Engineering mycobacteria artificial promoters and ribosomal binding sites for enhanced sterol production

Sun, Hong; Yang, Jingli; Song, Hao

doi:10.1016/j.bej.2020.107739

Cited by 17 publications

(15 citation statements)

References 61 publications

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“…The RBS sequence is a key factor in determining the translation initiation rate and protein expression level, and optimizing this region is considered an effective strategy to increase the yield of recombinant protein [ 17 , 41 , 42 , 43 ]. In order to better control the translation initiation rate and protein expression levels, the RBS calculator was developed and applied in the regulation of gene expression of recombinant strains [ 33 ].…”

Section: Resultsmentioning

confidence: 99%

“…In order to better control the translation initiation rate and protein expression levels, the RBS calculator was developed and applied in the regulation of gene expression of recombinant strains [ 33 ]. Previous studies have used the RBS calculator to generate the RBS sequence library, the purpose of which was to optimize protein expression levels; consequently, the expression levels of proteins have been significantly improved [ 17 , 40 ].…”

Section: Resultsmentioning

confidence: 99%

“…In the past few decades, single strong promoter [ 12 , 13 ], dual-promoter [ 14 ], and triple-promoter [ 15 , 16 ] expression systems were created to improve transcriptional levels. The ribosome binding site (RBS) and spacer between the RBS and the start codon were also components that allowed gene expression to be regulated at the translational level [ 17 , 18 , 19 , 20 ]. Pang et al, for example, used a mix of promoter and RBS engineering methods to greatly improve pullulanase yield [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

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Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Niu

Yan

Shen

et al. 2022

IJMS

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Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Niu

Yan

Shen

et al. 2022

IJMS

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“…For example, while combining multiple binding sites into one promoter has been shown to lead to increased promoter strength, as reviewed by Blazeck and Alper (2013), (Wang et al (2019a) report that introduction of multiple r-factor binding sites into one promoter can lead to decreased expression. Similarly, Sun et al (2020a) found that combining multiple strong promoters in tandem did not lead to changes in expression levels. Despite the limitations, the various methods outlined above are valuable approaches that enable the generation of promoter variants with various characteristics.…”

Section: Hybrid Mutated and (Semi-)artificial Promotersmentioning

confidence: 97%

“…Similarly, Sun et al . ( 2020a ) found that combining multiple strong promoters in tandem did not lead to changes in expression levels. Despite the limitations, the various methods outlined above are valuable approaches that enable the generation of promoter variants with various characteristics.…”

Section: ′ Regulatory Sequencesmentioning

confidence: 99%

Importance of the 5′ regulatory region to bacterial synthetic biology applications

Tietze

Lale

2021

Microbial Biotechnology

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The field of synthetic biology is evolving at a fast pace. It is advancing beyond single-gene alterations in single hosts to the logical design of complex circuits and the development of integrated synthetic genomes. Recent breakthroughs in deep learning, which is increasingly used in de novo assembly of DNA components with predictable effects, are also aiding the discipline. Despite advances in computing, the field is still reliant on the availability of precharacterized DNA parts, whether natural or synthetic, to regulate gene expression in bacteria and make valuable compounds. In this review, we discuss the different bacterial synthetic biology methodologies employed in the creation of 5 0 regulatory regionspromoters, untranslated regions and 5 0 -end of coding sequences. We summarize methodologies and discuss their significance for each of the functional DNA components, and highlight the key advances made in bacterial engineering by concentrating on their flaws and strengths. We end the review by outlining the issues that the discipline may face in the near future.

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High‐efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro‐emulsion system

Wang,

Ke,

Dong

et al. 2024

Biotechnology Journal

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Abstract21‐Hydroxy‐20‐methylpregn‐4‐en‐3‐one (4‐HBC, bisnoralcohol) is a crucial intermediate for the synthesis of steroidal drugs. Significant challenges including by‐products formation and poor substrate solubility were still confronted in its main synthetic route by microbial conversion from phytosterol. Construction of a direct bioconversion pathway to 4‐HBC and an efficient substrate emulsion system is therefore urgently required. In this study, three novel isoenzymes of 3‐ketosteroid‐Δ1‐dehydrogenase (KstD) and 3‐ketosteroid 9α‐hydroxylase (KsH) in Mycobacterium neoaurum were excavated and identified as KstD4, KstD5, and KsHA3. A strain capable of fully directing the synthesis of 4‐HBC was metabolically engineered via serial genetic deletion combined with enhanced expression of cholesterol oxidase (ChOx2) and enoyl‐CoA hydratase (EchA19). Moreover, a micro‐emulsion system combined with soybean oil and hydroxypropyl‐β‐cyclodextrin improved substrate solubility and bioavailability. In batch fermentation, molar yield of 96.7% with 39.5 g L−1 4‐HBC was obtained from 50 g L−1 phytosterol. Our findings demonstrate the potential for industrial‐scale biosynthesis of 4‐HBC.

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Engineering mycobacteria artificial promoters and ribosomal binding sites for enhanced sterol production

Cited by 17 publications

References 61 publications

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Importance of the 5′ regulatory region to bacterial synthetic biology applications

High‐efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro‐emulsion system

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