*2.5. The E*ff*ect of Mutant Proteins on the Virulence Factors and Survival of Pectobacterium Carotovorum* Subsp. *Carotovorum (Pcc)*

The inhibitory effects of mutant proteins on pectate lyase, the virulence factor of the plant pathogenic bacterium *Pcc*, was analyzed. Wild-type MomL, MomLI144V and MomLV149A inhibited the expression of the pectate lyase gene, and the inhibitory effect of MomLV149A was slightly higher than the wild-type MomL. We also analyzed the gene expression of pectate lyase when treated by MomLE238G, MomLK205E and MomLL254R. The mutation of these three amino acids resulted in the inability to inhibit pectate lyase gene expression (Figure 6A). In addition, the yield of pectate lyase

was determined and the results were consistent with the transcriptional analysis. MomLI144V and MomLV149A greatly reduced pectate lyase yield, while MomLE238G, MomLK205E and MomLL254R did not (Figure 6B). Besides, the presence of MomLI144V and MomLV149A reduced the *Pcc* survival rate under stress conditions to 30%–45% of the survival of *Pcc* alone. The presence of MomLE238G did not affect *Pcc* survival. Furthermore, the boiled MomL did not affect the *Pcc* survival rate (Figure 6C). We speculated that site-directed mutagenesis of *momL* led to changes in other function of the mutant proteins, such as the fold of the enzyme, stability, substrate interaction and many other performance parameters, and thus resulted in reduced survival of *Pcc*. However, the specific mechanism needs to be studied further.

**Figure 6.** Transcriptional level of pectate lyase encoding gene in *Pectobacterium carotovorum* subsp. *carotovorum* (*Pcc*) (**A**) and the production of pectate lyase (OD492). (**B**). Effects of MomL and mutant proteins towards the *Pcc* survival rate (**C**). All data are presented as mean ± standard deviation (SD, *n* = 3). An unpaired t-test was performed for testing significant differences between groups (\*\*\* *P* < 0.001, \*\* *P* < 0.01, \* *P* < 0.05).

#### *2.6. E*ff*ects of MomL and Mutant Proteins on Pcc Infection of Chinese Cabbage*

To further analyze MomL and its mutants, their treatment effect towards soft rot of Chinese cabbage was tested. When treated by *Pcc* alone, approximately 2/3 of the cabbage leaf area was infected and decomposed. Following infection with *Pcc* and treatment with MomL, only a small percentage of tissue was infected. However, inactivated MomL applied in combination with *Pcc* did not reduce the degree of decay in Chinese cabbage. The decay areas of the cabbage after treatment with MomLE238G, MomLK205E and *Pcc* were comparable to those obtained with *Pcc* infection alone. The application of MomLL254R and *Pcc* together reduced the decay area by approximately 50% compared with that treated by *Pcc* alone. The treatment effects of MomLI144V and MomLV149A were the most significant. After the application of MomLI144V and MomLV149A, the *Pcc* infection rate on Chinese cabbage decreased obviously (Figure 7). Overall, in infection experiments, the bacterial survival rate significantly decreased by more than 50% after adding MomL or active mutant proteins. The results

indicated that co-culture with MomL or mutant proteins can relieve the symptoms caused by *Pcc*, and this may be due to the decrease of virulence factors such as pectate lyase.

**Figure 7.** Effects of MomL and mutant proteins on *Pcc* infection of Chinese cabbage. (**A**) *Pcc*; (**B**) *Pcc* with MomLE238G; (**C**) *Pcc* with MomLK205E; (**D**) *Pcc* with MomLL254R; (**E**) *Pcc* with MomL; (**F**) *Pcc* with MomLI144V; (**G**) *Pcc* with MomLV149A; (**H**) *Pcc* with boiled MomL. The results shown are representative of biological duplicates.

