Red/ET recombineering enhances extracellular lipase production by Burkholderia glumae

Background : Microbial lipases are utilized widely in industrial fields, and the LipA lipase produced by Burkholderia glumae PG1 has been used for the production of enantiopure pharmaceuticals. However, efficient lipase expression and secretion are still problematic. LipA is encoded in an operon that also contains the lipase-specific foldase gene lipB . The purpose of this study was to enhance the production of active lipase by overexpressing lipAB in B. glumae PG1 using a host-vector system. Results : The lipAB operon, isolated from the genomic DNA of B. glumae PG1, was directly cloned into broad-host-range vectors, with pBBR1 or pRk2 backbones, using the Red/ET recombination system and then transformed into the original host strain. Lipase activity of the derivative strains, including PG1/Bl and PG1/Rl, were evaluated. When 1% olive oil was included in the growth medium, the maximum activities of the resultant strains were approximately 39.5- and 15.3-fold higher, respectively, than that of the wild-type PG1, indicating that increased lipase gene dosage resulted in high expression yields of lipase. To further enhance lipase expression, two recombinant strains, PG1/Ba and PG1/Ra, were generated with the plasmid-borne lipAB operon driven by the strong constitutive promoter P apra . PG1/Ba and PG1/Ra displayed 15.0- and 4.3-fold improvements in lipase production in 0.5% glucose-containing medium compared to PG1/Bl and PG1/Rl, respectively. However, glucose repressed lipase gene expression in strain PG1 when the native lipAB promoter was used. Conclusions : Homologous expression of lipase genes in B. glumae PG1 was achieved by the use of high-copy expression vectors, and the resulting presence of multiple copies of lipAB significantly improved extracellular lipase expression. Our results demonstrate that modification of B. glumae PG1 can improve this host system for lipase production.

