Improving the Herbicide Resistance of 4-Hydroxyphenylpyruvate Dioxygenase SpHPPD by Directed Evolution

Author(s):  
Haiyan Wang ◽  
Bin Liu ◽  
Peng Lei ◽  
Jianchun Zhu ◽  
Le Chen ◽  
...  
2020 ◽  
Vol 68 (44) ◽  
pp. 12365-12374
Author(s):  
Bin Liu ◽  
Haiyan Wang ◽  
Kaiyun Zhang ◽  
Jianchun Zhu ◽  
Qin He ◽  
...  

2021 ◽  
Author(s):  
Magdy Mahfouz ◽  
Haroon Butt ◽  
Jose Luis Moreno Ramirez

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated into the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as a selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.


2020 ◽  
Vol 31 (2) ◽  
pp. 90-92
Author(s):  
Rob Edwards

Herbicide resistance in problem weeds is now a major threat to global food production, being particularly widespread in wild grasses affecting cereal crops. In the UK, black-grass (Alopecurus myosuroides) holds the title of number one agronomic problem in winter wheat, with the loss of production associated with herbicide resistance now estimated to cost the farming sector at least £0.5 billion p.a. Black-grass presents us with many of the characteristic traits of a problem weed; being highly competitive, genetically diverse and obligately out-crossing, with a growth habit that matches winter wheat. With the UK’s limited arable crop rotations and the reliance on the repeated use of a very limited range of selective herbicides we have been continuously performing a classic Darwinian selection for resistance traits in weeds that possess great genetic diversity and plasticity in their growth habits. The result has been inevitable; the steady rise of herbicide resistance across the UK, which now affects over 2.1 million hectares of some of our best arable land. Once the resistance genie is out of the bottle, it has proven difficult to prevent its establishment and spread. With the selective herbicide option being no longer effective, the options are to revert to cultural control; changing rotations and cover crops, manual rogueing of weeds, deep ploughing and chemical mulching with total herbicides such as glyphosate. While new precision weeding technologies are being developed, their cost and scalability in arable farming remains unproven. As an agricultural scientist who has spent a working lifetime researching selective weed control, we seem to be giving up on a technology that has been a foundation stone of the green revolution. For me it begs the question, are we really unable to use modern chemical and biological technology to counter resistance? I would argue the answer to that question is most patently no; solutions are around the corner if we choose to develop them.


2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.


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