Current advances of biocontainment strategy in synthetic biology

Synthetic biotechnology has led to the widespread application of genetically modified organisms(GMOs)in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination by horizontal gene transfer, resulting in unpredictable biosafety risks. To deal with these challenges, many effective methods have been developed for biocontainment. In this article, we summarize and discuss recent advances in biocontainment strategies from three aspects: DNA replication, transcriptional regulation, and protein translation. We also briefly introduce the efforts in the biocontainment convention, such as the recent publication of the Tianjin Biosecurity Guidelines for the Code of Conduct for Scientists.

电子诱导金属多肽的超分子组装、化学响应释放及其催化应用

近年来,超分子组装在催化、制药、传感器、提纯、组织工程等领域获得广泛应用.为了实现超分子结构功能化,经常会将金属纳米颗粒或者金属活性位引入或共组装至有机超分子骨架中,由此获得金属化的纳米材料.例如,金属纳米颗粒修饰的多肽纤维、金属聚合物、金属负载的水凝胶和气凝胶.常见的金属化策略包括自组织、金属有机配位络合、聚合和电子诱导组装等.其中,由本课题组发展的电子诱导组装法已经被用于制备高效金属多肽催化剂、能源转化材料和非均相催化剂模板等.室温电子诱导组装利用了辉光等离子体富含的电子,是室温电子还原制备纳米金属颗粒及相关催化剂的进一步发展.该方法操作过程简单,仅需一步即可同时实现金属还原和有机物组装;且绿色环保、不需要添加还原剂、操作条件温和、反应时间短(在室温条件下几分钟内即可反应完全).所获得的二维多肽纳米薄膜含有高度分散的金属纳米颗粒.研究表明,电子诱导组装法是构建超分子催化剂强有力的工具.然而,电子诱导超分子组装的反应机理仍然不清楚.为了进一步开发应用新的超分子材料,开展电子诱导组装机理研究十分必要.本文选择β淀粉肽的五肽片段作为电子诱导组装的单体和贵金属(Pd,Pt和Au)离子通过电子还原得到金属多肽纳米膜.通过调控原料配比和浓度,获得了包覆有超细贵金属纳米颗粒(1–2 nm)的金属多肽纳米薄膜.通过扫描电子显微镜、透射电子显微镜、X射线衍射光谱、X射线光电能谱等表征手段,对金属多肽纳米薄膜的结构和成分进行了分析.通过原子力显微镜对金属多肽纳米薄膜的多级结构进行分析发现,电子诱导组装的组装单元为碟状组合体,与常规自组装有显著区别,通过傅里叶变换红外光谱、X射线光电能谱等表征方法,结合密度泛函(DFT)计算,对组装过程多肽分子的表面性质和变化进行了分析.发现多肽在电子诱导组装过程中会发生部分氧化.DFT研究给出两种可能的羟基自由基活化碳氢键过程,说明形成的羟基提供了额外的氢键相互作用,促进了组装的快速发生.多肽中含有苯环的侧链对多肽组装后二维结构的形成起到重要作用.本文还首次发现金属多肽薄膜能够在化学试剂刺激下响应释放金属纳米颗粒.如在硼氢化钠作用下可以快速释放金属纳米颗粒、在谷胱甘肽作用下可以缓慢释放金属纳米颗粒.硼氢化钠作用下释放后的金属颗粒对4-氨基苯酚还原反应具有良好的催化活性.金属多肽薄膜的快速响应释放可以为稳定纳米金属颗粒提供一个新方法,替代那些以往采用的稳定剂难于脱除的纳米金属稳定方法.而慢速响应释放则有潜力应用于药物缓释、光热治疗生物传感和成像等医学领域.

Enhanced Poly(ethylene terephthalate)Hydrolase Activity by Protein Engineering

Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.

Engineering the Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae with Heterologous Enzyme Combinations

Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.

Medium Optimization for Antifungal Active Substance Production from Streptomyces Lydicus Using Response Surface Methodology

Response surface methodology was used to optimize the medium for antifungal active substance production from Streptomyces lydicus E12 in flask cultivation.Initially,the component factors,which influence antifungal substance production,were studied by varying one factor at a time.Starch,soybean cake powder,K_2HPO_4·3H_2O and MgSO_4·7H_2O were found to have a significant effect on the production of antifungal substances by the traditional design.Then,a Box-Behnken design was applied for further optimization.A quadratic model was found to fit antifungal active substance production.The analysis revealed that the optimum values of the tested variable were starch 84.96 g/L,soybean cake powder 4.13 g/L,glucose 5 g/L,MgSO_4·7H_2O 1.23 g/L,K_2HPO_4·3H_2O2.14 g/L and NaCl 0.5 g/L.The test result of 67.44%antifungal inhibition agreed with the prediction and increased by 14.28%in comparison with the basal medium.

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

Xylose utilization is one of the key issues in lignocellulose bioconversion.Because of glucose repression,in most engineered yeast with heterogeneous xylose metabolic pathway,xylose is not consumed until glucose is completely utilized.Although simultaneous glucose and xylose utilization have been achieved in yeast by RPE1 deletion,we regulated ZWF1 and PGI1 transcription to improve simultaneous xylose and glucose utilization by controlling the metabolic flux from glucose into the PP pathway.Xylose and glucose consumption increased by approximately 80 and 72%,respectively,whereas ZWF1 was overexpressed by multi-copy plasmids with a strong transcriptional promoter.PGI1 expression was knocked down by promoter replacement; the glucose and xylose metabolism increased when PGI1p was replaced by weak promoters,SSA1p and PDA1p.ZWF1 overexpression decreased while PGI1 down-regulation increased the ethanol yield to some extent in the recombinant strains.

Enabling technology and core theory of synthetic biology

Synthetic biology provides a new paradigm for life science research(“build to learn”)and opens the future journey of biotechnology(“build to use”).Here,we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology,including synthesis and assembly of a genome,DNA storage,gene editing,molecular evolution and de novo design of function proteins,cell and gene circuit engineering,cell-free synthetic biology,artificial intelligence(AI)-aided synthetic biology,as well as biofoundries.We also introduce the concept of quantitative synthetic biology,which is guiding synthetic biology towards increased accuracy and predictability or the real rational design.We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.

Design and construction of microbial cell factories based on systems biology

Environmental sustainability is an increasingly important issue in industry.As an environmentally friendly and sustainable way,constructing microbial cell factories to produce all kinds of valuable products has attracted more and more attention.In the process of constructing microbial cell factories,systems biology plays a crucial role.This review summarizes the recent applications of systems biology in the design and construction of microbial cell factories from four perspectives,including functional genes/enzymes discovery,bottleneck pathways identification,strains tolerance improvement and design and construction of synthetic microbial consortia.Systems biology tools can be employed to identify functional genes/enzymes involved in the biosynthetic pathways of products.These discovered genes are introduced into appropriate chassis strains to build engineering microorganisms capable of producing products.Subsequently,systems biology tools are used to identify bottleneck pathways,improve strains tolerance and guide design and construction of synthetic microbial consortia,resulting in increasing the yield of engineered strains and constructing microbial cell factories successfully.

Loss of heterozygosity by SCRaMbLEing

Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as structural variation and aneuploidy is still limited and mainly based on the static, predetermined feature of individual genomes. Here, using SCRaMbLE,different levels of loss of heterozygosity(LOH) events including short-range LOH, long-range LOH and whole chromosome LOH were detected in evolved strains. By contrast, using rapid adaptive evolution, aneuploidy was detected in the adaptive strains. It was further found that deletion of gene GLN3, long-range LOH in the left arm of synthetic chromosome Ⅹ, whole chromosome LOH of synthetic chromosome Ⅹ, and duplication of chromosome Ⅷ(trisomy) lead to increased rapamycin resistance in synthetic yeast. Comparative analysis of genome stability of evolved strains indicates that the aneuploid strain has a higher frequency of degeneration than the SCRaMbLEd strain. These findings enrich our understanding of genetic mechanism of rapamycin resistance in yeast, and provide valuable insights into yeast genome architecture and function.

Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

The conversion of r-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps ofketolation are catalyzed by β-carotene hydroxylase （CrtZ） and β-carotene ketolase （CrtW） respectively. Here, astaxanthin biosynthesis path- way was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high r-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE Scl 18060 with CrtW from Brevundimonas vesicu- laris DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Scl 18060 was replaced from FBAlp to TEFlp, the astaxanthin yield was increased by 30.4% （from 3.4 to 4.5 mg/g DCW）. In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtWwere observed at early exponential phase in medium with 4% （w/v） glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% （w/v） glucose, the transcription level of both crtZ and crtW were actually decreased during the whole timecourse, consequently leading to no significant improve- ment on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance lation by employing the key desired compounds accumu- enzyme（s） in microbes.

Establishment of genomic library technology mediated by non-homologous end joining mechanism in Yarrowia lipolytica

Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits.The non-homologous end joining(NHEJ),which enables foreign DNA fragments to be randomly integrated into different chromosomal sites,shows prominent capability in genomic libraries construction.In this study,we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process,employment of defective Ura marker and optimization of iterative transformations,which enhanced genes integration efficiency by 4.67,22.74 and 1.87 times,respectively.We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE,CrtB and CrtI,with 23.8 times higher production than rDNA integration through homologous recombination(HR).The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites,with the copy number up to 65 and 53,respectively,creating potential for further application of recombinase mediated genome rearrangement in Y.lipolytica.This work provides a high-efficient NHEJ-mediated genomic library technology,which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h.This novel technology also lays an excellent foundation for the development of other genetic technologies in Y.lipolytica.

Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae

Metabolic engineering of heterologous resver- atrol production in Saccharomyces cerevisiae faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We over- expressed the main gene targets in the upstream pathways to investigate their influences on the downstream resver- atrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.144-3.74 mg.L-1, 2.27 times higher than the initial production in YPD medium （4% glucose）. The newly discovered gene targets TRP1 expressing phosphoribosy- lanthranilate isomerase, AR03 expressing 3-deoxy-D- arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae.

Enhancement and mapping of tolerance to salt stress and 5-fluorocytosine in synthetic yeast strains via SCRaMbLE

Varied environmental stress can affect cell growth and activity of the cellular catalyst.Traditional path of adaptive evolution generally takes a long time to achieve a tolerance phenotype,meanwhile,it is a challenge to dissect the underlying genetic mechanism.Here,using SCRaMbLE,a genome scale tool to generate random structural variations,a total of 222 evolved yeast strains with enhanced environmental tolerances were obtained in haploid or diploid yeasts containing six synthetic chromosomes.Whole genome sequencing of the evolved strains revealed that these strains generated different structural variants.Notably,by phenotypic-genotypic analysis of the SCRaMbLEd strains,we find that a deletion of gene YFR009W(GCN20)can improve salt tolerance of Saccharomyces cerevisiae,and a deletion of gene YER056C can improve 5-flucytosine tolerance of Saccharomyces cerevisiae.This study shows applications of SCRaMbLE to accelerate phenotypic evolution for varied environmental stress and to explore relationships between structural variations and evolved phenotypes.

Design, analysis and application of synthetic microbial consortia

The rapid development of synthetic biology has conferred almost perfect modification on single cells,and provided methodological support for synthesizing microbial consortia,which have a much wider application potential than synthetic single cells.Co-cultivating multiple cell populations with rational strategies based on interacting relationships within natural microbial consortia provides theoretical as well as experimental support for the successful obtaining of synthetic microbial consortia,promoting it into extensive research on both industrial applications in plenty of areas and also better understanding of natural microbial consortia.According to their composition complexity,synthetic microbial consortia are summarized in three aspects in this reviewand are discussed in principles of design and construction,insights and methods for analysis,and applications in energy,healthcare,etc.

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

Naturally occurring structural variations(SVs)are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes.Controllable methods aimed at introducing the complex SVs and their related molecular mechanisms have remained farfetched.In this study,an SV-prone yeast strain was developed using Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution(SCRaMbLE)technology with two synthetic chromosomes,namely synV and synX.The biosynthesis of astaxanthin is used as a readout and a proof of concept for the application of SVs in industries.Our findings showed that complex SVs,including a pericentric inversion and a trans-chromosome translocation between synV and synX,resulted in two neo-chromosomes and a 2.7-fold yield of astaxanthin.Also,genetic targets were mapped,which resulted in a higher astaxanthin yield,thus demonstrating the SVs’ability to reorganize genetic information along the chromosomes.The rational design of trans-chromosome translocation and pericentric inversion enabled precise induction of these phenomena.Collectively,this study provides an effective tool to not only accelerate the directed genome evolution but also to reveal the mechanistic insight of complex SVs for altering phenotypes.

