Background
Solutions
Future expectation
References
Shrimp

Design

Crab

Background

The spoilage of aquatic products poses an urgent challenge in the global food supply chain. This deterioration not only results in significant resource wastage but also leads to the histamine poisoning and the proliferation of pathogenic bacteria in the spoiled products. What's worse, consumption of such contaminated aquatic products by accident can cause acute food poisoning in consumers. Currently, chemical preservatives and cold chain systems are primarily used to maintain product freshness. However, chemical additives pose carcinogenic and other health risks, while cold chain systems generate high carbon emissions and plastic wastes, which not only incur substantial costs but also exert significant environmental pressure (Figure 1). Consequently, eco-friendly and safe biological preservatives have garnered increasing attention in recent years.

fig1
Figure 1 Current methods for preserving aquatic products.

Chitosan, a natural biological preservative, offers notable advantages in aquatic products preservation through its antibacterial, moisturizing, and antioxidant effects, providing an efficient and safe solution. However, excessive intake of chitosan may cause digestive discomfort, thus shifting research focus to its degradation products, chitooligosaccharides (COS) (1, 2). COS can also serve as natural preservatives for aquatic products, inhibiting microbial growth on food surfaces and delaying spoilage (3). Compared to chitosan, COS possesses smaller molecular structures with greater solubility and bioactivity, enabling them to prolong the shelf-life of aquatic products via multiple mechanisms. Although the conversion of chitin from shrimp and crab shells into chitosan is relatively well-established, challenges persist in degrading chitosan into COS (4, 5), which hampers the wide applications of COS as natural preservatives for aquatic products.

Therefore, we have established the CRUSTA (Circular Reutilization of Untapped Seafood-shell: Transformation into Antimicrobial-preservatives) project to address food safety concerns related to aquatic products spoilage and tackle environmental pollution caused by crustacean waste. In this project, we utilized error-prone PCR (EP-PCR) technology and designed a rapid screening system to identify chitosanase mutants capable of efficiently degrading chitosan into COS with enhanced antimicrobial properties. The resulting COS was applied for further aquatic products preservation (see Proof of Concepts for details), thereby promoting a circular economy of “sourced from aquatic products, used for aquatic products”. You can find more detailed information in our Project Description.

Solutions

1. Enzymatic degradation

In the current degradation process from chitosan to COS, the main preparation methods include chemical approaches (e.g., hydrogen peroxide oxidation, acid hydrolysis), physical methods (e.g., microwave treatment, ultraviolet irradiation, ultrasonic processing), enzymatic hydrolysis, electrochemical methods, and composite degradation techniques derived from these methods (6). These methods suffer from challenges such as difficult product separation and purification, low yields, high costs, rigorous equipment requirements, and even heavy pollution (7), posing substantial difficulties for practical COS production. Thus, in our CRUSTA project, to rapidly and efficiently obtain COS products while considering environmental sustainability, we decided to use secretion-based methods to obtain enzymes from E. coli and employ these enzymes to replace traditional methods for degrading chitosan. We selected various chitosanases as targets, constructed mutant libraries, and screened for chitosanases capable of efficiently degrading chitosan into COS with enhanced antimicrobial activity.

1.1 The Choice of Chitosanases

fig2
Figure 2 Schematic diagram of enzymatic degradation of chitosan via endo- and exo- chitosanase.
1.1.1 Exo-chitosanases

Exo-α-D-glucosidase (EC 3.2.1.165) catalyzes the hydrolysis of α(1-4) bonds in chitosan or COS, removing a glucosamine (GlcN) residue from the non-reducing end to produce monomeric GlcN, thereby degrading chitosan (8) (Figure 2).
GlmA, a kind of exo-chitosanase, belongs to the glycoside hydrolase 35 (GH35) family, and our focus is on the GlmATk enzyme from Thermococcus kodakaraensis. This homodimeric enzyme has been shown to exhibits hydrolytic activity toward reaction products derived from (GlcN)3-6 with varying chain lengths. It specifically cleaves the first β-1,4-glycosidic bond at the non-reducing end of oligosaccharides, yielding GlcN as the final product (9).

1.1.2 Endo-chitosanases

In addition to exo-chitosanases, we also investigated various endo-glycosidases capable of degrading chitosan.

