A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis

Abstract

Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals. Thus, its metabolic engineering attracts significant attention. Nevertheless, the limited availability of selective markers makes this process difficult and time-consuming, especially in the case of multistep biosynthetic pathways. Here, we employ CRISPR/Cas9 technology to build an easy cloning toolkit that addresses commonly encountered obstacles in the metabolic engineering of B. subtilis, including the chromosomal integration locus, promoter, terminator, and guide RNA (gRNA) target. Six promoters were characterized, and the promoter strengths ranged from 0.9- to 23-fold that of the commonly used strong promoter P₄₃. We characterized seven terminators in B. subtilis, and the termination efficiencies (TEs) of the seven terminators are all more than 90%. Six gRNA targets were designed upstream of the promoter and downstream of the terminator. Using a green fluorescent protein (GFP) reporter, we confirmed integration efficiency with the single-locus integration site is up to 100%. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important product, lycopene. By heterologous expression of the essential genes for lycopene synthesis on the B. subtilis genome, a total of 13 key genes involved in the lycopene biosynthetic pathway were manipulated. Moreover, our findings showed that the gene cluster ispG-idi-dxs-ispD could positively affect the production of lycopene, while the cluster dxr-ispE-ispF-ispH had a negative effect on lycopene production. Hence, our multilocus integration strategy can facilitate the pathway assembly for production of complex chemicals and pharmaceuticals in B. subtilis. IMPORTANCE We present a toolkit that allows for rapid cloning procedures and one-step subcloning to move from plasmid-based expression to stable chromosome integration and expression in a production strain in less than a week. The utility of the customized tool was demonstrated by integrating the MEP (2C-methyl-d-erythritol-4-phosphate) pathway, part of the pentose phosphate pathway (PPP), and the hetero-lycopene biosynthesis genes by stable expression in the genome. The tool could be useful to engineer B. subtilis strains through diverse recombination events and ultimately improve its potential and scope of industrial application as biological chassis.

Keywords: Bacillus subtilis; CRISPR; MEP pathway; genome editing; lycopene; microbial fermentation.

FIG 1

Major metabolic pathways associated with lycopene biosynthesis in B. subtilis and metabolic engineering manipulation applied to overproduce lycopene. Genes, the proteins they encode and pathway abbreviations are listed. EMP pathway: pgi, glucose-6-phosphate isomerase gene; fbp, fructose-1,6-bisphosphatase gene; fbaA, fructose-1,6-bisphosphate aldolase gene; gapA, glyceraldehyde-3-phosphate dehydrogenase gene; pgk, phosphoglycerate kinase gene; pyk, pyruvate kinase gene. MEP pathway: dxs, 1-deoxy-d-xylulose-5-phosphate synthase gene; dxr, 1-deoxy-d-xylulose-5-phosphate reductoisomerase gene; ispD, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase gene; ispE, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase gene; ispF, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase gene; ispG, flavodoxin-dependent (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase gene; ispH, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene; ispA, (2E,6E)-farnesyl diphosphate synthase gene; lycopene pathway, crtE_pa, geranylgeranyl pyrophosphate synthase gene; crtE_pa, geranylgeranyl pyrophosphate synthase gene; crtB_pa, phytoene synthase gene; crtI_pa, phytoene desaturase gene. PPP: zwf, glucose-6-phosphate dehydrogenase gene; pgl, 6-phosphogluconolactonase gene; gndA, NADP-dependent phosphogluconate dehydrogenase gene; ywlf, ribose 5-phosphate epimerase gene; rpe, ribulose-5-phosphate 3-epimerase gene; tkt, transketolase gene; tal, transaldolase gene; pos5P, NADH kinase; G-3-P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-d-xylulose phosphate; MEP, 2C-methyl-d-erythritol-4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-d-erythritol; CDP-MEP, 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate; MEC, 2C-methyl-d-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl-diphosphate; GGPP, geranylgeranyl diphosphate; MEP, the methylerythritol phosphate pathway; PPP, the pentose phosphate pathway; EMP, the Embden-Meyerhof-Parnas pathway. Blue arrows indicate the EMP pathway from glucose; green arrows indicate the MEP pathway; red arrows indicate the lycopene pathway; blank arrows indicate the PPP. Genes that are amplified are shown as purple, next to their corresponding reactions. An exogenous reaction (gray arrow) introduced to rewrite redox metabolism related to NADPH synthesis is also indicated.

