Synthetic biology of plant natural products: From pathway elucidation to engineered biosynthesis in plant cells

Plant natural products(PNPs)are the main sources of drugs,food additives,and new biofuels and have become a hotspot in synthetic biology.In the past two decades,the engineered biosynthesis of many PNPs has been achieved through the construction of microbial cell factories.Alongside the rapid development of plant physiology,genetics,and plant genetic modification techniques,hosts have now expanded from single-celled microbes to complex plant systems.Plant synthetic biology is an emerging field that combines engineering principles with plant biology.In this review,we introduce recent advances in the biosynthetic pathway elucidation of PNPs and summarize the progress of engineered PNP biosynthesis in plant cells.Furthermore,a future vision of plant synthetic biology is proposed.Although we are still a long way from overcoming all the bottlenecks in plant synthetic biology,the ascent of this field is expected to provide a huge opportunity for future agriculture and industry.

Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems

Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However,maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP)yields β-glucose 1-phosphate (β-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitrosynthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymaticreaction module comprised of MP, β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for thestoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this syntheticmodule, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP),respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6mW/cm^(2),whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0mM FDP from 50 g/L (139mM) maltose by adopting afed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novelstrategies for the high-efficient production of bioelectricity and value-added biochemicals.

Genome-scale CRISPRi screening:A powerful tool in engineering microbiology

Deciphering gene function is fundamental to engineering of microbiology.The clustered regularly interspaced short palindromic repeats(CRISPR)system has been adapted for gene repression across a range of hosts,creating a versatile tool called CRISPR interference(CRISPRi)that enables genome-scale analysis of gene function.This approach has yielded significant advances in the design of genome-scale CRISPRi libraries,as well as in applica-tions of CRISPRi screening in medical and industrial microbiology.This review provides an overview of the recent progress made in pooled and arrayed CRISPRi screening in microorganisms and highlights representative studies that have employed this method.Additionally,the challenges associated with CRISPRi screening are discussed,and potential solutions for optimizing this strategy are proposed.

Improving pathway prediction accuracy of constraints-based metabolic network models by treating enzymes as microcompartments

Metabolic network models have become increasingly precise and accurate as the most widespread and practical digital representations of living cells.The prediction functions were significantly expanded by integrating cellular resources and abiotic constraints in recent years.However,if unreasonable modeling methods were adopted due to a lack of consideration of biological knowledge,the conflicts between stoichiometric and other constraints,such as thermodynamic feasibility and enzyme resource availability,would lead to distorted predictions.In this work,we investigated a prediction anomaly of EcoETM,a constraints-based metabolic network model,and introduced the idea of enzyme compartmentalization into the analysis process.Through rational combination of reactions,we avoid the false prediction of pathway feasibility caused by the unrealistic assumption of free intermediate metabolites.This allowed us to correct the pathway structures of L-serine and L-tryptophan.A specific analysis explains the application method of the EcoETM-like model and demonstrates its potential and value in correcting the prediction results in pathway structure by resolving the conflict between different constraints and incorporating the evolved roles of enzymes as reaction compartments.Notably,this work also reveals the trade-off between product yield and thermodynamic feasibility.Our work is of great value for the structural improvement of constraints-based models.

Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde

Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to formaldehyde.Herein,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic activity.The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants.Mdh_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation libraries.The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity.The beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-helices.Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.

High-throughput base editing KO screening of cellular factors for enhanced GBE

Base editor techniques have been developed as a means of precisely converting bases without the need for double-stranded DNA breaks(DSBs)or editing templates.Currently,these techniques can be used for cytosine(C)to thymine(T)conversions(cytosine base editors,CBEs)(Komor et al.,2016;Nishida et al.,2016),adenine(A)to guanine(G)conversions(adenine base editors,ABEs)(Gaudelli et al.,2017),and cytosine(C)to guanine(G)conversions(glycosylase base editors,GBEs)(Zhao et al.,2021)in mammalian cells.GBE,in particular,is a promising base editing technique capable of correcting up to 11%of the 32,044 pathogenic single nucleotide polymorphisms(SNPs)known to date(Gaudelli et al.,2017).Despite its potential,the performance of GBE is still not optimal,and its editing outcomes exhibit a wider variation range than those of CBEs due to the dependence on cellular DNA repair systems(Jiang et al.,2021),which implies that efficient GBE performance remains a challenge.

