A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

Abstract

To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation. Graphical Abstract

Efficient Protein Expression and Biosynthetic Gene Cluster Regulation in Bacillus subtilis Driven by a T7-BOOST System

“…As shown in Figure S8, analysis of total intracellular protein by SDS-PAGE revealed that sfGFP was the most highly expressed protein in strains containing the T7-BOOST system in the low-copy-number plasmids, which is consistent with previous studies that expressed GFP in the genome or low-copy-number plasmid. 20,22 In the strain containing pHT-P T7lac , the sfGFP content and concentration were 19.4% and 0.18 g/L, and in the strain containing pHT-P T7xyl , the sfGFP content and concentration were 12.1% and 0.14 g/L, respectively. In order to assess potential misunderstandings regarding the characteristics of the expression systems, we randomly selected 20 colonies following the transformation of the plasmids depicted in Figure 3 into their respective B. subtilis strains.…”

“…18−20 In a recent study, the supercompetent SCK6 strain was utilized as the starting strain to establish the T7 expression system. 22 To enhance expression efficiency during high-density cell fermentation, two major protease genes (aprE and nprE), as well as genes associated with sporulation and fermentation foam generation (spoIIAC and srfAC), were knocked out. T7RNAP integration was accomplished through a plasmid-based approach, resulting in a T7 expression system that can be easily transferred to any other B. subtilis strain in a plug-and-play manner.…”

“…To facilitate the use of the upp gene encoding uracil phosphoribosyltransferase (UPRTase) as the negative selection marker in their seamless genome editing system, the upp gene on the B. subtilis genome was knocked out. 22 Eliminating UPRTase does not affect protein synthesis, but may impact bioproduction as it catalyzes the vital reaction in uracil salvage. 23 Compared with traditional genome editing technologies, CRISPR-based gene integration still relies on the double crossover event, but the selection is achieved through Cas nuclease cleavage, which avoids the use of antibiotic resistance and negative selection markers.…”

Synthesis of a Healthy Sweetener d-Tagatose from Starch Catalyzed by Semiartificial Cell Factories

“…The recombinant enzymes αGP, phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), tagatose 6-phosphate 4-epimerase (TPE), and tagatose 6-phosphate phosphatase (T6PP) were individually expressed in B. subtilis SCK 22 . TEOS and APTES were purchased from Shanghai McLean Biochemical Technology Co. Ltd. (Shanghai, China).…”

Isolation, identification, and biochemical characterization of a novel bifunctional phosphomannomutase/phosphoglucomutase from the metagenome of the brown alga Laminaria digitata

“…A recombinant PGM from E. coli has also been used to enhance UDP-galactose derived synthesis of the bioactive compound hyperoside (quercetin 3-O-galactoside) in a metabolically engineered E. coli strain (Li et al, 2022). In addition, PGM from T. kodakarensis has recently been heterologously expressed in B. subtilis thereby allowing for the recombinant enzyme to be produced at high levels in highdensity fermentations for subsequent potential synthesis of inositol from starch (Ye et al, 2022). Increased expression of PGM to generate increased polysaccharide production has recently been achieved in the fungus Cordyceps militaris, with homologous overexpression of PGM and UDP-glucose 6-dehydrogenase in the fungus resulting in an approximately 78% increase in polysaccharide levels.…”