#### **3. Discussion**

Marine metagenomic data revealed that QQ is a common activity in marine bacteria [34]. Many QQ enzymes have been identified from marine species, such as Aii20J from *Tenacibaculum* sp. strain 20J, QsdH from *Pseudoalteromonas byunsanensis* strain 1A01261 and AiiC from *Anabaena* sp. PCC 7120 [35,36]. QQ enzymes have broad application prospects in aquaculture disease control, biofouling prevention and drugs development [37,38]. Improving the degrading ability of QQ enzymes will lead to highly stable and efficient proteins for industrial use. Thus, further studies about marine aquatic QQ can expand marine QQ bioresource application and pave a way to solve problems related to aquaculture and agriculture that is conducted in a saline environment [39]. The marine-derived QQ enzyme MomL, a novel type of AHL lactonase with an unknown action mechanism, was investigated in this study. MomL demonstrates a wide antimicrobial spectrum and provides a promising alternative for disease control due to its ability to inhibit the pathogenicity induced by the AHL QS system. The amino acids and active site in MomL have not previously been explored, except for the "HXHXDH~H~D" motif. Hence, we focused on MomL to improve its bacteriostatic activity, explore its highly active mutant proteins, and identify amino acids involved in enzyme activity via site-directed mutagenesis, thus providing a theoretical basis for its mechanism of action.

Among protein engineering strategies, random mutagenesis methods are usually applied to study properties that are not understood rationally. EpPCR is standard method for random mutagenesis due to its robustness and simplicity in use [40]. A seamless cloning technique is used to insert a targeted fragment into any location in the vector without relying on an enzymatic site. The main factor affecting epPCR was the concentration of Mn2+, which can result in higher mutation frequencies at

higher concentrations. Other influential factors including the concentration of Mg2<sup>+</sup>, the proportion of deoxyribonucleoside triphosphates (dNTPs), and even the PCR reaction cycles. In this study, mutation frequencies were controlled at 1–3%. Thus, each protein contained 3-5 mutations. After multiple analyses, we ultimately determined the concentrations of dNTP, Mg2<sup>+</sup> and Mn2<sup>+</sup> for the use in next step. We screened amplification enzymes using epPCR to identify high-fidelity enzymes with improved cloning efficiency, but unsatisfactory mutation rates resulted in the low diversity of the random mutant library. Ultimately, Taq enzyme was chosen for epPCR. At the beginning of each reaction, the Taq enzyme produced higher mutant library diversity with 2–5 mutations per protein but achieved low seamless cloning efficiency, thus limiting the number of transformants. Presumably, the A-end of the Taq enzyme affected the efficiency of seamless cloning, which was optimized in our study. We removed the A-end using the HS DNA polymerase (Takara Primer STAR®). The entire experimental time was shortened to one-fifth of the time required for traditional experiment, and the efficiency of mutant library establishment was nearly 10 times higher than that achieved previously. Furthermore, the efficiency of positive cloning during mutant library construction was as high as 92%. Thus, our strategy demonstrated wide applications for establishing protein mutant libraries, and greatly improved the efficiency of seamless cloning.

The first two approaches involve large high-throughput selection, and only 10%–20% of bacteria on a parent plate can be transferred to a sub-plate in the traditional method. But IPTG in situ photocopying is a high-throughput screening system. By performing single colony dilution and counting the number of single colonies, we increased the transferred number of bacteria to 50%. This type of screening method holds great applicable value for other QQ enzymes' screening. By screening mutant proteins, we rapidly obtained two highly active mutants of MomL and identified seven amino acids which are involved in enzyme activity. However, given the lack of MomL crystals structure, the deep catalytic mechanism remains to be characterized. In infection experiments, the bacterial survival rate significantly decreased by more than 50% after adding highly active mutant proteins to *Pcc*. MomL and its mutant proteins also reduced the virulence factor pectate lyase produced by *Pcc*. We applied these proteins to infect Chinese cabbage and found that the infection symptoms were alleviated after adding MomL or its mutants, indicating MomL and its mutants can be an alternative strategy for disease control. We are currently characterizing the minimum concentration and maximum time required for MomL treatment to facilitate the application of MomL alike to actual utilization.

