Engineering modication of genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production

Background: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. Results: In this study, a genome-reduced strain GR167 lacking ~4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 harbored faster growth rate, higher transformation eciency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis cell GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PR suc and PR tpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. Conclusions: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis cell for further genetic modication. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

bacterial secondary metabolites [21]. For example, 14 endogenous promoters identi ed from Streptomyces albus J1074 by RNA-seq and reporter assays were successfully used to activate a cryptic gene cluster in S. griseu [22]. In another study, four endogenous promoters identi ed from S. coelicolor M145 by RNA-seq and reporter assays were used to activate cryptic biosynthetic clusters for jadomycin B production in S. venezuelae ISP5230 [5].
B. amyloliquefaciens LL3 was isolated initially for poly-γ-glutamic acid (γ-PGA) production by our lab, and whole genome of LL3 is currently available in the GenBank database (accession no. NC_017190.1) [23]. LL3 has a genomic size of 3,995,227 bp with an average G+C content of 45.7% and a circular plasmid (pMC1) of 6,758 bp. In particular, an intact srfA operon was found in the genome of LL3, suggesting the capability for surfactin biosynthesis. The essential genes and genomic islands (GIs) in LL3 were also identi ed by the Essential Genes Database (http://tubic.tju.edu.cn/deg/) and GIs Analysis Software (http://tubic.tju.edu.cn/GC-Pro le/). Previously, a marker-free large fragments deletion method was well established in LL3 [24]. Therefore, previous studies have laid a foundation for genome reduction and enhanced surfactin production in LL3.
In this study, a genome-reduced strain GR167 was constructed from B. amyloliquefaciens NK-1 (LL3 derivative, ΔuppΔpMC1) [25] and evaluated as a functional chassis cell for several physiological traits. Furthermore, GR167 was engineered using metabolic engineering strategies for enhanced surfactin production. Strategies designed for enhancing surfactin production in B. amyloliquefaciens are shown in Fig. 1.

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Construction of genome-reduced B. amyloliquefaciens strains To adapt to the adverse environmental conditions, there is a common mechanism horizontal gene transfer (HGT) among microorganisms, enabling host bacteria to acquire larger DNA segments, i.e., GIs, the G+C contents of which are signi cantly different from that of the core genome [26]. GIs usually carry some functional genes related to pathogenicity and antibiotic resistance, leading to the emergence of multiple resistant bacteria by HGT [27]. In addition, there are latent secondary metabolic biosynthesis gene clusters scattered across the LL3 genome, which may increase the metabolic burden on cells and the puri cation cost of target products [28]. Consequently, to streamline the genome of LL3, the GIs containing putative protein genes, antibiotic biosynthesis genes and prophage protein genes, where the G+C contents deviate signi cantly from 45.7%, were selected as the knockout targets. Besides, the gene clusters eps, bae and pgsBCA responsible for the biosynthesis of extracellular polysaccharides, bacillaene and γ-PGA, respectively, which consume more energy and substrates, were also deleted from the LL3 genome. The detailed information on the deleted regions is summarized in Tables S1 and S2. The schematic diagram for deletion of large genomic segments in LL3 is presented in Figure S1. Overall, a genome-reduced strain GR167 lacking ~4.18% of the LL3 genome was generated from NK-1 via a markerless deletion method [24]. The exact coordinates (G1 to G6) of the deleted regions on chromosome and the physical map of the endogenous plasmid pMC1 are shown in Figs. 2a and b, respectively.
Deleting redundant genes from a bacterial genome is expected to create superior chassis cells for the industrial production of valuable bio-based chemicals. Due to the existence of unannotated genes in the LL3 genome and lack of insight into the interactions among known genes, several industrially-relevant physiological traits were evaluated in GR167 to determine whether a chassis cell with excellent characteristics can be produced by genome reduction.
Genome reduction can improve the growth rate of LL3 To evaluate the effect of non-essential genomic sequences on cell growth, the growth pro les of GR167 and the parental NK-1 strain were detected by following the optical density (OD 600 ) of cells cultured in both poor (M9 medium) and rich (LB medium) conditions. As shown in Fig. 3a, obviously, whether incubated in LB or M9 medium, GR167 grew faster and yielded higher biomass with approximately 1.5 and 1.2-fold higher at the plateau phase than that of NK-1, respectively. The maximum speci c growth rate (μ max ) of the strains was further determined during exponential growth. When compared with NK-1, the GR167 showed a 23.7% and 67% increase in μ max when cultured in LB and M9 medium, respectively ( Fig. 3b).
During the evolution of the bacteria, various behaviors (e.g., horizontal gene transfer, HGT) enlarge the genome capacity, which may be unfavorable to cell growth because of the extra consumption for the expression of redundant metabolic pathways [29]. In current study, there was a positive correlation between cell growth and cumulative deletions, and deleting ~4.18% of the LL3 genome did not affect cellular viability of GR167. Moreover, the growth rate of GR167 outcompeted the parental strain under the tested culture conditions, making it a candidate chassis cell for further genetic engineering.

