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1. Overall

Our project aims to address perioperative gut microbiota dysbiosis through the use of engineered probiotics. To achieve this, we have designed or optimized several foundational genetic components that collectively form a complete system capable of intelligently sensing the intestinal environment, precisely executing therapeutic functions, and ensuring biosafety.First, we utilized the bile salt-responsive promoter P16090 (BBa_25S2TG5Z), derived from Lactobacillus. This promoter specifically responds to bile salt signals in the gut, ensuring that downstream genes are only activated in the target environment. We identified this promoter as the core regulatory switch of the entire system, establishing the foundation for the targeted functional expression of engineered probiotics within the gut.

In terms of metabolic pathways, we selected the accA gene (BBa_25ZF6KGL), also sourced from Lactobacillus. This gene encodes acetyl-CoA carboxylase, the rate-limiting enzyme in fatty acid biosynthesis, which significantly enhances the engineered bacteria\'s ability to synthesize short-chain fatty acids (SCFAs). Additionally, we introduced the OLE1 gene (BBa_25CA4F5T) from Saccharomyces cerevisiae and optimized its codons for expression in Lactobacillus. This gene encodes Δ9-fatty acid desaturase, which converts short-chain fatty acids into oleic acid.

To further enhance the antimicrobial activity of the engineered bacteria, we integrated the coding sequence of DEFB4A (BBa_25VPY4CG), a human defensin. This antimicrobial peptide effectively inhibits the growth of conditional pathogens, and its unique membrane-perforating mechanism minimizes the risk of resistance development in pathogenic bacteria.

More importantly, we innovatively combined these functional components to construct a multifunctional fused expression unit (BBa_25NBMOAR). This unit utilizes a 2A self-cleaving peptide to link OLE1 and DEFB4A, enabling the co-expression of these two genes within a single transcript. This allows the engineered probiotics to perform dual roles of \"repair\" and \"defense\" simultaneously.

Furthermore, we uploaded the stringent plasmid backbone PIP501 (BBa_251CFLR3) used in our system. This backbone, which was previously unavailable in the iDEC Registry, is expected to serve as a valuable resource for future teams working on similar projects.
BBa_25CA4F5T Coding The original sequence is sourced from the NCBI database (NC_001139.9) and has been modified by our team.
BBa_25ZF6KGL Coding The original sequence is sourced from the NCBI database, ID: 45549165
BBa_25S2TG5Z Promoter The original sequence was sourced from the genome of Lactobacillus casei in the NCBI database. Using the primer sequences described in the literature (DOI: 10.1007/s00253-019-09743-w), we retrieved and modified this fragment.
BBa_25S2TG5Z Coding The original sequence was sourced from the NCBI database (NC_000008.11) and was optimized by our team.
BBa_25NBMOAR Measurement This sequence was designed and measured by our team.
BBa_251CFLR3 Plasmid The original sequence was sourced from the ATCC database.

2. Basic Part

BBa_25CA4F5T

This part encodes stearoyl-CoA 9-desaturase (OLE1), which is a key component of our project. In Saccharomyces cerevisiae, OLE1 catalyzes the desaturation of 18-carbon saturated stearic acid, derived from the fatty acid metabolic pathway, to produce oleic acid.

The original sequence of OLE1 was obtained from the NCBI database. We first assessed its suitability for prokaryotic expression. Subsequently, certain codons were optimized based on the codon usage preference of Lactococcus lactis and the requirements for plasmid construction. Additionally, a 6xHis tag was added to the C-terminus of the protein to facilitate its identification and validation during protein expression. (For more details, please refer to BBa_25CA4F5T).

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Figure.1 The nucleotide sequences of OLE1 before and after optimization

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Figure.2 Expression of OLE1 with a 6xHis tag in engineered bacteria

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Figure.3 Expression of acetic acid, propionic acid, butyric acid, and oleic acid in engineered bacteria

BBa_25ZF6KGL

This part encodes the acetyl-CoA carboxylase carboxyltransferase subunit alpha (accA). In our engineered bacteria, accA plays a crucial role in enhancing the fatty acid synthesis pathway, resulting in the production of higher quantities of short-chain fatty acids (e.g., acetate, propionate, butyrate). These short-chain fatty acids serve as essential precursors, ensuring a sufficient substrate supply for downstream oleic acid synthesis. (For more details, please refer to BBa_25ZF6KGL).

BBa_25S2TG5Z

This part encodes the promoter 16090 (P16090) along with its upstream operator sequence. P16090 is a bile salt-responsive promoter derived from Lactobacillus. In Lactococcus lactis, it can be directly utilized for constructing expression vectors. Under bile salt conditions, transcription factors bind to the regulatory sequences upstream of the promoter, triggering the transcription of downstream genes. (For more details, please refer to BBa_25S2TG5Z).

BBa_25VPY4CG

This part encodes defensin DEFB4A, derived from the human genome. Defensins are natural peptides present in the gut with functions such as antimicrobial activity, inflammation clearance, and immune balance regulation. In our project, DEFB4A was successfully expressed in engineered bacteria, and an inflammatory cell model was established to evaluate the anti-inflammatory functions of the secreted products from the engineered bacteria. (For more details, please refer to BBa_25VPY4CG).

BBa_25NBMOAR

This part encodes our designed functional sequence, P16090-RBS-OLE1-2AA-DEFB4A-Terminator, which has been rigorously characterized. Integration of this sequence into an appropriate expression vector enables the engineered bacteria to express OLE1 and DEFB4A under bile salt conditions while maintaining their expected functionality. (For more details, please refer to BBa_25NBMOAR).

BBa_251CFLR3

This part represents the plasmid backbone sequence of PIP501, a stringent vector originating from Enterococcus faecium. It is capable of driving gene expression in various microorganisms, including Lactobacillus. Our team utilized this vector to construct the biosafety system. (For more details, please refer to BBa_251CFLR3).