#### **4. Materials and Methods**

#### *4.1. Bacterial Strains, Plasmids, Media, Growth Conditions, and Chemicals*

*Pectobacterium carotovorum* subsp. *carotovorum* (*Pcc*) was purchased from the CGMCC (China General Microbiological Culture Collection, Beijing, China) [41]. *E. coli* strain AHL882-5 was used to express the MomL protein. *E. coli* strain BL21(DE3) was used as a host for protein expression. Proteins were expressed following the cloning of random mutants of the *momL* gene into pET-24a(+). The strain *Chromobacterium violaceum* CV026 was used as an indicator in the AHL activity bioassay [42]. C6-HSL was purchased from the Cayman Chemical Company and prepared in dimethyl sulfoxide (DMSO). *M. olearia* Th120, CV026 and *Pcc* were routinely cultured on Luria-Bertani (LB) agar at 28 ◦C. *E. coli* strain AHL882-5 was cultured in LB medium at 37 ◦C. When required, 25 μg/mL kanamycin was added to the solid or liquid media.

#### *4.2. Random Mutant Library Construction and Identification of High-Activity Mutants*

The mutant library of the AHL lactonase MomL was constructed using error prone PCR (epPCR). The primers for epPCR are listed in Table S1. Each 100-μL epPCR reaction contained 10 μL of 10× PCR buffer (Takara, Shiga, Japan), 8 μL of dNTP mixture (2.5 mM dATP, 2.5 mM dGTP, 10 mM dCTP, and 10 mM dTTP), 1 μL of the primer *momL*-F (20 μM), 1 μL of the primer *momL*-R (20 μM), 1 μL of template plasmid from strain AHL882-5, 1 μL of Taq DNA Polymerase (Takara, 5 U/μL), appropriate

metal ions, and deionized water to a final volume of 100 μL. PCR was conducted using the following conditions: denaturation at 94 ◦C for 10 min, followed by 30 cycles of denaturation at 94 ◦C for 30 s, annealing at 55 ◦C for 30 s, extension at 72 ◦C for 60 s, and a final incubation at 72 ◦C for 10 min. The resulting PCR products were digested with Prime STAR® HS DNA Polymerase (Takara) to improve the ligation efficiency. They were then further digested with DpnI (NEB, Ipswich, MA, USA) to remove template plasmids and were finally purified using a PCR product purification kit (Biomed, Beijing, China) according to the manufacturer's instructions. Purified mutant *momL* genes were ligated into the linear vector pET-24a(+) via seamless cloning. Recombinant plasmids were transformed into *E. coli* BL21(DE3), diluted with fresh LB medium, plated on LB agar containing 25 μg/mL kanamycin, and cultured at 37 ◦C overnight.

### *4.3. High-Throughput Screening of High-Activity Mutants*

We added 1 mL of overnight cultured CV026, 7.5 μl C6-HSL (DMSO, 1 mM) and 0.5 mM IPTG (final concentration) to 15 mL of molten semisolid LB agar (1%, *w*/*v*) before the agar was poured into the plates. When the agar solidified, colonies growing on LB agar containing 25 μg/mL kanamycin were imprinted on the selection plate using sterile toothpicks. After the prescreening step, choosing mutants that produced a white halo for the next round screening. In second-round screening, the mutants were induced to expression in 0.5 mM IPTG condition, the supernatant of the cultures were collected after centrifugation at 12,000 rpm for 10 min at 4 ◦C and filtered through 0.22-μm-pore-size filter to test the AHL lactonase activity. The CV026 screening plate was prepared as described above without adding with 0.5 mM IPTG. The medium was punched using a sterile tip and the crude enzyme supernatant were added into the hole (with MomL crude enzyme supernatant as positive control and LB medium as negative control).
