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.

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.

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.

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.

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.

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.

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.

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.

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.

Engineering Escherichia coli to improve tryptophan production via genetic manipulation of precursor and cofactor pathways

Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds.Here,the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli.First,glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001,resulting in the best candidate strain TS-1.Then icd and gdhA were overexpressed in TS-1,which led to the accumulation of 1.060 g/L tryptophan.Subsequently,one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-D-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine,resulting in the accumulation of 1.380 g/L tryptophan.Finally,to maintain cofactor balance,sthA and pntAB,encoding transhydrogenase,were overexpressed.With sufficient amounts of precursors and balanced cofactors,the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation,which was 2.76-times higher than the titer of the parent strain.Taken together,our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan.Similar strategies could be applied to the production of other amino acids or related derivatives.

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.

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.

Discovery and modification of cytochrome P450 for plant natural products biosynthesis

Cytochrome P450s are widespread in nature and play key roles in the diversification and functional modification of plant natural products.Over the last few years,there has been remarkable progress in plant P450s identification with the rapid development of sequencing technology,“omics”analysis and synthetic biology.However,challenges still persist in respect of crystal structure,heterologous expression and enzyme engineering.Here,we reviewed several research hotspots of P450 enzymes involved in the biosynthesis of plant natural products,including P450 databases,gene mining,heterologous expression and protein engineering.

High-throughput metagenomic analysis of petroleum-contaminated soil microbiome reveals the versatility in xenobiotic aromatics metabolism

The soil with petroleum contamination is one of the most studied soil ecosystems due to its rich microorganisms for hydrocarbon degradation and broad applications in bioremediation. However, our understanding of the genomic properties and functional traits of the soil microbiome is limited. In this study, we used high-throughput metagenomic sequencing to comprehensively study the microbial community from petroleum-contaminated soils near Tianjin Dagang oilfield in eastern China. The analysis reveals that the soil metagenome is characterized by high level of community diversity and metabolic versatility. The metageome community is predominated by 7-Proteobacteria and a-Proteobacteria, which are key players for petroleum hydrocarbon degradation. The functional study demonstrates over-represented enzyme groups and pathways involved in degradation of a broad set of xenobiotic aromatic compounds, including toluene, xylene, chlorobenzoate, aminobenzoate, DDT, methylnaph- thalene, and bisphenol. A composite metabolic network is proposed for the identified pathways, thus consolidating our identification of the pathways. The overall data demon- strated the great potential of the studied soil microbiome in the xenobiotic aromatics degradation. The results not only establish a rich reservoir for novel enzyme discovery but also provide putative applications in bioremediation.

Development of a dual temperature control system for isoprene biosynthesis in Saccharomyces cerevisiae

Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter PSSA4 to control the expression of PGAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

The origin and evolution of the diosgenin biosynthetic pathway in yam

Diosgenin,mainly produced by Dioscorea species,is a traditional precursor of most hormonal drugs in the pharmaceutical industry.The mechanisms that underlie the origin and evolution of diosgenin biosynthesis in plants remain unclear.After sequencing the whole genome of Dioscorea zingiberensis,we revealed the evolutionary trajectory of the diosgenin biosynthetic pathway in Dioscorea and demonstrated the de novo biosynthesis of diosgenin in a yeast cell factory.First,we found that P450 gene duplication and neofunctionalization,driven by positive selection,played important roles in the origin of the diosgenin biosynthetic pathway.Subsequently,we found that the enrichment of diosgenin in the yam lineage was regulated by CpG islands,which evolved to regulate gene expression in the diosgenin pathway and balance the carbon flux between the biosynthesis of diosgenin and starch.Finally,by integrating genes fromplants,animals,and yeast,weheterologously synthesized diosgenin to 10mg/l in genetically-engineered yeast.Our study not only reveals the origin and evolutionary mechanisms of the diosgenin biosynthetic pathway in Dioscorea,but also introduces an alternative approach for the production of diosgenin through synthetic biology.

Metabolic engineering of Yarrowia lipolytica for scutellarin production

Scutellarin related drugs have superior therapeutic effects on cerebrovascular and cardiovascular diseases.Here,an optimal biosynthetic pathway for scutellarin was constructed in Yarrowia lipolytica platform due to its excellent metabolic potential.By integrating multi-copies of core genes from different species,the production of scutellarin was increased from 15.11 mg/L to 94.79 mg/L and the ratio of scutellarin to the main by-product was improved about 110-fold in flask condition.Finally,the production of scutellarin was improved 23-fold and reached to 346 mg/L in fed-batch bioreactor,which was the highest reported titer for de novo production of scutellarin in microbes.Our results represent a solid basis for further production of natural products on unconventional yeasts and have a potential of industrial implementation.

New xylose transporters support the simultaneous consumption of glucose and xylose in Escherichia coli

Glucose and xylose are two major components of lignocellulose.Simultaneous consumption of glucose and xylose is critical for engineered microorganisms to produce fuels and chemicals from lignocellulosic biomass.Although many production limitations have been resolved,glucose‐induced inhibition of xylose transport remains a challenge.In this study,a cell growthbased screening strategy was designed to identify xylose transporters uninhibited by glucose.The glucose pathway was genetically blocked in Escherichia coli so that glucose functions only as an inhibitor and cells need xylose as the carbon source for survival.Through adaptive evolution,omics analysis and reverse metabolic engineering,a new phosphoenolpyruvate:carbohydrate phosphotransferase system(PTS)galactitol transporter(GalABC,encoded by EcolC_1640,EcolC_1641,and EcolC_1642 genes)that is not inhibited by glucose was identified.Inactivation of adenylate cyclase led to increased expression of the EcolC_1642 gene,and a point mutation in gene EcolC_1642(N13S)further enhanced xylose transport.During the second round of gene mining,AraE and a new ABC transporter(AraFGH)of xylose were identified.A point mutation in the transcription regulator araC(L156I)caused increased expression of araE and araFGH genes without arabinose induction,and a point mutation in araE(D223Y)further enhanced xylose transport.These newly identified xylose transporters can support the simultaneous consumption of glucose and xylose and have potential use in producing chemicals from lignocellulose.

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).

Construction and analysis of an integrated biological network of Escherichia coli

Escherichia coli is a model organism with a clear genetic background that is widely used in metabolic engineering and synthetic biology research.To gain a complete picture of the complexly metabolic and regulatory interactions in E.coli,researchers often need to retrieve information from various databases which cover diferent types of interactions.A central one-stop service integrating various molecular interactions in E.coli would be helpful for the community.We constructed a database called E.coli integrated network(EcoIN)by integrating known molecular interaction information from databases and literature.EcoIN contains nearly 160,000 pairs of interactions and users can easily search the diferent types of interacting partners for a metabolite,gene or protein,and thus gain access to a more comprehensive interaction map of E.coli.To illustrate the application of EcoIN,we used the full path algorithm to identify metabolic feedback/feedforward regulatory loops having at least two diferent types of regulatory interactions.Applying this algorithm to analyze the regulatory loops for the amino acid biosynthetic pathways,we found some multi-step regulation loops which may afect the metabolic fux and are potential new engineering targets.The EcoIN database is freely accessible at http://ecoin.ibiodesign.net/and analysis codes are available at GitHub:https://github.com/maozhitao/EcoIN.