Draft Genome Sequence of Kozakia baliensis SR-745, the First Sequenced
            <i>Kozakia</i>
            Strain from the Family
            <i>Acetobacteraceae</i>

Schmid, Jochen; Koenig, Steven; Pick, André; Steffler, Fabian; Yoshida, Shosuke; Miyamoto, Kenji; Sieber, Volker

doi:10.1128/genomea.00594-14

Cited by 5 publications

(2 citation statements)

References 7 publications

(7 reference statements)

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“…In this section, we show that SIGER systems can facilitate the expression of difficult-to-produce proteins and yield soluble enzymes. To do so, we aimed to produce two proteins, the glycosyltransferase GumM from Kozakia baliensis , 72 involving the biosynthesis of xanthan gum, and the carbohydrate-binding module CBM73 of a lytic polysaccharide monooxygenase from Cellvibrio japonicus . 73 A previous investigation of GumM did not succeed in producing the protein using a pTYB1 expression vector, which is based on a C-terminal intein tag, for the IMPACT-CN purification system.…”

Section: Resultsmentioning

confidence: 99%

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

et al. 2023

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Coordination of multigene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features of the 5′UTR in combination with the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures. These features strongly influence the translation yield and protein quality by regulating the ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5′UTRs in combination with different target genes, leads to a wide variety of gene ramp compositions with irregular translation rates, leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramp (SIGER) system for controlling protein expression. The SIGER system makes use of inteins to decouple the translation initiation features from the gene of a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid the impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways. As a proof of concept of the potential of the system, we also used a SIGER system to express two difficult-toproduce proteins, GumM and CBM73.

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Section: Resultsmentioning

confidence: 99%

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

et al. 2023

View full text Add to dashboard Cite

show abstract

“…In this section, we show that SIGER systems can facilitate the expression of difficult-to-produce proteins and yield soluble enzymes. To do so, we aimed to produce two proteins, the glycosyltransferase GumM from Kozakia baliensis 72 , involved the biosynthesis of xanthan gum; and the carbohydrate-binding module CBM73 of a lytic polysaccharide monooxygenase from Cellvibrio japonicus 73 . A previous investigation of GumM did not succeed in producing the protein using a pTYB1 expression vector, which is based on a C-terminal intein tag, for the IMPACT-CN purification system 74 .…”

Section: Resultsmentioning

confidence: 99%

Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control

Fages-Lartaud

Mueller

Elie

et al. 2021

Preprint

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Coordination of multi-gene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features at the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures forming in relation with the 5'UTR. These features strongly influence translation yield and protein quality by regulating ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5'UTRs in combination with different target genes leads to a wide variety of gene ramp compositions with irregular translation rates leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramps (SIGER) system for controlling protein expression. The SIGER system uses inteins to uncouple a characterized gene ramp from a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways.

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Fermentative Production of Microbial Exopolysaccharides

Schmid

Sieber

2019

Bioprocessing for Biomolecules Production

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Draft Genome Sequence of Kozakia baliensis SR-745, the First Sequenced Kozakia Strain from the Family Acetobacteraceae

Cited by 5 publications

References 7 publications

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control

Fermentative Production of Microbial Exopolysaccharides

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