Qualitative analysis of chemical components in Lianhua Qingwen capsule by HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS

The Lianhua Qingwen(LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases.In particular,it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).However,due to its complex composition,little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule.This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry(HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry(GC-MS).An automated library alignment method with a high mass accuracy(within 5 ppm) was used for the rapid identification of compounds.A total of 104 compounds,consisting of alkaloids,flavonoids,phenols,phenolic acids,phenylpropanoids,quinones,terpenoids,and other phytochemicals,were successfully characterized.In addition,the fragmentation pathways and characteristic fragments of some representative compounds were elucidated.GC-MS analysis was conducted to characterize the volatile compounds present in LHQW.In total,17 compounds were putatively characterized by comparing the acquired data with that from the NIST library.The major constituent was menthol,and all the other compounds were terpenoids.This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS,coupled with GCMS,and the results of this study can be used for the quality control and standardization of LHQW capsules.

Evolutionary analysis and lineage designation of SARS-CoV-2 genomes

The pandemic due to the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the etiological agent of coronavirus disease 2019(COVID-19),has caused immense global disruption.With the rapid accumulation of SARS-CoV-2 genome sequences,however,thousands of genomic variants of SARSCoV-2 are now publicly available.To improve the tracing of the viral genomes’evolution during the development of the pandemic,we analyzed single nucleotide variants(SNVs)in 121,618 high-quality SARS-CoV-2 genomes.We divided these viral genomes into two major lineages(L and S)based on variants at sites 8782 and 28144,and further divided the L lineage into two major sublineages(L1 and L2)using SNVs at sites 3037,14408,and 23403.Subsequently,we categorized them into 130 sublineages(37 in S,35 in L1,and 58 in L2)based on marker SNVs at 201 additional genomic sites.This lineage/sublineage designation system has a hierarchical structure and reflects the relatedness among the subclades of the major lineages.We also provide a companion website(www.covid19evolution.net)that allows users to visualize sublineage information and upload their own SARS-CoV-2 genomes for sublineage classification.Finally,we discussed the possible roles of compensatory mutations and natural selection during SARS-CoV-2’s evolution.These efforts will improve our understanding of the temporal and spatial dynamics of SARS-CoV-2’s genome evolution.

Revolution of vitamin E production by starting from microbial fermented farnesene to isophytol

Vitamin E is one of the most widely used vitamins.In the classical commercial synthesis of vitamin E(a-tocopherol),the chemical synthesis of isophytol is the key technical barrier.Here,we establish a new process for isophytol synthesis from microbial fermented farnesene.To achieve an efficient pathway for farnesene production,Saccharomyces cerevisiae was selected as the host strain.First,b-farnesene synthase genes from different sources were screened,and through protein engineering and system metabolic engineering.

In vitro reconstitution guide for targeted synthetic metabolism of chemicals, nutraceuticals and drug precursors

With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an important barrier to traditional metabolic engineering,however,the identification of ratelimiting step(s)for the improvement of specific cellular functions is often difficult.Meanwhile,in the case of synthetic biology,more and more BioBricks could be constructed for targeted purposes,but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge.Owing to the lack of steady-state kinetic data for overall flux,balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials.A strategy called targeted engineering is proposed in an effort to solve this problem.Briefly,a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors,substrates and each enzyme will be analyzed systematically.Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays.To demonstrate its practical application,biosynthesis pathways for the production of important products,e.g.chemicals,nutraceuticals and drug precursors,have been engineered in Escherichia coli and Saccharomyces cerevisiae.These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.

