Proof of Concept | XMU-China

Initiated by the discovery that chitooligosaccharides (COS) with higher degrees of polymerization possess enhanced antibacterial properties, we designed experiments to identify chitosanases capable of producing degradation products with superior antibacterial efficacy. We further developed protocols to semi-rationally design signal peptides with improved secretion efficiency, intending to link these peptides with the most effective chitosanase mutants and bypass the protein purification step. Thus, chitosanases would traverse the cell membrane, be secreted into the supernatant and catalyze chitosan degradation. By building genetic circuits and conducting continuous tests and validations, we successfully confirmed the viability of our concept. We achieved chitosan degradation via a secretion system, enhancing the antibacterial performance of the degradation products by 7.96-fold compared to the wild-type. Looking ahead, we aim to leverage AI to conduct the learning step in the final stage of the DBTL cycle to obtain new LMT and chitosanase with enhanced efficacy.

Figure 1 Schematic diagram of exocrine, degradation, and product preservation effect testing.

1. Antibacterial

1.1 Introduction

Our understanding of human practices (see IHP for more details) reveals significant issues in the aquatic products preservation industry, such as high energy consumption and severe pollution. Biological preservatives, with their potential to ensure effective preservation while avoiding the environmental pollution caused by chemical reagents, emerge as a promising solution. However, the limited application of this method due to the low catalytic efficiency of enzymes and the high cost of enzymes restricts presents a challenge.

Inspired by our experimental results (see Results 3.2.1 for more details) showing that COS with higher degrees of polymerization exhibit stronger antibacterial properties, we aim to utilize the variation in antibacterial properties of COS with differing degrees of polymerization, using changes in CFU (colony-forming units) or OD (optical density), to screen for chitosanases from the enzyme mutation library capable of producing degradation products with superior antibacterial efficacy.

1.2 SaCsn46A mutant 2 exhibits outstanding antibacterial activity

First, we chose six kinds of Chitosanase BamCsn, CsnMY002, GlmA_Tk, CsnCA, McChoA and SaCsn46A from the reference. After the initial screening, we found that the degradation products of CsnMY002, CsnCA, McChoA, and SaCsn46A exhibited better antibacterial effects among the six enzymes. Following multiple rounds of screening and analysis, we found that the degradation products of six SaCsn46A mutants exhibited stronger antibacterial effects, subjecting them to more meticulous and rigorous characterization experiments for further selection (see Results for more details). We purified proteins from the lysate supernatant of these six SaCsn46A mutants and mixed the protein solution with chitosan powder for degradation experiments. And then we centrifuged the reaction product and incubated it with bacterial solution to measure the antibacterial ability of the degradation product.

Figure 2 Antibacterial effect of degradation products of SaCsn46A WT and its mutants under fixed protein concentration conditions.

The antibacterial activity of the enzyme degradation product was evaluated using the relative ΔOD₆₀₀ value (Figure 2). The degradation product of SaCsn46A mutant 2 (E151G D222G) (BBa_25C3NO7W) demonstrated superior antibacterial activity.

1.3 Conclusion

We have successfully established a screening method for chitosanases based on the antibacterial activity, leveraging the chitosanases SaCsn46A and its mutants reported in the literature. We identified a chitosanase with the most optimal antibacterial effect among the enzymatic products , marking a significant milestone in the production of the preservative chitooligosaccharides (see Design for more details). This, combined with a rapid screening method, structure-activity analysis, and random mutation, has led us to the SaCsn46A mutant 2, whose enzymatic hydrolysates exhibit optimal antibacterial efficacy.

2. Enzyme Secretion System

2.1 Introduction

In practical applications, the high-performance chitosanases we have identified are directly applied to the fermenter to degrade chitosan. To bypass the protein purification step and avoid the use of inducers, we utilize the signal peptide to directly secrete the enzyme outside the cell (see Design for more details). In this section, we aim to develop the signal peptide with enhanced efficiency and stability through semi-rational design and evaluate the effectiveness of releasing target proteins through a preservation experiment.

2.2 The LMT C14A has the best secretory efficiency

We constructed LMT-sfGFP fusion under the control of a constitutive promoter to enable continuous, inducer-free production and secretion. Subsequently, we adopted a semi-rational design strategy, experimented with different approaches (see Engineering Success for more details), and ultimately obtained the highly efficient mutant LMT C14A(BBa_25LUSMUT). As shown in Figure 3, the secretion efficiency of LMT C14A was significantly improved compared to the original LMT.

Figure 3 Fluorescence intensity (at 10 h) of culture and supernatant of C14A and LMT WT were compared. p-value: 0.0008 (***) and 0.0098 (**).

2.3 Secretion of SaCsn46A mutant 2 exhibits stronger antibacterial activity

We have demonstrated that the degradation products of SaCsn46A mutant 2 and 60 exhibit enhanced antibacterial effects, and LMT C14A shows promising secretion efficiency. After approved by the Safety and Security Committee (Safety Form), we aimed to verify the antibacterial efficacy of the degradation products of SaCsn46A mutants secreted into the supernatant by LMT C14A. First, we confirmed via SDS-PAGE that the strain could secrete the fusion protein into the supernatant (Figure 4). Subsequently, the collected supernatant was mixed with sterile chitosan powder and subjected to a degradation experiment. After centrifugation of the reaction solution, the supernatant was subjected to a preservation experiment (see Experiments for more details). Based on the bacterial CFU/g values (Figure 5), the degradation products of LMT C14A-SaCsn46A mutant 2 (BBa_25PUHM7F) in the supernatant exhibited the most effective preservation, showing a 7.96-fold increase compared with WT SaCsn46A.

