Improving Rhamnolipids Biosynthesis in Pseudomonas sp. L01 through Atmospheric and Room-Temperature Plasma (ARTP) Mutagenesis

Biosurfactants have significant applications in various industries, including microbial-enhanced oil recovery (MEOR). While the state-of-the-art genetic approaches can generate high-yield strains for biosurfactant production in fermenters, there remains a critical challenge in enhancing biosurfactant-producing strains for use in natural environments with minimal ecological risks. The objectives of this work are enhancing the strain’s capacity for rhamnolipids production and exploring the genetic mechanisms for its improvement. In this study, we employed atmospheric and room-temperature plasma (ARTP) mutagenesis to enhance the biosynthesis of rhamnolipids in Pseudomonas sp. L01, a biosurfactant-producing strain isolated from petroleum-contaminated soil. Following ARTP treatment, we identified 13 high-yield mutants, with the highest yield of 3.45 ± 0.09 g/L, representing a 2.7-fold increase compared to the parent strain. To determine the genetic mechanisms behind the enhanced rhamnolipids biosynthesis, we sequenced the genomes of the strain L01 and five high-yield mutants. A comparative genomic analysis suggested that mutations in genes related to the synthesis of lipopolysaccharides (LPS) and the transport of rhamnolipids may contribute to the improved biosynthesis. To the best of our knowledge, this is the first instance of utilizing the ARTP approach to improve rhamnolipid production in Pseudomonas strains. Our study provides valuable insights into the enhancement of biosurfactant-producing strains and the regulatory mechanisms of rhamnolipids biosynthesis.


Introduction
Biosurfactants are amphiphilic biomolecules produced during microbial growth and metabolism [1], and they play a crucial role in various industries, including petroleum recovery, soil remediation, pharmaceuticals, and food and beverage processing [2][3][4][5][6]. Unlike synthetic surfactants, biosurfactants are eco-friendly and sustainable, with unique properties such as high surface activity, low toxicity, degradability, and enhanced stability under extreme conditions [5,7]. Consequently, their use in the surfactant market has been to induce dose-dependent DNA damage [26]. This technique has been successfully applied to more than 40 microorganisms and plants, as well as complex microbial communities, to enhance their growth, productivity, and tolerance to environmental stresses [25]. Recent studies have also shown the potential of ARTP mutagenesis in improving biosurfactant production. For example, Bacillus amyloliquefaciens A3 treated with ARTP generated high lipopeptide-producing mutants with a yield of 1.6 g/L [27]. Similarly, ARTP-generated mutants of Starmerella bombicola increased total sophorolipids production by over 30% compared to the parent strain [28]. However, the application of ARTP on Pseudomonas species, one of the most attractive rhamnolipids producers, for improving rhamnolipids production remains limited.
Pseudomonas sp. L01 is a strain capable of producing rhamnolipids, recently isolated from oil-contaminated soil [10]. The objectives of this work are enhancing the strain's capacity for rhamnolipids production and exploring the genetic mechanisms for its improvement. Here, we utilized ARTP mutagenesis and identified high-yield mutants. Furthermore, we conducted a comparative genomic analysis of the high-yield mutants and the wild-type strain to investigate the underlying mechanisms behind the improved rhamnolipids production.

Mutagenesis by ARTP
To perform ARTP mutagenesis, the L01 strain was first cultured overnight in LB liquid medium by shaking at 220 rpm at 30 • C. The culture was then transferred to 30 mL of liquid LB medium with an initial OD 600 of 0.1 and incubated at 30 • C while shaking at 220 rpm. Cells were collected during the mid-exponential phase, washed three times with sterile physiological saline to remove the culture medium, and suspended in sterile physiological saline to a final concentration of 10 7 CFU/mL.
The ARTP mutagenesis was carried out using the ARTP breeding system (ARPT-IIS, Wuxi Tmaxtree Biotechnology Co., Ltd., Wuxi, China). The cell suspension was mixed with 10% (v/v) glycerol at a ratio of 1:1, and cells were treated with ARTP using the following operating parameters: radio frequency input of 120 W and helium flow rate of 10 L/min. To determine the optimal treatment time, cells treated with different times were eluted with sterile physiological saline to a new tube, and the total number of culturable cells after ARTP treatment was calculated using the traditional plate-counting method. The untreated cell suspension was used as the control, and the lethality rate, which is a widely used metric for assessing the impact of ARTP treatment, was calculated based on Equation (1).
where L is the lethality rate (%), Nc is the total colony count of the control sample without ARTP treatment, and Nt is that of the test sample after ARTP treatment. The optimal mutagenesis condition was determined according to the lethal curve with a lethality rate near 90%.

