Synthetic N-terminal coding sequences for fine-tuning gene expression and metabolic engineering in Bacillus subtilis

Tian, Rongzhen; Liu, Yanfeng; Chen, Jun-Rong; Li, Jianghua; Liu, Long; Du, Guocheng; Chen, Jian

doi:10.1016/j.ymben.2019.07.001

Cited by 57 publications

(49 citation statements)

References 43 publications

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“…Model simulation. MATLAB R2019a was used for kinetic modeling of cell growth, as well as for target metabolite (GlcNAc) and by-product (acetate) synthesis [61][62][63] . The development of the kinetic model was mainly based on four kinetics, including the growth kinetic model of acetic acid-producing E. coli developed by Luli et al, Monod equation describing nutrient limitation, acetate synthesis and specific growth rate equations determined by Basan et al, and Michaelis-Menten equation describing the biosynthesis pathway of GlcNAc 61,64,65 .…”

Section: Methodsmentioning

confidence: 99%

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

et al. 2020

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Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.

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Section: Methodsmentioning

confidence: 99%

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

et al. 2020

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“…Tian et al conducted and statistical analyzed 96 rationally selected N-terminal coding sequences (NCSs) from B. subtilis , which influenced gene expression at the translation level. They found that NCS substitution is more efficient and convenient than promoter substitution for gene expression improvement [ 37 ]. Naseri et al discussed the construction of complex libraries and combinatorial optimization strategies.…”

Section: Gene Expression In Bacillus Subtilismentioning

confidence: 99%

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine

Chuan²,

Fang³

et al. 2020

Microb Cell Fact

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Due to its clear inherited backgrounds as well as simple and diverse genetic manipulation systems, Bacillus subtilis is the key Gram-positive model bacterium for studies on physiology and metabolism. Furthermore, due to its highly efficient protein secretion system and adaptable metabolism, it has been widely used as a cell factory for microbial production of chemicals, enzymes, and antimicrobial materials for industry, agriculture, and medicine. In this mini-review, we first summarize the basic genetic manipulation tools and expression systems for this bacterium, including traditional methods and novel engineering systems. Secondly, we briefly introduce its applications in the production of chemicals and enzymes, and summarize its advantages, mainly focusing on some noteworthy products and recent progress in the engineering of B. subtilis . Finally, this review also covers applications such as microbial additives and antimicrobials, as well as biofilm systems and spore formation. We hope to provide an overview for novice researchers in this area, offering them a better understanding of B. subtilis and its applications.

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“…subtilis shuttle vector Lab stock pHT01 AMP, CmR, E. coli-B. subtilis shuttle vector Lab stock p43NMK-N gfp pP43NMK harboring gfp gene under the control of P 43 promoter Lab stock ( Tian et al., 2019 ) p43NMK- pfkA-alsSD pP43NMK harboring pfkA , alsS, and alsD gene under the control of P 43 promoter This study pHT- OVA - gfp pHT01 harboring gfp and OVA gene under the control of P 43 promoter This study p7S6 pMD18-T containing lox71-spe-lox66 cassette Lab stock …”

Section: Methodsmentioning

confidence: 99%

Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production

Liu

Tian

et al. 2020

Metabolic Engineering Communications

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Bacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered B. subtilis with faster growth phenotype is lacking. Here, fast-growing B. subtilis were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain B. subtilis 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of oppD , hag , and flgD genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting oppD , hag , and flgD genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h -1 in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing B. subtilis strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype.

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Synthetic N-terminal coding sequences for fine-tuning gene expression and metabolic engineering in Bacillus subtilis

Cited by 57 publications

References 43 publications

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine

Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production

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