A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in .

is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems for employ heterologous promoters, including P or AfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload.

MstH Is the Major High-Affinity Glucose Transporter with Pleiotropic Roles in Carbon Utilization Regulation and Citric Acid Production in .

is a versatile industrial workhorse for bulk biomanufacturing of organic acids and enzymic proteins owing to its remarkable capacity to utilize diverse crude carbon sources. Glucose, a primary carbon source, plays a critical role in industrial bioprocesses; however, the key components and mechanisms of its dual-affinity transport system in remain poorly characterized. Herein, we employed transcriptomic profiling and functional analysis of null mutants to comprehensively investigate the glucose transport system.

Comprehensive screening of industrially relevant components at genome scale using a high-quality gene overexpression collection of Corynebacterium glutamicum.

Development of efficient microbial strains for biomanufacturing requires deep understanding of the biology and functional components responsible for the synthesis, transport, and tolerance of the target compounds. A high-quality controllable gene overexpression strain collection was constructed for the industrial workhorse Corynebacterium glutamicum covering 99.7% of its genes.

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening.

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited.

Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids.

Biosensors are powerful tools for detecting, real-time imaging, and quantifying molecules, but rapidly constructing diverse genetically encoded biosensors remains challenging. Here, we report a method to rapidly convert enzymes into genetically encoded circularly permuted fluorescent protein-based indicators to detect organic acids (GECFINDER). ANL superfamily enzymes undergo hinge-mediated ligand-coupling domain movement during catalysis.

Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable.

Directed evolution of linker helix as an efficient strategy for engineering LysR-type transcriptional regulators as whole-cell biosensors.

Whole-cell biosensors based on transcriptional regulators are powerful tools for rapid measurement, high-throughput screening, dynamic metabolic regulation, etc. To optimize the biosensing performance of transcriptional regulator, its effector-binding domain is commonly engineered. However, this strategy is encumbered by the limitation of diversifying such a large domain and the risk of affecting effector specificity.

Engineering of the Translesion DNA Synthesis Pathway Enables Controllable C-to-G and C-to-A Base Editing in .

Expanding the base conversion type is expected to largely broaden the application of base editing, whereas it requires decipherment of the machinery controlling the editing outcome. Here, we discovered that the DNA polymerase V-mediated translesion DNA synthesis (TLS) pathway controlled the C-to-A editing by a glycosylase base editor (GBE) in . However, C-to-G conversion was surprisingly found to be the main product of the GBE in and subsequent gene inactivation identified the decisive TLS enzymes.

Base editor enables rational genome-scale functional screening for enhanced industrial phenotypes in .

Genome-scale functional screening accelerates comprehensive assessment of gene function in cells. Here, we have established a genome-scale loss-of-function screening strategy that combined a cytosine base editor with approximately 12,000 parallel sgRNAs targeting 98.1% of total genes in ATCC 13032.

CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction.

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering.

Development of a Hyperosmotic Stress Inducible Gene Expression System by Engineering the MtrA/MtrB-Dependent Promoter in .

is an important workhorse for industrial production of diversiform bioproducts. Precise regulation of gene expression is crucial for metabolic balance and enhancing production of target molecules. Auto-inducible promoters, which can be activated without expensive inducers, are ideal regulatory tools for industrial-scale application.

Pair Nodes Clock Synchronization Algorithm Based on Kalman Filter for Underwater Wireless Sensor Networks.

Time synchronization is the basis of many applications. Aiming at the limitations of the existing clock synchronization algorithms in underwater wireless sensor networks, we propose a pairwise synchronization algorithm called K-Sync, which is based on the Kalman filter. The algorithm does not need the assistance of the position sensor or the speed sensor, and the high time synchronization accuracy can be realized only by utilizing the time-stamps information in the process of message exchange.

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol-dependent growth of Corynebacterium glutamicum.

Methanol is a promising feedstock for biomanufacturing of fuels and chemicals. Although efforts have been made to engineer platform microorganisms for methanol bioconversion, the substrate uptake and cell growth rates on methanol are still unsatisfactory, suggesting certain limiting factors remain unsolved. Herein, we analysed the global metabolic regulation changes between an evolved methanol-dependent Corynebacterium glutamicum mutant and its ancestral strain by transcriptome analysis.

In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing.

Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms.

CRISPR-dCas9 Mediated Cytosine Deaminase Base Editing in .

Base editing technology based on clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from ) and activation-induced cytidine deaminase (AID).

Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum.

Synthetic methylotrophy has recently been intensively studied to achieve methanol-based biomanufacturing of fuels and chemicals. However, attempts to engineer platform microorganisms to utilize methanol mainly focus on enzyme and pathway engineering. Herein, we enhanced methanol bioconversion of synthetic methylotrophs by improving cellular tolerance to methanol.

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum.

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing.

GREACE-assisted adaptive laboratory evolution in endpoint fermentation broth enhances lysine production by Escherichia coli.

Background: Late-stage fermentation broth contains high concentrations of target chemicals. Additionally, it contains various cellular metabolites which have leaked from lysed cells, which would exert multifactorial stress to industrial hyperproducers and perturb both cellular metabolism and product formation. Although adaptive laboratory evolution (ALE) has been wildly used to improve stress tolerance of microbial cell factories, single-factor stress condition (i.

[Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis].

Promoter, an essential regulatory element, is widely used for metabolic engineering of industrial strains. Corynebacterium glutamicum is an important industrial workhorse to produce various amino acids. However, strong constitutive promoters that are applicable to C.

Mutations in Peptidoglycan Synthesis Gene Improve Electrotransformation Efficiency of ATCC 13869.

is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far.

Efficient production of trans-4-hydroxy-l-proline from glucose using a new trans-proline 4-hydroxylase in Escherichia coli.

trans-4-Hydroxy-l-proline (trans-4Hyp) is widely used as a valuable building block for the organic synthesis of many pharmaceuticals such as carbapenem antibiotics. The major limitation for industrial bioproduction of trans-4Hyp is the low titer and productivity by using the existing trans-proline 4-hydroxylases (trans-P4Hs). Herein, three new trans-P4Hs from Alteromonas mediterranea (AlP4H), Micromonospora sp.

MACBETH: Multiplex automated Corynebacterium glutamicum base editing method.

CRISPR/Cas9 or Cpf1-introduced double strand break dramatically decreases bacterial cell survival rate, which hampers multiplex genome editing in bacteria. In addition, the requirement of a foreign DNA template for each target locus is labor demanding and may encounter more GMO related regulatory hurdle in industrial applications. Herein, we developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID), without foreign DNA templates, achieving single-, double-, and triple-locus editing with efficiencies up to 100%, 87.

A Novel Corynebacterium glutamicum l-Glutamate Exporter.

Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyperproduction of biochemicals using microbial cell factories. The identification and characterization of transporters are therefore of great significance for the understanding and engineering of transport reactions. Herein, a novel l-glutamate exporter, MscCG2, which exists extensively in strains but is distinct from the only known l-glutamate exporter, MscCG, was discovered in an industrial l-glutamate-producing strain.