Endo-chitosanase is a glycoside hydrolase that specifically degrades chitosan. Based on amino acid sequence similarity, they are classified into seven GHs; according to the bonds of the reaction site, they can be further subdivided into four subclasses (10). All endo-chitosanases share a common hydrolysis mechanism (Figure 2), and the configuration of the end monomer unit is either retained or inverted. The reaction proceeds via proton-catalyzed mechanisms, which the glycosidic oxygen is protonated by an amino acid residue that acts as a proton donor within the catalytic core, while aglycone displacement is provided by the nucleophilic amino acid residue. In most glycoside hydrolases, glutamic acid (residue E) and aspartic acid (residue D) serve as proton donors and/or nucleophiles. In the retaining mechanism, the resulting glycosyl enzyme is hydrolyzed by a water molecule, and this second nucleophilic substitution at the anomeric carbon generates a product with the same stereochemistry as the substrate. In the inverting mechanism, the protonation of the glycosidic oxygen and aglycone displacement are accompanied by a concomitant attack by a water molecule, which is activated by the nucleophilic amino acid residue (11). The hydrolysis process of chitosanases is divided into two stages, with the overall rate starting fast and then decreasing. When long-chain substrates enter the active site, they undergo rapid hydrolysis, whereas the resulting short-chain products are degraded very slowly (10).

We selected five previously reported chitosanase enzymes (BamCsn, CsnMY002, McChoA, SaCsn46A, and CsnCA) as targets for testing their performance on degrading chitosan.

BamCsn is an endo-chitosanase belonging to the GH46 family discovered in Bacillus amyloliquefaciens. It degrades chitosan into higher-molecular-weight COS. Simultaneously, it cleaves highly polymerized COS, exhibiting cleavage activity at chito-tetraose and chito-pentaose residues but lacking the ability to cleave chito-triose. It may represent a novel class of chitosanase that features glycosyltransferase activity, COS cleavage, and the presence of W204 residue, whose primary function involves converting tetramers into dimers (12).

CsnMY002 from Bacillus subtilis MY002 is another endo-type chitosanase. Its hydrolysis products from reacting with chitosan are primarily (GlcN)2 and (GlcN)3. The crystal structure analysis reveals that this enzyme possesses a tunnel-like substrate-binding center, differing from the common open-type binding pocket structure. It was reported that wild-type CsnMY002 hydrolyzes 100 mg of chitosan to yield 20.75 mg of (GlcN)2 and 25.80 mg of (GlcN)3 (13).

McChoA is a GH80 family chitosanase from Mitsuaria chitosanitabida 3001 capable of complete hydrolysis of chitosan. Structural comparisons indicate that McChoA belongs to Class I enzymes and exhibits structural similarity to GH46 family chitosanases, suggesting that it employs the conserved flipping mechanism for catalysis (14).

SaCsn46A is a GH46 family chitosanase discovered in Streptomyces avermitilis. This enzyme exhibits an endolytic cleavage pattern, initially producing COS with higher degree of polymerization, which are then progressively degraded into GlcN and (GlcN)2 (15, 16).

CsnCA is an endo-chitosanase from Chromobacterium sp. ATCC 53434, which primarily hydrolyzes (GlcN)6 into a mixture of (GlcN)2, (GlcN)3, and (GlcN)4, with the final products being (GlcN)2 and GlcN. This enzyme exhibits diverse endolytic patterns but symmetric digestion is more likely to occur: (GlcN)6 predominantly yields (GlcN)3, while (GlcN)4 primarily produces (GlcN)2. Meanwhile, (GlcN)5 is primarily cleaved into (GlcN)2 and (GlcN)3. The final products (GlcN)2 and GlcN are yielded when (GlcN)3 serves as the substrate (17).

Through research and experiments on these five endo-chitosanases (see Results for details), we ultimately decided to focus our investigation mainly on SaCsn46A and its mutants. We constructed a mutant library to conduct subsequent high-throughput screening for chitosanases with degradation products of superior antibacterial efficacy.