included in the growth medium, the maximum activities of the resultant strains were approximately 39.5-and 15.3-fold higher, respectively, than that of the wild-type PG1, indicating that increased lipase gene dosage resulted in high expression yields of lipase.
To further enhance lipase expression, two recombinant strains, PG1/Ba and PG1/Ra, were generated with the plasmid-borne lipAB operon driven by the strong constitutive promoter P apra . PG1/Ba and PG1/Ra displayed 15.0-and 4.3-fold improvements in lipase production in 0.5% glucose-containing medium compared to PG1/Bl and PG1/Rl, respectively.
However, glucose repressed lipase gene expression in strain PG1 when the native lipAB promoter was used.
Conclusions: Homologous expression of lipase genes in B. glumae PG1 was achieved by the use of high-copy expression vectors, and the resulting presence of multiple copies of lipAB significantly improved extracellular lipase expression. Our results demonstrate that modification of B. glumae PG1 can improve this host system for lipase production. 4 Background Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) belonging to the family of α/β hydrolases are widely produced by organisms from animals to bacteria. Microbial lipases are important biocatalysts in the synthesis of detergents, food ingredients, agrochemicals and pharmaceuticals [1][2][3][4]. Many biotechnologically relevant lipases are relatively stable in organic solvents, do not require a cofactor, show broad substrate specificity and are readily available at low production costs [5]. Since lipases can catalyze a variety of synthetic and hydrolytic reactions under non-aqueous reaction conditions, they have received increasing attention over the past few years for their multiple potential applications in industry [6,7].
In recent years, the nucleotide and amino acid sequences of several microbial lipases have become available, and many bacterial lipases have been cloned and characterized, particularly those from the genera Pseudomonas and Burkholderia [8][9][10][11][12]. One of the most frequently used lipases, lipase A, is secreted by the Gram-negative bacterium Burkholderia glumae PG1 [5,13]. The regulation of lipase expression and secretion in B.
glumae is complex and requires effective cooperation of a variety of proteins. The Burkholderia lipase structural gene lipA is located in a single operon together with a secondary gene, lipB [13][14][15]. The LipA is synthesized as a precursor with an N-terminal signal peptide. This precursor is first transported across the inner membrane via the Sec secretion system and is then folded into an enzymatically active conformation by the lipase-specific foldase, encoded by lipB, in the periplasm [14,[16][17][18]. The mature lipase is then secreted into the extracellular medium via the type II secretion pathway [19].
Pseudomonas lipases have been studied extensively, but Pseudomonas is an opportunistic human pathogen and so is less suitable for bulk lipase production [2]. To enhance the production of extracellular lipase, many biotechnologically interesting lipases have been 5 cloned and expressed in Escherichia coli [20][21][22][23]. Unfortunately, the efficient production of active lipases in heterologous hosts and secretion into the culture medium under normal laboratory growth conditions can be difficult. Less than 3% of an expressed lipase may be active, with the remainder of the protein misfolding and accumulating as insoluble inclusion bodies in the E. coli cell [24]. In order to overcome the problem of insoluble protein arising with a heterologous host, we set out to use B. glumae as a homologous expression host. Compared with heterogeneous expression, homologous expression can take full advantage of the transcription and translation systems of the original host without any pretreatment and make the enzyme isolation and purification easier [25].
The lipase produced by B. glumae was found to perform well in several industrial applications, and several classical mutagenesis techniques have been applied to B. glumae PG1 to construct lipase-overproducing strains [2,5,9]. The aim of this study was to construct a host-vector system that would generate higher levels of active lipase production, in which PG1 was used as the host with multicopy expression vectors containing lipAB genes. The complete lipAB operon from PG1 was directly cloned into the expression vector p15A using homologous recombination (HR) in E. coli. We also utilized the full-length Rac prophage proteins RecE and RecT, which can mediate highly efficient HR between two linear DNA segments carrying homology arms at both ends [26][27][28]. HR mediated by Redαβ from lambda phage between a linear and circular DNA molecule in E.
coli was used for retrofitting the expression vector with a broad-host-range replication origin and a strong constitutive promoter [29][30][31] Effect of copy number of the lipAB gene on lipase production One of the common strategies to increase the expression level of a specific gene is to increase the copy number of the expression cassette [32]. B. glumae PG1 wild-type and two derivative strains, PG1/Bl and PG1/Rl, were cultivated in PG medium in the presence of 1% olive oil as a carbon source at 30 °C [8]. Culture supernatant and the whole-cell extract of each B. glumae strain were obtained after 72 h of cultivation and subjected to SDS-PAGE to investigate lipase expression. The mature lipase of PG1 has a calculated molecular mass of 33.1 kDa [9], and a protein of that size was visible in the extracts of all three strains and in the supernatants of PG1/Bl and PG1/Rl ( Fig. 2A, arrow). The secreted lipase was produced almost as a single band on SDS-PAGE, but the amount of it in the culture supernatant of wild-type B. glumae PG1 was markedly lower than in the supernatants from the recombinant strains, consistent with the fact that the wild-type strain harbors just one chromosomal copy of the lipAB operon. Additionally, PG1 harboring pBl (lane 5) secreted much more lipase than the host harboring pRl did (lane 3), which can be attributed to the lower copy number of pRK2 than of pBBR1. Real-time qPCR assays were performed to precisely quantify the transcription levels of lipA after 24 h of cultivation. As seen in Fig. 2B, the expression levels of lipA in the strains PG1/Rl and PG1/Bl were 12.9 times higher and 6.4 times higher than that of the PG1 wild-type strain, respectively.
The pBBR1 origin (about 30-40 copies per cell in E. coli) is functional in a wide range of Gram-negative bacterial genera, such as Burkholderia, Escherichia, Pseudomonas and Agrobacterium [35][36][37]. The copy number of the RK2 replicon is estimated to be 5 to 7 per cell in E. coli, and the origin delivers stable replication in more than 30 species of Gram-8 negative bacteria [38,39]. However, to the best of our knowledge, this is the first report of the use of the RK2 origin in B. glumae. The recombinant strains produced more lipase than did the wild-type strain, and no lipase was observed in the PG1 wild-type culture supernatant, which indicates that the increased dosage of the lipase gene enabled B.
glumae PG1 to secrete much more lipase. Therefore, overexpression of a lipase gene from a multicopy plasmid is a feasible strategy for enhancing lipase production, which is in accordance with a previous report [32].
Comparison of lipase activity and cell growth B. glumae PG1 variants secreted a considerable amount of lipase, but the lipase production and activity might have been influenced not only by the high-level expression of lipAB, but also by bacterial cell growth and density. Fang et al. demonstrated that cell density largely correlates with the yield of secreted protein and that optimized cultivation conditions are essential to obtain maximum cell growth and lipase activity [32]. The extracellular lipase activities of B. glumae PG1 and its variants were monitored during different growth stages in PG medium containing 1% olive oil as a carbon source at 30 °C ( Fig. 3A, B). We did not observe any growth defects in the strains harboring the plasmids with the lipAB operon when compared to the wild-type strain (Fig. 3B). However, there were marked strain differences in lipase activity as well as growth-associated changes in activity, with significantly increased lipase activity detected during the log phase Effect of the lipAB gene promoter on lipase production and activity 9 Next, to analyze the influence of the lipAB promoter on lipase production and activity, the native promoter P lipAB in the vectors was replaced with the strong constitutive promoter P apra , generating the two transformants PG1/Ba and PG1/Ra. Previous work has indicated that promoter optimization is an effective strategy for improving the expression levels of intracellular and secreted proteins by increasing transcription levels [40]. Wang et al. confirmed that the P apra promoter remarkably enhanced yields of target products in Burkholderia [ 41].
B. glumae PG1 strains carrying the four plasmids constructed in this study were grown in PG medium containing 1% olive oil as a carbon source at 30 °C. Secreted lipase was detected by the lipase activity assay (Fig. 4A) and SDS-PAGE in culture supernatants ( Fig. 4B) after 24 h of growth. Interestingly, the results revealed that strains PG1/Bl and PG1/Rl secreted much more lipase when compared to the two variants with the P apra promoter. In our test system, olive oil likely functions as an inducer when the lipase operon is under the control of the native promoter, consistent with the findings of Boekema et al. [2].
To increase the production of lipase, we attempted to optimize the culture conditions. Previous studies on the synthesis of natural products had emphasized that most constitutive promoters were active in a glucose-containing medium [42]. Therefore, 0.5% glucose was used as a carbon source in the growth medium in subsequent experiments.
The results revealed that the addition of 0.5% glucose in the PG medium stimulated the expression of the lipAB operon under the control of the P apra promoter. Lipase activity (Fig. 4C) and SDS-PAGE (Fig. 4D) analyses of culture supernatants were performed after 24 h of growth. The lipase activities of PG1/Ba (0.629 U/ml) and PG1/Ra (0.060 U/ml) were increased by 15.0-and 4.3-fold in comparison with those of PG1/Bl (0.148 U/ml) and PG1/Rl (0.004 U/ml), respectively. These results indicate that the P apra promoter is more active than the native promoter in a glucose-containing medium.
In summary, the lipAB operon under the P apra promoter was effective in enhancing active lipase production when 0.5% glucose was used as a carbon source, and the production of lipase using the P lipAB promoter was optimal when 1% olive oil was added. Olive oil exerted a negative effect on the expression levels of lipAB under the control of P apra , most probably by repressing transcriptional efficiency. The transcription levels of lipA were notably higher in strains PG1/Rl and PG1/Bl compared to PG1/Ra and PG1/Ba in the PG medium containing 1% olive oil (Fig S2). The effect of different carbon sources on promoters still need more exploration.

