Optimization of ribosomal binding site sequences for gene expression and 4-hydroxyisoleucine biosynthesis in recombinant corynebacterium glutamicum

Shi, Feng; Fan, Zhengyu; Zhang, Shuping; Wang, Yinghao; Tan, Shuyu; Li, Yongfu

doi:10.1016/j.enzmictec.2020.109622

Cited by 19 publications

(8 citation statements)

References 35 publications

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“…This indicates that the deletion of ddh could promote the accumulation of 4-HIL, but could not attenuate the synthesis of Lys. Although the 4-HIL titer of SL09 in this study was lower than that of some improved strains, such as SZ05 [19], SF12 [20] and ST17 [21], reported so far (Table 3), SL09 accumulated almost no other by-products except Lys, indicating its potential for further reconstruction. The remained Lys accumulation in SL09 is contrary to the result reported previously [15], in which the deletion of ddh in recombinant C. glutamicum ATCC13869 weakened the Lys synthesis.…”

Section: Effect Of Ddh and Lyse Deletion On 4-hil Productioncontrasting

confidence: 72%

“…So α-KG supply shall be enhanced in SY01 to increase the synthesis of 4-HIL. This can be achieved by increasing the expression of odhI using strong promoters or RBS sequences as reported previously [20]. The activity of OdhI can also be indirectly increased by deleting pknG or overexpressing ppp because PknG can phosphorylate and deactivate OdhI, while Ppp can dephosphorylate and activate OdhI [25,26].…”

Section: Transcriptional Difference In 4-hil and Lys Synthesis Pathwaymentioning

confidence: 94%

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Comparative transcriptome analysis of global effect of ddh and lysE deletion on 4-hydroxyisoleucine production in Corynebacterium glutamicum

Chen

Xiang

et al. 2022

Syst Microbiol and Biomanuf

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4-hydroxyisoleucine (4-HIL) is a potential drug for diabetes and weight control. 4-HIL was produced by expressing ido gene in L-isoleucine (Ile)-producing Corynebacterium glutamicum. But L-lysine (Lys) was also accumulated as the main by-product in this recombinant strain SN02. To attenuate Lys synthesis, two genes in Lys synthetic pathway, i.e., ddh encoding the diaminopimelic acid dehydrogenase and lysE encoding the specific Lys exporter were deleted in SN02. However, the deletion of ddh increased 4-HIL titer by 28.1%, but did not decrease Lys content; while the deletion of lysE significantly reduced Lys content by 66.7%, but 4-HIL titer also decreased by 19.3%. Therefore, we carried out transcriptome analysis to reveal the global variation in these mutants. Deletion of ddh and lysE (especially lysE) enhanced the transcription of key enzymes in succinylase branch of Lys synthesis pathway (DapD and DapC) and several enzymes involved in succinyl-CoA accessibility (SucC, SucD and OdhI), suggesting the compensatory synthesis of Lys via succinylase branch. In addition, the transcription of ilvBN in Ile synthesis pathway was improved, while the transcription of some genes in the 2-methylcitrate cycle and inositol metabolism pathway was weakened in these mutants. Mere deletion of ddh enhanced the transcription of aceA, ppc and pck, thus promoting oxaloacetate supply and 4-HIL synthesis. Deletion of lysE affected the transcription of some stress-related genes and transporter genes, suggesting that this mutant would be under stress, thus attenuating its 4-HIL synthesis. These findings will be helpful for systematic microbiology and bio-manufacturing of C. glutamicum.

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Section: Effect Of Ddh and Lyse Deletion On 4-hil Productioncontrasting

confidence: 72%

Section: Transcriptional Difference In 4-hil and Lys Synthesis Pathwaymentioning

confidence: 94%

Comparative transcriptome analysis of global effect of ddh and lysE deletion on 4-hydroxyisoleucine production in Corynebacterium glutamicum

Chen

Xiang

et al. 2022

Syst Microbiol and Biomanuf

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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%

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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“…For example, the start codon ATG of pyruvate dehydrogenase and glucose-6-phosphoisomerase was replaced by GTG, and the corresponding enzyme activity decreased by 60% and 40%, respectively, thereby increasing the l -lysine yield by 17% and 10%, respectively . In addition, the RBS engineering was widely used to regulate the gene translation and related enzyme activity, to increase the yield of the target product in C. glutamicum . Bicistron-modified RBSs were developed in C. glutamicum to minimize the context dependency of RBSs, which has been applied to regulate the arginine synthesis pathway .…”

Section: Synthetic Biological Device For Global Optimizationmentioning

confidence: 99%

“…27 In addition, the RBS engineering was widely used to regulate the gene translation and related enzyme activity, to increase the yield of the target product in C. glutamicum. 28 Bicistronmodified RBSs were developed in C. glutamicum to minimize the context dependency of RBSs, which has been applied to regulate the arginine synthesis pathway. 29 Additionally, the sequence between RBS and the start codon of downstream genes also affected the expression of target genes significantly.…”

Section: Synthetic Biological Device For Global Optimizationmentioning

confidence: 99%

Synthetic Biology Toolkits and Metabolic Engineering Applied in Corynebacterium glutamicum for Biomanufacturing

et al. 2021

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Corynebacterium glutamicum is an important workhorse in industrial white biotechnology. It has been widely applied in the producing processes of amino acids, fuels, and diverse value-added chemicals. With the continuous disclosure of genetic regulation mechanisms, various strategies and technologies of synthetic biology were used to design and construct C. glutamicum cells for biomanufacturing and bioremediation. This study mainly aimed to summarize the design and construction strategies of C. glutamicum-engineered strains, which were based on genomic modification, synthetic biological device-assisted metabolic flux optimization, and directed evolution-based engineering. Then, taking two important bioproducts (N-acetylglucosamine and hyaluronic acid) as examples, the applications of C. glutamicum cell factories were introduced. Finally, we discussed the current challenges and future development trends of C. glutamicumengineered strain construction.

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Optimization of ribosomal binding site sequences for gene expression and 4-hydroxyisoleucine biosynthesis in recombinant corynebacterium glutamicum

Cited by 19 publications

References 35 publications

Comparative transcriptome analysis of global effect of ddh and lysE deletion on 4-hydroxyisoleucine production in Corynebacterium glutamicum

Comparative transcriptome analysis of global effect of ddh and lysE deletion on 4-hydroxyisoleucine production in Corynebacterium glutamicum

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

Synthetic Biology Toolkits and Metabolic Engineering Applied in Corynebacterium glutamicum for Biomanufacturing

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