Designing artificial synthetic promoters for accurate,smart,and versatile gene expression in plants

With the development of high-throughput biology techniques and artificial intelligence,it has become increasingly feasible to design and construct artificial biological parts,modules,circuits,and even whole systems.To overcome the limitations of native promoters in controlling gene expression,artificial promoter design aims to synthesize short,inducible,and conditionally controlled promoters to coordinate the expression of multiple genes in diverse plant metabolic and signaling pathways.Synthetic promoters are versatile and can drive gene expression accurately with smart responses;they show potential for enhancing desirable traits in crops,thereby improving crop yield,nutritional quality,and food security.This review first illustrates the importance of synthetic promoters,then introduces promoter architecture and thoroughly summarizes advances in synthetic promoter construction.Restrictions to the development of synthetic promoters and future applications of such promoters in synthetic plant biology and crop improvement are also discussed.

Synthetic promoters consisting of defined cis-acting elements link multiple signaling pathways to probenazole-inducible system

Probenazole （3-allyloxy-l,2-benzisothiazole-1,1-dioxide, PBZ）, the active component of Oryzemate, could induce systemic acquired resistance （SAR） in plants through the induction of salicylic acid （SA） biosynthesis. As a widely used chemical inducer, PBZ is a good prospect for establishing a new chemical-inducible system. We first designed artificially synthetic promoters with tandem copies of a single type of cis-element （SARE, JERE, GCC, GST1, HSRE, and W-box） that could mediate the expression of the tS-glucuronidase （GUS） reporter gene in plants upon PBZ treatment. Then we combined different types of elements in order to improve inducibility in the PBZ-inducible system. On the other hand, we were surprised to find that the cis-elements, which are responsive to jasmonic acid （JA） and ethylene, also responded to PBZ, implying that SA, JA, and ethylene pathways also would play important roles in PBZ＇s action. Further analysis demonstrated that PBZ also induced early events of innate immunity via a signaling pathway in which Ca2＋ influx and mitogen-activated protein kinase （MAPK） activity were involved. We constructed synthesized artificial promoters to establish a PBZ chemical-inducible system, and preliminarily explored SA, JA, ethylene, calcium, and MAPK signaling pathways via PBZ-inducible system, which could provide an insight for in-depth study.

Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

AIM:To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.METHODS:We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes.We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter(rAd-SP-rINSfur) into diabetic Lepr db/db mice.A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control(rAd-CMV-βgal).Blood glucose levels and body weights were monitored for 50 d.Glucose and insulin tolerance tests were performed.Immunohistochemical staining was performed to investigate islet morphology and insulin content.RESULTS:Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d,whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic.Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve(AUC):21 508.80 ± 2248.18 vs 62 640.00 ± 5014.28,P < 0.01] and at 6 wk(AUC:29 956.60 ± 1757.33 vs 60 016.60 ± 3794.47,P < 0.01).In addition,insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice(AUC:9150.17 ± 1007.78 vs 11 994.20 ± 474.40,P < 0.05).The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice.CONCLUSION:Based on these results,we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.

Expression liver-directed genes by employing synthetic transcriptional control units

AIM： To generate and characcerize the synthetic transoiptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems. METHODS： Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS： A new bifunctionally-enhanced green fluorescent protein （EGFP）/neor fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to “naturally” derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 （APOB） promoter or the ornithin transcarbamoylase （OTC） enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor （HNF）, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein （C/EBP） only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xAPOB cassette construct, which fully complied with the dataobtained by initial FACS analysis CONCLUSION： Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.

Perspective on the use of synthetic biology in rudimentary food fermentations

Rudimentary food fermentation can be defined as a spontaneous process of conversion of food components through enzy-matic action.A great variety of fermented foods are produced using spontaneous approaches;however,cocoa and coffee represent the most important agricultural commodities on international markets.As a manner to increase the efficiency of these processes,starter cultures have been developed and applied under field conditions.The selection process begins with the recovery of microbial strains from spontaneous fermentation through phenotypic and metabolic traits.Next,mutation-based breeding is used to develop and improve well-adapted starter cultures.With advances in synthetic biology,especially in the last decade,the development of robust cellular fabrications with high fermentative capacity has become easier-largely due to the development of genomic approaches,such as next-generation sequencing techniques,CRISPR-Cas system and bioinformatics tools.This review brings prospects on the use of synthetic biology to design new robust strains for use in cocoa and coffee fermentations,but which can be extended to other rudimentary foods.In addition,metabolic traits and target genes(e.g.,UvrA,RecA,GPD1,and GPP2)are proposed as a starting point for the improvement of cocoa and coffee starters.Finally,the regulatory and safety requirements for these food crops are addressed.This review aims to stimulate research on the process of fermentation and the associated synthetic biology tools to produce fermented food efficiently and sustainably.

Superaligned carbon nanotubes guide oriented cell growth and promote electrophysiological homogeneity for synthetic cardiac tissues

With the support by the National Natural Science Foundation of China,the research team led by Professor Li Yigang(李毅刚)at Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Professor Peng Huisheng(彭慧胜)at Fudan University presented a new type of

Prolonged control of insulin-dependent diabetes via intramuscular expression of plasmid-encoded single-strand insulin analogue

Daily insulin injection is necessary for the treatment of the insulin-dependent diabetes. However, the process is painful and inconvenient. Accordingly, we have made exploratory efforts to establish an alternative method for continuous insulin supply via intramuscular injection of a designed plasmid encoding the single-strand insulin analogue (SIA), which provides safe, effective and prolonged control of insulin-dependent diabetes. To generate a SIA, a short flexible peptide was alternatively introduced into the natural proinsulin to replace its original long and rigid C-peptide. Then, the synthetic promoter SP301 was used to drive potent and specific expression of SIA in skeletal muscle cells. By combining the Pluronic L64 and low-voltage electropulse (L/E), the specialized gene delivery technique was applied to efficiently transfer the constructed plasmid into skeletal muscle cells via intramuscular injection. Through these efforts, a plasmid-based intramuscular gene expression system was established and improved, making it applicable for gene therapy. The plasmid-expressed SIA showed biological functions that were similar to that of natural insulin. A single L/E-pSP301-SIA administration provided sustained SIA expression in vivo for about 1.5 months. In addition, the diabetic mice treated with L/E-pSP301-SIA were much healthier than those with other treatments. This plasmid-based system was safe for the treatment of diabetes and did not cause immune responses or pathological damage. The results confirmed that, in a mouse model, long-term positive effects were achieved by a single intramuscular L/E-pSP301-SIA injection, which consequently provided reliable experimental basis for its clinical application for the treatment of diabetes mellitus with promising prospects.