Preliminary Screening of Biosurfactants High-Yield Mutants
Following ARTP treatment, colonies were isolated from the agar plate and transferred into a 96-well plate containing fresh LB medium. The plate was incubated at 30 • C for 24 h, with shaking at 900 rpm. Subsequently, 1 µL of the culture was dropped onto a blood agar plate and cultured at 30 • C for 72 h. The concentration of the biosurfactants was determined by measuring the diameter of the clear zones around the colonies. The diameters of both the clear zone and the colony were measured using Image J. The ratio of the clear zone diameter to the colony diameter was calculated to determine the hemolysis ability of the biosurfactants.

Crude Biosurfactant Extraction
After cultivation, the fermented broths were centrifuged at 5000× g rpm for 10 min. The resulting supernatant was acidified to pH 2 with 6 M of HCl and left at 4 • C overnight. The crude biosurfactant extract was obtained by further centrifuging the mixture at 8000× g rpm for 15 min at 4 • C to remove the supernatant. The extract was then dried at 90 • C to a constant weight for further analysis.

Evaluation of Genetic Stability of the Mutants
To evaluate the genetic stability of the mutants, subculturing was performed. The mutant strain was cultured in LB medium at 30 • C with shaking at 220 rpm for 72 h. Next, the culture was transferred to fresh LB medium at a 1:100 (v/v) ratio and grown under the same conditions for five generations. At the end of each generation, crude biosurfactants were extracted and measured to assess genetic stability.

Surface Tension Measurement
After cultivation, the cells were removed from the fermented broth by centrifugation at 8000× g rpm for 10 min, and the resulting supernatants from each strain were collected. Surface tension was measured using a video optical contact angle measuring device (Theta Flex, Biolin, Espoo, Finland) based on surface contact angle. Each sample was measured in triplicate, and LB medium with 1/10 peptone was used as the blank control.

Genome Sequencing and Comparative Genomic Analysis of the Strain L01 and the Mutants
The DNA of the strain L01 and the mutants was extracted using the AxyPrep TM Bacterial Genome DNA Miniprep kit, according to the manufacturer's instructions (Axygen Scientific, Union City, CA, USA). The extracted DNA was checked for quality through gel electrophoresis and quantified. Next, the qualified DNA was fragmented into 350 bp fragments at random using the Covaris M220 focused-ultrasonicator (Covaris, Woburn, MA, USA). The sequencing library was prepared using the DNA library preparation kit (Illumina, San Diego, CA, USA). Subsequently, DNA sequencing was carried out using the Illumina Novaseq 6000 platform with 150 bp paired-end reads at Magigene Co. Ltd. (Guangzhou, China). All genomic sequences are deposited in China National Microbiology Data Center (NMDC) with accession numbers NMDC60046434, NMDC60046440, NMDC60046441, NMDC60046442, NMDC60046443, and NMDC60046444.
The strain L01 genome was assembled using SPAdes 3.12 [29] with default parameters. Open reading frames (ORFs) were predicted with Glimmer 3 [30] and annotated against the NCBI Non-Redundant Protein Sequence Database (NR) and the Cluster of Orthologous Groups Database (COG). To identify mutation sites in the mutants, clean reads were mapped to the L01 genome using BWA [31] and SAMtools [32]. Single nucleotide polymorphism (SNP) and insertion and deletion (InDel) were detected using GATK4 [33].

