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1.
Biotechnol Lett ; 44(4): 571-580, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35254611

ABSTRACT

Patchoulol is a natural sesquiterpene, which is widely used in perfumes and cosmetics. In the work, the mitochondria of S. cerevisiae were engineered for patchoulol production. The patchoulol titer of mitochondria-compartmentalized strain (1.79 mg/L) was 2.71-fold higher than that of control strain (0.66 mg/L) using genome-integrated patchoulol synthase, indicating that mitochondria compartmentation resulted in higher concentration of FPP (farnesyl pyrophosphate) precursor for patchoulol production. Moreover, when fused FPP synthase and patchoulol synthase was overexpressed in the strain with a mitochondria-localized DMAPP (dimethylallyl diphosphate) pathway, the production of patchoulol increased significantly to 19.24 mg/L, indicating more precursors were provided for patchoulol production. Nevertheless, the introduction of excess foreign proteins into mitochondria might cause a certain stress on mitochondria and showed a negative effect on the growth of yeast cells, which could hinder the expression of foreign pathways and reduce the patchoulol production. In conclusion, mitochondria-engineered yeast cells showed important potential for the enhanced biosynthesis of patchoulol, and further engineering could be considered based on the present work.


Subject(s)
Saccharomyces cerevisiae Proteins , Sesquiterpenes , Metabolic Engineering/methods , Mitochondria/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sesquiterpenes/metabolism
2.
Bioresour Bioprocess ; 9(1): 70, 2022 Jun 27.
Article in English | MEDLINE | ID: mdl-38647553

ABSTRACT

Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π-π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.

3.
Mol Biotechnol ; 34(1): 55-68, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16943572

ABSTRACT

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).


Subject(s)
Cloning, Molecular/methods , Directed Molecular Evolution/methods , Evolution, Molecular , Gene Library , Genetic Engineering/methods , Mutagenesis , Polymerase Chain Reaction/methods , Animals , Directed Molecular Evolution/trends , Humans , Polymerase Chain Reaction/trends
4.
Sheng Wu Gong Cheng Xue Bao ; 22(2): 285-92, 2006 Mar.
Article in Chinese | MEDLINE | ID: mdl-16607958

ABSTRACT

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines


Subject(s)
Apoptosis Regulatory Proteins/biosynthesis , Apoptosis/drug effects , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/pharmacology , tat Gene Products, Human Immunodeficiency Virus/biosynthesis , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/pharmacology , Base Sequence , Cell Line, Tumor , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Survivin , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/pharmacology
5.
Biotechnol Lett ; 27(10): 725-30, 2005 May.
Article in English | MEDLINE | ID: mdl-16049742

ABSTRACT

Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l-1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni2+-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value.


Subject(s)
Antimicrobial Cationic Peptides/biosynthesis , Escherichia coli/metabolism , Recombinant Fusion Proteins/biosynthesis , Amphibian Proteins/biosynthesis , Amphibian Proteins/genetics , Animals , Antimicrobial Cationic Peptides/genetics , Anura , Bioreactors/microbiology , Cell Division/drug effects , Chromatography, High Pressure Liquid/methods , Electrophoresis, Polyacrylamide Gel , Escherichia coli/cytology , Escherichia coli/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Thioredoxins/genetics
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