FT5a interferes with the Dt1-AP1 feedback loop to control flowering time and shoot determinacy in soybean.

Flowering time and stem growth habit determine inflorescence architecture in soybean, which in turn influences seed yield. Dt1, a homolog of Arabidopsis TERMINAL FLOWER 1 (TFL1), is a major controller of stem growth habit, but its underlying molecular mechanisms remain unclear. Here, we demonstrate that Dt1 affects node number and plant height, as well as flowering time, in soybean under long-day conditions. The bZIP transcription factor FDc1 physically interacts with Dt1, and the FDc1-Dt1 complex directly represses the expression of APETALA1 (AP1). We propose that FT5a inhibits Dt1 activity via a competitive interaction with FDc1 and directly upregulates AP1. Moreover, AP1 represses Dt1 expression by directly binding to the Dt1 promoter, suggesting that AP1 and Dt1 form a suppressive regulatory feedback loop to determine the fate of the shoot apical meristem. These findings provide novel insights into the roles of Dt1 and FT5a in controlling the stem growth habit and flowering time in soybean, which determine the adaptability and grain yield of this important crop.

A Biological Route to Conjugated Alkenes: Microbial Production of Hepta-1,3,5-triene.

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.

Prenylated FMN: Biosynthesis, purification, and Fdc1 activation.

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors.

Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP+ oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

Overexpression of PAD1 and FDC1 results in significant cinnamic acid decarboxylase activity in Saccharomyces cerevisiae.

The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 µmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production.

Among industrial yeasts used for alcoholic beverage production, most wine and weizen beer yeasts decarboxylate ferulic acid to 4-vinylguaiacol, which has a smoke-like flavor, whereas sake, shochu, top-fermenting, and bottom-fermenting yeast strains lack this ability. However, the factors underlying this difference among industrial yeasts are not clear. We previously confirmed that both PAD1 (phenylacrylic acid decarboxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. In the present study, single nucleotide polymorphisms (SNPs) of PAD1 and FDC1 in sake, shochu, wine, weizen, top-fermenting, bottom-fermenting, and laboratory yeast strains were examined to clarify the differences in ferulic acid decarboxylation ability between these types of yeast. For PAD1, a nonsense mutation was observed in the gene sequence of standard top-fermenting yeast. Gene sequence analysis of FDC1 revealed that sake, shochu, and standard top-fermenting yeasts contained a nonsense mutation, whereas a frameshift mutation was identified in the FDC1 gene of bottom-fermenting yeast. No nonsense or frameshift mutations were detected in laboratory, wine, or weizen beer yeast strains. When FDC1 was introduced into sake and shochu yeast strains, the transformants exhibited ferulic acid decarboxylation activity. Our findings indicate that a positive relationship exists between SNPs in PAD1 and FDC1 genes and the ferulic acid decarboxylation ability of industrial yeast strains.