Statistical Analysis
To ensure the robustness and reproducibility of our findings, all experiments conducted in this work were performed at least in triplicate. All data in the experiment were expressed as mean ± standard deviation (SD). The statistical analysis of the data was performed using a two-tailed unpaired Student's t-test, and a p-value of 0.05 or less was considered statistically significant.

Lethality Rate of Pseudomonas sp. L01 with ARTP Treatment
In ARTP mutagenesis, the frequency of positive mutations is closely linked to the lethality rate of the strain, which depends on the dose of treatment. Achieving a lethality rate of approximately 90% is generally considered optimal for generating efficient mutations [34]. To determine the optimal exposure time for ARTP treatment, we assessed the lethality rate of the strain for various durations of exposure. We found that the lethality rate decreased with increasing exposure time ( Figure 1). Specifically, at an exposure time of 30 s, the lethality rate was 94.5%, while an exposure time of 90 s resulted in almost complete lethality (i.e., a lethality rate of nearly 100%). Based on these findings, we selected an exposure time of 30 s as the most appropriate duration for ARTP mutagenesis in our study. It is worth noting that the optimal exposure time varied for different strains, even within the same species, owing to their intrinsic properties. For instance, while a 20 s exposure time was found to be optimal for generating mutations in P. putida KT2440, a 10 s exposure time was identified as optimal for P. putida CGMCC3830 under the same operating conditions [35,36]. ducted in this work were performed at least in triplicate. All data in the experiment expressed as mean ± standard deviation (SD). The statistical analysis of the data wa formed using a two-tailed unpaired Student's t-test, and a p-value of 0.05 or less wa sidered statistically significant.

Lethality Rate of Pseudomonas sp. L01 with ARTP Treatment
In ARTP mutagenesis, the frequency of positive mutations is closely linked lethality rate of the strain, which depends on the dose of treatment. Achieving a let rate of approximately 90% is generally considered optimal for generating efficient tions [34]. To determine the optimal exposure time for ARTP treatment, we assesse lethality rate of the strain for various durations of exposure. We found that the let rate decreased with increasing exposure time ( Figure 1). Specifically, at an exposur of 30 s, the lethality rate was 94.5%, while an exposure time of 90 s resulted in a complete lethality (i.e., a lethality rate of nearly 100%). Based on these findings, w lected an exposure time of 30 s as the most appropriate duration for ARTP mutag in our study. It is worth noting that the optimal exposure time varied for different st even within the same species, owing to their intrinsic properties. For instance, whil s exposure time was found to be optimal for generating mutations in P. putida KT2 10 s exposure time was identified as optimal for P. putida CGMCC3830 under the operating conditions [35,36].

Screening and Verification of the Mutants with High-Yield Rhamnolipids Production
After conducting ARTP mutagenesis, we isolated the mutants and screened the marily using blood agar plates. Out of 854 colonies, we obtained 76 mutants with s cantly higher hemolytic activities. We then investigated the rhamnolipids producti pacity of each mutant by measuring the yield of crude biosurfactants. After culturin wild-type strain with 2% glucose as the sole carbon source for 60 h, the wild-type produced a crude biosurfactant yield of 1.15 ± 0.05 g/L ( Figure 2). Thirteen of the m strains had significantly higher yields than the wild-type strain. The mutant strain had the highest crude biosurfactants yield at 2.33 ± 0.53 g/L.

Screening and Verification of the Mutants with High-Yield Rhamnolipids Production
After conducting ARTP mutagenesis, we isolated the mutants and screened them primarily using blood agar plates. Out of 854 colonies, we obtained 76 mutants with significantly higher hemolytic activities. We then investigated the rhamnolipids production capacity of each mutant by measuring the yield of crude biosurfactants. After culturing the wild-type strain with 2% glucose as the sole carbon source for 60 h, the wild-type strain produced a crude biosurfactant yield of 1.15 ± 0.05 g/L ( Figure 2). Thirteen of the mutant strains had significantly higher yields than the wild-type strain. The mutant strain 8-135 had the highest crude biosurfactants yield at 2.33 ± 0.53 g/L.

