, the CRISPR toolbox has been significantly expanded through the exploration of new CRISPR systems, the engineering of Cas effectors, and the incorporation of other effectors, enabling multi-level regulation and gene editing free of double-strand breaks. This expanded CRISPR toolbox powerfully promotes MCF construction by facilitating pathway construction, enzyme engineering, flux redistribution, and metabolic burden control. In this article, we summarize different CRISPR tool designs and their applications in MCF construction for gene editing, transcriptional regulation, and enzyme modulation. Finally, we also discuss future perspectives for the development and application of the CRISPR toolbox.

Background Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. Results To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP production at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation ( glgc ) and succinate conversion to fumarate ( sdhA and sdh B). Transcription levels of glgc , sdhA and sdh B were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer ≈12.5-fold to ≈0.58–0.63 mg/L. Conclusions These data demonstrated that CRISPRi-mediated gene suppression allowed for re-directing the cellular carbon flow, thus paving a new avenue to rationally fine-tune the metabolic pathways in PCC 7942 for the production of biotechnological products.

Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid—a crucial precursor to artemisinin—is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.

Scopoletin is a typical example of coumarins, which can be produced in plants. Scopoletin acts as a precursor for pharmaceutical and health care products, and also possesses promising biological properties, including antibacterial, anti-tubercular, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperuricemic activity. Despite the potential benefits, the production of scopoletin using traditional extraction processes from plants is unsatisfactory. In recent years, synthetic biology has developed rapidly and enabled the effective construction of microbial cell factories for production of high value-added chemicals. Herein, this review summarizes the progress of scopoletin biosynthesis in artificial microbial cell factories. The two main pathways of scopoletin biosynthesis are summarized firstly. Then, synthetic microbial cell factories are reviewed as an attractive improvement strategy for biosynthesis. Emerging techniques in synthetic biology and metabolic engineering are introduced as innovative tools for the efficient synthesis of scopoletin. This review showcases the potential of biosynthesis of scopoletin in artificial microbial cell factories.