Efficient assembly of long <scp>DNA</scp> fragments and multiple genes with improved nickase‐based cloning and Cre/<scp><i>loxP</i></scp> recombination

Zhao, Yongchun; Han, Jingluan; Tan, Jiantao; Yang, Yugui; Li, Shuangchun; Gou, Yajun; Luo, Yuyu; Li, Tie; Xiao, Wenyu; Yang, Xue; Yu, Hao; Xie, Xianrong; Liu, Yao‐Guang; Zhu, Qinlong

doi:10.1111/pbi.13882

Cited by 9 publications

(6 citation statements)

References 52 publications

(88 reference statements)

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“…Recently, BvADHa was found to exhibit relaxed sensitivity to tyrosine and overexpression of this enzyme enhanced tyrosine content in tobacco leaves [ 27 ]. Simultaneous overexpression of this gene together with core biosynthesis genes in tomato and rice did increase betalain production [ 28 , 44 ]. Therefore, this enzyme may also be a critical tool to enhance the heterogenous production of betalains in carrots.…”

Section: Discussionmentioning

confidence: 99%

Generating colorful carrot germplasm through metabolic engineering of betalains pigments

Deng

Duan

Liu

et al. 2023

Horticulture Research

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Betalains are tyrosine-derived plant pigments exclusively found in the Caryophyllales order and some higher fungi and generally classified into two groups: red-violet betacyanins and yellow-orange betaxanthins. Betalains attract great scientific and economic interest because of their relatively simple biosynthesis pathway, attractive colors and health-promoting properties. Co-expressing two core genes BvCYP76AD1 and BvDODA1 with or without a glycosyltransferase gene MjcDOPA5GT allowed the engineering of carrot (an important taproot vegetable) to produce a palette of unique colors. The highest total betalains content, 943.2 μg·g-1 DW, was obtained in carrot taproot transformed with p35S:RUBY which produces all of the necessary enzymes for betalains synthesis. Root specific production of betalains slightly relieved tyrosine consumption revealing the possible bottleneck in betalains production. Furthermore, a unique volcano-like phenotype in carrot taproot cross section was created by vascular cambium specific production of betalains. The betalains-fortified carrot in this study is thus anticipated to be used as functional vegetable and colorful carrot germplasm in breeding to promote health.

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Section: Discussionmentioning

confidence: 99%

Generating colorful carrot germplasm through metabolic engineering of betalains pigments

Deng

Duan

Liu

et al. 2023

Horticulture Research

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“…The vector also included the hptII gene under the control of 35S promoter and poly(A) terminator from the cauliflower mosaic virus. Multiple genes were stacked in a single plasmid of pYLTAC380H or pYL1300H using the unique nucleotide sequence-guided nicking endonuclease mediated DNA assembly (UNiEDA) [ 34 ]. Each gene in the multigene construct was assembled into a cassette containing the promotor of the rRNA operon from tobacco ( Prrn ), the leader sequence from gene 10 of bacteriophage T7 ( T7Lg10 ), and the terminator of the chloroplast rbcL gene ( TrbcL ) [ 24 ].…”

Section: Methodsmentioning

confidence: 99%

Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum

Zhao,

Yang

et al. 2023

IJMS

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The sesquiterpene alkaloid dendrobine, widely recognized as the main active compound and a quality control standard of medicinal orchids in the Chinese Pharmacopoeia, demonstrates diverse biological functions. In this study, we engineered Dendrobium catenatum as a chassis plant for the production of dendrobine through the screening and pyramiding of key biosynthesis genes. Initially, previously predicted upstream key genes in the methyl-D-erythritol 4-phosphate (MEP) pathway for dendrobine synthesis, including 4-(Cytidine 5′-Diphospho)-2-C-Methyl-d-Erythritol Kinase (CMK), 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (DXR), 2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (MCT), and Strictosidine Synthase 1 (STR1), and a few downstream post-modification genes, including Cytochrome P450 94C1 (CYP94C1), Branched-Chain-Amino-Acid Aminotransferase 2 (BCAT2), and Methyltransferase-like Protein 23 (METTL23), were chosen due to their deduced roles in enhancing dendrobine production. The seven genes (SG) were then stacked and transiently expressed in the leaves of D. catenatum, resulting in a dendrobine yield that was two-fold higher compared to that of the empty vector control (EV). Further, RNA-seq analysis identified Copper Methylamine Oxidase (CMEAO) as a strong candidate with predicted functions in the post-modification processes of alkaloid biosynthesis. Overexpression of CMEAO increased dendrobine content by two-fold. Additionally, co-expression analysis of the differentially expressed genes (DEGs) by weighted gene co-expression network analysis (WGCNA) retrieved one regulatory transcription factor gene MYB61. Overexpression of MYB61 increased dendrobine levels by more than two-fold in D. catenatum. In short, this work provides an efficient strategy and prospective candidates for the genetic engineering of D. catenatum to produce dendrobine, thereby improving its medicinal value.

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“…Up to 10 gene cassettes in a single T‐DNA were successfully transferred into rice [64]. Unique nucleotide sequence‐guided nicking endonuclease (UNiE)‐mediated DNA assembly (UNiEDA) was introduced into TGSII system and developed TGSII‐UNiE to assemble more genes in parallel into acceptor/donor vectors [66]. 11 genes with three different synthetic pathways were successfully assembled into a T‐DNA region with the TGSII‐UNiE approach [66].…”

Section: Quantitative Plant Synthetic Biology Is Emergingmentioning

confidence: 99%

From qualitative to quantitative: the state of the art and challenges for plant synthetic biology

Tian,

Li,

Wang

2023

Quant. Biol.

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The flourishing plant science promotes the exploding number of data and the expansion of toolkits. Plant synthetic biology is still in its early stages and requires more quantitative and predictable study. Despite the challenges, some pioneering examples have been successfully demonstrated in model plants.BackgroundsAs an increasing number of synthetic switches and circuits have been created for plant systems and of synthetic products produced in plant chassis, plant synthetic biology is taking a strong foothold in agriculture and medicine. The ever‐exploding data has also promoted the expansion of toolkits in this field. Genetic parts libraries and quantitative characterization approaches have been developed. However, plant synthetic biology is still in its infancy. The considerations for selecting biological parts to design and construct genetic circuits with predictable functions remain desired.ResultsIn this article, we review the current biotechnological progresses in field of plant synthetic biology. Assembly standardization and quantitative approaches of genetic parts and genetic circuits are discussed. We also highlight the main challenges in the iterative cycles of design‐build‐test‐learn for introducing novel traits into plants.ConclusionPlant synthetic biology promises to provide important solutions to many issues in agricultural production, human health care, and environmental sustainability. However, tremendous challenges exist in this field. For example, the quantitative characterization of genetic parts is limited; the orthogonality and the transfer functions of circuits are unpredictable; and also, the mathematical modeling‐assisted circuits design still needs to improve predictability and reliability. These challenges are expected to be resolved in the near future as interests in this field are intensifying.

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Efficient assembly of long DNA fragments and multiple genes with improved nickase‐based cloning and Cre/loxP recombination

Cited by 9 publications

References 52 publications

Generating colorful carrot germplasm through metabolic engineering of betalains pigments

Generating colorful carrot germplasm through metabolic engineering of betalains pigments

Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum

From qualitative to quantitative: the state of the art and challenges for plant synthetic biology

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