The Genetic Stability of the Mutants
The stability of genetic mutations generated by mutagenesis can vary, which is paramount for their industrial application. The primary alterations in genes involved in DNA replication, DNA repair, signal transduction pathways, and alternative gene expression may lead toward destabilized genomes [37]. To address this, we tested the genetic stability of five high-yield mutants (8-41, 8-135, 8-303, 8-384, and 9-72) after five passages. All mutants displayed significantly higher yields than the wild-type strain ( Figure 3). Among the tested strains, , and 8-384 demonstrated high genetic stability, with no significant differences between crude biosurfactant yields in the first and fifth generations. The result indicate that these three mutants were genetically stable. Notably, the mutant strain 8-41 showed a continual increase in crude biosurfactant yield with each passage, reaching its highest yield in the fifth generation. In contrast, the mutant strain 9-72 showed a significant decrease in yield along with the passages. This demonstrates that the genetic stability of mutants can have a profound impact on their performance over time.

The Genetic Stability of the Mutants
The stability of genetic mutations generated by mutagenesis can vary, which is paramount for their industrial application. The primary alterations in genes involved in DNA replication, DNA repair, signal transduction pathways, and alternative gene expression may lead toward destabilized genomes [37]. To address this, we tested the genetic stability of five high-yield mutants (8-41, 8-135, 8-303, 8-384, and 9-72) after five passages. All mutants displayed significantly higher yields than the wild-type strain (Figure 3). Among the tested strains, , and 8-384 demonstrated high genetic stability, with no significant differences between crude biosurfactant yields in the first and fifth generations. The result indicate that these three mutants were genetically stable. Notably, the mutant strain 8-41 showed a continual increase in crude biosurfactant yield with each passage, reaching its highest yield in the fifth generation. In contrast, the mutant strain 9-72 showed a significant decrease in yield along with the passages. This demonstrates that the genetic stability of mutants can have a profound impact on their performance over time.

Comparison of the Growth and Biosurfactant Production between the Wild-type Strain and 8-135 Strain
To investigate differences in growth and biosurfactant production patterns between wild-type and mutant strains, we further characterized mutant strain 8-135 due to its high

Comparison of the Growth and Biosurfactant Production between the Wild-Type Strain and 8-135 Strain
To investigate differences in growth and biosurfactant production patterns between wild-type and mutant strains, we further characterized mutant strain 8-135 due to its high biosurfactant yield and genetic stability. The growth curves of the wild-type strain L01 and mutant strain 8-135 indicated that both strains had a similar growth rate, reaching the stationary phase at 36 h after incubation, and maintaining high biomass until 174 h ( Figure 4A). The crude biosurfactant production curve showed that the biosurfactant yield of both the wild-type strain and the mutant strain 8-135 was similar in the first 30 h after incubation. However, from 36 h after incubation, the biosurfactant yield rate of the wild-type strain decreased, while that of the mutant strain 8-135 remained high. Both the wild-type and the mutant strains reached their highest biosurfactant yield at 102 h after incubation, which accounted for 1.28 ± 0.01 g/L and 3.45 ± 0.09 g/L, respectively ( Figure 4A). These results suggest that ARTP mutagenesis caused genetic mutations in the biosynthesis of rhamnolipids, but not in strain growth and division. Both genetic engineering and random mutagenesis have been used to improve the yield of biosurfactants. For example, increasing the copy number of rhlAB genes in recombinant P. aeruginosa DAB resulted in a yield of 17.3 g/L of rhamnolipids, using crude oil as a carbon source [38]. Another mutant strain, P. aeruginosa SG∆rhlC, deficient in rhlC, produced 14.22 g/L of rhamnolipids using glycerol and nitrate [39]. Inserting RhlAB in a specific position in P. aeruginosa PEER02 facilitated the production of rhamnolipids with yields of 0.7-0.8 g/L with glucose as a substrate, and 1.7-1.9 g/L with soybean oil as a substrate, compared to the wild-type strain P. aeruginosa PAO1 [40]. In another study, gamma ray irradiation was used to generate a mutant strain of P. aeruginosa MR01-C, which demonstrated a more than one-and-a-half-fold increase in biosurfactant production compared to the parent strain (2.1 g/L) [23]. Similarly, the rhamnolipids yield of the gamma ray-induced mutant strain P. aeruginosa EBN-8 reached 8.5 g/L after optimizing the culture medium [22]. However, it is important to note that the fermentation conditions varied among the different studies, making it difficult to directly compare the efficiency of different approaches. Further optimization of conditions is necessary to maximize the yield of biosurfactants. note that the fermentation conditions varied among the different studies, making it difficult to directly compare the efficiency of different approaches. Further optimization of conditions is necessary to maximize the yield of biosurfactants. We also tested the critical micelle concentration (CMC) of the crude biosurfactants produced by both strains and found no significant difference, suggesting the chemical structure of the biosurfactants produced by the mutant strain was not altered ( Figure 4B). ARTP mutagenesis only influenced the metabolic flux of rhamnolipids synthesis, but not the synthetic metabolic pathway.

