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An effective cytokine combination for ex vivo expansion of porcine muscle stem cells

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food Bioscience 2022年第2期46卷 987-996页

作者： Qingzi Lei Mei Li Guocheng Du Jingwen Zhou Xin Guan Science center for Future foods Jiangnan University1800 Lihu RoadWuxiJiangsu214122China National engineering Laboratory for Cereal Fermentation Technology(NELCF) Jiangnan University1800 Lihu RoadWuxiJiangsu214122China engineering research center of ministry of education on food synthetic biotechnology Jiangnan University1800 Lihu RoadWuxiJiangsu214122China

Cultured meat technology is a novel and promising strategy for sustainable and effective meat *** stem cells are widely used as seed cell populations because of their ease of access and myogenic differentiation ***,muscle stem cells are difficult to continuously propagate ex vivo and are heavily dependent upon serum for survival and growth,which impedes their commercial use as a cultured meat ***,we identified an effective four-cytokine combination to promote long-term proliferation of porcine muscle stem cells using a serial elimination screening approach,which was consisted of long chain human insulin growth factor-1,platelet derived growth factor BB,basic fibroblast growth factor,and epidermal growth *** expansion of muscle stem cells with the four-cytokine combination achieved a 6.31×107-fold increase in cell number after 28 days of culture with retained cell myogenic differentiation potential,and most importantly,reduced the amount of fetal bovine serum required for cell culture by at least 5%.Furthermore,the four-cytokine combination exerted the pro-proliferative function by activating PI3K/Akt/mTOR and MEK/ERK signaling *** approach provides for a new means by which to industrialize the production of cultured meat.

关键词： Cellular agriculture Cytokine combination Cultured meat Cell proliferation Muscle stem cells

Highly efficient multiplex base editing:One-shot deactivation of eight genes in Shewanella oneidensis MR-1

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synthetic and Systems biotechnology 2023年第1期8卷 1-10页

作者： Yaru Chen Meijie Cheng Yan Li Lin Wang Lixia Fang Yingxiu Cao Hao Song Frontier Science center for synthetic Biology and Key Laboratory of Systems Bioengineering(ministry of education) School of Chemical Engineering and TechnologyTianjin UniversityTianjin300072China Key Laboratory of Systems Bioengineering(ministry of education) Tianjin UniversityTianjin300072China

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical ***,researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer *** editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis *** multiplexed application of base editing is still limited by its low throughput procedure,we thus here develop a rapid and efficient multiplex base editing system in *** approaches to express multiple gRNAs were assessed firstly,and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic ***,a smart scheme was designed to deliver one-pot assembly of multiple gRNAs.3,5,and 8 genes were deactivated using this system with editing efficiency of 83.3%,100%and 12.5%,*** offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette,different promoters,handles,and terminators were *** multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control *** maximum power density of the multiplex engineered strain HRF(8BE)in microbial fuel cells was 1108.1 mW/m2,which was 21.67 times higher than that of the wild-type *** highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella,and may provide valuable insights in fundamental and applied research of extracellular electron transfer.

关键词： Multiplex gene editing Electroactive microorganisms Base editing Multiplexed engineering CRISPR Extracellular electron transfer

Synthesis and assembly of full-length cyanophage A-4L genome

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synthetic and Systems biotechnology 2023年第1期8卷 121-128页

作者： Ting Zhang Bonan Xu Jia Feng Pingbo Ge Guorui Li Jiabao Zhang Jianting Zhou Jianlan Jiang School of Chemical engineering and Technology Tianjin UniversityTianjin300072China Key Laboratory of Systems Bioengineering(ministry of education) Tianjin UniversityTianjin300072China Frontier Science center for synthetic Biology(ministry of education) Tianjin UniversityTianjin300072China

Artificial cyanophages are considered to be an effective biological method to control harmful cyanobacterial ***,no synthetic cyanophage genome has been constructed and where its obstacles are ***,we survey a stretch of 16 kb length sequence of cyanophage A-4L that is unclonable in Escherichia *** test 12 predicted promoters of cyanophage A-4L which were verified all active in ***,we screen for eight ORFs that hindered the assembly of intermediate DNA fragments in *** and describe that seven ORFs in the 16 kb sequence could not be separately cloned in *** of unclonable ORFs in high-copy-number plasmid were successfully cloned using low-copy-number vector,suggesting that these ORFs were *** propose a clone strategy abandoned the promotor and the start codon that could be applied for unclonable ***,we de novo synthesized and assembled the full-length genome of cyanophage *** work deepens the understanding of synthetic cyanophages studies.

