Overexpression of Physcomitrium patens cell cycle regulators leads to larger gametophytes.

Regulation of cell division is crucial for the development of multicellular organisms, and in plants, this is in part regulated by the D-type cyclins (CYCD) and cyclin-dependent kinase A (CDKA) complex. Cell division regulation in Physcomitrium differs from other plants, by having cell division checks at both the G1 to S and G2 to M transition, controlled by the CYCD1/CDKA2 and CYCD2/CDKA1 complexes, respectively. This led us to hypothesize that upregulation of cell division could be archived in Bryophytes, without the devastating phenotypes observed in Arabidopsis. Overexpressing lines of PpCYCD1, PpCYCD2, PpCDKA1, or PpCDKA2 under Ubiquitin promotor control provided transcriptomic and phenotypical data that confirmed their involvement in the G1 to S or G2 to M transition control. Interestingly, combinatorial overexpression of all four genes produced plants with dominant PpCDKA2 and PpCYCD1 phenotypes and led to plants with twice as large gametophores. No detrimental phenotypes were observed in this line and two of the major carbon sinks in plants, the cell wall and starch, were unaffected by the increased growth rate. These results show that the cell cycle characteristics of P. patens can be manipulated by the ectopic expression of cell cycle regulators.

Connecting moss lipid droplets to patchoulol biosynthesis.

Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.

Insights into Heterologous Biosynthesis of Arteannuin B and Artemisinin in Physcomitrella patens.

: Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

Evaluation of synthetic promoters in Physcomitrella patens.

Securing a molecular toolbox including diverse promoters is essential for genome engineering. However, native promoters have limitations such as the available number or the length of the promoter. In this work, three short synthetic promoters were characterized by using the yellow fluorescent protein Venus. All of the tested promoters were active and showed higher mRNA expression than housekeeping gene PpAct7, and similar protein expression level to the AtUBQ10 promoter. This study shows that few cis-regulatory elements are enough to establish a strong promoter for continuous expression of genes in plants. Along with this, the study enhance the number of available promotors to be used in P. patens. It also demonstrates the potential to construct multiple non-native promoters on demand, which would aid to resolve one bottleneck in multiple pathway expression in P. patens and other plants.

Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens.

Malaria is a real and constant danger to nearly half of the world's population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year.

Enhancing alkane production in cyanobacterial lipid droplets: a model platform for industrially relevant compound production.

Cyanobacterial lipid droplets (LDs) are packed with hydrophobic energy-dense compounds and have great potential for biotechnological expression and the compartmentalization of high value compounds. Nostoc punctiforme normally accumulates LDs containing neutral lipids, and small amounts of heptadecane, during the stationary phase of growth. In this study, we further enhanced heptadecane production in N. punctiforme by introducing extrachromosomal copies of aar/adc genes, and report the discovery of a putative novel lipase encoded by Npun_F5141, which further enhanced alkane production. Extra copies of all three genes in high light conditions resulted in a 16-fold higher accumulation of heptadecane compared to the wild type strain in the exponential phase. LD accumulation during exponential phase also increased massively to accommodate the heptadecane production. A large number of small, less fluorescent LDs were observed at the cell periphery in exponential growth phase, whereas fewer number of highly fluorescent, much larger LDs were localized towards the center of the cell in the stationary phase. These advances demonstrate that cyanobacterial LDs are an ideal model platform to make industrially relevant compounds, such as alkanes, during exponential growth, and provide insight into LD formation in cyanobacteria.

Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme.

Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophylic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen-fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), α-tocopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids, whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location, these structures represent a novel sub-organelle in cyanobacteria.