Whole-Genome Sequencing of Recipient Microorganisms

Whole-Genome Sequencing of Recipient Microorganisms

1. Background Introduction

With the rapid development of synthetic biology and gene editing technologies, Genetically Modified Microorganisms (GMMs) have been widely applied in food processing, biomanufacturing, pharmaceutical production, and other fields, driving the innovation and iteration of "three new foods" (novel food ingredients, new varieties of food additives, new varieties of food-related products) and biological products [1]. As the original vector for genetic modification, the biological characteristics, genetic stability, and potential safety risks of recipient microorganisms directly determine the safety baseline of genetically modified products [2].

However, traditional safety evaluation of recipient microorganisms relies on conventional methods such as phenotypic observation and biochemical identification, which have significant detection blind spots: they cannot accurately identify potential risk factors at the genomic level, such as virulence genes, antibiotic resistance genes, and transposable elements, making it difficult to meet increasingly stringent regulatory requirements. The latest regulatory policies explicitly stipulate: the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing (Trial)" mandates that recipient microorganisms shall provide safety analysis data on virulence, antibiotic resistance, genetic variation, etc., based on whole-genome sequencing; the Codex Alimentarius Commission (CAC) and FDA have also designated Whole-Genome Sequencing (WGS) as the core technical method for GMM safety assessment [1,3].

To address the limitations of traditional methods, Zhuhai Shutong Medical Technology Co., Ltd. has developed a whole-genome sequencing and safety analysis technology platform for recipient microorganisms. Integrating high-depth sequencing, multi-dimensional bioinformatics analysis, and regulatory compliance interpretation, it provides comprehensive recipient safety data support for the registration and marketing of genetically modified microbial products.

2. Technical Principles and Detection Process

The whole-genome sequencing technology for recipient microorganisms is based on second/third-generation high-throughput sequencing platforms. Through unbiased, high-coverage sequence determination of the recipient microorganism genome, combined with bioinformatics analysis, it comprehensively deciphers genomic characteristics and safety risk loci.

2.1 Core Principles

Taking genomic DNA as the research object, whole-genome sequence data are obtained through library construction and sequencing. Using professional analysis software and databases, the following objectives are achieved:

(1) De novo assembly of complete genome/ scaffold genome to accurately decipher gene structures and functional elements;

(2) Comprehensive screening of risk genes such as virulence genes, antibiotic resistance genes, and toxin synthesis genes;

(3) Genetic stability analysis (identification of mobile elements such as plasmids and transposons);

(4) Sequence alignment with reference strains to clarify genetic variations and potential unintended effects.

2.2 Standardized Detection Process (Second-Generation WGS)

(1) Sample Processing: Receive cell pellets and extract genomic DNA; directly perform quality inspection if DNA samples are submitted;

(2) Library Construction: Construct PE150 sequencing libraries after end repair, A-tailing, adapter ligation, and fragment selection;

(3) High-Throughput Sequencing: Perform sequencing on the DNBSEQ-T7 platform to ensure genome coverage ≥ 100× and data quality Q30 ≥ 90%;

(4) Bioinformatics Analysis: Genome assembly, gene prediction, functional annotation, risk gene screening, and genetic stability assessment;

(5) Report Generation: Integrate sequencing data and analysis results to generate standardized reports meeting regulatory requirements.

Figure 1. Detection Process

3. Technological Innovations and Advantages

3.1 Core Technological Innovations

3.1.1 Unbiased Whole-Genome Deciphering

Breaks through the limitations of traditional targeted detection, covering all coding and non-coding regions of the genome without detection blind spots;

Simultaneously deciphers chromosomal and plasmid genomes, comprehensively identifying the distribution and risks of mobile genetic elements (transposons, insertion sequences, etc.).

3.1.2 Multi-Dimensional Risk Assessment System

Integrates 12 authoritative databases including the Virulence Factor Database (VFDB), Comprehensive Antibiotic Resistance Database (CARD), and Toxin Database (ToxinDB);

Implements parallel multi-dimensional analysis of virulence, antibiotic resistance, allergenicity, and genetic transfer risks to accurately locate potential safety hazards.

