Zanthoxylum-specific whole genome duplication and recent activity of transposable elements in the highly repetitive paleotetraploid Z.bungeanum genome

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites,which create a characteristic aroma and tingling sensation in the mouth.Owing to the high proportion of repetitive sequences,high heterozygosity,and increased chromosome number of Z.bungeanum,the assembly of its chromosomal pseudomolecules is extremely challenging.Here,we present a genome sequence for Z.bungeanum,with a dramatically expanded size of 4.23 Gb,assembled into 68 chromosomes.This genome is approximately tenfold larger than that of its close relative Citrus sinensis.After the divergence of Zanthoxylum and Citrus,the lineage-specific whole-genome duplication event q-WGD approximately 26.8 million years ago(MYA)and the recent transposable element(TE)burst~6.41 MYA account for the substantial genome expansion in Z.bungeanum.The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture.Integrative genomic and transcriptomic analyses suggested that prominent speciesspecific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools,terpenoids,and anthocyanins,which contribute to the special flavor and appearance of Z.bungeanum.In summary,the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

Agroinfection of sweet potato by vacuum infiltration of an infectious sweepovirus

Sweepovirus is an important monopartite begomovirus that infects plants of the genus Ipomoea worldwide. Development of artificial infection methods for sweepovirus using agroinoculation is a highly efficient means of studying infectivity in sweet potato. Unlike other begomoviruses, it has proven difficult to infect sweet potato plants with sweepoviruses using infectious clones. A novel sweepovirus, called Sweet potato leaf curl virus-Jiangsu(SPLCV-JS), was recently identified in China. In addition, the infectivity of the SPLCV-JS clone has been demonstrated in Nicotiana benthamiana. Here we describe the agroinfection of the sweet potato cultivar Xushu 22 with the SPLCV-JS infectious clone using vacuum infiltration. Yellowing symptoms were observed in newly emerged leaves. Molecular analysis confirmed successful inoculation by the detection of viral DNA. A synergistic effect of SPLCV-JS and the heterologous betasatellite DNA-β of Tomato yellow leaf curl China virus isolate Y10(TYLCCNV-Y10) on enhanced symptom severity and viral DNA accumulation was confirmed. The development of a routine agroinoculation system in sweet potato with SPLCV-JS using vacuum infiltration should facilitate the molecular study of sweepovirus in this host and permit the evaluation of virus resistance of sweet potato plants in breeding programs.

Experimental and Computational Study of the Effect of Temperature on the Electro-Polymerization Process of Thiophene

Temperature effect on the nucleation and growth mechanisms (NGM) of poly(thiophene) (PTh) was investigated through experimental and computational tools. The computational simulation method was based on a kinetic Monte Carlo algorithm. It reproduced key processes such as diffusion, oligomerization, and the precipitation of oligomers onto the electrode surface. Electrochemical synthesis conditions at temperatures between 263 and 303 K were optimized. The deconvolution of the i-t transients reflected two contributions: a progressive nucleation with three-dimensional growth controlled by diffusion and the other by charge transfer, PN3Ddif and PN3Dct, respectively. As temperature decreased, a diminution of the charge associated to each contribution was observed and the nucleation induction time increased. Experimental and computational evidence indicated that temperature does not change the nucleation and growth mechanism (NGM). This effect was ascribed to kinetic factors rather than to film conductivity. This work contrasts simulation and experimental evidence and demonstrates how computational simulations can help to understand the electrochemical process of conducting polymers formation.

Advances on systems metabolic engineering of Bacillus subtilis as a chassis cell

The Gram-positive model bacterium Bacillus subtilis,has been broadly applied in various fields because of its low pathogenicity and strong protein secretion ability,as well as its well-developed fermentation technology.B.subtilis is considered as an attractive host in the field of metabolic engineering,in particular for protein expression and secretion,so it has been well studied and applied in genetic engineering.In this review,we discussed why B.subtilis is a good chassis cell for metabolic engineering.We also summarized the latest research progress in systematic biology,synthetic biology and evolution-based engineering of B.subtilis,and showed systemic metabolic engineering expedite the harnessing B.subtilis for bioproduction.

Nucleotide Recognition by a Guanidinocalixarene Receptor in Aqueous Solution

Nucleotides participate in various physiological processes through their supramolecular interactions with biomolecules.Therefore,the molecular recognition of nucleotides became an important topic in supramolecular chemistry and exhibited many biomedical applications.Guanidinocalixarenes showed very strong binding affinities towards nucleotides,even reaching the nanomolar level.In this work,we systematically determined the binding constants between a typical guanidinocalixarene(guanidinium-modified calix[5]arene,GC5A)and various nucleotides and revealed the driving forces behind the molecular recognition using theoretical calculations.The electrostatic interactions and hydrogen bonding provided by the phosphate groups of the nucleotides dominated the binding between the nucleotides and GC5A.The lower rim alkyl chains and the skeleton of GC5A provide preorganized cavity and upper guanidinium groups.The difference in the type of nucleobase is also attributed to the different binding affinities.This work provides insight into the molecular recognition of nucleotides and facilitates the development of new supramolecular hosts for nucleotides and related biological applications.

