[Development and characterization of tobacco suspension cell chassis NBS-1].

Plant synthetic biology has significant theoretical advantages in exploration and production of plant natural products. However, its contribution to the field of biosynthesis is currently limited due to the lack of efficient chassis systems and related enabling technologies. Synthetic biologists often avoid tobacco as a chassis system because of its long operation cycle, difficulties in genetic and metabolic modification, complex metabolism and purification background, nicotine toxicity, and challenges in accurately controlling for agricultural production. Nevertheless, the tobacco suspension cell chassis system offers a viable solution to these challenges. The objective of this research was to develop a tobacco suspension cell chassis with high scientific and industrial potential. This chassis should exhibit rapid growth, high biomass, excellent dispersion, high transformation efficiency, and minimal nicotine content. Nicotiana benthamiana, which has high applicability in molecular technology, was used to induce suspension cells. The induced suspension cells, named NBS-1, exhibited rapid growth, excellent dispersion, and high biomass, reaching a maximum biomass of 476.39 g/L (fresh weight), which was significantly higher than that of BY-2. The transformation efficiency of the widely utilized pEAQ-HT transient expression system in NBS-1 reached 81%, which was substantially elevated compared to BY-2. The metabolic characteristics and bias of BY-2 and NBS-1 were analyzed using transcriptome data. It was found that the gene expression of pathways related to biosynthesis of flavonoids and their derivatives in NBS-1 was significantly higher, while the pathways related to alkaloid biosynthesis were significantly lower compared to BY-2. These findings were further validated by the total content of flavonoid and alkaloid. In summary, our research demonstrates NBS-1 possesses minimal nicotine content and provides valuable guidance for selecting appropriate chassis for specific products. In conclusion, this study developed NBS-1, a tobacco suspension cell chassis with excellent growth and transformation, high flavonoid content and minimal nicotine content, which has important guiding significance for the development of tobacco suspension cell chassis.

Synthesis of Crocin I and Crocin II by Multigene Stacking in Nicotiana benthamiana.

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer's disease. Crocins are mainly biosynthesized in the stigmas of food-medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.

Designing plant flavonoids: harnessing transcriptional regulation and enzyme variation to enhance yield and diversity.

Plant synthetic biology has emerged as a powerful and promising approach to enhance the production of value-added metabolites in plants. Flavonoids, a class of plant secondary metabolites, offer numerous health benefits and have attracted attention for their potential use in plant-based products. However, achieving high yields of specific flavonoids remains challenging due to the complex and diverse metabolic pathways involved in their biosynthesis. In recent years, synthetic biology approaches leveraging transcription factors and enzyme diversity have demonstrated promise in enhancing flavonoid yields and expanding their production repertoire. This review delves into the latest research progress in flavonoid metabolic engineering, encompassing the identification and manipulation of transcription factors and enzymes involved in flavonoid biosynthesis, as well as the deployment of synthetic biology tools for designing metabolic pathways. This review underscores the importance of employing carefully-selected transcription factors to boost plant flavonoid production and harnessing enzyme promiscuity to broaden flavonoid diversity or streamline the biosynthetic steps required for effective metabolic engineering. By harnessing the power of synthetic biology and a deeper understanding of flavonoid biosynthesis, future researchers can potentially transform the landscape of plant-based product development across the food and beverage, pharmaceutical, and cosmetic industries, ultimately benefiting consumers worldwide.

Optimising expression and extraction of recombinant proteins in plants.

Recombinant proteins are of paramount importance for research, industrial and medical use. Numerous expression chassis are available for recombinant protein production, and while bacterial and mammalian cell cultures are the most widely used, recent developments have positioned transgenic plant chassis as viable and often preferential options. Plant chassis are easily maintained at low cost, are hugely scalable, and capable of producing large quantities of protein bearing complex post-translational modification. Several protein targets, including antibodies and vaccines against human disease, have been successfully produced in plants, highlighting the significant potential of plant chassis. The aim of this review is to act as a guide to producing recombinant protein in plants, discussing recent progress in the field and summarising the factors that must be considered when utilising plants as recombinant protein expression systems, with a focus on optimising recombinant protein expression at the genetic level, and the subsequent extraction and purification of target proteins, which can lead to substantial improvements in protein stability, yield and purity.

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides.

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides.

Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in Nicotiana benthamiana.