Reconstruction and metabolic profiling of the genome-scale metabolic network model of Pseudomonas stutzeri A1501

Pseudomonas stutzeri A1501 is a non-fluorescent denitrifying bacteria that belongs to the gram-negative bacterial group.As a prominent strain in the fields of agriculture and bioengineering,there is still a lack of comprehensive understanding regarding its metabolic capabilities,specifically in terms of central metabolism and substrate utilization.Therefore,further exploration and extensive studies are required to gain a detailed insight into these aspects.This study reconstructed a genome-scale metabolic network model for P.stutzeri A1501 and conducted extensive curations,including correcting energy generation cycles,respiratory chains,and biomass composition.The final model,iQY1018,was successfully developed,covering more genes and reactions and having higher prediction accuracy compared with the previously published model iPB890.The substrate utilization ability of 71 carbon sources was investigated by BIOLOG experiment and was utilized to validate the model quality.The model prediction accuracy of substrate utilization for P.stutzeri A1501 reached 90%.The model analysis revealed its new ability in central metabolism and predicted that the strain is a suitable chassis for the production of Acetyl CoA-derived products.This work provides an updated,high-quality model of P.stutzeri A1501for further research and will further enhance our understanding of the metabolic capabilities.

Efficient enzymatic synthesis of(S)-1-(30-bromo-20-methoxyphenyl)ethanol,the key building block of lusutrombopag

(S)-1-(30-Bromo-20-methoxyphenyl)ethanol((S)-1b)is the key precursor for the synthesis of Lusutrombopag.The bioreduction of 1-(30-bromo-20-methoxyphenyl)ethanone(1a)offers an attractive method to access this important compound.Through screening the available carbonyl reductases,we obtained a carbonyl reductase from Novosphingobium aromaticivorans(CBR),which could completely convert 100 g/L of 1a to(S)-1b.Furthermore,a carbonyl reductase from Novosphingobium sp.Leaf2(NoCR)was identified to completely convert 200 g/L of 1a to(S)-1b with excellent enantioselectivity(>99%ee)and 77%isolated yield using FDH/formate system for NADH regeneration.The K_(m) and k_(cat) of recombinant NoCR towards 1a were 0.66 mmol/L and 7.5 s-1,and the catalytic efficiency k_(cat)/K_(m) was 11.3 mmol/s.L.Meanwhile,NoCR showed high catalytic activity and stereoselectivity towards acetophenone derivatives with halogen or methoxy substitution on the benzene ring,indicating that NoCR is a valuable biocatalyst with potential practical applications.

Effective electrocatalytic hydrodechlorination of 2,4,6-trichlorophenol by a novel Pd/MnO_(2)/Ni foam cathode

Pd modified electrodes possess problems such as easy agglomeration and low electrolytic ability,and the use of manganese dioxide(MnO_(2)) to facilitate Pd reduction of organic pollutants is just started.However,there is still a limited understanding of how to match the Pd load and MnO_(2) to realize optimal dechlorination efficiency at minimum cost.Here,a Pd/MnO_(2)/Ni foam cathode was successfully fabricated and applied for the efficient electrochemical dechlorination of 2,4,6-trichlorophenol(2,4,6-TCP).The optimal electrocatalytic hydrodechlorination(ECH)performance with 2,4,6-TCP dechlorination efficiency(92.58%in 180 min)was obtained when the concentration of PdCl_(2) precipitation was 1 mmol/L,the deposition time of MnO_(2) was 300 s and cathode potential was-0.8 V.Performance influenced by the exogenous factors(e.g.,initial pH and coexisted ions)were further investigated.It was found that the neutral pH was the most favorable for ECH and a reduction in dechlorination efficiency(6%~47.6%)was observed in presence of 5 mmol/L of NO_(2)^(-),NO_(3)^(-),S^(2-)or SO_(3)^(2-).Cyclic voltammetry(CV)and quenching experiments verified the existence of three hydrogen species on Pd surface,including adsorbed atomic hydrogen(H^(*)_(ads)),absorbed atomic hydrogen(H^(*)_(abs)),and molecular hydrogen(H_(2)).And the introduction of MnO_(2)promoted the generation of atomic H^(*).Only adsorbed atomic hydrogen(H^(*)_(ads)) was confirmed that it truly facilitated the ECH process.Besides H^(*)_(ads) induced reduction,the direct reduction by cathode electrons also participated in the 2,4,6-TCP dechlorination process.Pd/MnO_(2)/Ni foam cathode shows excellent dechlorination performance,fine stability and recyclable potential,which provides strategies for the effective degradation of persistent halogenated organic pollutants in groundwater.

Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli

This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6 T, T105 A and L239 M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid(99%). Lastly, the number of chromosomal copies of the balanced crtW*-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid(99%) with a specific titer of 0.88 g·L^(-1) without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.

Perturbation of clopyralid on bio-denitrification and nitrite accumulation:Long-term performance and biological mechanism

The contaminant of herbicide clopyralid(3,6-dichloro-2-pyridine-carboxylic acid,CLP)poses a potential threat to the ecological system.However,there is a general lack of research devoted to the perturbation of CLP to the bio-denitrification process,and its biological response mechanism remains unclear.Herein,long-term exposure to CLP was systematically investigated to explore its influences on denitrification performance and dynamic microbial responses.Results showed that low-concentration of CLP(<15 mg/L)caused severe nitrite accumulation initially,while higher concentrations(35e60 mg/L)of CLP had no further effect after long-term acclimation.The mechanistic study demonstrated that CLP reduced nitrite reductase(NIR)activity and inhibited metabolic activity(carbon metabolism and nitrogen metabolism)by causing oxidative stress and membrane damage,resulting in nitrite accumulation.However,after more than 80 days of acclimation,almost no nitrite accumulation was found at 60 mg/L CLP.It was proposed that the secretion of extracellular polymeric substances(EPS)increased from 75.03 mg/g VSS at 15 mg/L CLP to 109.97 mg/g VSS at 60 mg/L CLP,which strengthened the protection of microbial cells and improved NIR activity and metabolic activities.Additionally,the biodiversity and richness of the microbial community experienced a U-shaped process.The relative abundance of denitrification-and carbon metabolism-associated microorganisms decreased initially and then recovered with the enrichment of microorganisms related to the secretion of EPS and N-acyl-homoserine lactones(AHLs).These microorganisms protected microbe from toxic substances and regulated their interactions among interand intra-species.This study revealed the biological response mechanism of denitrification after successive exposure to CLP and provided proper guidance for analyzing and treating herbicide-containing wastewater.

A combined strategy for the overproduction of complex ergot alkaloid agroclavine

Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.

Ordered mesoporous carbon as an efficient heterogeneous catalyst to activate peroxydisulfate for degradation of sulfadiazine

Catalytic potential of carbon nanomaterials in peroxydisulfate(PDS)advanced oxidation systems for degradation of antibiotics remains poorly understood.This study revealed ordered mesoporous carbon(type CMK)acted as a superior catalyst for heterogeneous degradation of sulfadiazine(SDZ)in PDS sys-tem,with a first-order reaction kinetic constant(k)and total organic carbon(TOC)mineralization efficiency of 0.06 min^(–1) and 59.67%±3.4%within 60min,respectively.CMK catalyzed PDS system exhibited high degradation efficiencies of five other sulfonamides and three other types of antibiotics,verifying the broad-degradation capacity of antibiotics.Under neutral pH conditions,the optimal catalytic parameters were an initial SDZ concentration of 44.0mg/L,CMK dosage of 0.07g/L,and PDS dosage of 5.44mmol/L,respectively.X-ray photoelectron spectroscopy and Raman spectrum analysis confirmed that the defect structure at edge of CMK and oxygen-containing functional groups on surface of CMK were major active sites,contributing to the high catalytic activity.Free radical quenching analysis revealed that both SO_(4)•−and•OH were generated and participated in catalytic reaction.In addition,direct electron transfer by CMK to activate PDS also occurred,further promoting catalytic performance.Configuration of SDZ molecule was optimized using density functional theory,and the possible reaction sites in SDZ molecule were calculated using Fukui function.Combining ultra-high-performance liquid chromatography(UPLC)–mass spectrometry(MS)/MS analysis,three potential degradation pathways were proposed,including the direct removal of SO_(2)molecules,the 14S-17N fracture,and the 19C-20N and 19C-27N cleavage of the SDZ molecule.The study demonstrated that ordered mesoporous carbon could work as a feasible catalytic material for PDS advanced oxidation during removal of antibiotics from wastewater.