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.

Construction and application of high-quality genome-scale metabolic model of Zymomonas mobilis to guide rational design of microbial cell factories

High-quality genome-scale metabolic models(GEMs)could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies.Despite of the constant establishment and update of GEMs for model microorganisms such as Escherichia coli and Saccharomyces cerevisiae,high-quality GEMs for non-model industrial microorganisms are still scarce.Zymomonas mobilis subsp.mobilis ZM4 is a non-model ethanologenic microorganism with many excellent industrial characteristics that has been developing as microbial cell factories for biochemical production.Although five GEMs of Z.mobilis have been constructed,these models are either generating ATP incorrectly,or lacking information of plasmid genes,or not providing standard format file.In this study,a high-quality GEM iZM516 of Z.mobilis ZM4 was constructed.The information from the improved genome annotation,literature,datasets of Biolog Phenotype Microarray studies,and recently updated Gene-Protein-Reaction information was combined for the curation of iZM516.Finally,516 genes,1389 reactions,1437 metabolites,and 3 cell compartments are included in iZM516,which also had the highest MEMOTE score of 91%among all published GEMs of Z.mobilis.Cell growth was then predicted by iZM516,which had 79.4%agreement with the experimental results of the substrate utilization.In addition,the potential endogenous succinate synthesis pathway of Z.mobilis ZM4 was proposed through simulation and analysis using iZM516.Furthermore,metabolic engineering strategies to produce succinate and 1,4-butanediol(1,4-BDO)were designed and then simulated under anaerobic condition using iZM516.The results indicated that 1.68 mol/mol succinate and 1.07 mol/mol 1,4-BDO can be achieved through combinational metabolic engineering strategies,which was comparable to that of the model species E.coli.Our study thus not only established a high-quality GEM iZM516 to help understand and design microbial cell factories for economic biochemical production using Z.mobilis as the chassis,but also provided guidance on building accurate GEMs for other non-model industrial microorganisms.

Microbial L-malic acid production:History,current progress,and perspectives

L-malic acid(L-MA)is an important intermediate in the tricarboxylic acid cycle and a crucial bulk chemical with various applications in the food,pharmaceutical,and chemical industries.With the rapid advancements in metabolic engineering technology and the global commitment toward fostering a green economy and sustainable development,the large-scale production of L-MA is gradually transitioning from conventional petroleum-based approaches to microbial fermentation.This comprehensive review aims to provide a thorough overview of the historical background and recent advancements in the microbial fermentation production of L-MA,encompassing an in-depth introduction to diverse biosynthetic pathways and host strains.Moreover,this review elucidates the challenges encountered in the industrialization of microbial fermentation production of L-MA,offering a summary of potential solutions and prospects for future research directions.The anticipated outcome of this review is to contribute valuable theoretical guidance toward promoting technological innovation in L-MA production.

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).

Mutation of Oryza sativa CORONATINE INSENSITIVE 1b(OsCOI1b) delays leaf senescence

Jasmonic acid （JA） functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA‐responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice （Oryza sativa） genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant func-tions in development. Here, we use the oscoi1b‐1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence‐promoting conditions. oscoi1b‐1 mutants stayed green during dark‐induced and natural senescence, with substantial retention of chlorophylls and photosyn-thetic capacity. Furthermore, several senescence‐associated genes were downregulated in oscoi1b‐1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1‐1 plants containing 35S：OsCOI1a or 35S：OsCOI1b rescued the delayed leaf senescence during dark incubation, sug-gesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b‐1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1‐mediated JA signaling affects spikelet fertility and grain filling.

Rice tissue-specific promoters and conditiondependent promoters for effective translational application

Rice （Oryza sativa） is one of the most important staple food crops for more than half of the world＇s population. The demand is increasing for food security because of population growth and environmental challenges triggered by climate changes. This scenario has led to more interest in developing crops with greater productivity and sustainability. The process of genetic transformation, a major tool for crop improvement, utilizes promoters as one of its key elements. Those promoters are generally divided into three types： constitutive, spatiotemporal, and condition-dependent. Tran- scriptional control of a constitutive promoter often leads to reduced plant growth, due to a negative effect of accumu- lated molecules during cellular functions or energy consump- tion. To maximize the effect of a transgene on transgenic plants, it is better to use condition-dependent or tissue- specific promoters. However, until now, those types have not been as widely applied in crop biotechnology. In this review, we introduce and discuss four groups of tissue-specific promoters （5o promoters in total） and six groups of condition-dependent promoters （27 promoters）. These pro- moters can be utilized to fine-tune desirable agronomic traits and develop crops with tolerance to various stresses, enhanced nutritional value, and advanced productivity.

