Cell-free synthetic biology in the new era of enzyme engineering

With the gradual rise of enzyme engineering,it has played an essential role in synthetic biology,medicine,and biomanufacturing.However,due to the limitation of the cell membrane,the complexity of cellular metabolism,the difficulty of controlling the reaction environment,and the toxicity of some metabolic products in traditional in vivo enzyme engineering,it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds.Recently,cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies.This cell-free method has no limitation of the cell membrane and no need to maintain cell viability,and each biosynthetic pathway is highly flexible.This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize.This article aims to discuss the latest advances in cell-free enzyme engineering,assess the trend of this developing topical filed,and analyze its prospects.

Application of Enzyme Engineering in Pharmacy

Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmaceutical field.The application of enzymes in medicine is diverse,such as:diagnosis,prevention and treatment of diseases with enzymes,manufacture of various drugs with enzymes,etc.,mainly through manual operations,to obtain enzymes required by the pharmaceutical industry,and through various means Enzymes perform their catalytic functions.This article mainly introduces the application of enzyme engineering in the pharmaceutical field,and also prospects the development trend of enzyme engineering in the pharmaceutical field.

Enzyme engineering in microbial biosynthesis of terpenoids:progress and perspectives

Terpenoids,known for their structural and functional diversity,are highly valued,especially in food,cosmetics,and cleaning products.Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids.However,the natural enzymes involved in the synthesis of terpenoids have problems such as low activity,poor specificity,and insufficient stability,which limit the biosynthesis efficiency.Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids.By modifying the structures and functions of key enzymes,researchers have significantly improved the catalytic activity,specificity,and stability of enzymes related to terpenoid synthesis,providing strong support for the sustainable production of terpenoids.This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids,including improving enzyme activity and stability,changing specificity,and promoting mass transfer through multi-enzyme collaboration.Additionally,this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Book review of“Enzyme engineering:Selective catalysts for applications in biotechnology,organic chemistry,and life science”

This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.

Engineered microorganisms and enzymes for efficiently synthesizing plant natural products

Plant natural products are a kind of active substance widely used in pharmaceuticals and foods.However,the current production mode based on plant culture and extraction suffer complex processes and severe concerns for environmental and ecological.With the increasing awareness of environmental sustainability,engineered microbial cell factories have been an alternative approach to produce natural products.Many engineering strategies have been utilized in microbial biosynthesis of complex phytochemicals such as dynamic control and substructure engineering.Meanwhile,Enzyme engineering including directed evolution and rational design has been implemented to improve enzyme catalysis efficiency and stability as well as change promiscuity to expand product spectra.In this review,we discussed recent advances in microbial biosynthesis of complex phytochemicals from the following aspects,including pathway construction,strain engineering to boost the production.

Engineering Rubisco to enhance CO_(2) utilization

Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a pivotal enzyme that mediates the fixation of CO_(2).As the most abundant protein on earth,Rubisco has a significant impact on global carbon,water,and nitrogen cycles.However,the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency.This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco,which has been challenging due to its high conservation and limitations in chloroplast transformation techniques.However,recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms,including plant Rubisco,in microorganisms.This breakthrough facilitates the acquisition and evaluation of modified proteins,streamlining the measurement of their activity.Moreover,the establishment of a screening system in E.coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution.Finally,this review emphasizes the utilization of Rubisco in microorganisms,not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes.

Fatty acid hydratase for value-added biotransformation:A review

The synthesis of hydroxy fatty acids(HFAs) from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years. Given that selective asymmetric hydration of non-activated C_C bonds has been proven difficult to achieve with chemical catalysts, enzymatic catalysis by fatty acid hydratases(FAHs) presents an attractive alternative approach to produce value-added HFAs with high regio-, enantioand stereospecificity, as well as excellent atom economy. Even though FAHs have just been investigated as a potential biocatalyst for a decade, remarkable information about FAHs in different aspects is available;however, a comprehensive review has not been archived. Herein, we summarize the research progresses on biochemical characterization, structural and mechanistic determination, enzyme engineering, as well as biotechnological application of FAHs. The current challenges and opportunities for an efficient utilization of FAHs in organic synthesis and industrial applications are critically discussed.

Cofactor engineering in cyanobacteria to overcome imbalance between NADPH and NADH： A mini review

Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmo- spheric carbon dioxide by photosynthesis. Genetic manip- ulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches： （1） utilization of NADPH- dependent enzymes, （2） increasing NADH production, and （3） changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.

Methanol tolerance upgrading of Proteus mirabilis lipase by machine learning‑assisted directed evolution

For many crucial industrial applications,enzyme-catalyzed processes take place in harsh organic solvent environments.However,it remains a challenging problem to improve enzyme stability in organic solvents.This study utilized the MLDE(machine learning-assisted directed evolution)protocol to improve the methanol tolerance of Proteus mirabilis lipase(PML).The machine learning(ML)models were trained based on 266 combinatorial mutants.Using top 3 in 22 regression models based on evaluation of tenfold cross-validation,the fitness landscape of the 8000 full-space combinatorial mutants was predicted.All mutants in the restricted library showed higher methanol tolerance,among which the methanol tolerance of G202N/K208G/G266S(NGS)was up to 13-fold compared with the wild-type.Molecular dynamics(MD)simulation showed that reconstructing of critical hydrogen bond network in the mutant region of NGS provides a more stable local structure.This compact structure may improve the methanol tolerance by preventing organic solvent molecules into the activity site and resisting structural destruction.This work provides a successful case of evolution guided by ML for higher organic solvent tolerance of enzyme,and may also be a reference for broad enzyme modifications.

Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution‑guided mutagenesis

Streptomyces griseus trypsin(SGT)is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions.However,SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue.In this study,by engineering theαsignal peptide in Pichia pastoris,we increased the SGT amidase activity to 67.91 U mL^(−1)in shake flask cultures.Afterwards,we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S,V177I and E180M with increased catalytic efficiencies.On this basis,we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL^(−1)in shake flasks and 2506 U mL^(−1)in 3-L fed-batch cultures.Moreover,single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5,which was 2 times better than the animal-sourced trypsin.

Production of anthocyanins in metabolically engineered microorganisms:Current status and perspectives

Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology.Anthocyanins,the water-soluble colored pigments found in terrestrial plants that are responsible for the red,blue and purple coloration of many flowers and fruits,are extensively used in food and cosmetics industry;however,their current supply heavily relies on complex extraction from plant-based materials.A promising alternative is their sustainable production in metabolically engineered microbes.Here,we review the recent progress on anthocyanin biosynthesis in engineered bacteria,with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes,engineering of transportation,regulation of UDP-glucose supply,as well as process optimization.These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.

Structural Insights into the CatalyticMechanism of a Plant Diterpene Glycosyltransferase SrUGT76G1

Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.

Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution

Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that form active homo-oligomers.From a mechanistic as well as a structural point of view,they are similar to glycoside hydrolases or glycosyltransferases.As the majority of DSPs show strict stereo-and regiospecificities,these enzymes were used to synthesize specific disaccharides.Currently,protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes,to eventually generate a toolbox of new catalysts for glycoside synthesis.Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.

Isolation and mechanism analysis of a catalytic efciency improved L‑aspartateβ‑decarboxylase toward 3‑methylaspartic acid

L-Aspartateβ-decarboxylase from Acinetobacter radioresistens(ArASD)has been modifed to convert 3-methylaspartic acid into 2-aminobutyric acid,which activated a novel process for biosynthesis of 2-aminobutyric acid.However,the process is limited by the low activity of the ArASD.Here,the activity of ArASD was signifcantly improved by modifcation based on sequence alignment and structural analysis.The 38th residue of ArASD is speculated to be the key residue for regulating the conformation of the internal aldimine,and site-directed mutagenesis on R38 residue was carried out.A variant,K18A/R38K/V287I,with 2.2 times higher specifc activity was isolated.Molecular dynamics simulation indicated that the torsion angle of the imine bond of the variant decreased,which was benefcial to the protonation of the internal aldimine and the increase in the initial energy of the enzyme.Therefore,the energy barrier of the transition state was reduced,resulting in improved catalytic activity toward 3-methylaspartic acid.These results provide a reference and a new point of view for enzyme modifcation by increasing the energy of the initial state.

High-fructose corn syrup production and its new applications for 5-hydroxymethylfurfural and value-added furan derivatives:Promises and challenges

High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and commercial production of glucose isomerase have paved the way to explore more efficient variants of these enzymes.The 5-hydroxymethylfurfural can be produced from high fructose corn syrup catalytic dehydration,and it can be further converted into various furanic compounds chemically or biologically for various industrial applications as a promising platform chemical.Although the chemical conversion of 5-hydroxymethylfurfural into furanic compounds has been extensively investigated in recent years,bioconversion has shown promise for its mild conditions due to the harsh chemical reaction conditions.This review discusses pro-tein engineering potential for improving glucose isomerase production and recent advancements in bioconversion of 5-hydroxymethylfurfural into value-added furanic derivatives.It suggests bi-ological strategies for the industrial transformation of 5-hydroxymethylfurfural.

Microbial soluble aromatic prenyltransferases for engineered biosynthesis

Prenyltransferase(PTase)enzymes play crucial roles in natural product biosynthesis by transferring isoprene unit(s)to target substrates,thereby generating prenylated compounds.The prenylation step leads to a diverse group of natural products with improved membrane affinity and enhanced bioactivity,as compared to the nonprenylated forms.The last two decades have witnessed increasing studies on the identification,characterization,enzyme engineering,and synthetic biology of microbial PTase family enzymes.We herein summarize several examples of microbial soluble aromatic PTases for chemoenzymatic syntheses of unnatural novel prenylated compounds.

Structure-guided protein design of fluoroacetate dehalogenase for kinetic resolution of rac-2-bromobutyric acid

R-2-Bromobutyric acid is a very important intermediate for the synthesis of agrochemicals and pharmaceuticals.Bioresolution of rac-2-bromobutyric acid(rac-2-BBA)provides a promising process for R-2-bromobutyric acid(R-2-BBA)production.The fluoroacetate dehalogenase(FAcD)has been always studied in the defluorination process.We found that FAcD RPA1163 showed detectable activity but no enantioselectivity towards rac-2-BBA.The iterative saturation mutagenesis(ISM)of FAcD RPA1163 resulted in a mutant H155V/W156R/Y219M,which catalyzed the kinetic resolution of rac-2-BBA to produce R-2-BBA with enhanced activity and enantioselectivity(99.3%ee).The high preference for S-2-bromobutyric acid(S-2-BBA)is of synthetic value.Molecular docking analysis indicated that the H155V/W156R/Y219M mutation reduced steric hindrance and broadened the halide pocket.It is not only the steric hindrance but also the electrostatic environment that has an effect on the activity and enantioselectivity.