Explore the vast range of titles available.

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana . A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis. The biosynthesis of strictosidine, a key intermediate of monoterpene indole alkaloids, was successfully reconstructed in Nicotiana benthamiana, demonstrating the potential of Nicotiana benthamiana as a bioproduction chassis for small molecules.

Wheat (Triticum aestivum L.) is a major source of nutrients for populations across the globe, but the amino acid composition of wheat grain does not provide optimal nutrition. The nutritional value of wheat grain is limited by low concentrations of lysine (the most limiting essential amino acid) and high concentrations of free asparagine (precursor to the processing contaminant acrylamide). There are currently few available solutions for asparagine reduction and lysine biofortification through breeding. In this study, we investigated the genetic architecture controlling grain free amino acid composition and its relationship to other traits in a Robigus × Claire doubled haploid population. Multivariate analysis of amino acids and other traits showed that the two groups are largely independent of one another, with the largest effect on amino acids being from the environment. Linkage analysis of the population allowed identification of quantitative trait loci (QTL) controlling free amino acids and other traits, and this was compared against genomic prediction methods. Following identification of a QTL controlling free lysine content, wheat pangenome resources facilitated analysis of candidate genes in this region of the genome. These findings can be used to select appropriate strategies for lysine biofortification and free asparagine reduction in wheat breeding programs. Core Ideas High free asparagine and low lysine concentrations limit the nutritional value of wheat grain. Investigation of a biparental mapping population formed from the UK soft wheats Claire and Robigus. Breeding for lower free asparagine and higher lysine using Claire and Robigus diversity is possible but limited.

Extending the duration of the stem elongation (SE) phase between terminal spikelet (TS) and anthesis has often been proposed as an avenue to increase wheat yield. However, accurate determination of TS is labour intensive, and existing evidence is often based on a limited number of genotypes observed under controlled conditions. Here, a Buster x Charger population comprising 108 doubled haploid lines was grown across four year-sites under UK field conditions. TS was recorded through meristem dissection and SE duration was measured as the time between TS and ear emergence (EE). Mixed model analysis across year-sites revealed high heritabilities (H2yield = 0.66, H2TS = 0.93, H2SE = 0.89, H2EE = 0.95) and strong genetic correlations between yield and the phenology traits SE and EE (rg = 0.56 and rg = 0.6, respectively). While SE duration was mainly driven by EE, independent QTL for TS suggest that SE could be modified without affecting EE. The positive effect of SE duration on yield was attributed to increased grain number per area as well as increased grain weight, through independent QTL. Although validation in broader genetic backgrounds is needed, these QTL may offer opportunities for further yield increase in a physiological breeding framework.

We report highly efficient genome editing in Brassica species. We compare the efficiency of targeted mutagenesis using four Streptococcus pyogenes Cas9 (SpCas9) systems in Brassica oleracea and Brassica napus over 3 target genes and five guides to identify two which show a striking improvement to our first published system (Lawrenson et al., 2015). Targeted mutagenesis occurred in up to 100% of T0 plants with the improved systems, compared to 20% in the original system. This is the only reported comparison of SpCas9 systems we are aware of in Brassica species. Secondly, we report the first successful use of Lachnospiraceae bacterium Cas12a (LbCas12a) in Brassica oleracea. We test three LbCas12a coding sequences and two guide architectures against one target gene using four guides. From this we identify the best performing combination of our novel, multi-intron coding sequence and a ribozyme flanked guide expression cassette. In this case 68% of T0 plants contained targeted mutations. Heritability of LbCas12a mutations is shown. We show that our two useful and novel LbCas12a coding sequences have utility in Brassica species. Competing Interest Statement The authors have declared no competing interest.

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally significant compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. We used transcriptomic analysis to identify glycosyltransferases that were upregulated in response to biosynthetic intermediates and produced plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produced a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases did not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report the assembly of an improved N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated 3-4 times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and opens the possibility that BBLs are the sought-after coupling enzymes in nicotine biosynthesis.Competing Interest StatementThe authors have declared no competing interest.