Cancer progression is associated with the evolutionary accumulation of genetic mutations that are biologically significant. Mutations of the androgen receptor (AR) are associated with the development ...of prostate cancer (PCa) by responding to non-androgenic hormones, and the lack of annotations in their responsiveness to hormone ligands remains a daunting challenge. Here, we have used a yeast reporter system to quickly evaluate the responsiveness of all fifty clinical AR mutations to a variety of steroidal ligands including dihydrotestosterone (DHT), 17β-estradiol (E2), progesterone (PROG), and cyproterone acetate (CPA). Based on an AR-driven reporter that synthesizes histidine, a basic amino acid required for yeast survival and propagation, the yeast reporter system enabling clonal selection was further empowered by combining with a random DNA mutagenesis library to simulate the natural evolution of AR gene under the selective pressures of steroidal ligands. In a time-frame of 1-2 weeks, 19 AR mutants were identified, in which 11 AR mutants were validated for activation by tested steroidal compounds. The high efficiency of our artificial evolution strategy was further evidenced by a sequential selection that enabled the discovery of multipoint AR mutations and evolution directions under the pressure of steroidal ligands. In summary, our designer yeast is a portable reporter module that can be readily adapted to streamline high-throughput AR-compound screening, used as a PCa clinical reference, and combined with additional bioassay systems to further extend its potential.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Polyethylene terephthalate (PET) biodegradation is regarded as an environmentally friendly degradation method. In this study, an artificial microbial consortium composed of
,
and two metabolically ...engineered
was constructed to degrade PET. First, a two-species microbial consortium was constructed with two engineered
that could secrete PET hydrolase (PETase) and monohydroxyethyl terephthalate hydrolase (MHETase), respectively; it could degrade 13.6% (weight loss) of the PET film within 7 days. A three-species microbial consortium was further obtained by adding
to reduce the inhibition caused by terephthalic acid (TPA), a breakdown product of PET. The weight of PET film was reduced by 31.2% within 3 days, achieving about 17.6% improvement compared with the two-species microbial consortium. Finally,
was introduced to reduce the inhibition caused by ethylene glycol (EG), another breakdown product of PET, obtaining a four-species microbial consortium. With the four-species consortium, the weight loss of PET film reached 23.2% under ambient temperature. This study constructed and evaluated the artificial microbial consortia in PET degradation, which demonstrated the great potential of artificial microbial consortia in the utilization of complex substrates, providing new insights for biodegradation of complex polymers.
Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we ...successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.
Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step ...fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in
, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from
were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in
cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in
. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.
Biofoundries provide an integrated infrastructure to enable the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. Many ...biofoundries are being built and a Global Biofoundry Alliance has recently been established to coordinate activities worldwide.
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•This paper introduces current advances of biocontainment strategies.•This paper summarizes non-natural moleculars and orthogonal central dogma systems for biosafety application.•The ...Tianjin Biosecurity Guidelines focus on the prevention of misuse of bioscience research without interfering with beneficial biological research.
Synthetic biotechnology has led to the widespread application of genetically modified organisms (GMOs) in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination by horizontal gene transfer, resulting in unpredictable biosafety risks. To deal with these challenges, many effective methods have been developed for biocontainment. In this article, we summarize and discuss recent advances in biocontainment strategies from three aspects: DNA replication, transcriptional regulation, and protein translation. We also briefly introduce the efforts in the biocontainment convention, such as the recent publication of the Tianjin Biosecurity Guidelines for the Code of Conduct for Scientists.
Poly(ethylene terephthalate) hydrolase (PETase) from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate) (PET) at room temperature, and is thus regarded as a ...potential tool to solve the issue of polyester plastic pollution. Therefore, we explored the interaction between PETase and the substrate (a dimer of the PET monomer ethylene terephthalate, 2PET), using a model of PETase and its substrate. In this study, we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering. These PETase mutants were designed and tested. The enzymatic activities of the R61A, L88F, and I179F mutants, which were obtained with a rapid cell-free screening system, exhibited 1.4 fold, 2.1 fold, and 2.5 fold increases, respectively, in comparison with wild-type PETase. The I179F mutant showed the highest activity, with the degradation rate of a PET film reaching 22.5 mg per μmol·L−1 PETase per day. Thus, this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites, and has further illustrated the potential of biodegradable plastics.