关键词： Genome assembly Cyanophage A-4L Toxic ORFs screening

Characterization and implications of prokaryotic ribosome-binding sites across species

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Systems Microbiology and Biomanufacturing 2022年第4期2卷 676-684页

作者： Yanting Duan Xiaojuan Zhang Weiji Zhai Jinpeng Zhang Jiawei Ren Xiaomei Zhang Guoqiang Xu Jinsong Shi Zhenghong Xu biotechnology of ministry of education School of BiotechnologyJiangnan UniversityWuxiChina National engineering research center for Cereal Fermentation and food Biomanufacturing Jiangnan UniversityWuxiChina School of Life Science and Health engineering Jiangnan UniversityWuxi 214122China Jiangsu engineering research center for Bioactive Products Processing Technology Jiangnan University1800 Lihu AvenueWuxi 214122China

The ribosome-binding site(RBS)in the 5′untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression *** have been widely applied in regulating gene expression in various scenarios,including Gram-negative or Gram-positive ***,we first rationally designed and constructed an RBS mutant library containing 66 *** strength of these RBSs in *** and *** was characterized *** RBS strength spanned about 200 and 15 times in the two species,*** strength of RBSs in *** was generally lower than that of in ***.A total of 18 RBSs showed similar strength(within twofold differences)between the species in our study,and the correlation analysis of the strength of RBSs between *** and ***(R^(2)=0.7483)revealed that these RBSs can be used across *** sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function *** RBS characterized here can be used to precisely regulate gene expression in both hosts,and the characteristics of cross-species RBSs provide basic information for RBS rational design.

关键词： RBS library Cross-species RBSs Sequence characteristics Escherichia coli Corynebacterium glutamicum

Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

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synthetic and Systems biotechnology 2022年第4期7卷 1046-1055页

作者： Weiji Zhai Yanting Duan Xiaomei Zhang Guoqiang Xu Hui Li Jinsong Shi Zhenghong Xu Xiaojuan Zhang biotechnology of ministry of education School of BiotechnologyJiangnan UniversityWuxiChina National engineering research center for Cereal Fermentation and food Biomanufacturing Jiangnan UniversityWuxiChina School of Life Science and Health engineering Jiangnan UniversityWuxi214122China Jiangsu Provincial engineering research center for Bioactive Product Processing Jiangnan University1800 Lihu AvenueWuxi214122China

The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the ***,leaking transcription is common due to varied terminator *** of the representative hairpin and U-tract structure,detailed sequence and thermodynamic features of terminators were not completely clear,and the effect of terminator on the upstream gene expression was ***,it is still challenging to use terminator to control expression with higher ***,in ***,we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream ***,terminator mutation library was constructed,and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq *** result showed that under the regulation of terminators,a negative correlation was presented between the expression of upstream and downstream genes(r=􀀀0.60),and the terminators with lower free energy corelated with higher upstream gene ***,the terminator with longer stem length,more compact loop and perfect U-tract structure was benefit to the transcription ***,a terminator strength classification model was established,and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain *** results help to elucidate the role of terminators in gene expression,and the key factors identified are crucial for rational design of terminators,and the model provided a method for terminator strength prediction.