Comparative Genomic Analysis of the Wild-type Strain and the Mutants
To better understand the mechanisms underlying enhanced rhamnolipids production, we performed genome sequencing on five high-yield mutants (8-135, 8-303, 8-384, 8-41, and 9-72) and the wild-type strain L01.We found that the wild-type strain L01 contained the necessary genes for complete rhamnolipids synthesis, including algC, rmlABCD, and rhlABC, consistent with its rhamnolipids biosynthesis capacity. By comparing the genomes of the L01 and the mutant strains, we detected 151, 139, 42, 149, and 157 single nucleotide polymorphisms (SNPs) in strains 8-135, 8-303, 8-384, 8-41, and 9-72, We also tested the critical micelle concentration (CMC) of the crude biosurfactants produced by both strains and found no significant difference, suggesting the chemical structure of the biosurfactants produced by the mutant strain was not altered ( Figure 4B). ARTP mutagenesis only influenced the metabolic flux of rhamnolipids synthesis, but not the synthetic metabolic pathway.

Comparative Genomic Analysis of the Wild-Type Strain and the Mutants
To better understand the mechanisms underlying enhanced rhamnolipids production, we performed genome sequencing on five high-yield mutants (8-135, 8-303, 8-384, 8-41, and 9-72) and the wild-type strain L01.We found that the wild-type strain L01 contained the necessary genes for complete rhamnolipids synthesis, including algC, rmlABCD, and rhlABC, consistent with its rhamnolipids biosynthesis capacity. By comparing the genomes of the L01 and the mutant strains, we detected 151, 139, 42, 149, and 157 single nucleotide polymorphisms (SNPs) in strains 8-135, 8-303, 8-384, 8-41, and 9-72, respectively ( Table 1). The transition-to-transversion ratios for these mutant strains were between 1.16 and 1.33, except for strain 8-384, which had a ratio of 3.2, indicating a different genomic mechanism for high-yield rhamnolipids production. Furthermore, we found 35, 29, 6, 29, and 31 SNPs with non-synonymous mutations (nsSNPs) in the coding sequence regions of 8-135, 8-303, 8-384, 8-41, and 9-72, respectively, located in 33 different genes. Of these genes, four could be assigned to carbohydrate transport and metabolism according to COG annotation. Carbohydrates serve as the primary substrate for rhamnolipid biosynthesis. So, the transportation and metabolism of carbohydrates are essential processes that contribute to the availability of substrates necessary for rhamnolipid biosynthesis. Additionally, three genes were related to intracellular trafficking, secretion, and vesicular transport; three genes to signal transduction mechanisms; and two genes to secondary metabolites biosynthesis, transport, and catabolism ( Figure 5A). These findings suggest that ARTP mutagenesis can induce the improvement of rhamnolipids production through multiple functional pathways.   We conducted a comparative analysis of genes with non-synonymous single-nucleotide polymorphisms (nsSNPs) in the mutant strains 8-135, 8-303, 8-384, 8-41, and 9-72. Our findings reveal that only three genes with nsSNP mutations were shared by all five strains, two of which encode hypothetical proteins, while the other encodes ice nucleation protein ( Figure 5B). Ice-nucleating proteins (INPs) play a crucial role in directing ice nucleation to the extracellular space and are believed to counteract low-temperature damage [41]. Remarkably, certain glycolipid biosurfactants have been found to have a correlation with  We conducted a comparative analysis of genes with non-synonymous single-nucleotide polymorphisms (nsSNPs) in the mutant strains 8-135, 8-303, 8-384, 8-41, and 9-72. Our findings reveal that only three genes with nsSNP mutations were shared by all five strains, two of which encode hypothetical proteins, while the other encodes ice nucleation protein ( Figure 5B). Ice-nucleating proteins (INPs) play a crucial role in directing