Synthetic biology requires toolbox of promoters to finely tune gene expression levels for building up efficient cell factories. Yeast promoters owned variable core promoter regions between the ...TATA-box and transcriptional starting site (TSS) at the length mostly around 20-80 bases. This region allowed flexible design of artificial promoter but potentially demand special base motifs to maintain or enhance the promoter's strength.
Here, we designed and screened the base motifs and tested the activities of yeast artificial core promoters. Different 30 bases of artificial sequences led to variable expression levels of CrtY enzyme which determined the lycopene-carotene compositions, represented in the colony-color spectrum of red-orange-yellow. The upstream sequences of two strong promoter P
and P
and two starting strains with distinguishable lycopene production levels were utilized to characterize the promoter sequences. Different partition designs of T-rich or G/C-rich base motifs led to distinguishable colony-color distributions. Finally, we screened a champion promoter with a highest 5.5-fold enhancement of lycopene-carotene transformation. Another selected promoter generated a highest beta-carotene production as 7.4 mg/g DCW.
This work offered an approach to redesign promoter with artificial sequences. We concluded that the core promoter region could be designated as 30 bases and different base motifs would enhance or weaken the promoter's strength. Generally, more T-rich elements, higher %T and lower G/C percentage were beneficial to enhance the strength of artificial core promoter.
Increasingly complex synthetic environmental pollutants are prompting further research into bioremediation, which is one of the most economical and safest means of environmental restoration. From the ...current research, using microbial consortia to degrade complex compounds is more advantageous compared to using isolated bacteria, as the former is more adaptable and stable within the growth environment and can provide a suitable catalytic environment for each enzyme required by the biodegradation pathway. With the development of synthetic biology and gene-editing tools, artificial microbial consortia systems can be designed to be more efficient, stable, and robust, and they can be used to produce high-value-added products with their strong degradation ability. Furthermore, microbial consortia systems are shown to be promising in the degradation of complex compounds. In this review, the strategies for constructing stable and robust microbial consortia are discussed. The current advances in the degradation of complex compounds by microbial consortia are also classified and detailed, including plastics, petroleum, antibiotics, azo dyes, and some pollutants present in sewage. Thus, this paper aims to support some helps to those who focus on the degradation of complex compounds by microbial consortia.
Cancer progression is associated with the evolutionary accumulation of genetic mutations that are biologically significant. Mutations of the androgen receptor (AR) are associated with the development ...of prostate cancer (PCa) by responding to non-androgenic hormones, and the lack of annotations in their responsiveness to hormone ligands remains a daunting challenge. Here, we have used a yeast reporter system to quickly evaluate the responsiveness of all fifty clinical AR mutations to a variety of steroidal ligands including dihydrotestosterone (DHT), 17β-estradiol (E2), progesterone (PROG), and cyproterone acetate (CPA). Based on an AR-driven reporter that synthesizes histidine, a basic amino acid required for yeast survival and propagation, the yeast reporter system enabling clonal selection was further empowered by combining with a random DNA mutagenesis library to simulate the natural evolution of AR gene under the selective pressures of steroidal ligands. In a time-frame of 1-2 weeks, 19 AR mutants were identified, in which 11 AR mutants were validated for activation by tested steroidal compounds. The high efficiency of our artificial evolution strategy was further evidenced by a sequential selection that enabled the discovery of multipoint AR mutations and evolution directions under the pressure of steroidal ligands. In summary, our designer yeast is a portable reporter module that can be readily adapted to streamline high-throughput AR-compound screening, used as a PCa clinical reference, and combined with additional bioassay systems to further extend its potential.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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