The Construction of the Self-Induced Sal System and Its Application in Salicylic Acid Production.

The design and construction of more complex and delicate genetic control circuits suffer from poor orthogonality in quorum sensing (QS) systems. The Sal system, which relies on salicylic acid as a signaling molecule, is an artificially engineered regulatory system with a structure that differs significantly from that of natural QS signaling molecules. Salicylic acid is an important drug precursor, mainly used in the production of drugs such as aspirin and anti-HIV drugs. However, there have been no reports on the construction of a self-induced Sal system in single cells. In this study, a high-copy plasmid backbone was used to construct the regulatory proteins and a self-induced promoter of salicylic acid in E. coli by adjusting the precise regulation of key gene expression; the sensitivity and induction range of this system were improved. Subsequently, the exogenous gene pchBA was introduced in E. coli to extend the shikimate pathway and synthesize salicylic acid, resulting in the construction of the first complete self-induced Sal system. Finally, the self-induced Sal System was combined with artificial trans-encoded sRNAs (atsRNAs) to repress the growth-essential gene ppc and accumulate the precursor substance PEP, thereby increasing the titer of salicylic acid by 151%. This construction of a self-induced artificial system introduces a new tool for selecting communication tools and induction systems in synthetic biology and metabolic engineering, but also demonstrates a self-inducible pathway design strategy for salicylic acid biosynthesis.

Construction of cascade circuits for dynamic temporal regulation and its application to PHB production.

BACKGROUND: To maximize the production capacity and yield of microbial cell factories, metabolic pathways are generally modified with dynamic regulatory strategies, which can effectively solve the problems of low biological yield, growth retardation and metabolic imbalance. However, the strategy of dynamic regulating multiple genes in different time and order is still not effectively solved. Based on the quorum-sensing (QS) system and the principle of cascade regulation, we studied the sequence and time interval of gene expression in metabolic pathways. RESULTS: We designed and constructed a self-induced dynamic temporal regulatory cascade circuit in Escherichia coli using the QS system and dual regulatory protein cascade and found that the time intervals of the cascade circuits based on the Tra, Las system and the Lux, Tra system reached 200 min and 150 min, respectively. Furthermore, a dynamic temporal regulatory cascade circuit library with time intervals ranging from 110 to 310 min was obtained based on this circuit using promoter engineering and ribosome binding site replacement, which can provide more selective synthetic biology universal components for metabolic applications. Finally, poly-ß-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of the cascade circuit library. The content of PHB increased 1.5-fold. Moreover, circuits with different time intervals and different expression orders were found to have different potentials for application in PHB production, and the preferred time-interval circuit strain C2-max was identified by screening. CONCLUSIONS: The self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.

Creating Polyploid Escherichia Coli and Its Application in Efficient L-Threonine Production.

Prokaryotic genomes are generally organized in haploid. In synthetic biological research, efficient chassis cells must be constructed to produce bio-based products. Here, the essential division of the ftsZ gene to create functional polyploid E. coli is regulated. The artificial polyploid E. coli containing 2-4 chromosomes is confirmed through PCR amplification, terminator localization, and flow cytometry. The polyploid E. coli exhibits a larger cell size, and its low pH tolerance and acetate resistance are stronger than those of haploid E. coli. Transcriptome analysis shows that the genes of the cell's main functional pathways are significantly upregulated in the polyploid E. coli. These advantages of the polyploid E. coli results in the highest reported L-threonine yield (160.3 g L-1 ) in fed-batch fermentation to date. In summary, an easy and convenient method for constructing polyploid E. coli and demonstrated its application in L-threonine production is developed. This work provides a new approach for creating an excellent host strain for biochemical production and studying the evolution of prokaryotes and their chromosome functions.

Unique Raoultella species isolated from petroleum contaminated soil degrades polystyrene and polyethylene.