FIG 2

Genome editing in B. subtilis using the optimized CRISPR/Cas9 system with the customization of vectors. (a) Schematic view of the cleavage in the sgRNA-ccdB cassette. The BsaI restriction site is shown in bold letters, and nucleotides of the 4-bp single-stranded overhangs are colored in orange and blue. Arrows indicate the direction of the restriction site. A physical map is presented of vector pBAC9987—containing the ColE1 origin for E. coli, the temperature-sensitive replication origin from pE194^ts for B. subtilis, an ampicillin resistance gene (Amp^r) for E. coli, a chloramphenicol or spectinomycin resistance gene (Cm^r or Spe^r) for B. subtilis, cas9 under the control of the P_grac promoter and trpA terminator, the ccdB gene transcribed from the constitutive promoter P_ccdB and interrupted by the rrnB T1T2 terminator and the promoter P_veg—which is responsible for initiating sgRNA transcription. The plasmid pBAC9987-S was derived from pBAC9987-C and was endowed with the Spe^r gene. (b) Schematic diagram showing generation of knock-in customization of vectors. A pair of oligonucleotides were synthesized and annealed to generate double-stranded DNAs (dsDNAs) harboring a spacer sequence. The different sgRNA expression plasmids were constructed using the dsDNAs and pBAC9987. Two adjacent SfiI sites enable integration of these homology templates into the sgRNA expression plasmid to give the final integration plasmid. Only 1 step is required for any gene integration into the EGIB loci. However, for gene deletion or modification, two steps are required. gS, gRNA scaffold.

FIG 3

Construction and characterization of tailored promoters and terminators for the EGIB locus. (a) The expression vectors were constructed by the fusion of different promoters and the reporter gene gfp. (b) The expression vectors were transformed into B. subtilis and characterized by fluorescence imaging. (c) Structures of the expression cassettes with the two reporter genes gfp and mCherry for measurement of the terminators. The control without a terminator and the terminator were inserted between two fluorescent protein-encoding genes. RBS, ribosome-binding site. (d) GFP fluorescence of the terminators and control. The scale bar represents GFP fluorescence normalized by OD₆₀₀. (e) Red fluorescent protein (RFP) fluorescence of the terminators and control. The scale bar represents mCherry fluorescence normalized by OD₆₀₀. (f) TEs of terminators in time course; (g) organization of the EGIB locus. ORF, open reading frame. (h) Insertion of a 0.7-kb DNA fragment (gfp) using the all-in-one plasmid. Different sites of the EGIB locus were verified for integration efficiency. Error bars are standard deviations calculated from three independent experiments.

FIG 4

Design of a toolbox to tune genetic expression in B. subtilis. The toolkit includes 12 Cas9-sgRNA plasmids and the EGIB locus. (a) Architecture of the EGIB locus. The EGIB locus is integrated into the B. subtilis genome by our single-plasmid strategy at the amyE locus. The EGIB locus is composed of the standard regulatory elements (promoter, RBS, sgRNA target, terminator). Six customized gene expression cassettes flanked by spacers in the EGIB locus will promote gene expression from different promoters. Spacers of 900 bp were designed to facilitate the homologous recombination of genes into the EGIB locus of B. subtilis. The workflow of the toolkit for gene insertion is as follows. Step 1 includes building the repair plasmid pBAC9987-C-sgRNA1-G_x by replacing the ccdB-based counterselection cassette (dark green) in pBAC9987-C-sgRNA1-ccdB with a PCR fragment containing a gene/gene cluster of interest (here, G_x represents gene X). The repair plasmid pBAC9987-sgRNA-G_x harbors gene X, which will direct its integration into the EGIB locus by homologous recombination and facilitate expression from the customized promoter. IPTG (0.1 mM) was used for counterselection. (b) Architecture of the EGIB locus after insertion of gene X into the N20-1 site. In the case of multistep biosynthetic pathways, we can also select different gene combinations for expression on the genome, as step 2, a second gene/gene cluster of interest (here, G_Y represents gene Y) can be efficiently inserted into the plasmid pBAC9987-S-sgRNA2-ccdB by one-step Golden Gate, resulting in the repair plasmid pBAC9987-S-sgRNA2-G_Y. (c) Architecture of the EGIB locus after insertion of the two genes X and Y into the N20-1 and N20-2 sites. For multiple genes (GENE Z, GENE X1, GENE Y1, GENE Z1), the system can also select different gene combinations for expression on the EGIB locus. The knock-in customization of vectors for EGIB locus was divided into two groups: in group A, pBAC9987-ccdB-sgRNA1, -3, and -5 were endowed with the Cm^r gene; in group B, pBAC9987-ccdB-sgRNA2, -4, and -6 were endowed with the Spe^r gene.

FIG 5

Construction and lycopene production of engineered B. subtilis. (a) Schematic illustration of heterologous expressing lycopene biosynthesis genes by genomic integration. Colonies were used for genome editing and extraction of the genomic DNAs from the cells, PCR amplification, and Sanger sequence: GGPP synthase CrtE, phytoene desaturase CrtI, and phytoene synthase CrtB were from P. agglomerans. (b) HPLC analysis of lycopene content in engineering B. subtilis mutants and their color phenotypes. Error bars indicate standard deviations from three parallel experiments.