Genome reduction can enhance transformation e ciency
An ideal chassis cell is expected to possess the excellent capacity to take up exogenous plasmids. To eliminate the growth-rate bias of different strains, transformation e ciency was calculated by normalizing the colony number of bacteria transformed with plasmid pHT01 against the colony number of bacteria transformed without plasmid DNA. As shown in Fig. 3c, GR167 surpassed the transformation e ciency of the parental strain NK-1 by about 133%, indicating that the GR167 presented a better DNA uptake state during electroporation. For the transformation e ciency was a synergistic effect caused by many factors [30], it is di cult enough to pinpoint individual removed genes that are exactly affect the observed results.
Genome reduction can increase intracellular reducing power and the expression capacity of heterologous protein The intracellular reducing power (NADPH/NADP + ), which is indispensable for basic anabolic processes [31], was measured in this study. The intracellular NADPH/NADP + ratio of GR167 increased by 21.4% compared to the parental strain, (Fig. 3d), which may be attributed to the deletion of some NADPHconsuming biosynthesis pathways such as γ-PGA biosynthesis [32]. The improvement of intracellular reducing power level may be bene cial for GR167 to enhance the production of secondary metabolites.
Besides, an ideal chassis is expected to possess high heterologous protein expression capacity. In previous studies, green uorescent protein (GFP) was selected as a model heterologous protein in genome-reduced P. putida KT2440 mutants, and the expression capacity of heterologous protein was characterized by the GFP uorescence per biomass unit [9,33]. In this study, the production capability of GFP was evaluated in GR167 by transcriptional level and the uorescence intensity. As shown in Fig. 3e, when transformed with plasmid pHT-P 43 -gfp, the relative uorescence intensity of GR167 was about 50.4% higher than that of NK-1 and the increase in the transcriptional level of gfp was also observed in GR167. Overall, the reduced genome in GR167 has a positive effect on the expression capacity of heterologous protein.

Genome reduction can improve the metabolic phenotype
To further evaluate the difference in the metabolic potential of B. amyloliquefaciens, the GEN III MicroPlate TM was used to test and analyze the overall utilization of the substrates by both strains NK-1 and GR167. There are 71 carbon sources in the MicroPlates, 23 of which could be better utilized by both analyzed strains, especially L-Aspartic Acid, Citric Acid, L-Malic Acid, Glycerol, L-Glutamic Acid, and L-Lactic Acid, and among which, GR167 showed a better metabolic capacity than NK-1 except Glycerol (Table 1). These results indicated that these substrates were the preferred carbon sources for B. amyloliquefaciens LL3 and its derivatives, and that genome reduction could improve the capacity of LL3 to utilize certain substrates.
Use of genome-reduced strain GR167 as a starting strain for surfactin production Surfactin is synthesized by the non-ribosomal peptide synthetase (NRPS) encoded by srfA operon (srfAA, srfAB, srfAC, srfAD) in microbes, which uses four amino acids (L-glutamate, L-leucine, L-valine, and Laspartate) and fatty acids as precursors to form cyclic lipopeptide surfactin via a complex mechanism [34] ( Figure S4). For surfactin, it can hardly achieve a signi cant breakthrough in production only through traditional fermentation optimization because of its low yield in wild strains [16,35]. Engineering and modifying microbial chassis could maximize its practical application ranges and obtain maximum theoretical yields of bio-based products of interests. Such as B. subtilis BSK814, a genome-reduced strain, was endowed with the ability to hyperproduce guanosine as well as acetoin by modifying different metabolic pathways [4,19].
In this study, the chassis GR167 with the intact srfA operon and superior physiological traits was used as a starting strain for surfactin production. Because the fermentation broth of the NK-1 strain was too viscous to obtain a relatively purer surfactin product, the quanti cation of surfactin produced by NK-1 was very di cult. The γ-PGA production leads to the high viscosity and the limitation of dissolved oxygen of the culture broth [36] and competes with surfactin production for the common substrate glutamate (Glu) ( Figure S4). Moreover, both γ-PGA and surfactin are extracellular secretion products. Therefore, an extremely low yield of surfactin was detected with NK-1. Consequently, the mutant strain NK-ΔLP (NK-1 derivative, ΔpgsBCA) [37] without γ-PGA production was used as a control in the case of surfactin production. Surfactin produced by GR167 and NK-ΔLP was demonstrated by high-performance liquid chromatography-mass spectrometry (HPLC-MS). A slight increase (approximately 9.7%) in the surfactin titer was observed with GR167 ( Figure S2). Genome reduction seems to have little positive effects on the surfactin yield, however, the chassis GR167 constructed in this study shows superior genetic operability, e.g., higher transformation e ciency. In addition, higher growth rate of GR167 is also a critical factor for ensuring that further genetic modi cations are successfully performed. Therefore, it is interesting and necessary to explore whether microbial cell factories with high surfactin production capabilities can be constructed by further modi cation of GR167.