Development of Streptomyces sp. FR-008 as an emerging chassis

Microbial-derived natural products are important in both the pharmaceutical industry and academic research.As the metabolic potential of original producer especially Streptomyces is often limited by slow growth rate,complicated cultivation profile,and unfeasible genetic manipulation,so exploring a Streptomyces as a super industrial chassis is valuable and urgent.Streptomyces sp.FR-008 is a fast-growing microorganism and can also produce a considerable amount of macrolide candicidin via modular polyketide synthase.In this study,we evaluated Streptomyces sp.FR-008 as a potential industrial-production chassis.First,PacBio sequencing and transcriptome analyses indicated that the Streptomyces sp.FR-008 genome size is 7.26 Mb,which represents one of the smallest of currently sequenced Streptomyces genomes.In addition,we simplified the conjugation procedure without heat-shock and pre-germination treatments but with high conjugation efficiency,suggesting it is inherently capable of accepting heterologous DNA.In addition,a series of promoters selected from literatures was assessed based on GusA activity in Streptomyces sp.FR-008.Compared with the common used promoter ermE*-p,the strength of these promoters comprise a library with a constitutive range of 60e860%,thus providing the useful regulatory elements for future genetic engineering purpose.In order to minimum the genome,we also target deleted three endogenous polyketide synthase(PKS)gene clusters to generate a mutant LQ3.LQ3 is thus an“updated”version of Streptomyces sp.FR-008,producing fewer secondary metabolites profiles than Streptomyces sp.FR-008.We believe this work could facilitate further development of Streptomyces sp.FR-008 for use in biotechnological applications.

Increasing the heterologous production of spinosad in Streptomyces albus J1074 by regulating biosynthesis of its polyketide skeleton

Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain,and poor adaptability of the heterologous strain to the production of spinosyn.In this study,we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad(spinosyn A and spinosyn D)based on our previous work.We systematically investigated the result of overexpressing polyketide synthase genes(spnA,B,C,D,E)using a constitutive promoter on the spinosad titer in S.albus J1074.The supply of polyketide synthase precursors was then increased to further improve spinosad production.Finally,increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of~70 mg/L,which is the highest titer of spinosad achieved in heterologous Streptomyces species.This research provides useful strategies for efficient heterologous production of natural products.

Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification

With worldwide attention on renewable energy and climate change,metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research,with a view to enhance production of biofuels.Indeed,this pathway has already been extensively studied in Escherichia coli.Nevertheless,little is known about the absolute abundance of the enzymes involved,information that may be valuable for engineering,such as the optimal molar ratios of different proteins.In this study,we use protein standard absolute quantification(PSAQ)to measure the absolute abundance of proteins that catalyze fatty acid biosynthesis in E.coli.In addition,the changes of protein abundance were analyzed by comparing the differences between high-yield and the background strain.Our work highlights opportunities to enhance fatty acid production by measuring protein molar ratios and identifying catalytic and regulatory bottlenecks.More importantly,our results provide evidence that PSAQ is a generally valuable tool to investigate metabolic pathways.

Evaluation and optimization of analytical procedure and sample preparation for polar Streptomyces albus J1074 metabolome profiling

Metabolomics is an essential discipline in omics technology that promotes research on the biology of microbial systems.Streptomyces albus J1074 is a model organism used in fundamental research and industrial microbiology.Nevertheless,a comprehensive and standardized method for analyzing the metabolome of S.albus J1074 is yet to be developed.Thus,we comprehensively evaluated and optimized the analytical procedure and sample preparation for profiling polar metabolites using hydrophilic interaction liquid chromatography(HILIC)coupled with high-resolution mass spectrometry(HRMS).We systematically examined the HILIC columns,quenching solutions,sample-to-quenching ratios,and extraction methods.Then,the optimal protocol was used to investigate the dynamic intracellular polar metabolite profile of the engineered S.albus J1074 strains during spinosad(spinosyn A and spinosyn D)fermentation.A total of 3648 compounds were detected,and 83 metabolites were matched to the standards.The intracellular metabolomic profiles of engineered S.albus J1074 strains(ADE-AP and OE3)were detected;furthermore,their metabolomes in different stages were analyzed to reveal the reasons for their differences in their spinosad production,as well as the current metabolic limitation of heterologous spinosad production in S.albus J1074.The HILIC-HRMS method is a valuable tool for investigating polar metabolomes,and provides a reference methodology to study other Streptomyces metabolomes.