Polyolefin plastics, such as polyethylene (PE) and polystyrene (PS), are the most widely used synthetic plastics in our daily life. However, the chemical structure of polyolefin plastics is composed of carbon-carbon (C-C) bonds, which is extremely stable and makes polyolefin plastics recalcitrant to degradation. The growing accumulation of plastic waste has caused serious environmental pollution and has become a global environmental concern. In this study, we isolated a unique Raoultella sp. DY2415 strain from petroleum-contaminated soil that can degrade PE and PS film. After 60 d of incubation with strain DY2415, the weight of the UV-irradiated PE (UVPE) film and PS film decreased by 8% and 2%, respectively. Apparent microbial colonization and holes on the surface of the films were observed by scanning electron microscopy (SEM). Furthermore, the Fourier transform infrared spectrometer (FTIR) results showed that new oxygen-containing functional groups such as -OH and -CO were introduced into the polyolefin molecular structure. Potential enzymes that may be involved in the biodegradation of polyolefin plastics were analyzed. These results demonstrate that Raoultella sp. DY2415 has the ability to degrade polyolefin plastics and provide a basis for further investigating the biodegradation mechanism.

A Molecular Switch-Integrated Nanoplatform Enables Photo-Unlocked Antibacterial Drug Delivery for Synergistic Abscess Therapy.

Drug delivery systems (DDSs) capable of sequential multistage drug release are urgently needed for antibacterial applications. Herein, a molecular switch-integrated, photo-responsive nanoplatform is reported based on hollow mesoporous silica nanospheres (HMSN) loaded with silver nanoparticles (Ag NPs), vancomycin (Van), and hemin (HAVH) for bacteria elimination and abscess therapy. Upon near-infrared (NIR) light irradiation, the molecular switch, hemin, can effuse from the mesopores of HMSN, triggering the release of pre-loaded Ag+ and Van, which enables photothermal-modulated drug release and synergistic photothermal-chemo therapy (PTT-CHT). The HAVH_NIR irreversibly disrupts the bacterial cell membrane, facilitating the penetration of Ag+ and Van. It is found that these compounds restrain the transcription and translation of ribosomes and lead to rapid bacterial death. Furthermore, hemin can effectively inhibit excessive inflammatory responses associated with the treatment, promoting accelerated wound healing in a murine abscess model. This work presents a new strategy for antibacterial drug delivery with high controllability and extendibility, which may benefit the development of smart multifunctional nanomedicine for diseases not limited to bacterial infections.

[Advances in biodegradation of polyolefin plastics].

Polyolefin plastics are a group of polymers with C-C backbone that have been widely used in various areas of daily life. Due to their stable chemical properties and poor biodegradability, polyolefin plastic waste continues to accumulate worldwide, causing serious environmental pollution and ecological crises. In recent years, biological degradation of polyolefin plastics has attracted considerable attention. The abundant microbial resources in the nature offer the possibility of biodegradation of polyolefin plastic waste, and microorganisms capable of degrading polyolefin have been reported. This review summarizes the research progress on the biodegradation microbial resources and the biodegradation mechanisms of polyolefin plastics, presents the current challenges in the biodegradation of polyolefin plastics, and provides an outlook on future research directions.

A synthetic population-level oscillator in non-microfluidic environments.

Synthetic oscillators have become a research hotspot because of their complexity and importance. The construction and stable operation of oscillators in large-scale environments are important and challenging. Here, we introduce a synthetic population-level oscillator in Escherichia coli that operates stably during continuous culture in non-microfluidic environments without the addition of inducers or frequent dilution. Specifically, quorum-sensing components and protease regulating elements are employed, which form delayed negative feedback to trigger oscillation and accomplish the reset of signals through transcriptional and post-translational regulation. We test the circuit in devices with 1 mL, 50 mL, 400 mL of medium, and demonstrate that the circuit could maintain stable population-level oscillations. Finally, we explore potential applications of the circuit in regulating cellular morphology and metabolism. Our work contributes to the design and testing of synthetic biological clocks that function in large populations.

Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine.

L-threonine is an essential amino acid used widely in food, cosmetics, animal feed and medicine. The thrABC operon plays an important role in regulating the biosynthesis of L-theronine. In this work, we systematically analyzed the effects of separating thrAB and thrC in different proportions on strain growth and L-threonine production in Escherichia coli firstly. The results showed that higher expression of thrC than thrAB enhanced cell growth and L-threonine production; however, L-threonine production decreased when the thrC proportion was too high. The highest L-threonine production was achieved when the expression intensity ratio of thrAB to thrC was 3:5. Secondly, a stationary phase promoter was also used to dynamically regulate the expression of engineered thrABC. This strategy improved cell growth and shortened the fermentation period from 36 h to 24 h. Finally, the acetate metabolic overflow was reduced by deleting the ptsG gene, leading to a further increase in L-threonine production. With these efforts, the final strain P 2.1 -2901ΔptsG reached 40.06 g/L at 60 h fermentation, which was 96.85% higher than the initial control strain TH and the highest reported titer in shake flasks. The maximum L-threonine yield and productivity was obtained in reported fed-batch fermentation, and L-threonine production is close to the highest titer (127.30 g/L). In this work, the expression ratio of genes in the thrABC operon in E. coli was studied systematically, which provided a new approach to improve L-threonine production and its downstream products.

