Site directed mutagenesis as a precision tool to enable synthetic biology with engineered modular polyketide synthases

Modular polyketide synthases(PKSs)are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds,from new pharmaceuticals to high value commodity and specialty chemicals.Their colinear biosynthetic logic has been viewed as a promising platform for synthetic biology for decades.Due to this colinearity,domain swapping has long been used as a strategy to introduce molecular diversity.However,domain swapping often fails because it perturbs critical protein-protein interactions within the PKS.With our increased level of structural elucidation of PKSs,using judicious targeted mutations of individual residues is a more precise way to introduce molecular diversity with less potential for global disruption of the protein architecture.Here we review examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity,affect protein stability and interdomain communication,and promote more complex catalytic reactivity.

Development of an Engineered Sugar Aminotransferase with Simultaneously Improved Stability and Non-Natural Substrate Activity to Synthesize the Glucosidase Inhibitor Valienamine

Sugar aminotransferases(SATs)catalyze the installation of chiral amines onto specific keto sugars,pro-ducing bioactive amino sugars.Their activity has been utilized in artificial reactions,such as using the SAT WecE to transform valienone into the valuable a-glucosidase inhibitor valienamine.However,the low thermostability and limited activity on non-natural substrates have hindered their applications.Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity.A customized combinatorial active-site saturation test-iterative saturation mutagenesis(CAST-ISM)strategy was used to simultaneously enhance the stability and activity of WecE toward valienone.Fourteen hotspots related to improving the stability-\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE.Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis(SSM)and iterative evolution cycles.Compared with those of the wild-type(WT)WecE,the quadruple mutant M4(Y321F/K209F/V318R/F319V)displayed a 641.49-fold increase in half-life(t_(1/2))at 40℃ and a 31.37-fold increase in activity toward the non-natural substrate valienone.The tri-ple mutant M3(Y321F/K209F/V318R)demonstrated an 83.04-fold increase in(t_(1/2))at 40℃and a 37.77-fold increase in activity toward valienone.The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance,compared with those of the WT,which improved the stability and activity of the obtained mutants.Thus,we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.

In Situ Saturating Mutagenesis Screening Identifies a Functional Genomic Locus that Regulates Ucp1 Expression

A better understanding of the molecular mechanisms that control the UCP1 expression in brown and beige adipocytes is essential for us to modulate adipose cell fate and promote thermogenesis,which may provide a therapeutic view for the treatment of obesity and obesity-related diseases.To systematically identify cis-element(s)that transcriptionally regulates Ucp1,we here took advantage of the high-throughput CRIPSR-Cas9 screening system,and performed an in situ saturating mutagenesis screen,by using a customized sgRNA library targeting the~20 kb genomic region near Ucp1.Through the screening,we have identified several genomic loci that may contain key regulatory element for Ucp1 expression in cultured brown and white adipocytes in vitro,and in inguinal white adipose tissue in vivo.Our study highlights a broadly useful approach for studying cis-regulatory elements in a high-throughput manner.

Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.