1.2 Chitosanase Screening System

To obtain degradation products with enhanced bacteriostatic effects,we decided to employ error-prone PCR (EP-PCR) to randomly mutate the chitosanases. Subsequently, we rapidly screened the resulting large number of mutants using a method based on inhibiting the growth of E. coli to identify candidates capable of producing highly bacteriostatic products.

Since the amino group in chitosan is positively charged, it can interact with the negatively charged phosphoglycolic acid to change the rigidity of the cell wall; it can also interact with negatively charged cell membranes and then damage the cell membrane. Furthermore, chitosan forms a polymer layer on the cell surface to block the exchange of nutrients, which leads to bacterial cell death (18-20). Ardean et al. found that another bactericidal mechanism of COS may originate from their interaction with DNA, which inhibits mRNA translation and protein synthesis (21). In addition, it has been hypothesized that COS interferes with the TCA cycle in E. coli, thereby affecting the metabolism of E. coli and resulting in cell death (22) (Figure 3).

fig3
Figure 3 COS's antibacterial principles diagram.

Thus, by monitoring bacterial growth, we can screen for chitosanases capable of degrading chitosan into products with enhanced bacteriostatic effects for subsequent production of preservatives (Figure 4).

fig4
Figure 4 Screening process based on monitoring bacterial growth.

In addition, we conducted growth inhibition assays again by using purified enzymes to recheck the activity of the promising mutants.

1.3 Acquisition of chitosanase via secretion

In industrial production, the acquisition of enzymes is a complex problem. Conventional approaches that rely on cell disruption to extract intracellular proteins face several drawbacks such as discontinuous production, high cost, cumbersome operation and complicated protein purification. In contrast, signal peptide can make its connected protein secreted to out of the cell, without the need to obtain the target enzyme by means of cell lysis, which can largely simplify the process of obtaining the target protein (Figure 5). Therefore, inspired by our previous successes with LMT signal peptide (XMU-China 2021, XMU-China 2022, XMU-China 2023, XMU-China 2024), we tried to optimize this signal peptide in the hope of finding variants with higher secretion efficiency for the applications of our chitosanases (see Proof of Concept for details).

fig5
Figure 5 Schematic diagram of signal peptides at the cellular level.

In cells, there are multiple pathways for protein secretion, among which the Sec pathway is the most dominant secretion pathway, and the secretion directed by signal peptide LMT that we have used in previous years also belongs to this pathway. The signal peptide of Sec pathway is mainly composed of three parts: the positively charged region of N segment for membrane orientation, the hydrophobic core region of H segment for translocon engagement, and the polar region of C segment containing the signal peptidase (SPase) cleavage site for protein maturation (Figure 6 A). In the Sec pathway, SecB prevents premature folding, SecA directs the nascent protein into the SecYEG translocon using ATP, and SPase cleaves the signal peptide to release the mature protein (Figure 6 B) (23-25).

fig6
Figure 6 Sec pathway. (A) Typical sequence structure of a signal peptide. (B) Schematic diagram of signal peptides at the molecular level.

In Stefano's work (26), they tested tens of thousands of signal peptides using high-throughput analyses and performed correlation analyses of their structures. Based on their correlation analyses and combined with the signal peptide structure of LMT, we dedicated to the improvement of secretion performance of the existing signal peptide LMT, achieving higher concentrations of secreted recombinant proteins (see Engineering Success for details).

1.4 Characterization of degradation products

For the degradation products of chitosanases, we applied two common methods to characterize them: thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).

TLC enables qualitative and semi-quantitative analysis of COS products based on partition coefficient differences between stationary and mobile phases. By comparing Rf values and color intensities of standards and samples, the composition and relative content of low-degree polymerization COS in mutant products can be visualized (27).

To obtain accurate quantitative results, HPLC was used for sugar detection. Comparing the peak areas of degradation products and standards at different time points allows precise characterization and quantification of COS fractions, enabling accurate evaluation of degradation efficiency and product specificity of mutants (27).

2. Application of COS to food preservation

Since COS exerts strong bactericidal effect by disrupting cell membranes through electrostatic interaction (28), we hope that the COS products can be applied to the preservation of aquatic products, which contributes to the realization of circular economy of " sourced from aquatic products, used for aquatic products".