Characterization of surfactin produced by GR167
It was reported that surfactin produced by microorganisms is a mixture of several surfactin homologs [38]. In current study, by comparing the HPLC spectrogram of the produced surfactin by GR167 with that of the surfactin standard, there were four peaks to be detected within a retention time range of 6.4 to 7.2 min (Fig. 4a). To determine precisely the surfactin purity produced by GR167, each peak product of GR167 was puri ed from the culture supernatant and subjected to mass spectra (MS) analysis. The mass spectra of the product peaks 1, 2, 3, and 4 had the molecular ion peaks at m/z 995, 1009, 1023, and 1037, which were attributed to [C 13   Enhancing surfactin production by blocking the potential competitive pathways A transcriptional comparison between B. amyloliquefaciens LL3 and NK-ΔLP using RNA-seq revealed that the transcriptional levels of the gene clusters srfA, itu and fen, responsible for surfactin, iturin A and fengycin biosynthesis were all up-regulated when pgsBCA was removed ( Figure S3). Iturin A and fengycin belonging to CLP antibiotics are structural analogues of surfactin [39], possibly leading to the reduction of the purity of the extracted surfactin from the culture supernatant. Iturin A and fengycin are synthesized by NRPSs like surfactin [13]; thus, they may share similar biosynthesis mechanisms with surfactin and their biosynthesis may compete for NADPH, energy and direct precursors with surfactin biosynthesis. In this study, the gene clusters itu (37.2 kb) and fen (11.5 kb) were deleted to enhance surfactin production. The resulting mutants were designated as GR167I (Δitu), GR167D (Δfen) and GR167ID (Δitu, Δfen). The titer of surfactin was increased to 32.88 mg/L in GR167ID, with a 10% and 56% improvement in the titer and speci c productivity of surfactin compared to GR167, respectively ( Figure S2). The synthesis pathway of surfactin indicated that blocking the potential competitive pathways can eliminate the competition for the same amino acid precursors, allowing for the redistribution of substrates and precursors towards surfactin biosynthesis ( Figure S4).

Construction of endogenous promoter library of B. amyloliquefaciens LL3
Promoter engineering is considered as a promising approach for enhanced production of bacterial secondary metabolites [21,22]. FPKM (fragments per kilobase million) value is positively correlated with the transcriptional activity of a gene [40], which therefore can be regarded as an indicator for initial screening of promoters. Through RNA-seq analysis of LL3, all genes were ranked and classi ed into three groups based on their FPKM values, i.e., lower than 1,250, 1,250-4,000 and higher than 4,000. Then, the rst six genes with higher FPKM values in each group were selected, and their upstream regions were predicted and cloned as described in Methods, named PR x [x: the name of various related genes; PR: the sequences of predicted promoters with their ribosomal binding sites (RBSs)] and represented weak, moderate and strong promoters, respectively (Table S3). Subsequently, various reporter gene vectors derived from pHT01 containing fused fragments of the predicted promoters and gfp gene were used to assess the strengths of the tested promoters in LL3 ( Figure S5).

Characterization of the selected promoters via RT-qPCR and GFP uorescence measurement
As shown in Fig. 5a, the relative transcriptional levels of the candidate promoters measured with reporter gene vectors were PR ldh , PR ahp , PR hem , PR tpxi , PR clp , PR suc , PR accD , PR gltA , PR rpsu , PR nfrA , PR gltX , PR ydh , PR ugt , PR arg , PR nad , PR lac , PR alsD , PR hom and PR pgmi in a descending order, which were inconsistent with the strengths of the promoters shown by the FPKM values (Table S3), with similar results reported in a previous study [23]. We speculate that the transcription of a gene on chromosome may be affected and regulated by anking genes and regulatory sequences. However, this interference could be eliminated if a promoter is inserted into a plasmid.
To better evaluate these endogenous promoters, the relative uorescence intensities of GFP was measured. Among the 18 endogenous promoters, PR ahp , PR suc and PR tpxi showed superior production capacity of GFP, followed by PR rpsU , PR hem and PR ydh (Fig. 5b). However, the rst six promoters were PR ldh , PR ahp , PR hem , PR tpxi , PR clp and PR suc from high to low at the transcriptional levels (Fig. 5a). The different RBSs located upstream of the promoters may affect the translational initiation e ciencies of mRNA corresponding to GFP, leading to the different trends between the transcriptional level and production capacity of GFP.
Substitution of the native srfA promoter further enhanced surfactin production Considering the heterologous expression of srfA is challenging for which large genetic sequence (over 25 kb), substitution of the native srfA promoter by strong promoters is considered more bene cial for enhanced transcription of srfA operon [15,16,20]. In this study, two promoters PR suc and PR tpxi with better transcription and expression levels were integrated into upstream of the srfA operon in GR167ID, generating mutant strains GR167IDS and GR167IDT, respectively. The nucleotide sequences of the two selected promoters are shown in supplementary material. As expected, both the surfactin production and speci c productivity exhibited a signi cant elevation (Figs. 6a and b). In particular, the PR suc promotersubstituted strain GR167IDS produced 311.35 mg/L surfactin, which was about 9.5-fold higher than that of GR167ID (Fig. 6a). Meanwhile, the transcriptional level of srfA in GR167IDS was 678-fold higher than that in GR167ID (Fig. 6c). The melting curves of srfA and its internal standard gene rpsU indicated the absence of non-speci c products ( Figure S6).
The chassis strain GR167 does not differ in the surfactin yield compared to NK-ΔLP, however, the replacement of srfA promoter with PR suc promoter in GR167ID signi cantly increased the surfactin yield of the promoter-substituted strain GR167IDS compared with other modi cations (genome reduction and blocking of competitive pathways). Interestingly, PR suc was substituted for srfA promoter in NK-ΔLP to generate the mutant strain NK-ΔLPS, with a surfactin yield of 180.88 ± 2.87 mg/L (approximately 1.7-fold lower than that of GR167IDS). Therefore, genome reduction may contribute to the improvement of overall cellular metabolic activity, resulting in high-performance production of surfactin in GR167IDS.

Conclusions
In summary, a genome-reduced strain GR167 was constructed by deleting some non-essential genes accounting for ~4.18% of the LL3 genome and outcompeted the parental strain in several physiological traits assessed. GR167IDS, obtained from GR167 by promoter substitution, showed a 10.4-fold improvement in the titer of surfactin compared to GR167. The current results suggest that genome reduction in combination with promoter engineering may be a feasible strategy for the development of microbial cell factories capable of e ciently producing bacterial secondary metabolites.

Methods
Bacterial strains, media, and culture conditions Escherichia coli DH5α was employed for plasmid construction and propagation. For the subsequent successful electroporation of B. amyloliquefaciens strains, the E. coli JM110 was used as intermediate host to demethylate the desirable plasmids from E. coli DH5α. E. coli strains were incubated at 37 °C in Luria-Bertani (LB) broth. B. amyloliquefaciens LL3 was deposited in the China Center for Type Culture Collection (CCTCC) (accession number: CCTCC M 208109). B. amyloliquefaciens NK-1 was employed as the parental strain for genome reduction. GR167 was used as the starting strain for engineered highyielding surfactin producing mutants. M9 medium, which contains 3.4 g/L Na 2 HPO·12H 2 O, 0.6 g/L KH 2 PO 4 0.1 g/L NaCl 0.2 g/L NH 4 Cl supplemented with 20 g/L glucose, 50 mg/L tryptophan and 200 mM MOPS, was used for assessing growth of relevant strains. For lipopeptide surfactin production, B. amyloliquefaciens was incubated at 30 °C and 180 rpm for 48 h in Landy medium [41]. When appropriate, media were supplemented with ampicillin (Ap; 100 μg/mL), chloramphenicol (Cm; 5 μg/mL) or 5-uorouracil (5-FU; 1.3 mM).

Plasmid and strain construction
To construct the gene deletion vectors, the temperature-sensitive plasmid pKSU with an upp expression cassette was used [25]. The upstream and downstream fragments of the deleted genomic regions were ampli ed by PCR and then the two fragments were joined by overlap PCR. The generated fragment was ligated into pKSU via homologous recombination, to generate the gene deletion vectors. Introduction of plasmid into B. amyloliquefaciens was carried out using an optimized high osmolarity electroporation method [36]. To carry out multiple gene deletions on a single strain, a marker-less gene deletion method was used to construct the gene knockout mutants [24]. All the constructed plasmids and mutant strains were validated by PCR detection and DNA sequencing. All plasmids, strains, and primers used in this study are listed in Table S4, Table 2, and Table S5, respectively.

Physiological traits assessment
Growth pro les of GR167 and NK-1 were measured in both M9 mineral medium and LB medium. Overnight cultures (1 mL) were inoculated into 100 mL LB or M9 medium in 500-mL asks and then incubated for 20 h at 37 °C and 180 rpm. To determine the bacterial growth status, the OD 600 was monitored every 2 h using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan).
The metabolic phenotypic analyses were performed with a Biolog GEN Ш MicroPlate™ using a phenotype microarray system (Biolog Inc., California, USA) according to the manufacturer's instructions. The bacterial cells on the solid medium surface were collected by cotton swab, dissolved into the inoculating uid IF-B, and then the cell density was adjusted to a range of 80-86% Turbidity. Subsequently, 100 μl of bacterial suspensions were pipetted into the Biolog GEN Ш plates with different substrates. After the samples were incubated at 33 °C for 48 h, the absorbance at 590 nm was measured with the Biolog reader and the test data were analyzed by the Biolog system.
Electro-competent cells of GR167 and NK-1 (2 × 10 10 CFU/mL) were prepared according to previous methods [36]. Subsequently, approximately 100 ng of plasmid pHT01 was absorbed by 100 μL of electrocompetent cells via electroporation. After 3 h of recover at 37 °C and 180 rpm, the mixture was spread on LB agar plates supplemented with 5 μg/mL Cm. The numbers of colonies were calculated to evaluate the transformation e ciency.
Cells were cultured in LB medium at 37 °C for 18 h. The intracellular cofactors NADPH and NADP + were extracted and quanti ed by enzymatic methods [41] using an EnzyChrom™ assay kit (BioAssay Systems, USA) according to the manufacturer's protocols.
The heterologous protein productivity was determined by introducing plasmid pHT-P 43 -gfp into GR167 and NK-1. The detailed protocols for strain cultivation and uorescence intensity measurement refer to our previous work [9]. The relative uorescence intensity was normalized against per OD 600 of whole cells. The uorescence signal of NK-1 harboring pHT01 was set as background and was subtracted from overall uorescence.
RNA-seq, promoter prediction, and construction of reporter gene vectors RNA-seq analyses of LL3 were carried out according to our previous methods [32]. The expression levels of the predicted genes were quanti ed as the FPKM value [42]. The upstream regions of genes with different FPKM values were submitted online (http://www.fruit y.org/seq_tools/promoter) for promoter prediction.
Furthermore, each promoter sequence plus its native RBS and gfp gene were ampli ed by PCR from the LL3 genome and pHT-P 43 -gfp, respectively. Subsequently, 3′-end of a promoter sequence was fused with 5′-end of gfp gene and the fusion fragment was inserted into plasmid pHT01, to generate reporter gene vector pHT-PR x -gfp for promoter strength characterization ( Figure S5). Moreover, a control vector pHT-PR lac -gfp with gfp expression driven by lac promoter was similarly constructed with pHT01.  [43,44]. The relative transcriptional activity of a promoter was normalized against that of lac promoter. In addition, GFP uorescence measurement was performed as described previously [9].

Surfactin isolation and HPLC-MS analyses
All tested strains were cultured aerobically at 180 rpm in the Landy medium for 48 h. The culture supernatant and bacterial cell were separated by centrifugation at 4 °C and 14,000 rpm for 20 min. Subsequently, the bacterial cell was lyophilized for 24 h and weighed to obtain the cell dry weight (CDW). The supernatant was acidi ed to pH 2.0 with 6 M HCl and precipitated overnight at 4 °C. The precipitate formed was harvested by centrifugation and resuspended with 100 mL methyl alcohol. After which, 1 M NaOH was added to adjust the pH to 7.0 and further incubated for 48 h at 180 rpm and 37 °C. The supernatant containing surfactin extract was collected by centrifugation. The recovery and puri cation of surfactin homologues referred to the previous method [17]. Prior to HPLC-MS analysis, the supernatant was concentrated through a vacuum rotary evaporator and ltered via a 0.22-μm lter. Surfactin was analyzed and quanti ed by HPLC-MS equipped with a C18 column (Innoval ODS-2, 250 mm × 4.6 mm × 5 μm, Phenomenex, USA) using a validated method described previously [17,42].