Construction of a genome-reduced B. amyloliquefaciens strain GR167
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 significantly different from that of the core genome [24]. GIs usually carry some functional genes related to pathogenicity and antibiotic resistance, leading to the emergence of multiple resistant bacteria by HGT [25]. 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 purification cost of target products [26]. 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 significantly 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 Table S1. 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 [22]. The exact coordinates (G1 to G6) of the deleted regions on chromosome and the physical map of the endogenous plasmid pMC1 are shown in Fig. 1a 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 to determine whether GR167 is an ideal chassis for enhanced production of surfactin.
Genome reduction can improve the growth rate of LL3
To evaluate the effect of non-essential genomic sequences on cell growth, the growth profiles of GR167 and its parental strain NK-1 were detected by following the optical density (OD600) of cells cultured in both poor (M9 medium) and rich (LB medium) conditions. As shown in Fig. 2a, 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. To further quantify the growth parameters, the maximum specific growth rates (μmax) of both strains were determined during exponential growth (Fig. 2b). The μmax values of GR167 were 23.7% and 67% higher than that of NK-1 when cultured in LB and M9 medium, respectively. Due to the block of secondary metabolite biosynthesis pathways, more energy and substrates were used for basal metabolism and cell proliferation in GR167. When cultured in M9 medium, the μmax of NK-1 was only 0.185 ± 0.004 h-1, with a 30.7% decrease relative to that measured in LB, while GR167 showed a similar growth behavior in both media, suggesting that nutrition may be one of the main growth-limiting factors for NK-1 but not for GR167.
Deletion of non-essential genes may perturb cellular metabolism and thus impair cell growth [27, 28]. On the contrary, the genome-reduced strain GR167 acquired beneficial growth fitness, which was in agreement with previous studies [29, 30]. Overall, in this 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 rates of GR167 outcompeted the parental strain under the tested culture conditions, making it a promising chassis for further genetic engineering.
Genome reduction can broaden the range of carbon sources utilized by LL3
To further evaluate the changes in the metabolic potential of GR167 and NK-1, their ability to utilize various substrates was analyzed by a GEN III MicroPlate containing 23 carbon sources tested. As shown in Table 1, the substrates utilized by GR167 and NK-1 were significantly different with each other. Eight carbon sources could be efficiently metabolized by GR167, especially L-aspartate and methyl pyruvate, with a 30% and 43% increase in the utilization ratio compared to NK-1, respectively, suggesting that genome reduction may improve the capacity of LL3 to utilize certain substrates.
Genome reduction can improve transformation efficiency
An ideal chassis cell is expected to possess the excellent capacity to take up exogenous plasmids. As shown in Fig. 2c, when transformed with plasmid pHT01, GR167 surpassed the transformation efficiency of the parental strain NK-1 by about 137%. The GIs and BGCs deleted in this study may contain negative regulators related to transformation efficiency, making competent cells in the optimal DNA uptake state during electroporation. Li et al [4] found that the transformation efficiency of genome-reduced strains decreased with cumulative genomic deletions. In addition, similar to the results of the transformation efficiency, the growth rates of all mutants were inferior to the parental strain [4]. In this study, on the contrary, both the growth rate and transformation efficiency of GR167 were obviously higher than that of NK-1 (Figs. 2a and 2c). Similarly, in another study, an E. coli mutant MDS12 lacking 8.1% of the genome of the parental strain also displayed a positive correlation between the growth parameters and transformation efficiency [26]. We therefore speculate that higher transformation efficiency may be associated with the improved growth fitness of GR167, notwithstanding which may be a synergistic effect caused by many physiological characteristics [31].
Genome reduction can increase intracellular reducing power and the productivity of heterologous proteins
The intracellular reducing power (NADPH/NADP+), which is indispensable for basic anabolic processes [32], was measured in this study. The intracellular NADPH/NADP+ ratio of GR167 increased by 21.4% compared to the parental strain NK-1 (Fig. 2d), which may be attributed to the deletion of some NADPH-consuming biosynthesis pathways such as γ-PGA biosynthesis [33]. The improvement of intracellular reducing power level may be beneficial for GR167 to act as an ideal chassis for enhanced production of secondary metabolites.
Also, an optimal chassis is expected to possess high heterologous protein productivity. In a previous study, prophage and hypothetical proteins accounting for 45.6% and 54.4% of the genome of Lactococcus lactis NZ9000, respectively, were deleted, resulting in a significant increase in the production capability of red fluorescent protein [5]. In another study, a genome-reduced strain EM383 was constructed from P. putida KT2440 by deleting flagellar operon and prophage protein genes, leading to a 40% increase in the production capability of foreign proteins [34].
In this study, GFP was selected as a model protein to determine the heterologous protein productivity. As shown in Fig. 2e, when transformed with plasmid pHT-P43-gfp, the relative fluorescence intensity of GR167 was 50.4% higher than that of NK-1, indicating that the productivity of heterologous proteins was significantly improved by genome reduction.