Recent Advances in Dissecting Stress-Regulatory Crosstalk in Rice

Biotic and abiotic stresses impose a serious limitation on crop productivity worldwide. Prior or simultaneous exposure to one type of stress often affects the plant response to other stresses, indicating extensive overlap and cross-talk between stress-response signaling pathways. Systems biology approaches that integrate large genomic and prot-eomic data sets have facilitated identification of candidate genes that govern this stress-regulatory crosstalk. Recently, we constructed a yeast two-hybrid map around three rice proteins that control the response to biotic and abiotic stresses, namely the immune receptor XA21, which confers resistance to the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae; NH1, the rice ortholog of NPR1, a key regulator of systemic acquired resistance; and the ethylene-responsive transcription factor, SUBIA, which confers tolerance to submergence stress. These studies coupled with transcriptional profiling and co-expression analyses identified a suite of proteins that are positioned at the interface of biotic and abiotic stress responses, including mitogen-activated protein kinase 5 （OsMPK5）, wall-associated kinase 25 （WAK25）, sucrose non-fermenting-l-related protein kinase-1 （SnRK1）, SUBIA binding protein 23 （SAB23）, and several WRKY family tran- scription factors. Emerging evidence suggests that these genes orchestrate crosstalk between biotic and abiotic stresses through a variety of mechanisms, including regulation of cellular energy homeostasis and modification of synergistic and/or antagonistic interactions between the stress hormones salicylic acid, ethylene, jasmonic acid, and abscisic acid.

Conservation and Diversification of the SHR-SCR- SCL23 Regulatory Network in the Development of the Functional Endodermis in Arabidopsis Shoots

Development of the functional endodermis of Arabidopsis thaliana roots is controlled, in part, by GRAS transcription factors, namely SHORT-ROOT （SHR）, SCARECROW （SCR）, and SCARECROW-LIKE 23 （SCL23）. Recently, it has been shown that the SHR-SCR-SCL23 regulatory module is also essential for spec- ification of the endodermis （known as the bundle sheath） in leaves. Nevertheless, compared with what is known about the role of the SHR-SCR-SCL23 regulatory network in roots, the molecular interactions of SHR, SCR, and SCL23 are much less understood in shoots. Here, we show that SHR forms protein com- plexes with SCL23 to regulate transcription of SCL23 in shoots, similar to the regulation mode of SCR expression. Our results indicate that SHR acts as master regulator to directly activate the expression of SCR and SCL23. In the SHR-SCR-SCL23 network, we found a previously uncharacterized negative feed- back loop whereby SCL23 modulates SHR levels. Through molecular, genetic, physiological, and morpho- logical analyses, we also reveal that the SHR-SCR-SCL23 module plays a key role in the formation of the endodermis （known as the starch sheath） in hypocotyls. Taken together, our results provide new insights into the regulatory role of the SHR-SCR-SCL23 network in the endodermis development in both roots and shoots.

Why mosaic? Gene expression profiling of African cassava mosaic virus-infected cassava reveals the effect of chlorophyll degradation on symptom development

Cassava mosaic disease, caused by cassava bego- moviruses, is the most serious disease for cassava in Africa. However, the pathogenesis of this disease is poorly under- stood. We employed high throughput digital gene expression profiling based on the Illumina Solexa sequencing technology to investigate the global transcriptional response of cassava to African cassava mosaic virus infection. We found that 3,21o genes were differentially expressed in virus-infected cassava leaves. Gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that genes implicated in photosynthesis were most affected, consistent with the chlorotic symptoms observed in infected leaves. The upregu- lation of chlorophyll degradation genes, including the genes encoding chlorophyUase, pheophytinase, and pheophorbide a oxygenase, and downregulation of genes encoding the major apoproteins in light-harvesting complex II were confirmed by qRT-PCR. These findings, together with the reduction of chlorophyll b content and fewer grana stacks in the infected leaf cells, reveal that the degradation of chlorophyll plays an important role in A^rican cassava mosaic virus symptom development. This study will provide a road map for future investigations into viral pathogenesis.