Biodegradation of polyurethane by the microbial consortia enriched from landfill.

Polyurethanes (PU) are one of the most used categories of plastics and have become a significant source of environmental pollutants. Degrading the refractory PU wastes using environmentally friendly strategies is in high demand. In this study, three microbial consortia from the landfill leachate were enriched using PU powder as the sole carbon source. The consortia efficiently degraded polyester PU film and accumulated high biomass within 1 week. Scanning electron microscopy, Fourier transform infrared spectroscopy, and contact angle analyses showed significant physical and chemical changes to the PU film after incubating with the consortia for 48 h. In addition, the degradation products adipic acid and butanediol were detected by high-performance liquid chromatography in the supernatant of the consortia. Microbial composition and extracellular enzyme analyses revealed that the consortia can secrete esterase and urease, which were potentially involved in the degradation of PU. The dominant microbes in the consortia changed when continuously passaged for 50 generations of growth on the PU films. This work demonstrates the potential use of microbial consortia in the biodegradation of PU wastes. KEY POINTS: • Microbial consortia enriched from landfill leachate degraded polyurethane film. • Consortia reached high biomass within 1 week using polyurethane film as the sole carbon source. • The consortia secreted potential polyurethane-degrading enzymes.

Genetically encoded ATP and NAD(P)H biosensors: potential tools in metabolic engineering.

To date, many metabolic engineering tools and strategies have been developed, including tools for cofactor engineering, which is a common strategy for bioproduct synthesis. Cofactor engineering is used for the regulation of pyridine nucleotides, including NADH/NAD+ and NADPH/NADP+, and adenosine triphosphate/adenosine diphosphate (ATP/ADP), which is crucial for maintaining redox and energy balance. However, the intracellular levels of NADH/NAD+, NADPH/NADP+, and ATP/ADP cannot be monitored in real time using traditional methods. Recently, many biosensors for detecting, monitoring, and regulating the intracellular levels of NADH/NAD+, NADPH/NADP+, and ATP/ADP have been developed. Although cofactor biosensors have been mainly developed for use in mammalian cells, the potential application of cofactor biosensors in metabolic engineering in bacterial and yeast cells has received recent attention. Coupling cofactor biosensors with genetic circuits is a promising strategy in metabolic engineering for optimizing the production of biochemicals. In this review, we focus on the development of biosensors for NADH/NAD+, NADPH/NADP+, and ATP/ADP and the potential application of these biosensors in metabolic engineering. We also provide critical perspectives, identify current research challenges, and provide guidance for future research in this promising field.

Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering Pseudomonas Putida.

Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.

Random genome reduction coupled with polyhydroxybutyrate biosynthesis to facilitate its accumulation in Escherichia coli.

Genome reduction has been emerged as a powerful tool to construct ideal chassis for synthetic biology. Random genome reduction couple genomic deletion with growth and has the potential to construct optimum genome for a given environment. Recently, we developed a transposon-mediated random deletion (TMRD) method that allows the random and continuous reduction of Escherichia coli genome. Here, to prove its ability in constructing optimal cell factories, we coupled polyhydroxybutyrate (PHB) accumulation with random genome reduction and proceeded to reduce the E. coli genome. Five mutants showed high biomass and PHB yields were selected from 18 candidates after ten rounds of genome reduction. And eight or nine genomic fragments (totally 230.1-270.0 Kb) were deleted in their genomes, encompassing 4.95%-5.82% of the parental MG1655 genome. Most mutants displayed better growth, glucose utilization, protein expression, and significant increase of electroporation efficiency compared with MG1655. The PHB content and concentration enhanced up to 13.3%-37.2% and 60.2%-102.9% when batch fermentation was performed in M9-glucose medium using the five mutants. Particularly, in mutant H16, lacking 5.28% of its genome, the increase of biomass and PHB concentration were more than 50% and 100% compared with MG1655, respectively. This work expands the strategy for creating streamlined chassis to improve the production of high value-added products.