Use of genome-reduced strain GR167 as an optimal chassis for surfactin production
For surafctin, it can hardly achieve a significant breakthrough in production only through traditional fermentation optimization because of its low yield in wild strains [14, 35]. Strategies for surfactin overproduction were focused on strain modification recent years, such as substitution of the native promoter Psrf of srfA operon [13, 14], overexpressing transporters to enhance surfactin efflux [17], and modifying the regulators ComX and PhrC [35]. However, most modifications were performed in existing strains. In our study, a genome-reduced strain GR167 with intact surfactin synthase operon was evaluated as an ideal chassis for its superior physiological characteristics. Engineering and modifying microbial chassis may maximize its practical application ranges and obtain maximum theoretical yields of bioproducts of interests. In a previous study, by deleting and co-overexpressing specific genes conducive to guanosine accumulation in a genome-reduced strain B. subtilis BSK814, the guanosine titer in the final strain was 4.4-fold higher than that in the control strain bearing the same genetic modifications [4]. In another study, BSK814 was also endowed with the ability to produce acetoin using xylose as carbon source by modifying xylose utilization related pathways [36]. Therefore, genome reduction may provide a desirable chassis for further strain modification, and metabolic engineering of genome-reduced strains may be more beneficial to the development of microbial cell factories.
As shown in Fig. 3, surfactin production by GR167 was demonstrated by high-performance liquid chromatography (HPLC). Compared with NK-ΔLP (NK-1 derivative, ΔpgsBCA) [37], a slight increase in the surfaction titer was observed with GR167. Consequently, it is interesting and necessary to explore whether microbial cell factories with high surfactin production capabilities can be constructed by further modification of GR167.
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 (unpublished data). Iturin A and fengycin belonging to CLP antibiotics are structural analogues of surfactin [38], 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 [11]; 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 specific productivity of surfactin compared to GR167, respectively (Fig. 4). We speculate that blocking the potential competitive pathways may eliminate the competition for the same amino acid precursors, allowing for the redistribution of substrates towards surfactin biosynthesis.
Construction of endogenous promoter library of B. amyloliquefaciens LL3
Promoter engineering is considered as a promising approach for enhanced production of bacterial secondary metabolites [9, 19, 20]. FPKM (fragements per kilobase million) value is positively correlated with the transcriptional activity of a gene [39], which therefore can be regarded as an indicator for initial screening of promoters. Through RNA-seq analysis of LL3, all genes were ranked and classified 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 first six genes with higher FPKM values in each group were selected, and their upstream regions were predicted and cloned as described in Methods, named PRx [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 2). 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.
Characterization of the selected promoters via qPCR (quantitative real-time PCR) and GFP fluorescence measurement
As shown in Fig. 5a, the relative transcriptional levels of the candidate promoters measured with reporter gene vectors were PRldh, PRahp, PRhem, PRtpxi, PRclp, PRsuc, PRaccD, PRgltA, PRrpsu, PRnfrA, PRgltX, PRydh, PRugt, PRarg, PRnad, PRlac, PRalsD, PRhom and PRpgmi in a descending order, which were inconsistent with the strengths of the promoters shown by the FPKM values (Table 2), with similar results reported in a previous study [19]. We speculate that the transcription of a gene on chromosome may be affected and regulated by flanking genes and regulatory sequences. However, this interference can be eliminated if a promoter is inserted into a plasmid.
To further determine the production capabilities of GFP, in this study, the relative fluorescence intensities of GFP were also measured in LL3. Among the 18 endogenous promoters, PRahp showed the strongest production capacity of GFP, followed by PRsuc, PRtpxi, PRrpsU, PRhem and PRydh (Fig. 5b). However, the first six promoters were PRldh, PRahp, PRhem, PRtpxi, PRclp and PRsuc from high to low at the transcriptional levels (Fig. 5a). Considering the different RBSs located upstream of the promoters evaluated in this study, we speculate that the different RBSs may affect the translational initiation efficiencies 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 enhanced surfactin production
Considering the heterologous expression of srfA is challenging for which large genetic sequence (over 25 kb) [40], substitution of the native srfA promoter by strong promoters is considered more beneficial for enhanced transcription of srfA operon [13, 14, 18]. For example, Sun et al [13] replaced the native Psrf promoter of B. subtilis, resulting in a 10-fold improvement in the titer of surfactin. In this study, two strong promoters PRsuc and PRtpxi, of which nucleotide sequences are shown in supplementary material, derived from endogenous promoter library of LL3, were integrated into upstream of the srfA operon in GR167ID to construct surfactin hyperproducers GR167IDS and GR167IDT. As expected, both the surfactin production and specific productivity exhibited a significant elevation (Fig. 6a and b). In particular, the PRsuc promoter-substituted 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 operon in GR167IDS was 678-fold higher than that in GR167ID (Fig. 6c), indicating that the endogenous promoter PRsuc could significantly improve surfactin production by enhancing the transcription of srfA operon in B. amyloliquefaciens LL3.