Exploiting genetic diversity by directed evolution: molecular breeding of type III polyketide synthases improves productivity

2008 ◽  
Vol 4 (3) ◽  
pp. 246 ◽  
Author(s):  
Wenjuan Zha ◽  
Sheryl B. Rubin-Pitel ◽  
Huimin Zhao
2009 ◽  
Vol 284 (17) ◽  
pp. 11160-11170 ◽  
Author(s):  
Yohei Katsuyama ◽  
Tomoko Kita ◽  
Nobutaka Funa ◽  
Sueharu Horinouchi

2020 ◽  
Vol 33 ◽  
Author(s):  
Zhiqing Wang ◽  
Aarti Doshi ◽  
Ratul Chowdhury ◽  
Yixi Wang ◽  
Costas D Maranas ◽  
...  

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.


2019 ◽  
Vol 294 (41) ◽  
pp. 15121-15136 ◽  
Author(s):  
Hiroyuki Morita ◽  
Chin Piow Wong ◽  
Ikuro Abe

Plant Disease ◽  
2012 ◽  
Vol 96 (2) ◽  
pp. 193-203 ◽  
Author(s):  
F. J. Jaciani ◽  
J. A. Ferro ◽  
M. I. T. Ferro ◽  
C. Vernière ◽  
O. Pruvost ◽  
...  

Exclusion and eradication or management based on an integrated approach with less susceptible varieties, copper-based bactericides, and windbreaks are the two main strategies used to prevent or control citrus canker. Field tolerance or resistance to citrus canker is not found in the most important commercial sweet orange cultivars, and pathogen-derived resistance has been developed and applied in different crops to obtain resistant genotypes to plant pathogens. We describe the development of DNA primers and probes based on the type III effector genes avrXacE1, avrXacE2, avrXacE3, avrBs2, pthA4, hpaF, and XAC3090 (leucine rich protein), and their application in the evaluation of the genetic diversity of the pathogen. A total of 49 haplotypes were identified in 157 strains by Southern blot analysis. No genetic polymorphism was detected by BOX elements - and enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR) analysis, nor with the genes avrBs2, XAC3090, and hpaF. Nei's genetic diversity indexes varied from 0.65 to 0.96 for subcollections of the pathogen. One or few haplotypes were most frequent in the strain collection, but several haplotypes were represented by solely one or few strains. The PthA4 probe resulted in the higher number of haplotypes identified in the Brazilian subcollections. Greater variation in the frequency of haplotypes occurred within subcollections (93.7%) than among subcollections. Only some haplotypes were genetically distant from all others, especially those originated from Rio Grande do Sul and Santa Catarina states. These bacterial effectors are widely spread in the collections and are useful for a better understanding of the host–pathogen interaction and the search for resistance genes in host and nonhost plants.


2012 ◽  
Vol 288 (4) ◽  
pp. 2767-2777 ◽  
Author(s):  
Sun Young Kim ◽  
Che C. Colpitts ◽  
Gertrud Wiedemann ◽  
Christina Jepson ◽  
Mehrieh Rahimi ◽  
...  

2013 ◽  
Vol 7 (3) ◽  
pp. 371-381 ◽  
Author(s):  
Yan-Wu Guo ◽  
Hui-Li Guo ◽  
Xing Li ◽  
Li-Li Huang ◽  
Bo-Ning Zhang ◽  
...  

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