Design

To overcome the inherent metabolic and regulatory constraints of native B. velezensis and develop high-titer, genetically stable CLP producers, we implemented an integrated mutagenesis and high-throughput screening pipeline. Atmospheric and Room-Temperature Plasma (ARTP) was selected as the core mutagenesis method due to its high mutation rate, operational safety, and ability to generate large, diverse mutant libraries in a single treatment.

The ARTP mechanism involves reactive plasma species that transiently increase membrane permeability and cause DNA damage. This activates cellular error‑prone repair systems (e.g., SOS response), resulting in diverse and stable genomic mutations ^[1]. Coupled with our custom high-throughput screening platform, this DBTL‑compatible workflow allowed rapid identification of improved strains, establishing a reusable pipeline for continuous microbial improvement.

Build

To enhance antimicrobial lipopeptide production in Bacillus velezensis, we established an integrated mutagenesis and screening workflow comprising four key stages: ARTP mutagenesis ^[2]→ high-throughput screening ^[3]→ CLPs extraction→ quantitative validation.

Following ARTP treatment of wild-type cells, we then implemented a two-tiered screening strategy, utilizing the high-throughput Oxford cup method for initial selection followed by the minimum inhibition concentration (MIC) assay to identify mutants with significantly improved CLP yields.

Validated high-yield mutants were processed through an acid precipitation-based extraction protocol to obtain crude lipopeptide powders. These samples were dissolved in methanol, sterile-filtered, and subjected to quantification using HPLC.

Test

We successfully isolated a series of mutant strains exhibiting significantly enhanced antibacterial activity compared to the wild-type HMBY. Among these, mutant strain HMBY-106 demonstrated the most potent antibacterial performance (Figure 4).

This finding underscores its potential for agricultural biocontrol, enabling reduced chemical pesticide use and enhanced food security, in line with Sustainable Development Goal 2 (Zero Hunger).

HPLC detection of the fermentation product revealed that HMBY-106 had around 2 times higher CLPs production compared to HMBY (Figure 5).

Design

To identify the genetic regulators responsible for enhanced CLP production in mutant strain HMBY-106, we designed a systematic gene discovery and validation strategy. This approach begins with whole-genome sequencing to identify acquired mutations in HMBY-106 compared to the wild-type HMBY.

We then implemented a targeted genome editing strategy using CRISPR-Cas9 to reconstruct each HMBY-106 mutation individually in the wild-type strain. The resulting isogenic mutant strains underwent phenotypic characterization, including CLP yield quantification and physiological profiling.

By correlating specific genetic mutations with changes in CLP production and cellular physiology, we established a data-driven framework to: (1) dentify key regulatory genes influencing CLP biosynthesis; (2) determine their individual contributions to yield enhancement and (3) construct an integrated regulatory model for CLP production.

This systematic design enables direct linkage between genotypic changes and phenotypic outcomes, forming a complete DBTL cycle for genetic engineering optimization.

Build

To investigate the specific genes that are responsible for the higher-yield CLPs of HMBY-106 than the wild-type strain, we sequenced and analyzed the whole genomes of HMBY and HMBY-106 to identify the mutant genes. Then, we employed a CRISPR-Cas9 system to construct mutants of each gene in the wild-type HMBY. These engineered strains were then subjected to a comprehensive phenotypic analysis, including growth profiling, CLP yield quantification, and expression analysis of key Non-ribosomal Peptide Synthetase (NRPS) genes. The resulting data allowed us to elucidate the specific regulatory function of mutant gene and integrate these findings into a proposed mechanistic model for CLP biosynthesis.

Test

Comparative genomic analysis of HMBY-106 and HMBY revealed three genes that acquire missense mutations in their protein-coding regions cdaA, relA and cheB. The individual mutant strain of these three gene was successfully reconstruct in HMBY.

