METABOLIC ENGINEERING
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2018
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IF 7.674
Shubin Li,Tao Sun,Chunxiao Xu,Lei Chen,Weiwen Zhang
The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973) has been considered a good chassis candidate for “microbial cell factory” as it can perform oxygenic photosynthesis and its doubling time can be as short as 1.9 h. However, the limited genetic tools currently restrict its further research and application efforts using synthetic biology approaches. In this study, a series of genetic tools were systematically developed and optimized for Synechococcus 2973. First, the introduction of Tfp pilus assembly protein encoding gene pilN into Synechococcus 2973 successfully recovered its natural transformability, which greatly simplified the DNA transformation process. Second, a series of promoters with different strengths were evaluated and the super-strong promoters including P cpc560 from Synechocystis sp. PCC 6803, native P psbA2 and P psbA3 of Synechococcus 2973 were found with the highest activity of β -galactosidase among those evaluated by miller values. Some promoters related to photosystems (i.e., P psbA2 , P psbA3 , P 6803psbA2 and P cpc560 ) were also demonstrated to be induced by high intensity of light. Third, three lactose induction systems were evaluated, among which P lac combined with lacI q showed the best application prospect with great induction capacity, low leakage and middle induced expression. Fourth, the translational on riboswitch theoE* , the transcriptional off riboswitches theo/yitJ and xpt(C74U)/metE and an artificial inducing system combining theoE* with T7 RNA polymerase were successfully developed and characterized in Synechococcus 2973. Finally, by using T7 induction system to control the expression of both small RNA and chaperone Hfq, a small RNA regulatory tool was developed and optimized to be a strictly inducible off system for gene regulation in Synechococcus 2973. The work here presented valuable genetic toolboxes necessary for metabolic engineering and synthetic biology research in Synechococcus 2973, which will facilitate the future application of the fast growing cyanobacterial chassis.
研究领域:
合成生物学
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代谢工程
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微生物学
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PLANT BIOTECHNOLOGY JOURNAL
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2026
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IF 12.8
Shubin Li,Tao Sun,Dailin Liu,Tong Zhang,Lei Chen,Weiwen Zhang
The single crossover occurring via homologous recombination is a common phenomenon existing among microbes like Escherichia coli , Bacillus subtilis , Vibrio natriegens , Gluconobacter oxydans and most cyanobacteria species, threatening the stability of engineered strains and challenging iterative genetic engineering. Among them, we take the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) as a representative study due to its promising roles for CO 2 fixation and bioconversion. We established three marker-free platforms to achieve stable genome recombination: (i) T4CROSS, which employs two plasmids and four rounds of single crossover; (ii) TRIPLEARM, which uses a single plasmid containing three homologous arms for three rounds of single crossover; and (iii) CRISPRARM, which integrates CRISPR/Cpf1-mediated genome editing with homologous recombination. As proof of concept, we employed the CRISPRARM platform for a three-step sequential engineering of the sucrose biosynthetic pathway. The final engineered strain produced 7.12 g L −1 of sucrose within 4 days.
✓ 已验证
DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2020
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IF 3.644
Sun Tao,Zhang Yingying,Zhang Chaonan,Wang Hanjie,Pan Huizhuo,Liu Jing,Li Zhixiang,Chen Lei,Chang Jin,Zhang Weiwen
Hypoxia not only alters tumor microenvironment but leads to the tumor progression and metastasis as well as drug resistance. As a promising strategy, photodynamic therapy (PDT) can inhibit tumor by catalyzing O2 to cytotoxic reactive oxygen species. However, its effects were limited by hypoxia and in turn deteriorate hypoxia due to O2 consumption. Hereon, aiming to alleviate hypoxia and promote PDT, a bio-oxygen pump was created based on cyanobacteria, which are the only prokaryotic organisms performing oxygenic photosynthesis. Detailly, controlled-release PDT via loading indocyanine green into mesoporous silica nanoparticles was established. Then bio-oxygen pump based on a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 was tested and further packaged together with PDT to create an injectable hydrogel. The packaged hydrogel showed stable oxygen production and synergetic therapy effect especially toward hypoxia 4T1 cells in vitro. More importantly, strong in vivo therapeutic effect reaching almost 100% inhibition on tumor tissues was realized using PDT equipped with oxygen pump, with only negligible in vivo side effect on healthy mice from S. elongatus UTEX 2973. The new photo-oxygen-dynamic therapy presented here provided a promising strategy against hypoxia-resistant tumor and may worth further modifications for therapeutic application.
研究领域:
生物医学工程
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肿瘤学
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纳米技术
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药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2022
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IF 16.744
Tao Sun,Zhixiang Li,Shubin Li,Lei Chen,Weiwen Zhang
Cyanobacteria have been considered as an ideal photosynthetic chassis for CO 2 recycle and bioconversion, which could make a significant contribution to carbon neutrality. However, several inherited issues associated with current cyanobacterial strains need to be addressed before their biotechnological application, such as slow growth, uneven of carbon distribution, biased production of reducing power and imbalance between growth and biosynthesis. To address the issues, myo -inositol (MI) was selected as an example target and a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 was utilized as chassis to improve cell growth. Second, several genes involved in MI synthesis were evaluated. Third, the MI biosynthesis was further increased by cofactors enhancement and down regulation of the competing pathways using multiplex artificial small RNAs (PTRNAs). Forth, a MI sensor was developed and optimized to trigger the automatic expression PTRNAs to achieve real-time intelligent regulation, avoiding the growth defect caused by blocking essential genes at early growth stage. Fifth, carbon re-direction was achieved using rhythmical cultivation, resulting in a final production of 262.6 mg/L MI in shaking flask, which represents the highest production from CO 2 in cyanobacteria. Finally, the metabolomic analysis was applied to elucidate the shifted metabolic flux and predicted targets for future optimization. Together of all efforts, we can achieve the CO 2 -based MI biosynthesis to the same order of magnitude as heterogenous microorganisms with suitable scale-up in the future.
研究领域:
生物技术
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微生物学
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代谢工程
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合成生物学
✓ 已验证
DOI
高影响因子
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