Designing a whole-cell biosensor applicable for S-adenosyl-l-methionine-dependent methyltransferases.

This study was undertaken to develop a high-throughput screening strategy using a whole-cell biosensor to enhance methyl-group transfer, a rate-limiting step influenced by intracellular methyl donor availability and methyltransferase efficiency. An l-homocysteine biosensor was designed based on regulatory protein MetR from Escherichia coli, which rapidly reported intracellular l-homocysteine accumulation resulted from S-adenosyl-l-homocysteine (SAH) formation after methyl-group transfer. Using S-adenosyl-l-methionine (SAM) as a methyl donor, this biosensor was applied to caffeic acid 3-O-methyltransferase derived from Arabidopsis thaliana (AtComT).

Engineering of -Based Tryptophan Biosensors for Dynamic Control of Violacein Production.

Tryptophan not only serves as a fundamental building block for protein synthesis but also acts as a metabolic precursor for a diverse array of high-value chemicals. Although a few tryptophan-responsive biosensors are currently available, there is a growing interest in developing high-performance biosensors. In this study, we create a miniature toolkit of tryptophan biosensors based upon the leader regulatory region of the operon, which is responsible for tryptophan catabolism in .

Designing a highly efficient type III polyketide whole-cell catalyst with minimized byproduct formation.

Background: Polyketide synthases (PKSs) are classified into three types based on their enzyme structures. Among them, type III PKSs, catalyzing the iterative condensation of malonyl-coenzyme A (CoA) with a CoA-linked starter molecule, are important synthases of valuable natural products. However, low efficiency and byproducts formation often limit their applications in recombinant overproduction.

Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol.

Eugenol, the main component of essential oil from the clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway.

Engineering and application of LacI mutants with stringent expressions.

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the P /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties.

Atezolizumab Plus Bevacizumab Combined with Transarterial Embolization Plus Hepatic Arterial Infusion Chemotherapy for Unresectable Hepatocellular Carcinoma with a Diameter >8 Cm: A Retrospective Study.

Purpose: Local in combination with systemic therapy might be an option for patients with advanced unresectable hepatocellular carcinoma (uHCC). This study examined the clinical benefits and adverse events (AEs) of first-line transarterial embolization (TAE) and hepatic arterial infusion chemotherapy (HAIC) combined with atezolizumab (Atezo) and bevacizumab (Bev) in patients with uHCC of a diameter larger than 8 cm.

Patients And Methods: This retrospective study included patients with uHCC of a diameter larger than 8 cm who were treated with first-line Atezo-Bev and TAE+HAIC at the First Affiliated Hospital of Sun Yat-Sen University between September 30, 2019, and September 30, 2022.

Anlotinib combined with transarterial chemoembolization for unresectable hepatocellular carcinoma associated with hepatitis B virus: a retrospective controlled study.

Purpose: To investigate the efficacy and safety of combined treatment of anlotinib and transarterial chemoembolization (TACE) in patients with unresectable hepatocellular carcinoma (uHCC) associated with hepatitis B virus (HBV) infection.

Methods: We retrospectively collected the data of 96 uHCC patients associated with HBV infection who received either TACE only (TO group; n = 64) or anlotinib combined with TACE (TA group; n = 32) from January 2017 to January 2021. The primary endpoint was overall survival (OS).

Comprehensive analysis of ZNF692 as a potential biomarker associated with immune infiltration in a pan cancer analysis and validation in hepatocellular carcinoma.

Currently, the roles of ZNF692 have been documented exclusively in lung, colon, and cervical cancers. However, its involvement in pan cancer remains unknown. In this study, we employed bioinformatics analysis and experimental validation to investigate the role of ZNF692 in pan cancer.

Designing glucose utilization "highway" for recombinant biosynthesis.

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRP mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRP.

Engineering an SspB-mediated degron for novel controllable protein degradation.

Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory.

[Enzymatic properties of α-L-rhamnosidase and the factors affecting its activity: a review].

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range.

Whole-Cell Biosensors Aid Exploration of Vanillin Transmembrane Transport.

Transcriptional regulatory protein (TRP)-based whole-cell biosensors are widely used nowadays. Here, they were demonstrated to have great potential application in screening cell efflux and influx pumps for small molecules. First, a vanillin whole-cell biosensor was developed by altering the specificity of a TRP, VanR, and strains with improved vanillin productions that were selected from a random genome mutagenesis library by using this biosensor as a high-throughput screening tool.

Biosynthesis of Chlorogenic Acid Using an Artificial Microbial Community.

Engineering an artificial microbial community for natural product production is a promising strategy. As mono- and dual-culture systems only gave non-detectable or minimal chlorogenic acid (CGA) biosynthesis, here, a polyculture of three recombinant strains, acting as biosynthetic modules of caffeic acid (CA), quinic acid (QA), and CGA, was designed and used for CGA biosynthesis. An influx transporter of 3-dehydroshikimic acid (DHS)/shikimic acid (SA), ShiA, was introduced into the QA module-a DHS auxotroph.

Metabolic Engineering for Improved Curcumin Biosynthesis in .

The biosynthetic efficiency of curcumin, a highly bioactive compound from the plant , needs to be improved. In this study, we performed host cell and biosynthetic pathway engineering to improve curcumin biosynthesis. Using -directed evolution, the expression level of curcuminoid synthase (CUS), the rate-limiting enzyme in the curcumin biosynthetic pathway, was significantly improved.

CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli.

Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host.

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps.

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps.

Development of a highly efficient and specific L-theanine synthase.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied.

Dynamic control of toxic natural product biosynthesis by an artificial regulatory circuit.

To mimic the delicately regulated metabolism in nature for improved efficiency, artificial and customized regulatory components for dynamically controlling metabolic networks in multiple layers are essential in laboratory engineering. For this purpose, a novel regulatory component for controlling vanillin biosynthetic pathway was developed through directed evolution, which was responsive to both the product vanillin and substrate ferulic acid, with different capacities. This regulatory component facilitated pathway expression via dynamic control of the intracellular substrate and product concentrations.

Promiscuous enzymatic activity-aided multiple-pathway network design for metabolic flux rearrangement in hydroxytyrosol biosynthesis.

Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate.

Engineering the effector specificity of regulatory proteins for the in vitro detection of biomarkers and pesticide residues.

Transcriptional regulatory proteins (TRPs)-based whole-cell biosensors are promising owing to their specificity and sensitivity, but their applications are currently limited. Herein, TRPs were adapted for the extracellular detection of a disease biomarker, uric acid, and a typical pesticide residue, carbaryl. A mutant regulatory protein that specifically recognizes carbaryl as its non-natural effector and activates transcription upon carbaryl binding was developed by engineering the regulatory protein TtgR from Pseudomonas putida.

Towards the construction of high-quality mutagenesis libraries.

Objectives: To improve the quality of mutagenesis libraries in directed evolution strategy.

Results: In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency.

Biosensor-aided high-throughput screening of hyper-producing cells for malonyl-CoA-derived products.

Background: Malonyl-coenzyme A (CoA) is an important biosynthetic precursor in vivo. Although Escherichia coli is a useful organism for biosynthetic applications, its malonyl-CoA level is too low.

Results: To identify strains with the best potential for enhanced malonyl-CoA production, we developed a whole-cell biosensor for rapidly reporting intracellular malonyl-CoA concentrations.

Monitoring in vivo metabolic flux with a designed whole-cell metabolite biosensor of shikimic acid.

Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications.