Taxonomic Identification of Recipient Microorganisms

Taxonomic Identification of Recipient Microorganisms

1. Background Introduction

In the safety risk assessment of Genetically Modified Microorganisms (GMMs), the accurate taxonomic positioning of recipient microorganisms is the primary link in regulatory review—their species affiliation, phylogenetic relationships, and taxonomic status directly determine the scientificity of safety classification, risk baseline setting, and subsequent evaluation of genetic modification effects [1]. The "Measures for the Safety Evaluation of Agricultural Genetically Modified Organisms" clearly stipulates that the determination of the safety level of recipient organisms must be based on "species biological characteristics, taxonomic status, and known safety records"; the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing (Trial)" further specifies that recipient microorganisms shall provide an "identification report identified to the species or subspecies level based on phenotype, genotype, and the latest sequencing technology", supplemented by core data such as biological characteristics (including physicochemical properties) [2].

Traditional taxonomic identification relies on single phenotypic observation or 16S rRNA gene sequencing, with significant limitations: phenotypic identification is susceptible to culture conditions and low accuracy; single-gene sequencing cannot distinguish closely related species, failing to meet the regulatory requirement of "species or subspecies level". With the development of microbial taxonomic technology, Average Nucleotide Identity (ANI) analysis has become the internationally recognized gold standard for species delineation. Multi-dimensional integrated identification combining phenotype, genotype, and physicochemical properties has emerged as the core technical solution to meet regulatory compliance [3].

To address industry pain points and regulatory needs, Zhuhai Shutong Medical Technology Co., Ltd. has developed a four-dimensional integrated taxonomic identification technology platform for recipient microorganisms. Integrating traditional microbiological detection with modern high-throughput sequencing technology, it achieves comprehensive identification from phenotypic characteristics to genomic level, providing authoritative and compliant taxonomic data support for GMM product registration and safety assessment.

2. Technical Principles and Detection Process

The taxonomic identification technology platform for recipient microorganisms is based on a four-dimensional integrated strategy of "phenotype - genotype - physicochemical properties - ANI analysis", realizing accurate species delineation and taxonomic positioning through multi-level data cross-validation.

2.1 Core Principles

The four-dimensional identification system complements and validates each other hierarchically:

(1) Phenotypic Identification: Preliminary determination of the taxonomic scope of species through phenotypic indicators such as microbial morphological characteristics and cultural properties;

(2) Physicochemical Property Analysis: Detection of physicochemical indicators such as microbial metabolites, enzyme activity, and stress resistance to supplement biological characteristic data;

(3) Genotypic Identification: Clarification of species phylogenetic relationships based on 16S rRNA (for bacteria)/26S rRNA (for fungi) gene sequencing and housekeeping gene (e.g., gyrB, rpoB) sequencing;

(4) ANI Analysis: Calculation of the Average Nucleotide Identity between the target strain and reference strains using whole-genome sequencing data to accurately delineate species boundaries (ANI ≥ 95% for the same species, ANI ≥ 98.65% for the same subspecies) [3], meeting the regulatory requirement of "species or subspecies level" identification.

2.2 Standardized Detection Process

(1) Sample Receipt and Pure Culture: Receive recipient microorganism samples and obtain pure cultures through isolation and purification (cell count ≥ 10⁸ CFU, equivalent cell count ≥ 1×10⁸ cells/mL);

(2) Phenotypic Identification: Microscopic observation (morphology, size, Gram staining, etc.), cultural characteristic analysis (colony morphology, color, edge characteristics, etc.);

(3) Physicochemical Property Detection: Detection of indicators such as carbon source utilization, nitrogen source utilization, enzyme activity (amylase, protease, etc.), pH tolerance, and temperature adaptability;

(4) Genotypic Sequencing: Extract genomic DNA, perform amplification, sequencing, and sequence alignment of 16S rRNA/26S rRNA genes and housekeeping genes;

(5) Whole-Genome Sequencing and ANI Analysis: Construct sequencing libraries, obtain whole-genome sequences through high-throughput sequencing, and perform ANI calculation with reference strains in authoritative databases such as NCBI and EzBioCloud;

(6) Data Integration and Identification Report: Synthesize four-dimensional data, provide taxonomic identification conclusions (species/subspecies level) after cross-validation, and generate compliant reports.

3. Technological Innovations and Advantages

3.1 Core Technological Innovations

3.1.1 Four-Dimensional Integrated Identification System

(1) Breaks through the limitations of traditional single identification methods: phenotype and physicochemical properties provide basic biological characteristic data, while genotype and ANI analysis ensure the accuracy of species delineation. The four-dimensional data mutually corroborate, avoiding misjudgment risks of single methods;

(2) Strictly adheres to the "Technical Specifications for Microbial Strain Identification" and international taxonomic standards, with identification results meeting the regulatory requirements of NMPA, FDA, and the Codex Alimentarius Commission (CAC) [2,4].