Sequence motifs and prediction model of GBE editing outcomes based on target library analysis and machine learning

Base editor techniques were developed for precise base conversion without requiring double-stranded DNA breaks(DSBs) or an editing template(Komor et al., 2016;Nishida et al., 2016).

Data-Driven Synthetic Cell Factories Development for Industrial Biomanufacturing

Revolutionary breakthroughs in artificial intelligence (AI) and machine learning (ML) have had a profound impact on a widerange of scientific disciplines, including the development of artificial cell factories for biomanufacturing. In this paper, wereview the latest studies on the application of data-driven methods for the design of new proteins, pathways, and strains. Wefirst briefly introduce the various types of data and databases relevant to industrial biomanufacturing, which are the basis fordata-driven research. Different types of algorithms, including traditional ML and more recent deep learning methods, are alsopresented. We then demonstrate how these data-based approaches can be applied to address various issues in cell factorydevelopment using examples from recent studies, including the prediction of protein function, improvement of metabolicmodels, and estimation of missing kinetic parameters, design of non-natural biosynthesis pathways, and pathway optimization.In the last section, we discuss the current limitations of these data-driven approaches and propose that data-driven methodsshould be integrated with mechanistic models to complement each other and facilitate the development of synthetic strains forindustrial biomanufacturing.

二氧化碳到己糖的人工合成

土地短缺及环境恶化导致依赖光合作用的糖制造技术面临诸多挑战.当前人工CO_(2)-糖合成技术在立体选择性转化、降低能量消耗、扩展产物谱等方面亟待突破.本文设计了低ATP消耗、路线短且普适性强的CO_(2)-糖路线,能量转化效率高于传统制糖方法.结合酶工程技术获得了催化性能优异的酶元件,构建了化学-酶转化体系,实现转化CO_(2)合成葡萄糖、阿洛酮糖等己糖分子,具有较高的产物选择性和碳转化率,CO_(2)-糖合成效率为目前报道最高水平.本研究为创制结构多样性糖及其衍生物,解决人类食糖与健康、工业发酵等糖需求提供蓝图和方案.

Genomic allele-specific base editing with imperfect gRNA

CRISPR base editor(BE)techniques are a promising tool for precise cytosine(C)to thymine(T),adenine(A)to guanine(G),and C to G base editing(CBE,ABE,and GBE,respectively)without the use of a donor DNA template conversion(Komor et al.,2016;Nishida et al.,2016;Gaudelli et al.,2017;Kurt et al.,2021;Zhao et al.,2021).

Thirteen Dipterocarpoideae genomes provide insights into their evolution and borneol biosynthesis

Dipterocarpoideae,the largest subfamily of the Dipterocarpaceae,is a dominant component of Southeast Asian rainforests and is widely used as a source of wood,damar resin,medicine,and essential oil.However,many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance.Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes,providing clues to the function and evolution of these species.Here,we report on 13 high-quality Dipterocarpoideae genome assemblies,ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae.Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae.Based on evolutionary analysis,we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes.We discovered an expansion of genes encoding cellulose synthase(CesA),which is essential for cellulose biosynthesis and secondary cell-wall formation.We functionally identified five bornyl diphosphate synthase(BPPS)genes,which specifically catalyze the biosynthesis of borneol,a natural medicinal compound extracted from damar resin and oils,thus providing a basis for large-scale production of natural borneol in vitro.

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.

Carboxylic acid reductases enable intramolecular lactamization reactions

As a versatile type of enzyme,carboxylic acid reductases(CAR)can not only reduce various carboxylic acids to aldehydes in cooperation with cofactors ATP and NADPH but also catalyze the synthesis of amides and esters in the absence of NADPH.Here,we report an intramolecular cyclization catalyzed by CAR only with the use of ATP to transform amino acids into diverse lactams,includingγ-/δ-/ε-lactams and chiral derivatives thereof.The observed wide substrate scope and selectivity enable potential applications to be implemented.Our results demonstrate that CAR-catalyzed lactamization is a promising approach for the synthesis of chiral lactam com-pounds under mild conditions,thereby enriching the toolbox for the biosynthesis of lactams as a viable alternative to purely chemical procedures.