Bacillus subtilis is a model strain for studying the physiological and biochemical mechanisms of microorganism, and is also a good chassis cell for industrial application to produce biological agents such as small molecule compounds, bulk chemicals, industrial enzymes, precursors of drugs and health product. In recent years, studies on metabolic engineering methods and strategies of B. subtilis have been increasingly reported, providing good tools and theoretical references for using it as chassis cells to produce biological agents. This review provides information on systematically optimizing the Bacillus subtilis chassis cell by regulating global regulatory factors, simplifying and optimizing the genome, multi-site and multi-dimensional regulating, dynamic regulating through biosensors, membrane protein engineering. For producing the protein reagent, the strain is optimized by optimizing the promoters, signal peptides, secretion components and building the expression system without chemical inducers. In addition, this review also prospects the important issues and directions that need to be focused on in the further optimization of B. subtilis in industrial production.
Bacillus subtilis/genetics* ; Bacterial Proteins/genetics* ; Biotechnology ; Metabolic Engineering ; Promoter Regions, Genetic ; Protein Sorting Signals/genetics*

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SP_SacC, SP_AmyL, SP_AprE, SP_YwbN and SP_WapA) were screened, among which SP_SacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, P_ykzA-P43, P_Ubay and P_bacA) from Bacillus were screened, and tandem promoter P_ykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/P_ykzA-P43-SP_SacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
Bacillus licheniformis/metabolism* ; Asparaginase/genetics* ; Bacillus/genetics* ; Protein Sorting Signals ; Promoter Regions, Genetic/genetics* ; Bacillus subtilis/genetics* ; Bacterial Proteins

To prolong serum half-life of human kallikrein (hK) and enhance its secretion rate, we modified hK gene and constructed a new form of recombinant hK protein (hK'-Fc). We amplified hK gene and Fc sequence, replaced the signal peptide of hK gene with murine signal peptide, constructed native expression plasmid of pcDNA-hK-Fc and modified expression plasmid of pcDNA-hK'-Fc, then transfected to CHO cells respectively. After the stable cell lines were screened, we compared the secretion rate between native fusion protein and modified fusion protein, purified fusion protein through Protein A+G affinity chromatography column and investigated the bioactivity of fusion protein. The results showed that recombinant vectors encoding fusion protein hK-Fc and hK'-Fc were constructed successfully; CHO cell lines stably secreting fusion protein were obtained, the yield is higher than 11 mg/L; Secretion rate was enhanced by 5-10 times after the signal peptide of fusion protein was modified; Fusion protein has enzymatic activity in vitro. The above results could promote the following researches on serum half-life of the fusion protein and develop a new stroke medicine with better clinical efficacy.
Animals ; CHO Cells ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cricetinae ; Cricetulus ; Humans ; Mice ; Protein Sorting Signals ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; physiology ; Tissue Kallikreins ; biosynthesis ; genetics ; Transfection

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Animals ; Antibodies, Viral ; genetics ; immunology ; Cricetinae ; Cytomegalovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin M ; immunology ; Mice ; Protein Sorting Signals ; Recombinant Fusion Proteins ; immunology

Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3, SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.
Nitrate Transporters ; Nitrates/metabolism* ; Sorghum/metabolism* ; Anion Transport Proteins/metabolism* ; Phylogeny ; Protein Sorting Signals/genetics* ; Nitrogen/metabolism* ; DNA ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*

OBJECTIVETo predict the exported proteins of the novel bacterium Phenylobacterium zucineum HLK1(T) using genome-wide computational identification by searching the export signals including N-terminal signal peptides and alpha-transmembrane helices.

METHODSThe computational identification of signal sequences was based on a consensus between multiple predictive tools, including SignalP V3.0, LipoP V1.0, Phobius and TMHMM 2.0. Type IV signal peptides and proteins exported via TAT machinery were searched manually based on the conservative motifs. All the predicted proteins were classified according to the Cluster of Orthologous Group (COG) standard.