3.1.3 Regulatory Compliance Analysis Process

Strictly adheres to domestic and foreign regulatory standards such as NMPA, FDA, and CAC, with analysis modules fully aligned with registration requirements;

Built-in specialized modules for off-target effect analysis and genetic stability prediction to meet the regulatory requirement of "unintended effect assessment" [2].

3.2 Service Advantages

(1) Technical Compliance: The analysis process fully complies with regulatory documents such as the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing", and the report can be directly used for registration;

(2) Qualification Guarantee: The laboratory is certified by ISO9001 and CNAS quality management systems, ensuring complete traceability and compliance of data;

(3) Professional Interpretation: Provides one-on-one technical consultation to assist enterprises in interpreting report data and responding to regulatory reviews;

(4) Rich Experience: Has completed sequencing and analysis projects for more than 50 strains of recipient microorganisms, covering bacteria, yeasts, fungi, and other categories;

(5) Efficient Delivery: The standardized process completes detection and report delivery within 15-20 working days, with expedited service available in as fast as 10 working days.

4. Application Scenarios

4.1 Core Application Scenarios

(1) GMM Registration Support: Provide whole-genome safety data of recipient microorganisms for the registration of "three new foods", biological products, etc., meeting regulatory registration requirements;

(2) Recipient Strain Screening and Optimization: Assist enterprises in screening low-risk recipient strains and evaluating the feasibility and safety of genetic modification;

(3) Preclinical Safety Evaluation: Support preclinical research of genetically modified microbial products and provide recipient-related risk assessment data;

(4) Post-Marketing Monitoring: Long-term tracking of the genetic stability of recipient microorganisms to support post-marketing safety monitoring of products;

(5) Scientific Research and Technology Transformation: Provide genomic data support for research such as synthetic biology and microbial breeding.

5. Core Content of Example Report

Shutong Technology provides professional whole-genome sequencing reports for recipient microorganisms, including the following core modules to fully meet registration and scientific research needs:

5.1 Basic Genomic Characteristics Analysis

Provides basic information such as genome size, GC content, number of coding genes, and number of plasmids. Visual charts display the gene distribution and functional element localization of chromosomes and plasmids.

5.2 Risk Gene Screening Results

Detailedly lists the name, sequence location, homology, functional annotation, and risk level of risk factors such as virulence genes, antibiotic resistance genes, and toxin synthesis genes (as shown in Table 1).

Table 1. Example of Risk Gene Screening Results

5.3 Genetic Stability Assessment

Analyzes plasmid stability, transposon distribution, gene recombination hotspots, etc., predicts the genetic variation risk of recipient microorganisms during passage, and provides passage stability recommendations.

5.4 Regulatory Compliance Summary

Compares with NMPA/FDA registration requirements, itemizes whether the test results meet the standards, clarifies safety level recommendations (e.g., "Safety Level Class I"), and provides risk control suggestions.

6. Service Content and Process

Service Cycle: Standard process: 15-20 working days; Expedited service: Need to communicate in advance.

7. Sample Requirements and Information to Be Provided by Clients

8. References

[1]国家卫生健康委员会。食品加工用遗传修饰微生物安全性评价申报材料要求（试行）[S].2023.

[2]国家药品监督管理局药品审评中心。体内基因治疗产品药学研究与评价技术指导原则（试行）[S].2022.

[3] Food and Drug Administration (FDA). Guidance for Industry: Safety Assessment of Genetically Modified Microorganisms Used in Food Production [S]. 2021.

[4] Chen L, Wang Y, Li J, et al. Whole-genome sequencing-based safety assessment of genetically modified microorganisms: A review [J]. Food Control, 2022, 139: 109152.

[5] Zhu Y, Zhang H, Liu X, et al. Comprehensive risk assessment of recipient microorganisms using high-throughput sequencing [J]. Frontiers in Microbiology, 2023, 14: 1089245.