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Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection

A technology of genetic engineering, cyanobacteria, applied in the field of metabolically engineering cells to increase their ability to compete with contaminating microorganisms without antibiotics, which can solve problems such as the risk of horizontal gene transfer of antibiotic resistance cassettes

Pending Publication Date: 2021-07-23
NANYANG TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if this proves that these pathways confer advantages to organisms that carry them, the risk of horizontal gene transfer of one or more antibiotic resistance cassettes remains

Method used

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  • Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection
  • Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection
  • Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1: method

[0114] Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0115] cell growth conditions

[0116] Synechococcus PCC 7002 (a gift from Prof. Donald Bryant, Penn State University, USA) using the D7 micronutrient [Arnon et al., Biochim BiophysActa.357:231-45 (1974)], according to Supplemented with 12mM sodium nitrate (AD7-NO 3 ), 4mM cyanurate (AD7-Cya) or 2mM melamine (AD7-Mel), and vitamin B 12 (0.01 mg / L) in medium A [Stevens et al., J Phycol. 9:427-430 (1973)] in photoautotrophic growth. For phosphite-utilizing strains, potassium dihydrogen phosphate (Pho) was replaced by potassium dihydrogen phosphite (Phi, Rudong Huayun Chemical Co., Ltd., Jiangsu, China) at 0.370 mM (Pho 1x or Phi1x). Solid medium was prepared by supplementing the above medium w...

Embodiment 2

[0142] Introduction of melamine degradation pathway requires evolutionary adaptation for efficient utilization

[0143] The melamine degradation pathway utilized in this study was based on the optimal pathway reported by Shaw and colleagues (genes triA, guaD, trzC, atzD, trzE, and DUR1,2, including the R352S mutation described in the guaD gene product) [Shaw et al. People, Science.353:583-6(2016)]( figure 1 ). In our case we used codon optimized genes (triA, SEQ ID NO:70; guaD, SEQ ID NO:80; trzC, SEQ ID NO:74; atzD, SEQ ID NO:78; trzE SEQ ID NO:78; NO:72; and DUR1,2, SEQ ID NO:76), a synthetic strong cyanobacterial promoter P c223 (SEQ ID NO:82) [Markley et al., ACS Synth Biol.4:595 (2015)] and the strongest RBS sequence (AGGAGA) tested in Syn7002 [Markley et al., ACS Synth Biol.4:595 (2015) )] upstream of all 6 genes. The intergenic region (21bp in total), including the space before (7bp) and after (8bp) the RBS sequence, was generated by a random DNA sequence generator ...

Embodiment 3

[0150] Phosphite and PtxD can be used as efficient selection systems in Synechococcus sp. PCC7002

[0151] Although phosphite (Phi) was previously shown to be able to maintain Synechocystis sp. PCC 6803 [Polyviou et al., Environmental microbiology reports. 7:824-30 (2015)] and Synechocystis sp. PCC 7942 [Motomura et al., ACS Synth Biol. 10:1021 (2018)] Growth of modified strains of both, in both cases genetic manipulation (integration of operons containing ptxD as well as specific Phi transporters) is driven by antibiotic selection pressure . Also, neither strain (wild-type Synechocystis PCC 6803 or wild-type Synechococcus PCC 7942) appears to be unable to take up Phi without including the specific transporter, thus making the construct too large to be practical as a selection marker. use. Since no data on the ability of Syn7002 to grow on Phi as the sole P source exists in the literature, the growth of the WT strain was tested using different concentrations of Phi ( Image...

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Abstract

The present invention relates to methods of metabolic engineering cells to increase their ability to compete with contaminating microorganisms without the need for antibiotics. More particularly, the invention provides methods to engineer cyanobacteria to utilize melamine as nitrogen source, phosphite as phosphorous source, optionally also utilizing NADP<+> over NAD<+>, and also provides genetically engineered cyanobacteria made using such methods. In a particular embodiment, the genetically engineered cells are cyanobacterium transformed by at least one polynucleotide molecule comprising heterologous melamine utilization pathway genes, atzD, trzE, DUR1,2, trzC, guaD and triA operably linked to at least one promoter and / or comprising a further phosphite dehydrogenase (ptxD ) gene.

Description

[0001] field of invention [0002] The present invention relates to methods of metabolically engineering cells to increase their ability to compete with contaminating microorganisms without the need for antibiotics. More particularly, the present invention provides a method of engineering cyanobacteria to utilize melamine as a nitrogen source, phosphite as a phosphorus source, optionally also utilizing NADP+ instead of NAD+, and also provides genetically engineered cells made using this method . Background technique [0003] Cyanobacteria are increasingly being used for metabolic engineering as part of efforts to advance a carbon-neutral economy. They are photoautotrophs, able to grow with fairly simple requirements - minimal medium with inorganic nitrogen and phosphorus sources, use of light for energy generation and conversion of CO 2 as the only carbon input. In recent years, it has been demonstrated that cyanobacteria are capable of producing a large number of different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52
CPCC12N15/52C12N9/78C12N9/80C12Y305/01084C12Y305/02015C12N9/86C12Y305/04003C12Y120/01001C12N9/0004C12N1/38C12N15/74
Inventor 蒂亚戈·德索萨·若热·托斯卡诺·塞里奥英厄·比吉塔·诺林彼得·朱利安·尼克松
Owner NANYANG TECH UNIV
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