In-Depth Computational Analysis of Natural and Artificial Carbon Fixation Pathways

In the recent years,engineering new-to-nature CO_(2)-and C1-fixing metabolic pathways made a leap forward.New,artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham(CBB)cycle.The question remains how to best predict their in vivo performance and what actually makes one pathway“better”than another.In this context,we explore aerobic carbon fixation pathways by a computational approach and compare them based on their specific activity and yield on methanol,formate,and CO_(2)/H_(2)considering the kinetics and thermodynamics of the reactions.Besides pathways found in nature or implemented in the laboratory,this included two completely new cycles with favorable features:the reductive citramalyl-CoA cycle and the 2-hydroxyglutarate-reverse tricarboxylic acid cycle.A comprehensive kinetic data set was collected for all enzymes of all pathways,and missing kinetic data were sampled with the Parameter Balancing algorithm.Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway-specific activities.The specific activities of the reductive glycine pathway,the CETCH cycle,and the new reductive citramalyl-CoA cycle were predicted to match the best natural cycles with superior productsubstrate yield.However,the CBB cycle performed better in terms of activity compared to the alternative pathways than previously thought.We make an argument that stoichiometric yield is likely not the most important design criterion of the CBB cycle.Still,alternative carbon fixation pathways were paretooptimal for specific activity and product-substrate yield in simulations with C1 substrates and CO_(2)/H_(2)and therefore hold great potential for future applications in Industrial Biotechnology and Synthetic Biology.

Interplay between ABA and GA Modulates the Timing of Asymmetric Cell Divisions in the Arabidopsis Root Ground Tissue

In multicellular organisms, controlling the timing and extent of asymmetric cell divisions （ACDs） is crucial for correct patterning. During post-embryonic root development in Arabidopsis thaliana, ground tissue （GT） maturation involves an additional ACD of the endodermis, which generates two different tissues： the endo- dermis （inner） and the middle cortex （outer）. It has been reported that the abscisic acid （ABA） and gibberellin （GA） pathways are involved in middle cortex （MC） formation. However, the molecular mechanisms under- lying the interaction between ABA and GA during GT maturation remain largely unknown. Through transcriptome analyses, we identified a previously uncharacterized C2H2-type zinc finger gene, whose expression is regulated by GA and ABA, thus named GAZ （GA- AND ABA-RESPONSIVE ZINC FINGER）. Seedlings ectopically overexpressing GAZ （GAZ-OX） were sensitive to ABA and GA during MC formation, whereas GAZ-SRDX and RNAi seedlings displayed opposite phenotypes. In addition, our results indicated that GAZ was involved in the transcriptional regulation of ABA and GA homeostasis. In agreement with pre- vious studies that ABA and GA coordinate to control the timing of MC formation, we also confirmed the unique interplay between ABA and GA and identified factors and regulatory networks bridging the two hor- mone pathways during GT maturation of the Arabidopsis root.

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.

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway

Taxus,commonly known as yew,is a well-known gymnosperm with great ornamental and medicinal value.In this study,by assembling a chromosome-level genome of the Himalayan yew(Taxus wallichiana)with 10.9 Gb in 12 chromosomes,we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome,resulting in the main genes for paclitaxel biosynthesis,i.e.those encoding the taxadiene synthase,P450s,and transferases,being clustered on the same chromosome.The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization.Furthermore,we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway.The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodi-versity of taxoids in gymnosperms.

Expanding application of CRISPR-Cas9 system in microorganisms

The development of CRISPR-Cas9 based genetic manipulation tools represents a huge breakthrough in life sciences and has been stimulating research on metabolic engineering,synthetic biology,and systems biology.The CRISPR-Cas9 and its derivative tools are one of the best choices for precise genome editing,multiplexed genome editing,and reversible gene expression control in microorganisms.However,challenges remain for applying CRISPR-Cas9 in novel microorganisms,especially those industrial microorganism hosts that are intractable using traditional genetic manipulation tools.How to further extend CRISPR-Cas9 to these microorganisms is being an urgent matter.In this review,we first introduce the mechanism and application of CRISPR-Cas9,then discuss how to optimize CRISPR-Cas9 as genome editing tools,including but not limited to how to reduce off-target effects and Cas9 related toxicity,and how to increase on-target efficiency by optimizing crRNA and sgRNA design.

Raising the production of phloretin by alleviation of by-product of chalcone synthase in the engineered yeast

Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of phloretin production is still not enough for industrial application. Here, we established an artificial pathway in the yeast to produce phloretin via assembling two genes of p-coumaroyl-CoA ligase(4CL) and chalcone synthase(CHS). CHS is a key enzyme which conventionally condenses a CoA-tethered starter with three molecules of malonyl-CoA to form the backbone of flavonoids. However, there was 33% of byproduct generated via CHS by condensing two molecules of malonyl-CoA during the fermentation process. Hence, we introduced a more efficient CHS and improved the supply of malonyl-CoA through two pathways;the by-product ratio was decreased from 33% to 17% and the production of phloretin was improved from 48 to 83.2 mg L^(-1). Finally, a fed-batch fermentation process was optimized and the production of phloretin reached 619.5 mg L^(-1), which was 14-fold higher than that of the previous studies. Our work established a platform for the biosynthesis of phloretin from the low-cost raw material 3-(4-hydroxyphenyl) propanoic acid and also illustrated the potential for industrial scale bio-manufacturing of phloretin.