Carbene chemistry for unnatural biosynthesis

Previously,chemists working in synthesis were jealous of biologists since the enzymes found in nature usually exhibited great catalytic properties(e.g.,high catalytic efficiency,excellent selectivity such as substrate selectivity,regioselectivity and stereoselectivity).However,biological systems lack many counterparts of reactions widely used in chemical synthesis.

Tobacco as a promising crop for low-carbon biorefinery

Energy crops play a vital role in meeting future energy and chemical demands while addressing climate change.However,the idealization of lowcarbon workflows and careful consideration of cost-benefit equations are crucial for their more sustainable implementation.Here,we propose tobacco as a promising energy crop because of its exceptional water solubility,mainly attributed to a high proportion of water-soluble carbohydrates and nitrogen,less lignocellulose,and the presence of acids.We then designed a strategy that maximizes biomass conversion into bio-based products while minimizing energy and material inputs.By autoclaving tobacco leaves in water,we obtained a nutrient-rich medium capable of supporting the growth of microorganisms and the production of bioproducts without the need for extensive pretreatment,hydrolysis,or additional supplements.Additionally,cultivating tobacco on barren lands can generate sufficient biomass to produce approximately 573 billion gallons of ethanol per year.This approach also leads to a reduction of greenhouse gas emissions by approximately 76%compared to traditional corn stover during biorefinery processes.Therefore,our study presents a novel and direct strategy that could significantly contribute to the goal of reducing carbon emissions and global sustainable development compared to traditional methods.

Engineering of Yarrowia lipolytica for production of astaxanthin

Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.

The secondary metabolite bioinformatics portal: Computational tools to facilitate synthetic biology of secondary metabolite production

Natural products are among the most important sources of lead molecules for drug discovery.With the development of affordable whole-genome sequencing technologies and other‘omics tools,the field of natural products research is currently undergoing a shift in paradigms.While,for decades,mainly analytical and chemical methods gave access to this group of compounds,nowadays genomics-based methods offer complementary approaches to find,identify and characterize such molecules.This paradigm shift also resulted in a high demand for computational tools to assist researchers in their daily work.In this context,this review gives a summary of tools and databases that currently are available to mine,identify and characterize natural product biosynthesis pathways and their producers based on‘omics data.A web portal called Secondary Metabolite Bioinformatics Portal(SMBP at http://www.secondarymetabolites.org)is introduced to provide a one-stop catalog and links to these bioinformatics resources.In addition,an outlook is presented how the existing tools and those to be developed will influence synthetic biology approaches in the natural products field.

CRISPy-web: An online resource to design sgRNAs for CRISPR applications

CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology,allowing the targeted engineering of a wide range of genomes.The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections.It now sees widespread application in genome engineering workflows,especially using the Streptococcus pyogenes endonuclease Cas9.To utilize Cas9,so-called single guide RNAs(sgRNAs)need to be designed for each target gene.While there are many tools available to design sgRNAs for the popular model organisms,only few tools that allow designing sgRNAs for non-model organisms exist.Here,we present CRISPy-web(http://crispy.secondarymetabolites.org/),an easy to use web tool based on CRISPy to design sgRNAs for any userprovided microbial genome.CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs.After checks for potential off-target matches,the resulting sgRNA sequences are displayed graphically and can be exported to text files.All steps and information are accessible from a web browser without the requirement to install and use command line scripts.

A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes

Rapid and sensitive detection of various analytes is in high demand.Apart from its application in genome editing,CRISPR-Cas also shows promises in nucleic acid detection applications.To further exploit the potential of CRISPR-Cas for detection of diverse analytes,we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12 a.We demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background,i.e.,the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 lmol/L,respectively,highlighting the advantages of simplicity,sensitivity,short detection time,and low cost compared with the state-of-the-art biosensing approaches.Altogether,this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes.

Designing sgRNAs for CRISPR-BEST base editing applications with CRISPyweb 2.0

CRISPR/Cas9 systems are an established tool in genome engineering.As double strand breaks caused by the standard Cas9-based knock-out techniques can be problematic in some organisms,new systems were developed that can efficiently create knock-outs without causing double strand breaks to elegantly sidestep these issues.The recently published CRISPR-BEST base editor system for actinobacteria is built around a C to T or A to G base exchange.These base editing systems however require additional constraints to be considered for designing the sgRNAs.Here,we present an updated version of the interactive CRISPy-web single guide RNA design tool https://crispy.secondarymetabolites.org/that was built to support“classical”CRISPR and now also CRISPRBEST workflows.

