Biomedical subjects

Zhilei Chen

Publications and source records attributed to Zhilei Chen.

7 recordsLinked to original sources

PubMed2026

Biocontrol effect of a solid-state fermentation-derived extract mixture of Trichoderma asperellum on sunflower Sclerotinia rot and associated host defense responses.

Sclerotinia disease is a destructive fungal disease of sunflowers, soybeans, and other economically important crops, causing substantial yield loss and quality deterioration. Long-term reliance on dose-dependent broad-spectrum fungicides is constrained by resistance risks and potential environmental burdens, creating tension with the sustainability goal of "reducing pesticide use while improving efficacy." Here, we explore a Trichoderma spp.-based microbial disease management strategy. Whole-genome sequencing of Trichoderma asperellum TCS007 isolated from Antarctic marine sediments, coupled with genome mining, predicted diverse biosynthetic gene clusters putatively associated with siderophores, polyketides, nonribosomal peptides, and terpenoids; the corresponding metabolites are not chemically confirmed and require further validation. Using a solid-state fermentation workflow, we prepared a fermentation-derived extract mixture (TCS007-SSF-Ex). In vitro assays showed dose-dependent inhibition of Sclerotinia sclerotiorum by TCS007-SSF-Ex (EC50 = 1.252 mg/L), and microscopy revealed cellular damage-consistent changes, including organelle disruption and plasmolysis. Pathogen transcriptomic and metabolism-related analyses indicated broad perturbations in organelle biogenesis and metabolic processes, with significant alterations in pathways associated with succinate, D-glucose, and phenylacetate; these results are consistent with growth inhibition and reduced pathogenicity, but specific molecular targets and causal links remain to be validated. In vivo, under certain application conditions, triple applications increased APX activity (+492.5%) and β-1,3-glucanase activity (+419.6%). Collectively, this work supports a "pathogen suppression-host defense induction" framework and facilitates subsequent identification of active components and mechanistic validation.IMPORTANCESclerotinia diseases cause recurrent and economically important losses in oilseed crops, while long-term fungicide use is constrained by resistance risks and environmental burdens. Trichoderma-based biocontrol is a promising complementary strategy, yet evidence supporting metabolite-containing Trichoderma-derived preparations as immune elicitors remains less consolidated than that for living inoculants, and scalable production routes are still needed. Here, we examine an Antarctic marine sediment-derived strain, Trichoderma asperellum TCS007, and a solid-state fermentation (SSF)-derived extract mixture (TCS007-SSF-Ex) produced via solid-state fermentation. We combine in vitro antifungal assays, pathogen ultrastructural observations, and correlative omics analyses with in vivo measurements of sunflower defense enzymes (APX and β-1,3-glucanase) to evaluate a "pathogen suppression-host defense induction" framework. Our findings support the potential of SSF-derived Trichoderma metabolite mixtures for greener management of Sclerotinia disease and provide a foundation for future chemical identification of active components and mechanistic validation.

Ascomycota↗

PubMed2005

A highly sensitive selection method for directed evolution of homing endonucleases.

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80-100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.

Deoxyribonucleases, Type II Site-Specific↗

PubMed2005

Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches.

Despite their versatility and power in controlling gene regulation in nature, nuclear hormone receptors (NHRs) have largely eluded utility in heterologous gene regulation applications such as gene therapy and metabolic engineering. The main reason for this void is the pleiotropic interference of the receptor-ligand combination with regulatory networks in the host organism. In recent years, numerous strategies have been developed to engineer ligand-receptor pairs that do not cross-interact with host regulatory pathways. However, these strategies have either met with limited success or cannot be readily extended to other ligand-receptor pairs. Here, we present a simple, effective, and readily generalizable strategy for reengineering NHRs to respond specifically to a selected synthetic ligand. The method involves generation of genetic diversity by stepwise individual site saturation mutagenesis of a fixed set of ligand-contacting residues and random point mutagenesis, followed by phenotypic screening based on a yeast two-hybrid system. As a test case, this method was used to alter the specificity of the NHR human estrogen receptor alpha in favor of the synthetic ligand 4,4'-dihydroxybenzil, relative to the natural ligand 17beta-estradiol, by >10(7)-fold. The resulting ligand-receptor pair is highly sensitive to the synthetic ligand in human endometrial cancer cells and is essentially fully orthogonal to the wild-type receptor-natural ligand pair. This method should provide a powerful, broadly applicable tool for engineering receptors/enzymes with improved or novel ligand/substrate specificity.