Construction of synthetic microbial consortia for 2-keto-L-gulonic acid biosynthesis

Currently,the establishment of synthetic microbial consortia with rational strategies has gained extensive attention,becoming one of the important frontiers of synthetic biology.Systems biology can offer insights into the design and construction of synthetic microbial consortia.Taking the high-efficiency production of 2-keto-L-gulonic acid(2-KLG)as an example,we constructed a synthetic microbial consortium“Saccharomyces cerevisiae-Ketogulonigenium vulgare”based on systems biology analysis.In the consortium,K.vulgare was the 2-KLG pro-ducing strain,and S.cerevisiae acted as the helper strain.Comparative transcriptomic analysis was performed on an engineered S.cerevisiae(VTC2)and a wild-type S.cerevisiae BY4741.The results showed that the up-regulated genes in VTC2,compared with BY4741,were mainly involved in glycolysis,TCA cycle,purine metabolism,and biosynthesis of amino acids,B vitamins,and antioxidant proteases,all of which play important roles in pro-moting the growth of K.vulgare.Furthermore,Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG.Therefore,VTC2 would be of great advantage in working with K.vulgare.Thus,the synthetic microbial consortium"VTC2-K.vulgare"was constructed based on transcriptomics analyses,and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K.vulgare,reaching 13.2±0.52 g/L.In addition,the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes(sod,cat and gpd)in K.vulgare.The results indicated that the oxida-tive stress in the synthetic microbial consortium was efficiently reduced.Thus,systems analysis confirmed a favorable symbiotic relationship between microorganisms,providing guidance for further engineering synthetic consortia.

Collaborations of China with the world in Synbio

The emerging field of synthetic biology has gained great attractions in recent years, both in China and around the world. As a result, the number of publications in this field has grown exponentially in the past decade. Analysis of this trend can be found in the article ＂Bibliometrics analysis of synthetic biology based on Web of Science＂ [1], which is authored in 2015 by researchers from Peking Union Medical College/Chinese Academy of Medical Sciences with support from the Consulting Project of Chinese Academy of Engineedng. Just using ＂synthetic biology＂ as the ＂topic＂ in Web of Science Core Collection, we can get a coarse estimate of the speed of its growth （Fig. 1）. Only 56 papers were published in 1998 globally and in 2016 the number reached more than 1000. In China, only 10 papers were published in 2007 and the numbers reached over 150 each year in 2015 and 2016.

Simultaneous saccharification and fermentation of sweet potato powder for the production of ethanol under conditions of very high gravity

Due to its merits of drought tolerance and high yield,sweet potatoes are widely considered as a potential alterative feedstock for bioethanol production.Very high gravity(VHG)technology is an effective strategy for improving the efficiency of ethanol fermentation from starch materials.However,this technology has rarely been applied to sweet potatoes because of the high viscosity of their liquid mash.To overcome this problem,cellulase was added to reduce the high viscosity,and the optimal dosage and treatment time were 8 U/g(sweet potato powder)and 1 h,respectively.After pretreatment by cellulase,the viscosity of the VHG sweet potato mash(containing 284.2 g/L of carbohydrates)was reduced by 81%.After liquefaction and simultaneous saccharification and fer-mentation(SSF),thefinal ethanol concentration reached 15.5%(v/v),and the total sugar conversion and ethanol yields were 96.5%and 87.8%,respectively.

Synthetic genome with recoding

With the development of DNA synthesis techniques,synthetic biology has enabled redesign and construction of genome on purposes(Luo et al.,2018).The whole-genome recoding of Escherichia coli could be used to enhance incorporation of non-natural amino acids into proteins and to construct safer and multi-virus-resistant strains,which has always been a long-cherished wish in synthetic biology.In recent years,many research teams around the world have been engaged in genetic recoding.The Church group in Harvard University used the MAGE technique to convert 314 TAG stop codons into TAA,generating a first E.coli that is completely recoding for TAG codon(Isaacs et al.,2011).