A Gene Circuit Combining the Endogenous I-E Type CRISPR-Cas System and a Light Sensor to Produce Poly-ß-Hydroxybutyric Acid Efficiently.

'Metabolic burden,' which arises when introducing exogenic synthesizing pathways into a host strain, remains a challenging issue in metabolic engineering. Redirecting metabolic flux from cell growth to product synthesis at an appropriate culture timepoint is ideal for resolving this issue. In this report, we introduce optogenetics-which is capable of precise temporal and spatial control-as a genetic switch, accompanied by the endogenous type I-E CRISPRi system in Escherichia coli (E. coli) to generate a metabolic platform that redirects metabolic flux. Poly-ß-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of this platform. A two-to-three-fold increase in PHB content was observed under green light when compared with the production of PHB under red light, confirming the regulatory activity of this platform and its potential to redirect metabolic flux to synthesize target products.

The Expression Modulation of the Key Enzyme Acc for Highly Efficient 3-Hydroxypropionic Acid Production.

3-Hydroxypropionic acid (3-HP) is a promising high value-added chemical. Acetyl-CoA carboxylase (Acc) is a vital rate-limiting step in 3-HP biosynthesis through the malonyl-CoA pathway. However, Acc toxicity in cells during growth blocks its ability to catalyze acetyl-CoA to malonyl-CoA. The balancing of Acc and malonyl-CoA reductase (MCR) expression is another an unexplored but key process in 3-HP production. To solve these problems, in the present study, we developed a method to mitigate Acc toxicity cell growth through Acc subunits (AccBC and DtsR1) expression adjustment. The results revealed that cell growth and 3-HP production can be accelerated through the adjustment of DtsR1 and AccBC expression. Subsequently, the balancing Acc and MCR expression was also employed for 3-HP production, the engineered strain achieved the highest titer of 6.8 g/L, with a high yield of 0.566 g/g glucose and productivity of 0.13 g/L/h, in shake-flask fermentation through the malonyl-CoA pathway. Likewise, the engineered strain also had the highest productivity (1.03 g/L/h) as well as a high yield (0.246 g/g glucose) and titer (up to 38.13 g/L) in fed-batch fermentation, constituting the most efficient strain for 3-HP production through the malonyl-CoA pathway using a cheap carbon source. This strategy might facilitate the production of other malonyl-CoA-derived chemical compounds in the future.

Development of Optogenetic Dual-Switch System for Rewiring Metabolic Flux for Polyhydroxybutyrate Production.

Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.

Construction of Synthetic Microbial Ecosystems and the Regulation of Population Proportion.

With the development of synthetic biology, the design and application of microbial consortia have received increasing attention. However, the construction of synthetic ecosystems is still hampered by our limited ability to rapidly develop microbial consortia with the required dynamics and functions. By using modular design, we constructed synthetic competitive and symbiotic ecosystems with Escherichia coli. Two ecological relationships were realized by reconfiguring the layout between the communication and effect modules. Furthermore, we designed inducible synthetic ecosystems to regulate subpopulation ratios. With the addition of different inducers, a wide range of strain ratios between subpopulations was achieved. These inducible synthetic ecosystems enabled a larger volume of population regulation and simplified culture conditions. The synthetic ecosystems we constructed combined both basic and applied functionalities and expanded the toolkit of synthetic biology research.

Computational design of a cutinase for plastic biodegradation by mining molecular dynamics simulations trajectories.