ice nucleation to the extracellular space and are believed to counteract low-temperature damage [41]. Remarkably, certain glycolipid biosurfactants have been found to have a correlation with cold adaptation. Firstly, it has been observed that cold-adapted microorganisms often produce biosurfactants [42,43]. Additionally, glycolipid biosurfactants can act as ice recrystallisation inhibitors. They can prevent water from crystallizing into ice and form a cage surrounding the proteins that slows the water dynamics in its proximity [43]. These findings suggest that these biosurfactants and INPs are functionally complementary, with the activity of INPs potentially influencing the biosurfactant synthesis process. Although the relationship between INPs and the biosynthesis of rhamnolipids has not been reported, further research is necessary to determine if INPs are involved in rhamnolipids biosynthesis.
Furthermore, we compared genes with nsSNP mutations in strains 8-135, 8-303, 8-41, and 9-72, excluding 8-384 due to its low number of nsSNP mutations and different transitionto-transversion ratios compared to the other strains. Our results indicate that these four strains share 18 genes with nsSNP mutations, of which 13 are annotated as hypothetical proteins with unknown functions. The remaining five genes have varied annotated functions, including Lipid-A-disaccharide synthase (LpxB) and signal recognition particle receptor FtsY. Specifically, lpxB is involved in the biosynthesis of the lipid A component of lipopolysaccharide (LPS) [44], which has a metabolic link to rhamnolipids [14]. 3-hydroxyalkanoate and dTDP-L-rhamnose are the common precursors of both LPS and rhamnolipids [45,46]. Disrupting lpxB could enhance the metabolic flux of the rhamnolipids biosynthesis pathway by accumulating 3-hydroxyalkanoate and dTDP-L-rhamnose. Additionally, a shared gene encoding an XRE family transcriptional regulator is located downstream of a gene-encoding major facilitator superfamily (MFS) transporter. Members of the MFS transporter family have been implicated in rhamnolipids transport [47,48], and disrupting this XRE family regulatory gene might increase the secretion of rhamnolipids. We also identified a range of 25~93 mutations in the intergenic regions (ncSNPs), which may influence the transcription of flanking genes. Detailed molecular mechanisms for improving rhamnolipids biosynthesis require further investigation.
Mutant strains generated through ARTP mutagenesis exhibit genetic stability and a reduced risk of introducing heterogenous genes, making them highly versatile and suitable for a wide range of applications. These strains can be utilized in various areas such as MEOR and in situ bioremediation in open and natural environments. However, optimizing conditions for in situ application may be more complex compared to those in fermenters, and further research is necessary to understand these conditions better.

Conclusions
In this study, we employed ARTP mutagenesis to enhance the biosynthesis of rhamnolipids in the biosurfactant-producing strain Pseudomonas sp. L01. To our knowledge, this is the first time that the ARTP approach has been used to enhance rhamnolipid production in Pseudomonas strains. We identified a total of 13 high-yield mutants, with the maximum yield being 3.45 ± 0.09 g/L, representing a 2.7-fold increase compared to the parent strain. Through a comparative genomic analysis, we observed mutations in genes linked to LPS synthesis and rhamnolipids transport, indicating that they may have contributed to the enhancement of rhamnolipids biosynthesis. Further exploration of the molecular mechanisms involved in rhamnolipids biosynthesis would allow for a more informed approach towards enhancing its biosynthesis. Our findings offer an alternative method for the mutagenesis of biosurfactant strains, as well as insights into the regulation of rhamnolipids biosynthesis.