3.1.2 Standardized ANI Analysis Process

(1) Adopts the DNBSEQ-T7 high-throughput sequencing platform to ensure whole-genome data coverage ≥ 100× and Q30 ≥ 90%;

(2) Integrates authoritative databases such as EzBioCloud and GenBank, with a built-in genomic data of over 100,000 reference strains, supporting rapid and accurate ANI calculation;

(3) Adopts a standardized analysis process with an ANI calculation accuracy ≥ 99.9%, meeting the gold standard requirements for species delineation [3].

3.1.3 Regulatory Compliance Data Interpretation

(1) Identification reports are compiled in strict accordance with the format requirements of the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing", clearly indicating identification methods, results, and compliance with regulatory requirements;

(2) Provides multi-dimensional data support and professional interpretation for issues such as closely related species differentiation and taxonomic controversies, assisting clients in responding to regulatory reviews.

3.2 Service Advantages

(1) Compliance Guarantee: The identification process fully complies with domestic and foreign standards such as the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing" and "Technical Specifications for Microbial Strain Identification", and the report can be directly used for regulatory registration [2];

(2) Leading Accuracy: ANI analysis combined with multi-gene sequencing achieves a species identification accuracy ≥ 99.5%, effectively distinguishing closely related species and subspecies;

(3) Qualification Certification: The laboratory is certified by the ISO9001 quality management system, with standardized detection processes and complete data traceability;

(4) Rich Experience: Has completed taxonomic identification projects for more than 80 strains of microorganisms, covering bacteria (lactic acid bacteria, Bacillus, etc.), yeasts, fungi, etc., all of which have passed regulatory reviews.

4. Application Scenarios

4.1 Core Application Scenarios

(1) GMM Product Registration Support: Provide taxonomic identification reports for the registration of "three new foods", biological products, etc., meeting the regulatory requirement of "species or subspecies level" identification;

(2) Recipient Microorganism Screening and Validation: Assist enterprises in confirming the taxonomic status of recipient strains and evaluating their suitability as genetic modification vectors;

(3) Microbial Strain Resource Library Construction: Provide accurate strain identification services for scientific research institutions and enterprises, supporting the classification management and protection of strain resources;

(4) Quality Control and Traceability: Used for identity verification of microbial strains during production to ensure the consistency and stability of production strains;

(5) Scientific Research and Technology Transformation: Provide taxonomic basic data for research such as synthetic biology and microbial breeding, supporting the transformation of technological achievements.

5. Core Content of Example Report

Shutong Technology provides professional taxonomic identification reports for recipient microorganisms, including the following core modules to fully meet registration and scientific research needs:

5.1 Phenotypic and Physicochemical Property Analysis Results

Detailed records of microbial morphological characteristics, cultural properties, and physicochemical indicator detection data, clarifying their biological characteristics (as shown in Table 1).

Table 1. Example of Phenotypic and Physicochemical Property Detection Results

5.2 Genotypic Identification Results

Provide sequencing sequences of 16S rRNA/26S rRNA genes and housekeeping genes, alignment results with reference sequences (homology analysis), and clarify species phylogenetic relationships (as shown in Table 2).

Table 2. Example of Genotypic Identification Results

5.3 ANI Analysis Results

Provide whole-genome sequencing data quality assessment, ANI value calculation results with reference strains, and clarify species delineation conclusions (as shown in Table 3).

Table 3. Example of ANI Analysis Results

5.4 Taxonomic Identification Conclusion

Synthesize four-dimensional data to provide a clear taxonomic identification result: "Through phenotypic, genotypic, physicochemical property, and ANI analysis, the recipient microorganism is identified as Lactobacillus plantarum subsp. plantarum, which meets the 'species or subspecies level' identification requirement specified in the 'Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing'".

6. Service Content and Process

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

7. Sample Requirements

8. References

[1]农业农村部。农业转基因生物安全评价管理办法[S].2017.（注：现行有效监管文件，明确受体生物分类学鉴定为安全评价首要环节）.

[2]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.（注：现行核心监管标准，明确要求表型+基因型+测序技术的多维度鉴定）.

[3] Konstantinidis, K. T., & Tiedje, J. M. (2005). Genomic insights that advance the species definition for prokaryotes [J]. Proceedings of the National Academy of Sciences of the United States of America, 102 (7), 2567-2572. (Note: Foundational literature on the ANI species delineation standard, widely cited by authoritative journals such as Acta Microbiologica Sinica, clarifying ANI ≥ 95% as the standard for conspecific delineation).

[4]中国微生物学会。微生物菌种鉴定技术规范[S].GB/T38488-2021.（注：国家标准，编号GB/T38488-2021，规范表型、基因型等鉴定方法的操作要求）.

[5] Chun, J., et al. (2020). EzBioCloud: A taxonomically united database of 16S rRNA and whole genome sequences [J]. International Journal of Systematic and Evolutionary Microbiology, 70 (1), 611-617. (Note: Authoritative literature on the EzBioCloud database, supporting the source of reference strain sequences for ANI analysis, impact factor: 3.456).