Conclusions
To enhance active lipase production, the lipase gene lipA and its chaperone gene, lipB, were cloned from the genomic DNA of B. glumae PG1 into high-copy vectors using the Red/ET systems followed by overexpressing in the homologous host. When the plasmidborne lipase gene was expressed under native promoter control, the derivative strains, PG1/Bl and PG1/Rl, yielded 39-and 15-fold higher production of the active lipase, respectively, in comparison with the PG1 wild-type strain in the presence of 1% olive oil.
This finding indicates that use of a multicopy expression vector had a positive effect on the expression level of lipase. To achieve higher lipase production, the lipase operon was expressed under the control of a strong constitutive promoter, yielding two transformants PG1/Ba and PG1/Ra. The lipase activities of PG1/Ba and PG1/Ra were 15-and 4-fold higher in the presence of 0.5% glucose as a carbon source, respectively, than that of PG1/Bl and PG1/Rl. Overall, the lipase produced by PG1/Ba in the medium containing glucose was less than that of the PG1/Bl in the medium using olive oil as substrate, but lipase gene 11 expression driven by a native promoter in PG1 was repressed by glucose.
Plasmid pRk2-km-apra carries the broad-host-range Rk2 origin and conveys kanamycin and apramycin resistance. Plasmid pBBR1-km has the broad-host-range pBBR1 origin and conveys kanamycin resistance. Appropriate antibiotics were added when necessary at the following concentrations: for E. coli strains, kanamycin, chloramphenicol and apramycin were each used at 15 µg/ml; for B. glumae strains, kanamycin and apramycin were each used at 30 µg/ml. The concentration of L-arabinose used for induction was 1.4 mg/ml. Plasmids used in this work are listed in Table S1.

Direct cloning of the lipAB operon
The p15A-cm vector was amplified from p15A-cm-tetO-tetR-hyg-ccdB with PCR using the PAGE-purified oligonucleotides PG1-lipAB-A and PG1-lipAB-S, which contain 80 nt homology arms to the target DNA sequence, and 20 nt standard PCR primers at the 3' end.
DNA oligonucleotides are listed in Table S2. The template plasmid and PCR products were purified with the Qiagen plasmid mini kit and eluted from the column with autoclaved ddH 2 O. The genomic DNA of B. glumae PG1 was prepared from the lysate by phenolchloroform-isoamyl alcohol extraction (25:24:1, pH 8.0) and ethanol precipitation, and digested with EcoR V and EcoR I to expose the terminal homology arms [36]. Ten micrograms of digested genomic DNA and 200 ng of p15A-cm linear vectors were coelectroporated into arabinose-induced, RecET-expressing GB05-dir cells for direct cloning of the lipAB operon, which was expressed under the control of its native promoter, P lipAB .
Transformants were selected after growth on LB agar plates containing chloramphenicol at 37 °C overnight, and correct recombinants were identified by restriction analysis. The final construct, p15A-cm-P lipAB -lipAB, was electroporated into GB08-red cells for further engineering.

