Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I–IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.

Summary: Thiamine pyrophosphate (TPP), an essential co‐factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3′UTR of some thiamine metabolism‐related genes, but little information is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT‐qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine‐related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4‐amino‐5‐hydroxymethyl‐2‐methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP‐P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B₁₂ (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B₁₂ uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B₁₂ uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B₁₂. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B₁₂ uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B₁₂-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B₁₂ acquisition, a process that likely underpins many interactions in aquatic microbial communities.

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.

Summary: The green microalga Chlamydomonas reinhardtii is a promising host organism for the production of valuable compounds. Engineering the Chlamydomonas chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the Chlamydomonas chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B₁₂‐responsive METE promoter and a modified thiamine (vitamin B₁) riboswitch, along with nucleus‐encoded chloroplast‐targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine‐tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.

SUMMARY: For advanced metabolic engineering strategies, it is crucial to be able to regulate transgene expression, to prevent potential deleterious effects in the host organism during growth and allow optimisation of production levels. Here, we identified vitamin B ₁₂ (cobalamin)‐responsive promoters in the diatom Phaeodactylum tricornutum, a promising biotechnological chassis that readily absorbs this metabolite with minimal physiological impact. Using promoter–reporter constructs, the promoters of the cobalamin acquisition protein 1 (CBA1) and the B ₁₂ ‐independent form of methionine synthase (METE) were shown to regulate transgene expression in a B ₁₂ ‐dependent manner. Further characterisation of the METE promoter ( P _METE ) demonstrated that it exhibited significantly higher expression levels than several previously characterised promoters, but could be repressed by nanomolar amounts of B ₁₂ , with a dynamic range >100‐fold. Tight regulation was demonstrated by the suppression of the lethal ribonuclease, barnase at 1 μg L ⁻¹ B ₁₂ . Reporter expression was doubled when P _METE was paired with its cognate terminator, compared with the widely used FCPA terminator. Promoter truncations resulted in decreased expression, but no loss of B ₁₂ regulation. A 14 nucleotide motif, present in four copies in P _METE , was found to be necessary for expression, and when fused to the constitutive FCPA promoter, enhanced expression levels. Transgenic lines expressing the heterologous diterpenoid enzyme, casbene synthase, produced casbene titres of approximately 2 mg L ⁻¹ and this was tuneable by B ₁₂ . This demonstrates the utility of P _METE in efforts to establish P. tricornutum as an industrial biotechnology production platform.