蜡状葡糖杆菌

Transposon Library of Gluconobacter cerinus

1. Gluconobacter cerinus

Gluconobacter cerinus is a group of acetic acid bacteria with high biosafety and superior oxidative metabolic properties, holding significant application value in the fields of biocatalytic transformation, industrial synthesis of vitamin C, food processing and natural product preparation. Its unique incomplete oxidative metabolic mechanism, abundant dehydrogenase systems and high-efficiency catalytic capacity under mild conditions make it a distinctive functional strain for research on industrial biotransformation, fine chemical synthesis and microbial fermentation. Gradually refined genetic modification and metabolic regulation technologies serve as the core approaches to exploit its catalytic potential and construct high-efficiency biocatalytic engineered strains.

(1) Gram-staining characteristic: Gluconobacter cerinus is a Gram-negative bacterium.

(2) Morphological characteristics: Cells are short rod-shaped without endospores; colonies exhibit waxy luster and moist texture. As an obligate aerobic microorganism, it adapts well to mild fermentation environments and shows prominent tolerance to weak acids and osmotic pressure. The strain possesses a stable genome and abundant functional enzyme systems including pyrroloquinoline quinone (PQQ)-dependent dehydrogenases and flavoprotein dehydrogenases. It efficiently catalyzes the incomplete oxidation of saccharides and alcohols to specifically synthesize various organic acids and ketonic compounds, featuring abundant secondary metabolites and high catalytic specificity.

(3) Industrial significance: It is a classic functional strain in industrial biocatalysis and an important research model for exploring microbial incomplete oxidation mechanisms and substrate-specific catalytic pathways. Industrially, it is primarily applied to the biosynthesis of 2-keto-L-gulonic acid, the precursor of vitamin C, which greatly simplifies chemical synthesis processes and cuts production costs. It is also widely utilized in sugar-alcohol conversion, fine production of organic acids, food flavor modulation and the development of functional biological preparations. Additionally, it displays favorable in vitro anti-alcohol activity, promising broad prospects in food and healthcare industries.

(4) Genetic transformation: Stable and feasible gene delivery methods are available, including conjugation transformation, chemical competent cell transformation and electroporation. By utilizing compatible shuttle expression vectors, constitutive and inducible promoters combined with CRISPR-Cas gene editing technology, a mature system for gene knockout, overexpression and metabolic pathway engineering has been established. This system enables precise tuning of dehydrogenase expression levels and elimination of rate-limiting metabolic bottlenecks, supporting targeted construction of high-performance biocatalytic engineered strains and enhancement of metabolic capacity.

2. Construction of Gluconobacter cerinus Transposon Library

Shutong Biotechnology has achieved efficient and random insertion of resistance genes in Gluconobacter cerinus using the Mariner transposon system, and established a high-quality genome-wide transposon mutant library. This system offers the following prominent advantages:

Outstanding transposition efficiency: Rigorous tests confirm that the transposition efficiency of the Mariner transposon in Gluconobacter cerinus is stably above 80%, ensuring broad and random insertion events and providing a reliable basis for subsequent functional screening.

Large library size and high coverage: The constructed transposon library contains more than 5×10⁵ mutants, achieving high-density coverage of non-essential genomic regions. This scale is sufficient for systematic screening of key genes related to specific traits such as stress tolerance and metabolic enhancement.

Standardization and reproducibility: Standardized workflows for transposition, screening and validation have been established. Custom library construction is available for different Gluconobacter cerinus strain backgrounds, covering industrial scenarios including stress resistance improvement and product synthesis optimization.

Figure 1 PCR detection: Transposition validation and resistance gene insertion verification

Figure 2 PCR detection: Plasmid residual verification

3. Example Tn-Seq Report for Gluconobacter cerinus Transposon Library

The Tn-Seq report first presents statistics of raw sequencing data and quality-controlled filtered data.

Figure 3 Schematic diagram of sample data volume statistics

To ensure accurate identification of integration sites, all initially detected sites are strictly filtered. Only sites supported by at least 3 unique molecular identifiers (UMIs) are retained for subsequent statistical analysis.

Figure 4 Schematic diagram of insertion site statistics

A Circos plot displays the distribution of transposon insertion sites across the host genome; each line points to a specific integration locus.

Figure 5 Schematic diagram of integration site distribution on the host genome

Genome-wide coverage and gene insertion density are two core indicators for evaluating the quality and reliability of transposon insertion mutation screening. Genome-wide coverage reflects the saturation and screening breadth of the mutant library, helping exclude false-positive essential genes caused by incomplete coverage. Gene insertion density directly quantifies the tolerance of individual genes to insertion mutations and serves as a key basis for systematic identification of essential genes.

Figure 6 Schematic diagram of genome-wide coverage

To explore the functional impacts of essential genes, the report performs KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. The KEGG database characterizes gene interaction networks in metabolic and signaling pathways.

Figure 7 Schematic diagram of KEGG pathway enrichment

To comprehensively understand essential gene functions, the report further conducts GO (Gene Ontology) functional classification analysis covering three categories: Biological Process (BP), Cellular Component (CC) and Molecular Function (MF).

Figure 8 Schematic diagram of GO term enrichment

4. Services Provided by Shutong Biotechnology

You only need to provide the glycerol stock of the target strain and relevant information, and we will provide a full-process service for you.

Table 1 Service Content and Cycle

Table 2 Deliverables and Quality Control Standards