COS can disrupt the structure of bacterial cell membranes, triggering leakage of cell contents and ultimately leading to bacterial death (29). COS inhibits protein oxidation and lipid degradation, thereby delaying the loss of fish fillet firmness and reducing the whiteness decline, which helps maintain the texture and color of aquatic products. These properties make COS a highly promising biological preservative (30). As a kind of cryoprotectant, COS can reduce the ice crystal formation, stabilize the secondary structure of myofibrillar proteins, reduce thawing losses and enhance the water-holding capacity, thus improving overall water retention (31).

After approved by the Safety and Security Committee (Safety Form), we selected grass carp (freshwater fish) to evaluate the effects of COS preservative on microbial proliferation of fish during storage. The preservation performance was assessed by determining the total colony count and other key indicators (Figure 7).

fig7
Figure 7 Freshness testing process and changes in colony forming unit (CFU).

In addition, given that the broad-spectrum antibacterial properties of COS have been fully demonstrated in fish preservation experiments, we further tested it on various fruits and vegetables (e.g., blueberries, peaches, strawberries, and cherry tomatoes) to evaluate its potential in post-harvest preservation of fruits and vegetables (see Proof of Concept for details). And it should be noted that the fruits and fish used in our preservation experiment have passed the safety review.

3. Biosafety

To prevent engineered bacteria from contaminating other production stages during chitosanase production and causing biosafety issues (32), we designed two independent sets of kill switches to ensure biosafety: one is an IPTG-induced overexpression of tyrS gene, the other is ccdB/ccdA-based toxin-antitoxin system induced by cuminic acid (Figure 8).

fig8
Figure 8 Schematic diagram of kill switches.

3.1 tyrS System

Overexpression of some essential genes showed severe growth defects to E. coli (33). tyrS gene is an essential gene encoding tyrosyl-tRNA synthetase (TyrS), and by placing the tyrS gene under the regulation of the T5 phage promoter embedded with the lac operator (lacO), overexpression is suppressed in the absence of IPTG (OFF state) and the bacteria remain viable and grow. While in the presence of IPTG (ON state), the tyrS essential gene is overexpressed and kills the host bacteria (34). IPTG is a commonly used inducer in the laboratory, which is easy to obtain and stable.

3.2 ccdB/ccdA System

The ccdB gene encodes a toxin that interferes with DNA topoisomerase activity (35) and is placed under the control of the pCymRC promoter to achieve the killing effect induced by cuminic acid (36). Cuminic acid is a naturally organic acid that is specific, relatively inexpensive, can be extracted from natural sources or chemically synthesized, and is generally regarded as safe and low in toxicity. In addition, to reduce the growth burden caused by potential leaky expression of the toxin, the antitoxin gene ccdA was introduced, which was designed to be controlled by a weak constitutively engineered promoter. Therefore, once cuminic acid is added and the expression level of ccdB exceeds that of ccdA, the engineered bacteria will be killed due to the toxicity of CcdB.

In our designed plasmid (Figure 9), two mutually independent biocontainment systems guarantee that in the event of a mutational loss of function on one side of ccdB/tyrS, there is still another killing mechanism to prevent the engineered bacteria from escaping. In addition, most of the natural amino acid misincorporation phenomena caused by misacylated aminoacyl-tRNAs are tolerable (37).

fig9
Figure 9 Design of gene circuit of kill switches.

Future expectation

Considering the excellent antibacterial ability of COS, we envision that COS can be applied not only in the preservation of aquatic products, but also across a broader range of applications within the food industry. In the preservation of fruits and vegetables, COS can be coated or soaked to form a layer of selectively permeable membrane that regulate respiration intensity and reduces water loss. By disrupting microbial cell membranes and causing the leakage of intracellular substances, COS exerts a strong inhibitory effect on a wide range of scavenging bacteria at the same time. In meat products, COS effectively inhibits fat oxidation and protein degradation, slows down the generation of off-flavors and helps maintain product freshness. In the future, with the increasing demand for green consumption and innovations in food processing technology, COS applications may expand to prepare vegetables and functional food packaging. Furthermore, advanced approaches such as compound emulsification and nano-encapsulation can enhance its stability and targeting (38), paving the way for a safer and more efficient food preservation system that promotes a transition toward natural and sustainable food production.

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