FIG 6

Effect of different combinations of MEP pathway genes in the EGIB locus on lycopene production in B. subtilis. (a) Schematic of the gene overexpression in the EGIB locus, denoting the corresponding strains. The crtE gene and crtIB gene from P. agglomerans were overexpressed by insertion into the N20-1 and N20-5 sites in the EGIB locus under the control of P_shuttle-09 and P₄₃, respectively, achieving the recombinant strains CrtE and CrtEIB. The ispA gene was overexpressed by inserting into the N20-4 site in the EGIB locus of strain CrtEIB under the control of P_HpaII, achieving the recombinant strain CrtEIB-IspA. The GISD operon and the FHCE operon were overexpressed by insertion into the N20-6 and N20-2 sites in the EGIB locus of strain CrtEIB-IspA under the control of P_gapDH and P_spoVG, respectively, achieving the recombinant strains CrtEIB-IspA-GISD and CrtEIB-IspA-FHCE. Finally, the strain CrtEIB-IspA-GISD-FHCE was obtained by the cooverexpression of GISD and FHCE in the EGIB locus of strain CrtEIB-IspA. (b and c) Lycopene production by the starting strain and the recombinant strains after 24 h of fermentation. The error bar represents the standard deviation (n = 3). Two-tailed t tests indicate significance: *, P < 0.05; **, P < 0.01. CrtE, CrtI, and CrtB from P. agglomerans are responsible for lycopene biosynthesis; MEP pathway-related genes were integrated into EGIB sites in the B. subtilis genome. S, C, D, E, F, G, H, I, and A represent dxs, dxr, ispD, ispE, ispF, ispG, ispH, idi (fni), and ispA, respectively.

FIG 7

Effect of single-gene overexpression of NADPH regeneration-related enzymes on lycopene production and the cell mass of recombinant strains. (a) Schematic of the gene overexpression in the EGIB locus, denoting the corresponding strains. The pos5P gene from S. cerevisiae was overexpressed by insertion into the N20-3 site in the EGIB locus of strain CrtEIB-IspA-GISD-FHCE under the control of P_yvyD promoter, achieving the recombinant strain CrtEIB-IspA-GISD-FHCE-Pos5P. The zwf gene and gndA gene from B. subtilis were overexpressed by insertion into the N20-3 site in the EGIB locus of strain CrtEIB-IspA-GISD-FHCE under the control of the P_yvyD promoter, achieving the recombinant strains CrtEIB-IspA-GISD-FHCE-Zwf and CrtEIB-IspA-GISD-FHCE-GndA, respectively. (b and c) Lycopene production by the starting strain and the recombinant strains after 24 h of fermentation. The error bar represents the standard deviation (n = 3). Two-tailed t tests indicate significance: *, P < 0.05. CrtE, CrtI, and CrtB from P. agglomerans are responsible for lycopene biosynthesis; MEP pathway-related genes were integrated into EGIB sites in the B. subtilis genome. S, C, D, E, F, G, H, I, and A represent dxs, dxr, ispD, ispE, ispF, ispG, ispH, idi (fni), and ispA, respectively.

FIG 8

Fermentation times of the lycopene-producing candidates by cultivation in flask fermentation. (a) Effects of ispG-idi-dxs-ispD cluster overexpression on lycopene production. Strain CrtEIB-IspA-GISD was constructed by adding a second copy of ispG, idi, dxs, and ispD controlled by the constitutive promoter P_gapDH in the chromosome of strain CrtEIB-IspA. (b) Effects of cluster ispF-ispH-dxr-ispE overexpression on lycopene production. Strain CrtEIB-IspA-GISD-FHCE was constructed by adding a second copy of ispF, ispH, dxr, and ispE controlled by the constitutive promoter P_spoVG in the chromosome of strain CrtEIB-IspA-GISD. (c) Effects of zwf overexpression on lycopene production. Strain CrtEIB-IspA-GISD-FHCE-Zwf was constructed by adding a second copy of zwf controlled by the constitutive promoter P_yvyD in the chromosome of strain CrtEIB-IspA-GISD-FHCE. (d) Effects of gndA overexpression on lycopene production. Strain CrtEIB-IspA-GISD-FHCE-GndA was constructed by adding a second copy of gndA controlled by the constitutive promoter P_yvyD in the chromosome of strain CrtEIB-IspA-GISD-FHCE. Data are presented as mean values ± SD (n = 3 independent experiments). All Student’s two-tailed t tests compare the lycopene production levels of strains with production at the adjacent time points.