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Bacteria with streamlined genomes, that harbor full functional genes for essential metabolic networks, are able to synthesize the desired products more effectively and thus have advantages as production platforms in industrial applications. To obtain streamlined chassis genomes, a large amount of effort has been made to reduce existing bacterial genomes. This work falls into two categories: rational and random reduction. The identification of essential gene sets and the emergence of various genome-deletion techniques have greatly promoted genome reduction in many bacteria over the past few decades. Some of the constructed genomes possessed desirable properties for industrial applications, such as: increased genome stability, transformation capacity, cell growth, and biomaterial productivity. The decreased growth and perturbations in physiological phenotype of some genome-reduced strains may limit their applications as optimized cell factories. This review presents an assessment of the advancements made to date in bacterial genome reduction to construct optimal chassis for synthetic biology, including: the identification of essential gene sets, the genome-deletion techniques, the properties and industrial applications of artificially streamlined genomes, the obstacles encountered in constructing reduced genomes, and the future perspectives. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II‐A CRISPR‐Cas9, by delivering a self‐targeting CRISPR array with DNA repair template into S. thermophilus LMD‐9. We achieved 785‐bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1‐kb DNA repair template was provided. While providing with 1.5‐kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234‐bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%–100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single‐gene knockout mutants with efficiencies of 29%–80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD‐9. Altogether, repurposing the native type II‐A CRISPR‐Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications. [ABSTRACT FROM AUTHOR]

To date, many metabolic engineering tools and strategies have been developed, including tools for cofactor engineering, which is a common strategy for bioproduct synthesis. Cofactor engineering is used for the regulation of pyridine nucleotides, including NADH/NAD+ and NADPH/NADP+, and adenosine triphosphate/adenosine diphosphate (ATP/ADP), which is crucial for maintaining redox and energy balance. However, the intracellular levels of NADH/NAD+, NADPH/NADP+, and ATP/ADP cannot be monitored in real time using traditional methods. Recently, many biosensors for detecting, monitoring, and regulating the intracellular levels of NADH/NAD+, NADPH/NADP+, and ATP/ADP have been developed. Although cofactor biosensors have been mainly developed for use in mammalian cells, the potential application of cofactor biosensors in metabolic engineering in bacterial and yeast cells has received recent attention. Coupling cofactor biosensors with genetic circuits is a promising strategy in metabolic engineering for optimizing the production of biochemicals. In this review, we focus on the development of biosensors for NADH/NAD+, NADPH/NADP+, and ATP/ADP and the potential application of these biosensors in metabolic engineering. We also provide critical perspectives, identify current research challenges, and provide guidance for future research in this promising field. [ABSTRACT FROM AUTHOR]