Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering

Kang, Xue-Mei; Cai, Xue; Huang, Zihan; Liu, Zhiqiang; Zheng, Yu‐Guo

doi:10.1016/j.ijbiomac.2019.09.144

Cited by 43 publications

(26 citation statements)

References 55 publications

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“…37 These enzymes were also produced using the P sodA -fusA -amyE triple promoter. 91,92 Kang et al 46 used a system containing a P amyE-cdd dual promoter and a (Pac) signal peptide to increase the expression of a recombinant amidase (Bm-Ami). The extracellular activity of Bm-Ami containing the plasmid pBSHdd2-20 reached 10.72 U/mg À1 DCW after 52 h in a scaling fermentation that, according to the authors, was the highest secretion obtained from Bm-Ami to date.…”

Section: The Dual Promoters In B Subtilismentioning

confidence: 99%

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The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

Souza

Guimarães

Pereira

et al. 2021

Exp Biol Med (Maywood)

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Bacillus subtilis is a successful host for producing recombinant proteins. Its GRAS (generally recognized as safe) status and its remarkable innate ability to absorb and incorporate exogenous DNA into its genome make this organism an ideal platform for the heterologous expression of bioactive substances. The factors that corroborate its value can be attributed to the scientific knowledge obtained from decades of study regarding its biology that has fostered the development of several genetic engineering strategies, such as the use of different plasmids, engineering of constitutive or double promoters, chemical inducers, systems of self-inducing expression with or without a secretion system that uses a signal peptide, and so on. Tools that enrich the technological arsenal of this expression platform improve the efficiency and reduce the costs of production of proteins of biotechnological importance. Therefore, this review aims to highlight the major advances involving recombinant expression systems developed in B. subtilis, thus sustaining the generation of knowledge and its application in future research. It was verified that this bacterium is a model in constant demand and studies of the expression of recombinant proteins on a large scale are increasing in number. As such, it represents a powerful bacterial host for academic research and industrial purposes.

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Section: The Dual Promoters In B Subtilismentioning

confidence: 99%

“…The extracellular activity of Bm-Ami containing the plasmid pBSHdd2-20 reached 10.72 U/mg À1 DCW after 52 h in a scaling fermentation that, according to the authors, was the highest secretion obtained from Bm-Ami to date. 46 The functional synthetic promoters in B. subtilis…”

Section: The Dual Promoters In B Subtilismentioning

confidence: 99%

The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

Souza

Guimarães

Pereira

et al. 2021

Exp Biol Med (Maywood)

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“…Several standardized techniques are currently designed to regulate the B. subtilis gene expression ( Popp et al, 2017 ; Yang et al, 2017 ). For instance, the dual-promoter system ( Rao et al, 2020 ), the auto-inducible expression systems ( Sun et al, 2020 ), and the highly active secretory expression system ( Kang et al, 2020 ) are common genetic engineering used for B. subtilis gene modifications. These gene expression systems without markers have limited capability, which the CRISPR-Cas9 system can resolve.…”

Section: Future Perspectives On Gene Editing Tools For B Subtilismentioning

confidence: 99%

Advances in Enhanced Menaquinone-7 Production From Bacillus subtilis

Liao

Ayansola

et al. 2021

Front. Bioeng. Biotechnol.

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The production of nutraceutical compounds through biosynthetic approaches has received considerable attention in recent years. For example, Menaquinone-7 (MK-7), a sub-type of Vitamin K2, biosynthesized from Bacillus subtilis (B. subtilis), proved to be more efficiently produced than the conventional chemical synthesis techniques. This is possible due to the development of B. subtilis as a chassis cell during the biosynthesis stages. Hence, it is imperative to provide insights on the B. subtilis membrane permeability modifications, biofilm reactors, and fermentation optimization as advanced techniques relevant to MK-7 production. Although the traditional gene-editing method of homologous recombination improves the biosynthetic pathway, CRISPR-Cas9 could potentially resolve the drawbacks of traditional genome editing techniques. For these reasons, future studies should explore the applications of CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) system gene-editing tools in the MK-7 anabolism pathway.

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“… 7 To increase the titer of recombinant proteins in bacteria, yeast, or mammalian cells, a number of studies used methods that screened either small sets of native, heterologous SPs, 8 or combinations of such SPs and promoters. 9–12 Other studies involved creating random or site-specific mutagenic libraries of SPs via PCR, and then testing the mutants for their ability to improve titer. 13–16…”

Section: Introductionmentioning

confidence: 99%

In silico high throughput mutagenesis and screening of signal peptides to mitigate N-terminal heterogeneity of recombinant monoclonal antibodies

Conyne

Lake

et al. 2022

mAbs

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N-terminal heterogeneity resulting from non-uniform signal peptide (SP) cleavage can potentially affect biologics property attributes and result in extended product development timelines. Few studies are available on engineering SPs systematically to address miscleavage issues. Herein, we developed a novel high throughput computational pipeline capable of generating millions of SP mutant sequences that uses the SignalP 5.0 deep learning model to predict which of these mutants are likely to alleviate the N-terminal miscleavage in antibodies. We optimized the parameters to target mutating one or two amino acids at the C-terminus of 84 unique SPs, exhausting all theoretically possible combinations and resulting in a library of 296,077 unique wildtype and mutant signal peptides for in silico screening of each antibody. We applied this method to five antibodies against different targets, with various extent of miscleavage (2.3% to 100%) on their Lambda light chains. In each case, multiple SP mutants were generated, with miscleavage reduced to a non-detectable level and titers comparable with or better than that of the original SPs. Pairwise mutational analysis using an in silico library enriched with high-scoring mutants revealed patterns of amino acids at the C-terminus of SPs, providing insights beyond the “Heijne rule”. To our knowledge, no similar approach that combines high throughput in silico mutagenesis and screening with SP cleavage prediction has been reported in the literature. This method can be applied to both the light chain and heavy chain of antibodies, regardless of their initial extent of miscleavage, provides optimized solutions for individual cases, and facilitates the development of antibody therapeutics. Abbreviations: Aa, amino acids; CHO, Chinese hamster ovary; CNN, convolutional neural network; CSscore, cleavage site score; CSV, comma-separated values; HC, heavy chain; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; IGLV, immunoglobulin G Lambda variable; LC, light chain; LCMS, liquid chromatography–mass spectrometry; MS, mass spectrometry; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PEI, polyethylenimine; SP, signal peptide; SPase, signal peptidase; TCEP, tris(2-carboxyethyl) phosphine; TOF, time-of-flight.

show abstract

Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering

Cited by 43 publications

References 55 publications

The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

Advances in Enhanced Menaquinone-7 Production From Bacillus subtilis

In silico high throughput mutagenesis and screening of signal peptides to mitigate N-terminal heterogeneity of recombinant monoclonal antibodies

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