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae

The synthetic biology toolkit for baker’s yeast,Saccharomyces cerevisiae,includes extensive genome engineering toolkits and parts repositories.However,with the increasing complexity of engineering tasks and versatile applications of this model eukaryote,there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR(findable,accessible,interoperable,and reproducible)compliance.In this study,we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements.Moreover,we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements,respectively.Additionally,all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts.We expect the data,parts,and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Expression of fungal biosynthetic gene clusters in S. cerevisiae for natural product discovery

Fungi are well known for production of antibiotics and other bioactive secondary metabolites,that can be served as pharmaceuticals,therapeutic agents and industrially useful compounds.However,compared with the characterization of prokaryotic biosynthetic gene clusters(BGCs),less attention has been paid to evaluate fungal BGCs.This is partially because heterologous expression of eukaryotic gene constructs often requires replacement of original promoters and terminators,as well as removal of intron sequences,and this substantially slow down the workflow in natural product discovery.It is therefore of interest to investigate the possibility and effectiveness of heterologous expression and library screening of intact BGCs without refactoring in industrial friendly microbial cell factories,such as the yeast Saccharomyces cerevisiae.Here,we discuss the importance of developing new research directions on library screening of fungal BGCs in yeast without refactoring,followed by outlooking prominent opportunities and challenges for future advancement.

Introduction to the Special Issue “Bioinformatic tools and approaches for Synthetic Biology of natural products”

Microorganisms are the major source for the identification of novel bioactive natural products,which may serve as future lead molecules for drug development to treat infectious and other diseases.However,the research efforts to find such molecules have been hampered by high re-discovery rates of known biomolecules and a lack of new innovative screening technologies resulting in reduced screening efforts by large parts of the pharmaceutical industry.With recent advances in whole-genome sequencing technologies,mass spectrometry,cheminformatics and Synthetic Biology,new technologies are now available that could become game-changers in the field.All these technologies require strong computational efforts to analyze and mine the large datasets,and/or design and optimize new pathways for Synthetic Biology applications.

Corrigendum to“Antibiotic Treatment Drives the Diversification of the Human Gut Resistome”[Genomics Proteomics Bioinformatics 17(1)(2019)39-51]

The editors regret there was an error in“Shotgun metagenome library construction and sequencing”section.“The raw sequences can be found in BGID(CRA000815)”should be corrected to“The raw metagenome sequencing data have been deposited in the Genome Sequence Archive at Beijing Institute of Genomics,Chinese Academy of Sciences(GSA:CRA000815),and are publicly accessible at https://bigd.big.ac.cn/gsa/”.The correct section is shown below.The editors would like to apologize for any inconvenience caused.

Programmable polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms,development of new,versatile,bio-based production platforms is required.Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides.The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

A chromosome-scale genome assembly of Rauvolfia tetraphylla facilitates identification of the complete ajmaline biosynthetic pathway

Dear Editor,Rauvolfia tetraphylla(aka the Devil pepper)(Supplemental Figure 1)is a well-known medicinal plant that produces monoterpenoid indole alkaloids(MIAs).This MIA biosynthesis occurs in several organs,including leaves,stems,fruit,and roots,which accumulate the famous antiarrhythmic ajmaline(Kumar et al.2016a,2016b;Kumara et al.,2019).MIAs are natural products notably involved in plant adaptation to the environment and defense against aggressors.This mainly results from their high biological activities,which also explain their pharmacological properties.

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that shortterm antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was ～3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota.

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001

For a safe and sustainable environment,effective microbes as biocontrol agents are in high demand.We have isolated a new Bacillus velezensis strain DTU001,investigated its antifungal spectrum,sequenced its genome,and uncovered the production of lipopeptides in HPLC-HRMS analysis.To test the antifungal efficacy,extracts of B.velezensis DTU001 was tested against a range of twenty human or plant pathogenic fungi.We demonstrate that inhibitory potential of B.velezensis DTU001 against selected fungi is superior in comparison to single lipopeptide,either iturin or fengycin.The isolate showed analogous biofilm formation to other closely related Bacilli.To further support the biocontrol properties of the isolate,coculture with Candida albicans demonstrated that B.velezensis DTU001 exhibited excellent antiproliferation effect against C.albicans.In summary,the described isolate is a potential antifungal agent with a broad antifungal spectrum that might assist our aims to avoid hazardous pathogenic fungi and provide alternative to toxicity caused by chemicals.