Polyethylene terephthalate (PET) has caused serious environmental concerns but could be degraded at high temperature. Previous studies show that cutinase from Thermobifida fusca KW3 (TfCut2) is capable of degrading and upcycling PET but is limited by its thermal stability. Nowadays, Popular protein stability modification methods rely mostly on the crystal structures, but ignore the fact that the actual conformation of protein is complex and constantly changing. To solve these problems, we developed a computational approach to design variants with enhanced protein thermal stability by mining Molecular Dynamics simulation trajectories using Machine Learning methods (MDL). The optimal classification accuracy and the optimal Pearson correlation coefficient of MDL model were 0.780 and 0.716, respectively. And we successfully designed variants with high ΔT m values using MDL method. The optimal variant S121P/D174S/D204P had the highest ΔT m value of 9.3 °C, and the PET degradation ratio increased by 46.42-fold at 70℃, compared with that of wild type TfCut2. These results deepen our understanding on the complex conformations of proteins and may enhance the plastic recycling and sustainability at glass transition temperature.

[Degradation of petroleum-based plastics by microbes and microbial consortia].

Along with the increasingly serious environmental pollution, dealing with the "white pollution" issue, which is caused by the worldwide use of not readily-degradable or non-degradable synthetic plastics, has become a great challenge. It is an environmentally friendly strategy to degrade synthetic plastics using microorganisms that exist in nature or evolved under selection pressure. Based on the NSFC-EU International Cooperation and Exchanges Project "Bio Innovation of a Circular Economy for Plastics", this review summarized the screening of bacteria, fungi and microbial consortia capable of degrading synthetic plastics such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyurethane (PUR), and polyethylene terephthalate (PET). We also analyzed the role of various microorganisms played in the degradation of petroleum-based plastics. Moreover, we discussed the pros and cons of using microorganisms and enzymes for degradation of synthetic plastics.

Identification and Characterization of the Mitochondrial Replication Origin for Stable and Episomal Expression in Yarrowia lipolytica.

Episomal plasmids are crucial expression tools for recombinant protein production and genome editing. In Saccharomyces cerevisiae, 2-µm artificial plasmids with a high copy number have been developed and used in metabolic engineering and synthetic biology. However, in unconventional yeasts such as Yarrowia lipolytica, episomal expression relies on a chromosome replication system; this system has the disadvantages of genetic instability and low copy numbers. In this study, we identified and characterized replication origins from the mitochondrial DNA (mtDNA) of Y. lipolytica. A 516-bp mtDNA sequence, mtORI, was confirmed to mediate the autonomous replication of circular plasmids with high protein expression levels and hereditary stability. However, the nonhomologous end-joining pathway could interfere with mtORI plasmid replication and engender genetic heterogeneity. In the Po 1f ΔKu70 strain, the homogeneity of the mtORI plasmid was significantly improved, and the highest copy number reached 5.0 per cell. Overall, mitochondrial-origin sequences can be used to establish highly stable and autonomously replicating plasmids, which can be a powerful supplement to the current synthetic biology tool library and promote the development of Y. lipolytica as a microbial cell factory.

Enhancing the Glucose Flux of an Engineered EP-Bifido Pathway for High Poly(Hydroxybutyrate) Yield Production.

BACKGROUND: As the greenhouse effect becomes more serious and carbon dioxide emissions continue rise, the application prospects of carbon sequestration or carbon-saving pathways increase. Previously, we constructed an EP-bifido pathway in Escherichia coli by combining Embden-Meyerhof-Parnas pathway, pentose phosphate pathway and "bifid shunt" for high acetyl-CoA production. There is much room for improvement in the EP-bifido pathway, including in production of target compounds such as poly(hydroxybutyrate) (PHB). RESULT: To optimize the EP-bifido pathway and obtain higher PHB yields, we knocked out the specific phosphoenolpyruvate phosphate transferase system (PTS) component II Cglc, encoded by ptsG. This severely inhibited the growth and sugar consumption of the bacterial cells. Subsequently, we used multiple automated genome engineering (MAGE) to optimize the ribosome binding site (RBS) sequences of galP (galactose: H (+) symporter) and glk (glucokinase gene bank: NC_017262.1), encoding galactose permease and glucokinase, respectively. Growth and glucose uptake were partially restored in the bacteria. Finally, we introduced the glf (UDP-galactopyranose) from Zymomonas mobilis mutase sugar transport vector into the host strain genome. CONCLUSION: After optimizing RBS of galP, the resulting strain L-6 obtained a PHB yield of 71.9% (mol/mol) and a 76 wt% PHB content using glucose as the carbon source. Then when glf was integrated into the genome strain L-6, the resulting strain M-6 reached a 5.81 g/L PHB titer and 85.1 wt% PHB content.