Binding Sites↗

PubMed2005

Rapid creation of a novel protein function by in vitro coevolution.

We have developed a simple and efficient method for creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves design of a hypothetical pathway for the target function followed by stepwise directed evolution of the corresponding protein along the pathway. As a test case, this strategy was used to engineer variants of human estrogen receptor alpha ligand-binding domain (hERalphaLBD) with novel corticosterone activity. Two steroids, testosterone and progesterone, that provide a progressive structural bridge between 17beta-estradiol and corticosterone, were chosen to assist the directed evolution of hERalphaLBD. A total of approximately 10(6) variants were screened in four rounds of random mutagenesis, resulting in two hERalphaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches and also shed light on the natural evolution of nuclear hormone receptors. This in vitro coevolution approach should provide a powerful, broadly applicable tool for engineering biological molecules and systems with novel functions.

Corticosterone↗

PubMed2004

Directed evolution of human estrogen receptor variants with significantly enhanced androgen specificity and affinity.

Human estrogen receptor alpha (hERalpha) and human androgen receptor exhibit exquisite ligand specificity, which underlies their remarkable ability to effect ligand-regulated gene transcription in a highly distinctive and specific manner. Here we used a directed evolution approach to create hERalpha variants with enhanced androgen specificity and affinity with the goal to better understand the molecular basis of ER ligand specificity and the evolutionary mechanism of nuclear receptors. We developed a sensitive yeast two-hybrid system to screen for hERalpha variants with increased transactivation potency toward testosterone. After two rounds of directed evolution, we identified five hERalpha variants with dramatically improved transactivation potency toward testosterone in both yeast and mammalian cells. These variants showed up to 7,600-fold improvement in the binding affinity for testosterone and only slightly reduced affinity toward 17beta-estradiol. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo led to several unexpected findings including the following. 1) Only two beneficial mutations were needed to create hERalpha variants with near nanomolar affinity for testosterone. 2) Some beneficial mutations were synergistic, context-dependent, or non-additive. 3) Of the five identified beneficial mutations, four of them were not in the ER ligand binding pocket and yet exerted important action on ligand specificity. 4) The single ligand-contacting mutation E353Q plays a dominant role in discriminating androgens and estrogens. These results, viewed in conjunction with the ligand exploitation model of nuclear receptor evolution, suggest that the mutation E353Q may represent a key event in the evolution of androgen receptors from an ancestral estrogen receptor and that ligand promiscuity may play an important role in the creation of new nuclear receptors via divergent evolution.

Amino Acid Sequence↗

PubMed2003

A highly efficient and sensitive screening method for trans-activation activity of estrogen receptors.

We describe a highly efficient and sensitive yeast-based screening method for isolating human estrogen receptor alpha (ERalpha) mutants with altered trans-activation activity. This method takes advantage of the fact that estrogen receptor is a ligand-activated transcription factor, and links the transactivation activity of estrogen receptor to the growth rate of yeast cells. We used this method to screen a library of human ERalpha mutants created by random mutagenesis of the ligand binding domain of human ERalpha in the presence of ligand 17beta-estradiol (E(2)). We isolated several human ERalpha mutants with significantly altered trans-activation activity toward E(2) in yeast cells. We also used this method to screen a library of chemical compounds and showed that it can be used to rapidly identify estrogenic compounds and the different cell growth rates for these estrogenic compounds correlated well with their relative binding affinities. Thus, this method is suitable for selecting novel estrogenic compounds and estrogen receptor mutants. In principle, this method might also be used to isolate mutants of any nuclear receptors with altered trans-activation activity, which may greatly facilitate their structural and functional studies.

Amitrole↗

PubMed2002

Directed evolution of enzymes and pathways for industrial biocatalysis.

Directed evolution has become a powerful tool for developing enzyme and whole cell based biocatalysts. Significant recent advances include the creation of novel enzyme functions and the development of several new efficient directed evolution methods. The combination of directed evolution and rational design promises to accelerate the development of biocatalysts for applications in the pharmaceutical, chemical and food industries.

Catalysis↗