Nicotiana benthamiana is a valuable plant chassis for heterologous production of medicinal plant natural products. This host is well suited for the processing of organelle-localized plant enzymes, and the conservation of the primary metabolism across the plant kingdom often provides required plant-specific precursor molecules that feed a given pathway. Despite this commonality in metabolism, limited precursor supply and/or competing host pathways can interfere with yields of heterologous products. Here, we use transient transcriptional reprogramming of endogenous N. benthamiana metabolism to drastically improve flux through the etoposide pathway derived from the medicinal plant Podophyllum spp. Specifically, coexpression of a single lignin-associated transcription factor, MYB85, with pathway genes results in unprecedented levels of heterologous product accumulation in N. benthamiana leaves: 1 mg/g dry weight (DW) of the etoposide aglycone, 35 mg/g DW (-)-deoxypodophyllotoxin, and 3.5 mg/g DW (-)-epipodophyllotoxin─up to two orders of magnitude above previously reported biosynthetic yields for the etoposide aglycone and eight times higher than what is observed for (-)-deoxypodophyllotoxin in the native medicinal plant. Unexpectedly, transient activation of lignin metabolism by transcription factor overexpression also reduces the production of undesired side products that likely result from competing N. benthamiana metabolism. Our work demonstrates that synthetic activation of lignin biosynthesis in leaf tissue is an effective strategy for optimizing the production of medicinal compounds derived from phenylpropanoid precursors in the plant chassis N. benthamiana. Furthermore, our results highlight the engineering value of MYB85, an early switch in lignin biosynthesis, for on-demand modulation of monolignol flux and support the role of MYB46 as a master regulator of lignin polymer deposition.

Biosynthetic approaches to efficient assimilation of CO2 via photorespiration modification in plant chassis.

Plant chassis has emerged as the platform with great potential for bioproduction of high value-added products such as recombinant protein, vaccine and natural product. However, as the primary metabolic pathway, photorespiration results in the loss of photosynthetically fixed carbon compounds and limits the exploration of plant chassis. People are endeavored to reduce the photorespiration energy or carbon loss based on variation screening or genetic engineering. Insomuch as protein engineering of Rubisco has not resulted in the significant improvement of Rubisco specificity which is linked to the direct CO2 fixation, the biosynthetic approaches of photorespiration bypass are gaining much more attention and manifested great potentiality in conferring efficient assimilation of CO2 in plant chassis. In this review, we summarize the recent studies on the metabolic pathway design and implementation of photorespiration alternative pathway aiming to provide clues to efficiently enhance carbon fixation via the modification of photorespiration in plant chassis for bioproduction. These will benefit the development of plant synthetic metabolism for biorefineries via improvement of artificial carbon sequestration cycle, particularly for the mitigation of serious challenges such as extreme climate change, food and energy shortages in the future.

Heterologous Biosynthesis of Health-Promoting Baicalein in Lycopersicon esculentum.

Baicalein is a valuable flavonoid isolated from the medicinal plant Scutellaria baicalensis Georgi, which exhibits intensive biological activities, such as anticancer and antiviral activities. However, its production is limited in the root with low yield. In this study, In-Fusion and 2A peptide linker were developed to assemble SbCLL-7, SbCHI, SbCHS-2, SbFNSII-2 and SbCYP82D1.1 genes driven by the AtPD7, CaMV 35S and AtUBQ10 promoters with HSP, E9 and NOS terminators, and were used to engineer baicalein biosynthesis in transgenic tomato plants. The genetically modified tomato plants with this construct synthesized baicalein, ranging from 150 ng/g to 558 ng/g FW (fresh weight). Baicalein-fortified tomatoes have the potential to be health-promoting fresh vegetables and provide an alternative source of baicalein production, with great prospects for market application.

Plant synthetic biology for producing potent phyto-antimicrobials to combat antimicrobial resistance.

Inappropriate and injudicious use of antimicrobial drugs in human health, hygiene, agriculture, animal husbandry and food industries has contributed significantly to rapid emergence and persistence of antimicrobial resistance (AMR), one of the serious global public health threats. The crisis of AMR versus slower discovery of newer antibiotics put forth a daunting task to control these drug-resistant superbugs. Several phyto-antimicrobials have been identified in recent years with direct-killing (bactericidal) and/or drug-resistance reversal (re-sensitization of AMR phenotypes) potencies. Phyto-antimicrobials may hold the key in combating AMR owing to their abilities to target major microbial drug-resistance determinants including cell membrane, drug-efflux pumps, cell communication and biofilms. However, limited distribution, low intracellular concentrations, eco-geographical variations, beside other considerations like dynamic environments, climate change and over-exploitation of plant-resources are major blockades in full potential exploration phyto-antimicrobials. Synthetic biology (SynBio) strategies integrating metabolic engineering, RNA-interference, genome editing/engineering and/or systems biology approaches using plant chassis (as engineerable platforms) offer prospective tools for production of phyto-antimicrobials. With expanding SynBio toolkit, successful attempts towards introduction of entire gene cluster, reconstituting the metabolic pathway or transferring an entire metabolic (or synthetic) pathway into heterologous plant systems highlight the potential of this field. Through this perspective review, we are presenting herein the current situation and options for addressing AMR, emphasizing on the significance of phyto-antimicrobials in this apparently post-antibiotic era, and effective use of plant chassis for phyto-antimicrobial production at industrial scales along with major SynBio tools and useful databases. Current knowledge, recent success stories, associated challenges and prospects of translational success are also discussed.