In this project, our objective is to further increase plant formaldehyde uptake and metabolism ability using synthetic biology methods. We find some genes encoding key enzymes related to formaldehyde metabolic pathways from microorganism. They are 3-hexulose-6-phosphate(HPS), 6-phospho-3-hexuloisomerase(PHI), formaldehyde dehydrogenase(FALDH) and formate-dehydrogenase(FDH). These genes are inserted into plants and will work to promote formaldehyde metabolism. For security reasons, we also add gene AdCP into our project because of its capability to lead to pollen abortion. At the same time, chloroplast transformation is taken into consideration to decrease the probability of gene flow.

HPS-PHI

The ribulose monophosphate (RuMP) pathway is one of the HCHO-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes HCHO to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose 6-phosphate (Hu6P), and 6-phospho-3- hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P).The two key enzymes work in chloroplast both.We will use fusion expression to conduct heterologous expression in tobacco( Li-mei Chen et al,2010). Here are some datas from the paper.

Fig.1 Schematic Representation of the Bacterial RuMP Pathway and the Plant Calvin-Benson Cycle. HPS and PHI denote 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase respectively. The abbreviations for several sugar phosphates are as follows: Ru5P, ribulose 5-phosphate; Hu6P, hexulose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate;RuBP,ribulose1,5-bisphosphate;3-PGA,3-phosphoglyce-rate. The other metabolites in the pathway are symbolized merely by their carbon numbers for simplicity.

FALDH

The glutathione-dependent formaldehyde dehydrogenase(FALDH) plays a key role in formaldehyde metabolism(Fig.3). FALDH is identified as an enzyme expressed in the cytoplasm. If we make FALDH over-express in plants, we can enhance plants’ tolerance to HCHO and increase the ability of plants to absorb HCHO. In the process of metabolism of formaldehyde, the formaldehyde may first combined with glutathione (GSH) to form the product of S-hydroxymethyl glutathione (HM-GSH), then FALDH in cytoplasm will catalyzes the formation of a S-formyl glutathione(F-GSH). Next the F-GSH will be hydrolyzed to formate(HCOOH) and GSH by S-formyl glutathione hydrolase (FGH).

Fig.2 a. spectra from leaf extracts of transgenic tobacco plant treated with gaseous for 2 h. b. spectra from leaf extracts of WT treated with gaseous for 2 h. c. The extract from WT plant leaves without treatment was used to monitor the background signal levels

FDH

Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial，FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity.In our project, the heterologous expression of FDH from arabidopsis thaliana in tobacco was completed.

Fig.3 The abbreviations are as follows: CAT:catalase; FALDH:glutathione-dependent formaldehyde dehydrogena-se; FGH: S-formylglutathione hydrolase; FDH: Formate dehydrogenase; SYN: 10-Formyl-THF synthetase; FTD: 10-formyltetrahydrofolate dehydrogenase MTD: 5,10-methylenetetrahydrofolate dehydrogenase; MTC: 5,10-methylenetetrahydrofolate cyclohydrolase; SHMT: Serine hydroxymethyl transferase; GDC: Glycine decarboxylase complex; GXS: Glyoxalic acid synthetase; GXDC: Glyoxalic acid decarboxylase; HM-GSH: S-Hydroxymethyl glutathione; Forml-GSH: Formyl glutathione; SMM cycle: Methionine cycle.

Stomatal opening

Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Yin Wang, et al (2014) showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening (Fig. 4). Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore,in order to strengthen the ability of absorbing formaldehyde, we overexpressed H+-ATPase(At AHA2) in transgenic tobacco guard cells,resulting in a significant effect on light-induced stomatal opening.

Fig.4 Typical stomata in the epidermis illuminated with light for 30 min(Yin Wang,et al.2014).

Biosafty

In order to promise biology safety, we use male sterility systems which can be used as a biological safety containment to prevent horizontal transgene flow. Pawan Shukla et al (2014) has used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachisdiogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsispersonata.Arachisdiogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion.

Fig.5 Pollen germination of untransformed control plant and sterile transgenic plantsin vitro. Pollen grains were germinated on sucrose-boric acid medium and over 500 pollen grains were observed. a. Untansformed control plant pollen, b. Sterile pollen.Scale bar 25 μm (Pawan Shukla et al 2014).

Vectors

The production of HPS, PHI, and FDH are located in chloroplast, while the production of FALDH are located in cytoplast. So we constructed different vectors with and without transit peptide. We hope to compare the ability of metabolizing HCHO of transgenic tobacco between different transgenic lines. We planned to constructed 11 vectors (Fig. 6), including two backbones, six mono-gene expression vectors and three multi-gene expression vectors (Fig. 7). piGEM003, piGEM004 and piGEM005 are individual mono-gene expression vectors with transit peptides, while piGEM006, piGEM006, piGEM008 are individual multi-gene expression vectors without transit peptides. piGEM009 is a multi-gene expression vector without any transit peptides, while piGEM011 is a multi-gene expression vector with three peptides. piGEM010 is a multi-gene expression vector with two transit peptides.

Fig.6 The procedure we constructed our vectors.
piGEM001: P35S+P2A+T2A+F2A+nptII+T-HSP+T-35S+T-nos
piGEM002: piGEM001+AtAHA2+PGC1+PTA29+AdCP
piGEM003: piGEM002+TCP02-HPS-PHI
piGEM004: piGEM002+TCP03-AtFDH
piGEM005: piGEM002+TCP01-BbFALDH
piGEM006: piGEM002+HPS-PHI
piGEM007: piGEM002+AtFDH
piGEM008: piGEM002+BbFALDH
piGEM009: piGEM002+HPS-PHI+AtFDH+BbFALDH
piGEM010: piGEM002+TCP02-HPS-PHI+TCP03-AtFDH+FALDH
piGEM011: piGEM002+TCP02-HPS-PHI+TCP03-AtFDH+TCP01-BbFALDH
piGEM012: piGEM001+TCP02-HPS-PHI+TCP03-AtFDH+TCP01-BbFALDH

2A peptide Sequence

2A peptide sequences were found in Picornaviruses to mediate ‘cleavage’ between two proteins. We use 2A peptide linked multi-cistronicvectors to express multiple proteins from a single open reading frame (ORF) effectively.

The 18-22 amino acids 2A self-cleaving oligo peptides can be used for co-expression of multiple, discrete proteins from a single ORF. Based on highly inefficient peptide bond formation between glycine and proline residues within the 2A peptide, placement of 2A peptide sequence as a linker region between tandem cDNA’s allows the stoichiometric translation of multiple unfused protein products. These sequences were first discovered in the foot-and-mouth disease virus (FMDV). And since then many 2A-like sequences have been identified in other viruses and some parasites. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants.In our project,we use F2A(from foot-and-mouth disease virus), P2A(from porcine teschovirus-1) and T2A (from Thoseaasigna virus) to achieve our goal.