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Plastic contamination currently threatens a wide variety of ecosystems and presents damaging repercussions and negative consequences for many wildlife species. Sustainable plastic waste management is an important approach to environmental protection and a necessity in the current life cycle of plastics in nature. Plastic biodegradation by microorganisms is a notable possible solution. This opinion article includes a proposal to use hypothetical P450 enzymes with an engineered active site as potent trigger biocatalysts to biodegrade polyethylene (PE) via in-chain hydroxylation into smaller products of linear aliphatic alcohols and alkanoic acids based on cascade enzymatic reactions. Furthermore, we propose the adoption of P450 into plastic-eating synthetic bacteria for PE biodegradation. This strategy can be applicable to other dense plastics, such as polypropylene (PP) and polystyrene (PS). Plastic pollution causes harmful environment effects, but the development of biocatalysts can solve them.Cascading enzymatic systems that are started via in-chain hydroxylation and catalyzed by a hypothetical P450 enzyme with an engineered active site can depolymerize polyethylene into small degradation molecules.Synthetic artificial bacteria are emerging interdisciplinary technologies that can solve plastic issues by applying degrading enzymes that contribute to the breakdown of plastics.This biodegradation enzyme cascade system will require directed evolution and rational engineering to enhance its degradation activity.

Biocatalytic cyclization is highly desirable for efficient synthesis of biologically derived chemical substances, such as the commodity chemicals ε-caprolactam and δ-valerolactam. To identify biocatalysts in lactam biosynthesis, we develop a caprolactam-detecting genetic enzyme screening system (CL-GESS). The Alcaligenes faecalis regulatory protein NitR is adopted for the highly specific detection of lactam compounds against lactam biosynthetic intermediates. We further systematically optimize the genetic components of the CL-GESS to enhance sensitivity, achieving 10-fold improvement. Using this highly sensitive GESS, we screen marine metagenomes and find an enzyme that cyclizes ω-amino fatty acids to lactam. Moreover, we determine the X-ray crystal structure and catalytic residues based on mutational analysis of the cyclase. The cyclase is also used as a helper enzyme to sense intracellular ω-amino fatty acids. We expect this simple and accurate biosensor to have wide-ranging applications in rapid screening of new lactam-synthesizing enzymes and metabolic engineering for lactam bio-production. Efficient biosynthesis of lactams is still undesirable due to lacking of suitable enzyme. Here, the authors develop a sensitive transcription factor-based biosensor for high-throughput screening of marine metagenome and find a cyclase that can cyclize ω-amino fatty acids to lactam.

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx.

Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8-1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.

Aldol chemicals are synthesized by condensation reactions between the carbon units of ketones and aldehydes using aldolases. The efficient synthesis of diverse organic chemicals requires intrinsic modification of aldolases via engineering and design, as well as extrinsic modification through immobilization or combination with other catalysts. This review describes the development of aldolases, including their engineering and design, and the selection of desired aldolases using high-throughput screening, to enhance their catalytic properties and perform novel reactions. Aldolase-containing catalysts, which catalyze the aldol reaction combined with other enzymatic and/or chemical reactions, can efficiently synthesize diverse complex organic chemicals using inexpensive and simple materials as substrates. We also discuss the current challenges and emerging solutions for aldolase-based catalysts. Aldolases catalyze condensation reactions between donor ketones and acceptor aldehydes by forming C–C bonds to produce aldol chemicals with high selectivity.Aldolases must be engineered, or de novo aldolases must be designed, to enhance their activity, stability, and selectivity, or to catalyze novel reactions.High-throughput screening techniques, such as phage display screening and microfluidic droplet screening, can be applied to rapidly acquire desired aldolases.Aldolase-containing catalysts are used to synthesize not only aldol chemicals but also complex organic chemicals via multiple sequence reactions using simple materials.Synthetic chemistry can be applied to develop novel artificial aldolases, and chemo- and biocatalysis can be combined for the efficient synthesis of organic chemicals for industrial applications.

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

Summary Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease‐deficient Cas9 gene and a designed single guide RNA, under control of l‐rhamnose‐inducible PrhaBAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi‐mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi‐mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products. We present an l‐rhamnose‐inducible single‐plasmid CRISPRi system for achieving the simple and efficient regulation of target genes in Pseudomonas putida KT2440. This regulatable CRISPRi system was able to control the expression of exogenous and endogenous genes in P. putida KT2440. We further provide examples of its application for metabolic flux alteration to enhance the production of mevalonate (MVA), a key intermediate metabolite for the biosynthesis of a myriad of terpenoids.

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ 54 )-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ 70 ) TFs. Here, we show that these unique characteristics of σ 54 -dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes ( relA , spoT ) and nutrient conditions, to link the σ 54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ 54 -dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.

Cytosolic lipid droplets (LDs) derived from the endoplasmic reticulum (ER) mainly contain neutral lipids, such as triacylglycerols (TAGs) and sterol esters, which are considered energy reserves. The metabolic pathways associated with LDs in eukaryotic species are involved in diverse cellular functions. TAG synthesis in plants is mediated by the sequential involvement of two subcellular organelles, i.e., plastids - plant-specific organelles, which serve as the site of lipid synthesis, and the ER. TAGs and sterol esters synthesized in the ER are sequestered to form LDs through the cooperative action of several proteins, such as SEIPINs, LD-associated proteins, LDAP-interacting proteins, and plant-specific proteins such as oleosins. The integrity and stability of LDs are highly dependent on oleosins, especially in the seeds, and oleosin degradation is critical for efficient mobilization of the TAGs of plant LDs. As the TAGs mobilize in LDs during germination and post-germinative growth, a plant-specific lipase—sugar-dependent 1 (SDP1)—plays a major role, through the inter-organellar communication between the ER and peroxisomes. In this review, we briefly recapitulate the different processes involved in the biogenesis and degradation of plant LDs, followed by a discussion of future perspectives in this field.

Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia. The major metabolites in humans are 2-OH and 4-OH atorvastatin, which are active metabolites known to show highly inhibiting effects on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Producing the hydroxylated metabolites by biocatalysts using enzymes and whole-cell biotransformation is more desirable than chemical synthesis. It is more eco-friendly and can increase the yield of desired products. In this study, we have found an enzymatic strategy of P450 enzymes for highly efficient synthesis of the 4-OH atorvastatin, which is an expensive commercial product, by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide without NADPH. We obtained a set of CYP102A1 mutants with high catalytic activity toward atorvastatin using enzyme library generation, high-throughput screening of highly active mutants, and enzymatic characterization of the mutants. In the hydrogen peroxide supported reactions, a mutant, with nine changed amino acid residues compared to a wild-type among tested mutants, showed the highest catalytic activity of atorvastatin 4-hydroxylation (1.8 min−1). This result shows that CYP102A1 can catalyze atorvastatin 4-hydroxylation by peroxide-dependent oxidation with high catalytic activity. The advantages of CYP102A1 peroxygenase activity over NADPH-supported monooxygenase activity are discussed. Taken together, we suggest that the P450 peroxygenase activity can be used to produce drugs’ metabolites for further studies of their efficacy and safety.