Engineered polyketides:Synergy between protein and host level engineering

Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities.Polyketides encompass one of the largest classes of chemically diverse natural products.With thousands of known polyketides,modular polyketide synthases(PKSs)share a particularly attractive biosynthetic logic for generating chemical diversity.The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds.In this review,we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures,and we examine future applications of PKSs in the production of medicines,fuels and other industrially relevant chemicals.

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p

Clinical use of antimicrobials faces great challenges from the emergence of multidrug-resistant pathogens. The overexpression of drug efflux pumps is one of the major contributors to multidrug resistance(MDR). Reversing the function of drug efflux pumps is a promising approach to overcome MDR. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily(MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to MDR. Here we report a counterintuitive case of reversing MDR in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. This results in the selective elimination of Mdr1 p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time of mice with blood-borne dissemination of Mdr1p overexpressed multidrug-resistant candidiasis. This study provides a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

Nat Methods:利用CRISPR-Cas9组合筛选发现癌症弱点

导致癌症的基因突变也会削弱癌细胞，从而使得人们有机会开发选择性地杀死它们同时不影响正常细胞的药物。这一概念被称作”合成致死性（synthetic lethality）”，这是因为这些药物仅对发生突变的（或者说合成的）细胞是致命性的。在一项新的研究中。来自美国加州大学圣地亚哥分校、加州大学旧金山分校、斯坦福大学和癌细胞图谱项目（Cancer Cell Map Initiative）的研究人员开发出一种新的方法来寻找合成致死性的基因组合。

An aurora of natural products-based drug discovery is coming

Natural products(NPs),a nature's reservoir possessing enormous structural and functional diversity far beyond the current ability of chemical synthesis,are now proving themselves as most wonderful gifts from mother nature for human beings.Many of them have been used successfully as medicines,as well as the most important sources of drug leads,food additives,and many industry relevant products for millennia.Most notably,more than half of the antibiotics and anti-cancer drugs currently in use are,or derived from,natural products.However,the speed and outputs of NP-based drug discovery has been slowing down dramatically after the fruitful harvest of the“low-hanging fruit”during the golden age of 1950s-1960s.With recent scientific advances combining metabolic sciences and technology,multi-omics,big data,combinatorial biosynthesis,synthetic biology,genome editing technology(such as CRISPR),artificial intelligence(AI),and 3D printing,the“high-hanging fruit”is becoming more and more accessible with reduced costs.We are now more and more confident that a new age of natural products discovery is dawning.

Beauvericin counteracted multi-drug resistant Candida albicans by blocking ABC transporters

Multi-drug resistance of pathogenic microorganisms is becoming a serious threat,particularly to immunocompromised populations.The high mortality of systematic fungal infections necessitates novel antifungal drugs and therapies.Unfortunately,with traditional drug discovery approaches,only echinocandins was approved by FDA as a new class of antifungals in the past two decades.Drug efflux is one of the major contributors to multi-drug resistance,the modulator of drug efflux pumps is considered as one of the keys to conquer multi-drug resistance.In this study,we combined structure-based virtual screening and whole-cell based mechanism study,identified a natural product,beauvericin(BEA)as a drug efflux pump modulator,which can reverse the multi-drug resistant phenotype of Candida albicans by specifically blocking the ATP-binding cassette(ABC)transporters;meantime,BEA alone has fungicidal activity in vitro by elevating intracellular calcium and reactive oxygen species(ROS).It was further demonstrated by histopathological study that BEA synergizes with a sub-therapeutic dose of ketoconazole(KTC)and could cure the murine model of disseminated candidiasis.Toxicity evaluation of BEA,including acute toxicity test,Ames test,and hERG(human ether-a-go-go-related gene)test promised that BEA can be harnessed for treatment of candidiasis,especially the candidiasis caused by ABC overexpressed multi-drug resistant C.albicans.