Subcloning the lipAB operon into a broad-host-range vector
Subcloning vectors pBBR1-km and pRk2-km were prepared by PCR amplification from plasmids pBBR1-km and pRk2-km-apra with the pRlmer pairs RK2-lipAB-A/S and BBR1-lipAB-A/S, respectively. PCR products were purified using the Qiagen plasmid mini kit. The pBBR1-km and pRk2-km cassettes, flanked by the 80 nt homology arms, contain a broadhost-range origin and a kanamycin-selection marker and were electroporated into arabinose-induced, redαβ-expressing GB08-red cells harboring p15A-cm-P lipAB -lipAB to subclone the lipAB genes. Transformants were selected with kanamycin, and correct recombinants were identified by restriction digestion. The plasmid-borne lipase was 13 expressed under an native promoter. The final constructs, pBl and pRl, were electroporated into B. glumae PG1 for further studies.

Promoter exchange of the lipAB genes
The promoter of the lipAB was exchanged with the constitutive promoter P apra via Redαβ recombineering. P apra segments, flanked by homology arms, were amplified with PCR from the template plasmid pRK2-km-apra using the 100 nt primers P apra -A and P apra -S. Purified PCR products were recombined with plasmids pBl or pRl in arabinose-induced, Redαβexpressing GB08-red cells to exchange the P lipAB promoter. Following confirmation by restriction analysis, the final constructs, pBa and pRa, were electroporated into B. glumae PG1 to examine lipase activity.
Electroporation of the expression plasmid into B. glumae PG1 For B. glumae PG1, overnight cultures were diluted into 1.3 ml fresh LB medium without any antibiotics (OD 600 = 0.1) and incubated at 30 °C, 950 rpm, until the OD 600 was around 0.6. Cells were pelleted by centrifugation at 9500 rpm for 1 min at room temperature, washed, and then 10 µg of resuspended plasmid was added to the electrocompetent cells.
Electroporation was conducted using an Eppendorf 2510 electroporator at 1250 V with a 1 mm electroporation cuvette. The cells were resuspended in 1 ml LB medium and transferred into a 1.5 ml punctured micro-centrifuge tube. The culture was incubated at 30 °C, 950 rpm, for 2 h before plating on LB plates containing kanamycin (30 µg/ml) followed by incubation at 30 °C for 48 h. LB agar plates contained 1.2% (wt/vol) of agar.
Recombineering in E. coli was performed as previously described [41].

Lipase activity assay
Lipase activity in culture supernatants was assayed with para-nitrophenylpalrnitate (pNPP) as the substrate [44]. First, 100 µl acetonitrile containing 0.9 mg pNPP was mixed with 14 900 µl of 50 mM Tris-HCl buffer (pH 8.0). After 5 min of prewarming at 37 °C, freshly prepared substrate solution was mixed with 50 µl of 5000-fold dilutions of supernatant, and then incubated at 37 °C for 5 min. Next, 200 µl of the complex was taken to measure the OD 410 against an enzyme-free control using a spectrophotometer. Relative lipase activity was calculated as units/ml. One unit (U) was defined as the amount of lipase that liberates 1 mmol of para-nitrophenol per minute.

SDS-PAGE
Lipase proteins in whole-cell extracts and supernatants were analyzed with SDS-PAGE [20]. Culture supernatants obtained by centrifugation (4 °C, 8000 rpm, 1 min) were mixed with SDS sample buffer (5×), and then 20 µl was subjected to SDS-PAGE. Cell pellets were resuspended by sonication, and 20 µl cell suspension was mixed with SDS sample buffer for analysis. SDS-PAGE was carried out using 12% polyacrylamide gels, and proteins were stained with Coomassie brilliant blue R-250.

Quantitative real-time PCR
The analysis of transcriptional levels of lipA was performed with real-time qPCR in an ABI PRISM 7500 System (Applied Biosystems, USA) using SYBR green. Primers used for qPCR are listed in Table S2. The constitutively expressed gene rpoD was used as the internal reference. The amount of PCR product was calculated using the ΔΔCT-method, as previously described [45].

Ethics approval and consent to participate
Not applicable.

Availability of data and materials
(1) The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
(2) All data generated or analyzed during this study are included in this published article.

Competing interests
A Chinese patent application (ZL 201810571727.3) has been filed and granted for the technology/ invention disclosed in the present work.  Retrofitting the cloned genes with a broad-host-range vector. The broad-host-range pBBR1 and pRk2 origins flanked by homology arms, were used for retrofitting of the p15A vectors using the Redαβ recombination system, and then a strong constitutive promoter (Papra) was exchanged with the native promoter of lipAB to regulate expression of lipAB. A kanamycin resistance gene was used as a selectable marker.