Abstract:
BACKGROUND:Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins. RESULTS:The shuttle vector pBSMul1 containing the strong constitutive promoter PHpaII and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and β-glucuronidase, an increase of spacer lengths from 4 to 7-9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7-10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10-12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out. CONCLUSION:Our results confirm the importance of the 5' end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5' sequence encoding the signal peptide for Sec-depended secretion should also be considered.
journal_name
Microb Cell Factjournal_title
Microbial cell factoriesauthors
Volkenborn K,Kuschmierz L,Benz N,Lenz P,Knapp A,Jaeger KEdoi
10.1186/s12934-020-01404-2subject
Has Abstractpub_date
2020-07-29 00:00:00pages
154issue
1issn
1475-2859pii
10.1186/s12934-020-01404-2journal_volume
19pub_type
杂志文章abstract:BACKGROUND:Escherichia coli has emerged as a promising platform microorganism to produce biofuels and fine chemicals of industrial interests. Certain obstacles however remain to be overcome, among which organic-solvent tolerance is a crucial one. RESULTS:We used global transcription machinery engineering (gTME) to imp...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0368-4
更新日期:2015-11-05 00:00:00
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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doi:10.1186/s12934-018-0982-z
更新日期:2018-08-29 00:00:00
abstract:: Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been th...
journal_title:Microbial cell factories
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doi:10.1186/1475-2859-7-11
更新日期:2008-04-04 00:00:00
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journal_title:Microbial cell factories
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doi:10.1186/1475-2859-11-117
更新日期:2012-08-31 00:00:00
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更新日期:2018-12-26 00:00:00
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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更新日期:2018-03-22 00:00:00
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-5-38
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-8-41
更新日期:2009-07-24 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-10-15
更新日期:2011-03-03 00:00:00
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journal_title:Microbial cell factories
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doi:10.1186/1475-2859-7-5
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doi:10.1186/s12934-019-1158-1
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-11-72
更新日期:2012-06-01 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0830-6
更新日期:2017-11-28 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-014-0142-z
更新日期:2014-10-01 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-020-01454-6
更新日期:2020-10-19 00:00:00
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doi:10.1186/1475-2859-11-11
更新日期:2012-01-20 00:00:00
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journal_title:Microbial cell factories
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更新日期:2012-06-11 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-018-0977-9
更新日期:2018-08-21 00:00:00
abstract:BACKGROUND:cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of ...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0319-0
更新日期:2015-09-15 00:00:00
abstract:BACKGROUND:The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and cho...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-6-30
更新日期:2007-09-19 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0381-7
更新日期:2015-11-26 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-10-36
更新日期:2011-05-18 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
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更新日期:2018-03-26 00:00:00
abstract:BACKGROUND:The oleaginous yeast Yarrowia lipolytica is an organism of choice for the tailored production of various compounds such as biofuels or biopolymers. When properly engineered, it is capable of producing medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biobased and biodegradable polymer that can be used as...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1140-y
更新日期:2019-05-31 00:00:00