关键词： Intrinsic terminator Transcription termination Machine learning Structure-activity relationship FlowSeq Free energy

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

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synthetic and Systems biotechnology 2023年第1期8卷 168-175页

作者： Xiuru Shen Lingtong Liao Guoqiang Zhang Jingwen Zhou Jianghua Li Guocheng Du Science center for Future foods Jiangnan UniversityWuxi214122China National engineering research center for Cereal Fermentation and food Biomanufacturing Jiangnan University1800 Lihu RoadWuxiJiangsu214122China School of biotechnology and Key Laboratory of Industrial biotechnology Ministry of EducationJiangnan University1800 Lihu RoadWuxiJiangsu214122China

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and ***,extracellular lactase production was still restricted to the process of cell *** this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-*** purified enzyme was identified as a neutral lactase with an optimum pH of *** facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of *** was knocked *** could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme *** optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient *** JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

关键词： Kluyveromyces lactis Lactase Screening Enzymatic properties Mannoprotein Fermentation optimization Secretion

Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin

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synthetic and Systems biotechnology 2023年第2期8卷 273-280页

作者： Peng Zhang Jin-Song Gong Zhi-Hao Xie Chang Su Xiao-Mei Zhang Zhi-Ming Rao Zheng-Hong Xu Jin-Song Shi Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of Life Sciences and Health EngineeringJiangnan UniversityWuxi214122PR China National engineering research center for Cereal Fermentation and food Biomanufacturing School of BiotechnologyJiangnan UniversityWuxi214122PR China Jiangsu Provincial engineering research center for Bioactive Product Processing Jiangnan UniversityWuxi214122PR China Yixing Institute of food and biotechnology Co. LtdYixing214200PR China School of Chemical and Material engineering Jiangnan UniversityWuxi214122PR China

Phospholipase D(PLD)is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid ***,the efficient heterologous expression of PLD is limited by its cell *** this study,a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL.A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was estab-lished,and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation,191.30-fold higher than that of the *** optimum reaction conditions,a 61.61%conversion ratio and 21.07 g/L of phosphatidylserine production were ***,the synthesis system of PL derivates was established,which could efficiently synthesis novel PL *** results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application,and laid the foundation for the biosynthesis of phosphatidylserine and other PL *** far as we know,this work re-ports the highest level of extracellular PLD expression to date and the enzymatic production of several PL der-ivates for the first time.

关键词： Phospholipase D Secretion expression Bacillus subtilis Biosynthesis Enzymatic PLs modification

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

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synthetic and Systems biotechnology 2023年第1期8卷 61-68页

作者： Guobin Yin Anqi Peng Luyao Zhang Yang Wang Guocheng Du Jian Chen Zhen Kang The Science center for Future foods Jiangnan UniversityWuxi214122China The Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China The Key Laboratory of Industrial biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for *** are still not fully ***,a bottom-up approach is proposed for designing artificial trans-acting *** engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing *** applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin *** sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for *** and *** but also enhanced our understanding of trans-acting sRNAs.

关键词： Gene knockdown Posttranscriptional regulation Small noncoding RNA synthetic biology Cross-species Bacillus subtilis

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

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synthetic and Systems biotechnology 2023年第1期8卷 86-96页

作者： Yang Wang Guobin Yin Huanjiao Weng Luyao Zhang Guocheng Du Jian Chen Zhen Kang The Science center for Future foods Jiangnan UniversityWuxi214122China The Key Laboratory of Industrial biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi214122China The Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationJiangnan UniversityWuxiChina

Gene regulation by trans-acting small RNAs(sRNAs)has considerable advantages over other gene regulation ***,synthetic sRNAs mainly take natural sRNAs(MicC or SgrS)as backbones and comprise three functional elements folding into two or more stem-loop structures:an mRNA base pairing region,an Hfq-binding structure,and a rho-independent *** to limited numbers of natural sRNAs and complicated backbone structures,synthetic sRNAs suffer from low activity programmability and poor structural ***,natural sRNA backbone sequences may increase the possibility of unwanted ***,we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs(ssl-sRNAs),which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight ***,ssl-sRNA requires no independent Hfq-binding structure,as the polyU tail fulfills the roles of binding Hfq.A thermodynamic-based scoring model and a web server sslRNAD(http://***/)were developed for automated design of ssl-sRNAs with well-defined structures and programmable ***-sRNAs displayed weak polar effects when regulating polycistronic *** ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis.A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA *** examples,the *** cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ***-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

关键词： Regulatory RNA Metabolic engineering synthetic biology De novo design Ergothioneine