These engineered strains were then subjected to a comprehensive phenotypic analysis, including growth profiling, CLP yield quantification, and expression analysis of key Non-ribosomal Peptide Synthetase (NRPS) genes. The comparison of growth curves for the wild type (HMBY), HMBY-106, and mutants in the key genes (HMBY-rel, HMBY-cdaA, HMBY-cheB) showed that the mutant strain HMBY-rel and HMBY-cdaA exihibited a lower growth rate than HMBY. NRPS gene expression was up-regulated in HMBY-rel and HMBY-cdaA strains. Consistently, the amount of CLPs produced by the mutants of HMBY-rel and HMBY-cdaA were higher than that of wide-type strain HMBY. Among the individual lipopeptide components, fengycin displayed the most pronounced increase.

Learn

This round identified two key regulatory genes that significantly enhance CLPs production, with both mutants consistently showing upregulated NRPS gene expression. The gene rel synthesis (p)ppGpp that regulates amino acid biosynthesis, and protects cells during nutrient limitation that the mutation in rel may optimize precursor supply for CLPs assembly by reprogramming amino acid metabolism.

Integrating these findings with our phenotypic data, we propose a regulatory model wherein these genetic mutations coordinately activate NRPS expression and optimize metabolic precursor availability (Figure 8.). This model (Figure 9.) provides a mechanistic foundation for the observed yield improvement and establishes new targets for future engineering cycles.

Design

Guided by industrial partner feedback indicating that fengycin demonstrates superior antifungal efficacy without affecting crop development, we engineered B. velezensis to redirect metabolic flux toward fengycin biosynthesis. Since native CLP pathways compete for common precursors, we designed a CRISPR-Cas9 mediated strategy to delete the surfactin (srfA-D) and iturin (ituA-D) biosynthetic clusters.

This targeted pathway refactoring achieved:

(1) Elimination of substrate competition between parallel CLP pathways

(2) Enhanced precursor and energy channeling toward fengycin synthesis

(3) Significant increase in fengycin yield and purity

This engineering advance supports multiple Sustainable Development Goals:

(1) SDG 12.2 & 12.5: Optimized resource efficiency through precise metabolic engineering, minimizing raw material waste

(2) SDG 12.4: Replacement of chemical pesticides with targeted biopesticides

Our engineered strain represents a sustainable platform for specialized biopesticide production, demonstrating how synthetic biology can address both agricultural and environmental challenges.

Build

During our engineering process, we identified a critical technical bottleneck: conventional single-guide RNA CRISPR-Cas9 systems showed limited efficiency when targeting long, repetitive genomic regions such as NRPS gene clusters, primarily due to low homologous recombination efficiency.

To overcome this challenge, we designed and constructed a novel dual-gRNA ^[4] CRISPR vector system (Figure 11.). This innovative tool is designed to express two guide RNAs simultaneously, targeting two specific sites, significantly improving the editing efficiency for long fragment.

Design

Efficient nonribosomal peptide biosynthesis requires precise coordination between multiple NRPS modules, where COM domain serves as the essential structural interface mediating module-module interaction and assembly line connectivity ^[5].

Guided by this understanding, we designed a rational strategy to reprogram the fengycin NRPS machinery in our high-yield chassis strain. By systematically redesigning COM domain pairs, we aim to alter peptide chain length and composition while maintaining high production levels. This approach enables simultaneous optimization of both titer and bioactivity, establishing a versatile platform for developing novel lipopeptide variants with enhanced antimicrobial properties.

This NRPS engineering framework demonstrates how synthetic biology can expand natural product diversity while maintaining industrial relevance, advancing both fundamental understanding and applied bioproduction capabilities.

Build

Our software was designed to offer a comprehensive computational solution for reprogramming NRPS communication (COM) domain. It was builded by integrating multiple bioinformatic tools—including MAFFT for sequence alignment, IQ-TREE for phylogenetic analysis, and InterProScan for domain annotation—into a unified and automated workflow. Key functionalities include fen sequence integration, functional domain mapping, and automated extraction of COM junction regions. Through multi-dimensional data analysis, the platform successfully identified four critical residue sites, providing precise targets for experimental validation. The predicted fen COM reprogramming will further be validated by our wet-lab results.