RESULTIn the total 3861 proteins encoded by P. zucineum HLK1(T) 1 378 (35.7%) were predicted to be exported proteins, most of which (totally 735, 19.0% of the proteome and 53.3% of all the exported proteins) were uncleavable transmembrane helices. In addition, 499 type I signal peptides (12.9%, 36.2%), 101 lipoproteins (2.6%, 7.3%) were also identified. Four Type IV signal peptides and 12 TAT proteins were detected as well. According to the COG classification standard, most of these exported proteins were P proteins related to inorganic ion transport and metabolism and S proteins whose functions were unknown.

CONCLUSIONThe genome of HLK1(T) coded various types of exported proteins which may play an important role in the interaction between P. zucineum and the host cell, and facilitate the strain to invade into the cell.

Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Caulobacteraceae ; genetics ; metabolism ; Computational Biology ; methods ; Consensus Sequence ; genetics ; Genome, Bacterial ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Protein Transport ; genetics ; Proteome ; metabolism

A shuttle promoter-probe vector pNW33N-mpd was constructed with the E. coli-B. subtilis shuttle vector pNW33N and the mature mpd gene without it' s signal peptide-encoding sequence. The promoter fragments of B. subtilis ytkA and ywoF gene were cloned from plasmid pMPDP3 and pMPDP29 then generated the shuttle expression vector pNYTM and pNYWM. Expression vectors pNYTM and pNYWM were transformed into B. subtilis 1A751 to construct the expression strain 1A751 (pNYTM) and 1A751 (pNYTM), in these strains, under the control of the promoters and signal peptides of ytkA and ywoF gene, mpd gene was expressed and secreted with its biological activity; the result showed that the promoter of ytkA gene is much stronger than that of ywoF gene. Then a new shuttle expression-secretion vector pYNMK was constructed using the ytkA gene promoter and the signal peptide-encoding sequence of B. subtilis nprB gene, the expression of mpd gene achieved a higher level using the B. subtilis WB800 as the host, the methyl parathion hydrolase activity accumulated to a maximum level of 10.40 u/mL after 84 h of cultivation at the late stationary phase, which was 10.8-fold higher than the expression level of the original Plesiomonas strain M6, about 91.4% of the recombinant expression production was secreted into the culture medium.
Bacillus subtilis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genes, Bacterial ; Genetic Vectors ; Phosphoric Monoester Hydrolases ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Protein Sorting Signals ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Selection of suitable signal peptides is an important factor for efficient secretion of heterologous proteins. We defined structural fusion degree (SFD) as the compatibility degree of target proteins and signal peptides by a bioinformatics approach. We mathematically analyzed the interaction of fused signal peptides and adjacent residues of proteins, and proposed a mathematical model of extended signal region and the protein. SFD Features was extracted from this model to characterize the secretability of heterologous proteins. Simulation tests showed that SFD features can effectively discriminate high secretory proteins from poor ones in the host Bacillus subtilis. Results from this research will be useful in signal peptide selection and have a better guiding significance for the optimization of heterologous protein secretion.
Amino Acid Sequence ; Bacillus subtilis ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biotechnology ; methods ; Membrane Transport Proteins ; genetics ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Proteins ; secretion ; Recombinant Fusion Proteins ; genetics ; metabolism

The glycoprotein 3 (GP3) of type II porcine reproductive and respiratory syndrome virus has the characteristic domains of a membrane protein. However, this protein has been reported to be retained in the endoplasmic reticulum (ER) rather than transported to the plasma membrane of the cell. In this study, we performed confocal laser scanning microscopy analysis of variants of GP3 and foundthat the signal sequence of the GP3 led to confinement of GP3 in the ER, while the functional ortransmembrane domain did not affect its localization. Based on these results, we concludedthat the signal sequence of GP3 contains the ER retention signal, which might play an important role in assembly of viral proteins.
Animals ; Cell Line ; Cell Membrane/*metabolism/virology ; Cricetinae ; Endoplasmic Reticulum/*metabolism/virology ; Microscopy, Confocal/veterinary ; Plasmids/genetics/metabolism ; Porcine respiratory and reproductive syndrome virus/*genetics/metabolism ; *Protein Sorting Signals ; Sequence Analysis, Protein/veterinary ; Viral Envelope Proteins/chemistry/*genetics/metabolism

The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.
Amino Acid Sequence ; Animals ; Baculoviridae ; genetics ; Codon ; Gene Expression Profiling ; Gene Expression Regulation ; Glycosylation ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sf9 Cells ; Spodoptera ; genetics