scholarly journals Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

2020 ◽  
Author(s):  
Zhiqing Wang ◽  
Aarti Doshi ◽  
Ratul Chowdhury ◽  
Yixi Wang ◽  
Costas D. Maranas ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Sensitivity And Specificity ◽  
Negative Selection ◽  
Regulatory Protein ◽  
Polyketide Synthases ◽  
Computational Framework ◽  
Orsellinic Acid ◽  
Acid Lactone ◽  
Triacetic Acid Lactone ◽  
Background Expression

AbstractWe previously described the design of triacetic acid lactone (TAL) biosensor “AraC-TAL1”, based on the AraC regulatory protein. While useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity, and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a “decoy” ligand during negative selection, resulting in isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the IPRO computational framework was employed, followed by two rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

scholarly journals Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

2020 ◽  
Vol 33 ◽  
Author(s):  
Zhiqing Wang ◽  
Aarti Doshi ◽  
Ratul Chowdhury ◽  
Yixi Wang ◽  
Costas D Maranas ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Negative Selection ◽  
Regulatory Protein ◽  
Polyketide Synthases ◽  
Computational Framework ◽  
Orsellinic Acid ◽  
Acid Lactone ◽  
Triacetic Acid Lactone ◽  
Protein Redesign ◽  
Background Expression

Abstract We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

scholarly journals Control of Expression of DivergentPseudomonas putida put Promoters for Proline Catabolism

2000 ◽  
Vol 66 (12) ◽  
pp. 5221-5225 ◽  
Author(s):  
Susana Vı́lchez ◽  
Maximino Manzanera ◽  
Juan L. Ramos
Keyword(s):  
Sequence Analysis ◽  
Transcription Initiation ◽  
Mrna Level ◽  
Regulatory Protein ◽  
Protein Sequence Analysis ◽  
Regulatory Activity ◽  
Proline Catabolism ◽  
C And N ◽  
Background Expression ◽  
Control Of Expression

ABSTRACT Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that theputA and putP genes are transcribed divergently. Expression from the putP and putAgenes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from theput promoters was induced by proline. The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes. The PutA protein acts as a repressor of put gene expression in P. putida because expression from theput promoters is constitutive in a host background with a knockout putA gene. This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896→Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity. Expression from theput promoters in the presence of proline in aputA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be ς54 dependent because the put genes were not expressed in a ς54-deficient background. Expression of the putA and putP genes was equally high in the presence of proline in ς38- and ihf-deficientP. putida backgrounds.

scholarly journals Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation

2018 ◽  
Vol 115 (9) ◽  
pp. 2096-2101 ◽  
Author(s):  
Kelly A. Markham ◽  
Claire M. Palmer ◽  
Malgorzata Chwatko ◽  
James M. Wagner ◽  
Clare Murray ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Building Blocks ◽  
Specific Productivity ◽  
Chemical Production ◽  
Conversion Yield ◽  
Acetyl Coa ◽  
Type Iii Polyketide Synthase ◽  
Chemical Catalysis ◽  
Acid Lactone ◽  
Triacetic Acid Lactone

Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica) can overcome such production limitations owing to a natural propensity for high flux through acetyl–CoA. By exploring three distinct metabolic engineering strategies for acetyl–CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression (PEX10) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.

scholarly journals Cd-Specific Mutants of Mercury-Sensing Regulatory Protein MerR, Generated by Directed Evolution

2011 ◽  
Vol 77 (17) ◽  
pp. 6215-6224 ◽  
Author(s):  
Kaisa M. Hakkila ◽  
Pia A. Nikander ◽  
Sini M. Junttila ◽  
Urpo J. Lamminmäki ◽  
Marko P. Virta
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Metal Binding ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Screening Strategy ◽  
Mercury Resistance ◽  
Content Type ◽  
Green Fluorescent ◽  
Mercury Sensing

ABSTRACTThe mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via themeroperator/promoter. AnEscherichia colicell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.

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