New Set of Yeast Vectors for Shuttle Expression in <i>Escherichia coli</i>

Mo, Qiwen; Fan, Cong

doi:10.1021/acsomega.1c00339

Cited by 17 publications

(11 citation statements)

References 33 publications

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“…The nding suggests that the eukaryotic km.TEF1 promoter from K. marxianus has the ability to allow gene expressions in the prokaryotic host E. coli. A similar study reports that the eukaryotic promoter GAL1/10 from S. cerevisiae could direct express gene in the E. coli [45]. In Fig.…”

Section: Yeast Shuttle Vectors Expressed In Escherichia Colimentioning

confidence: 53%

“…The feasibility of some heterologous yeast promoters in different expression systems have been characterized. For example, Kluyveromyces marxianus TPI and Hansenula polymorpha PMA promoters in P. pastoris [43], GAL1/2 promoters from other Saccharomyces species in S. cerevisiae [34], S. cerevisiae promoters (P GPD , P ADH , P TEF , and P CYC ) in K. marxianus [44] and the eukaryotic promoter GAL1/10 from S. cerevisiae direct expressing gene in E. coli [45]. The development of promoters with broad host properties could enable rapid phenotyping of genetic constructs in different hosts.…”

Section: Introductionmentioning

confidence: 99%

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Design and construction of shuttle expression vectors for non- conventional yeasts and bacteria

Zhang

et al. 2023

Preprint

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Synthetic biology seeks to engineer microbial cells for sustainable efficient production of value-added biofuels and bioproducts from low-cost renewable feedstocks. In order to resolve the conflicts of carbon flux between cell growth and bioproducts synthesis, the dynamic up-regulation on the bioproduct synthesis pathways and down-regulation on the competitive pathways simultaneously could be adjusted by promoter sets with diverse strengths. The development of broad-spectrum promoter libraries comprising promoters of varying strengths for different hosts without tedious reconstruction processes are attractive for biosynthetic engineers. In this study, we observed that five K. marxianus promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) can all express genes in Y. lipolytica and that five Y. lipolytica promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) can all express genes in K. marxianus with variable expression strengths. Interestingly, we also found two yeast promoters could shuttle express reporter genes in P. pastoris, E. coli and C. glutamicum. The yl.TEF1 promoter can also strongly express amylase and RFP in yeast P. pastoris and the eukaryotic promoter km.TEF1 can constitutively strong express RFP in bacterium E. coli and C. glutamicum. The RFP expression strength of the promoter km.TEF1 reached ∼20% to that of the T7 promoter in E. coli and was much stronger (more than 10 times) than in K. marxianus. Our work will expand the future development of broad host acceptable dynamic regulated systems with these broad-spectrum promoters for dynamically orchestrate the carbon flux to maximize target bioproduct synthesis.

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Section: Yeast Shuttle Vectors Expressed In Escherichia Colimentioning

confidence: 53%

Section: Introductionmentioning

confidence: 99%

Design and construction of shuttle expression vectors for non- conventional yeasts and bacteria

Zhang

et al. 2023

Preprint

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“…However, the current fungal expression systems mainly vary in their type of promoter activation, but most of them require are a pre-cloning of the GOI into a specific vector, which limits their applicability to long SM biosynthetic genes. Effort was done with the yeast/ E.coli shuttle vectors in yeast, allowing a multi-gene assembly and co-expression of smaller biosynthetic genes in yeast and Aspergillus [ 68 – 70 ]. Alternatively, reiterative recombination systems assemble multigene constructs in S. cerevisiae , but this endonuclease-induced recombination requires multiple rounds of transformation to recycle selectable markers and is hence a robust, but more laborious technique [ 71 ].…”

Section: Discussionmentioning

confidence: 99%

A genetic tool to express long fungal biosynthetic genes

Kirchgaessner

Wurlitzer

Seibold

et al. 2023

Fungal Biol Biotechnol

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Background Secondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts. Results An expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆pyrG, resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus. We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina. Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF. Conclusions The system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi.

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“…GapC was PCR ampli ed from the genomic DNA of C. acetobutylicum ATCC 824. Plasmid pRS423-AroZ/OMT, pRS423-AsbF/OMT, pRS425-CAR/PPTase, pRS423-HpaB/HpaC, pRS425-BFD/HMO, pRS426-HmaS/OMT, pRS423-MetK/OMT, pRS423-MetK I303V /OMT, pRS423-Xfpk/Pta, pRS426-UbiC/PobA, and pRS425-Sfp/EntD, were all constructed via the golden-gate approach [50]. All the plasmids used in this study are provided in Supplementary Table S3.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Multidimensional engineering of Saccharomyces cerevisiae for improved vanillin synthesis

Mo,

Yuan

2023

Preprint

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Background Vanillin represents one of the most widely used flavoring agents in the world. However, microbial synthesis of vanillin is hindered by the host native metabolism that could rapidly degrade vanillin to the byproducts. Results Here, we report that the industrial workhorse Saccharomyces cerevisiae was engineered by systematic deletion of oxidoreductases to improve the vanillin accumulation. Subsequently, we harnessed the reduced aromatic aldehyde reduction (RARE) yeast platform for de novo synthesis of vanillin from glucose. We investigated multiple coenzyme-A free pathways to improve vanillin production in yeast. The vanillin productivity in yeast was enhanced by multidimensional engineering to optimize the supply of cofactors (NADPH and S-adenosylmethionine) together with metabolic reconfiguration of yeast central metabolism. The final yeast strain with overall 24 genetic modifications produced 365.55 ± 7.42 mg l-1 under shake-flasks, which represents the highest vanillin titer from glucose achieved to date. Conclusions The success of vanillin overproduction in budding yeast showcases the great potential of synthetic biology for the creation of suitable biocatalysts to meet the requirement in industry. Our work lays a foundation for the future implementation of microbial production of aromatic aldehydes in budding yeast.

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New Set of Yeast Vectors for Shuttle Expression in Escherichia coli

Cited by 17 publications

References 33 publications

Design and construction of shuttle expression vectors for non- conventional yeasts and bacteria

Design and construction of shuttle expression vectors for non- conventional yeasts and bacteria

A genetic tool to express long fungal biosynthetic genes

Multidimensional engineering of Saccharomyces cerevisiae for improved vanillin synthesis

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