Figure 4 SDS-PAGE analysis of LMT C14A-SaCsn46A WT (A), LMT C14A-SaCsn46A mutant 2 (B), and LMT C14A-SaCsn46A mutant 60 (C).

Figure 5 Fish preservation performance of degradation products from LMT C14A-SaCsn46A WT, LMT C14A-SaCsn46A mutant 2, and LMT C14A-SaCsn46A mutant 60. (A) Colony counts after direct treatment. (B) Comparison between the degradation products group and the LB control group on Day 2 post-direct-treatment.

We performed HPLC and TLC analyses on the degradation products of LMT C14A-SaCsn46A WT and its mutants to investigate the changes in their product profiles and analyze the reasons for the improvement in their product antibacterial efficiency.

Figure 6 HPLC analysis of the chitosan degradation products of LMT C14A-SaCsn46A WT (A), LMT C14A-SaCsn46A mutant 2 (B) and LMT C14A-SaCsn46A mutant 60 (C) with TLC analysis (D).

As shown in Figure 6, LMT C14A-SaCsn46A mutant 2 and 60 significantly increased the production of glucosamine (GlcN) and its dimer (GlcN)₂ through enhanced degradation compared to the wild-type, indicating superior chitosan degradation capability. This result is consistent with the observed stronger antibacterial effect of the degradation products of LMT C14A-SaCsn46A mutant 2 and 60 in our preservation experiments.

2.4 Conclusion

Through these rounds of screening, we identified that products from the LMT C14A-SaCsn46A mutant 2 exhibit optimal antibacterial performance. We successfully integrated the chitosanases SaCsn46A mutant 2 and 60 with a secretion system, establishing a highly efficient chitosan degradation system. Our work provides an environmentally friendly biological approach for producing COS preservatives, promotes the efficient production of natural biological preservatives, and offers valuable references for subsequent teams exploring COS production processes and chitosanase optimization strategies.

3. Life Cycle Assessment

3.1 Introduction

Life Cycle Assessment (LCA) is a comprehensive method for evaluating the environmental impact of a product from raw material extraction to disposal. We conducted a cradle-to-gate LCA to compare our enzymatically produced COS preservatives with both chemically produced COS and the traditional chemical preservative potassium sorbate. Our goal was to quantify the advantages of our method in carbon emission reduction, cost savings, and safety.

3.2 Enzymatic production of COS contributes to low carbon emissions

We calculated the carbon emissions during the production stage of our enzymatically produced COS and chemically synthesized potassium sorbate, with the functional unit defined as a preservative solution of equivalent antimicrobial efficacy. Our assessment considered carbon emissions from raw material acquisition, chemical production, and electricity consumption. The results show that, in the aspect of production of chitosan oligosaccharides, the enzymatic approach decreases about 99.1% of CO₂ emissions and 96.4% of cost compared to the chemical one. Additionally, our method saves about 61.0% of CO₂ emissions and 71.7% of cost compared to the chemical production of traditional preservative, potassium sorbate, in the same functional performance . (Figure 7 and 8,see LCA for more details). It is worth emphasizing that the enzymatic pathway demonstrates significant advantages in both carbon emission reduction and cost savings, holding promise as a more sustainable solution in the field of aquatic product preservation.

Figure 7 Comparison of the carbon emissions and production costs of enzymatic methods versus chemical methods.

Figure 8 Comparison of the carbon emissions and production costs of COS preservative versus potassium sorbate preservative.

3.3 Conclusion

Through this Life Cycle Assessment, we have successfully validated the core concept of our project: creating a circular economy for crustacean wastes. By developing an enzymatic process to convert shrimp and crab shells into an effective bio-preservative, our solution dramatically reduces carbon emissions and production costs while being significantly safer than both chemical oligosaccharide production and the traditional preservative potassium sorbate. Therefore, our project presents a sustainable, economically attractive, and safer alternative for the industry, demonstrating a strong proof of concept for its future application in promoting a circular blue economy.

3.4 Future

We have demonstrated that the chitosanase SaCsn46A mutant 2 possesses strong degradation capability, and its degradation products exhibit excellent antibacterial and preservative effects. By utilizing a constitutive promoter, we can achieve low-cost and high-efficiency requirements on an industrial scale. We have also characterized the combination of the highly efficient secretion peptide mutant LMT C14A and SaCsn46A mutant 2, proving that the fusion protein LMT C14A-linker-SaCsn46A mutant 2 enables the coupling of secretion and production of COS preservatives. In the future, we will apply the produced COS preservatives to a wider variety of seafood and agricultural products and actively establish collaborations with aquatic product processing, agricultural preservation, and bio-product enterprises to achieve the industrial implementation of the project and finally contribute tangible solutions to the green circular economy.