Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in <i>Bacillus subtilis</i>

You, Jiajia; Du, Yuxuan; Pan, Xuewei; Zhang, Xian; Yang, Taowei; Rao, Zhiming

doi:10.1021/acssynbio.1c00640

Cited by 6 publications

(7 citation statements)

References 39 publications

(84 reference statements)

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“…The fluorescence values of eGFP of different strains were measured to further identify strong constitutive promoters in S. marcescens as described previously ( You et al, 2022 ). In brief, strain JNB5-1/pUCP18 and recombinant strains containing plasmids pUCP18-promoter-egfp were inoculated in sterile 96 black-well plates (corning 3603) containing 200 μl LB medium and incubated at 37°C for 10 h. Then eGFP fluorescence was determined at the end of the incubation with a microplate Multi-Mode Reader (BIOTEK, Cytation 3) at the absorption wavelengths of 490 nm and 530 nm.…”

Section: Methodsmentioning

confidence: 99%

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens

et al. 2022

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Prodigiosin (PG), a red linear tripyrrole pigment produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. Although many studies have been used to dissect the biosynthetic pathways and regulatory network of prodigiosin production in S. marcescens, few studies have been focused on improving prodigiosin production through metabolic engineering in this strain. In this study, transcription factor engineering and promoter engineering was used to promote the production of prodigiosin in S. marcescens JNB5-1. Firstly, through construing of a Tn5G transposon insertion library of strain JNB5-1, it was found that the DNA-binding response regulator BVG89_19895 (OmpR) can promote prodigiosin synthesis in this strain. Then, using RNA-Seq analysis, reporter green fluorescent protein analysis and RT-qPCR analysis, the promoter P17 (PRplJ) was found to be a strong constitutive promoter in strain JNB5-1. Finally, the promoter P17 was used for overexpressing of prodigiosin synthesis activator OmpR and PsrA in strain JNB5-1 and a recombinant strain PG-6 was obtained. Shake flask analysis showed that the prodigiosin titer of this strain was increased to 10.25 g/L, which was 1.62-times that of the original strain JNB5-1 (6.33 g/L). Taken together, this is the first well-characterized constitutive promoter library from S. marcescens, and the transcription factor engineering and promoter engineering can be also useful strategies to improve the production of other high value-added products in S. marcescens.

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Section: Methodsmentioning

confidence: 99%

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens

et al. 2022

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“…Fluorescence intensities of EGFP and mCherry were determined as previously reported [36]. Single colonies were selected and inoculated into 96-well black plates (Corning 3603), each containing 200 µL of LB medium.…”

Section: Fluorescence Assaysmentioning

confidence: 99%

Utilizing 5′ UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis

You,

Wang,

Wang

et al. 2024

Biology

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The application of synthetic biology tools to modulate gene expression to increase yield has been thoroughly demonstrated as an effective and convenient approach in industrial production. In this study, we employed a high-throughput screening strategy to identify a 5′ UTR sequence from the genome of B. subtilis 168. This sequence resulted in a 5.8-fold increase in the expression level of EGFP. By utilizing the 5′ UTR sequence to overexpress individual genes within the rib operon, it was determined that the genes ribD and ribAB serve as rate-limiting enzymes in the riboflavin synthesis pathway. Constructing a 5′ UTR library to regulate EGFP expression resulted in a variation range in gene expression levels exceeding 100-fold. Employing the same 5′ UTR library to regulate the expression of EGFP and mCherry within the operon led to a change in the expression ratio of these two genes by over 10,000-fold. So, employing a 5′ UTR library to modulate the expression of the rib operon gene and construct a synthetic rib operon resulted in a 2.09-fold increase in riboflavin production. These results indicate that the 5′ UTR sequence identified and characterized in this study can serve as a versatile synthetic biology toolkit for achieving complex metabolic network reconstruction. This toolkit can facilitate the fine-tuning of gene expression to produce target products.

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“…Zhang et al overexpressed gntP , which encodes gluconate permease, to increase riboflavin production [ 54 ]. By constructing the tunable intergenic regions library, the gene expression of zwf , ribBA and ywlF were adjusted to increase the intracellular Ru5P and produced 2.7 g/L riboflavin in shaking flask, which increased the yield by 64.35% [ 55 ].…”

Section: Metabolic Engineering Strategies To Increase Riboflavin Prod...mentioning

confidence: 99%

Metabolic Engineering of Bacillus subtilis for Riboflavin Production: A Review

Liu

Zhang

et al. 2023

Microorganisms

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Riboflavin (vitamin B2) is one of the essential vitamins that the human body needs to maintain normal metabolism. Its biosynthesis has become one of the successful models for gradual replacement of traditional chemical production routes. B. subtilis is characterized by its short fermentation time and high yield, which shows a huge competitive advantage in microbial fermentation for production of riboflavin. This review summarized the advancements of regulation on riboflavin production as well as the synthesis of two precursors of ribulose-5-phosphate riboflavin (Ru5P) and guanosine 5′-triphosphate (GTP) in B. subtilis. The different strategies to improve production of riboflavin by metabolic engineering were also reviewed.

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Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis

Cited by 6 publications

References 39 publications

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens

Utilizing 5′ UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis

Metabolic Engineering of Bacillus subtilis for Riboflavin Production: A Review

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