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Engineering microbes to synthesize molecules of societal value has historically been a time consuming and artisanal process, with the synthesis of each new non-native molecule typically warranting its own separate publication. Because most microbial strain engineering efforts leverage a finite number of common metabolic engineering design tactics, we reasoned that automating these design steps would help create a pipeline that can quickly, cheaply, and reliably generate so-called microbial factories. In this work we describe the design and implementation of a computational system, an Automated Scientist we call Lila, which handles all metabolic engineering design and optimization through the design-build-test-learn (DBTL) paradigm. Lila generates metabolic routes, identifies relevant genetic elements for perturbation, and specifies the design and re-design of microbial strains in a matter of seconds to minutes. Strains specified by Lila are then built and subsequently phenotyped as part of a largely automated in-house pipeline. Humans remain in-the-loop to curate choices made by the system, helping for example to refine the metabolic model or suggest custom protein modifications. Lila attempted to build strains that could produce 454 biochemically diverse molecules with precursors located broadly throughout the metabolism of two microbial hosts,Saccharomyces cerevisiaeandEscherichia coli. Notably, we observed the highest published titers for the molecule naringenin, the metabolic precursor to flavonoids. In total we created hundreds of thousands of microbial strains capable of overproducing 242 molecules, of which 180 are not native toS. cerevisiaeorE. coli.

Accommodating equipment for controlling train movements, railway signal boxes are surviving representatives of functional buildings, a building category emerging during the Industrial Revolution sp...

We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

Modern computerised railway control methods are making traditional railway signal boxes obsolete and most signal boxes owned by Network Rail in Great Britain will close by 2026. Many of these signal boxes have a listing as buildings of architectural or historical significance. Listed buildings should ideally remain in their original location and this particularly applies to signal boxes, where the railway environment is an intrinsic aspect of the listing. However, there is pressure to relocate redundant listed and heritage signal boxes. Primary research methodology is by focusing upon key exemplars to determine the actual situation against theoretical conservation policy and practice. Findings are that while relocation affects the building’s conservation integrity, presentation of relocated signal boxes in a heritage railway environment provides for interpretation of railway history. The conclusions identify that there are contradictory requirements and pressures in conserving heritage signal boxes. These pressures materially affect the conservation process and there is a need to redefine accepted conservation theory to cope with the realties of signal box preservation. This will necessitate engagement by all interested parties and a systematic identification of all affected signal boxes.

Successful elucidation and optimization of the biosynthetic pathways for isoprenoids can lead to an array of natural products with a wide range of properties, including biofuels, pharmaceuticals, flavors, and fragrances. In order to maximize the potential of these pathways in high-performing microbial vehicles, considerations like codon usage, promoter strength, pathway bottlenecks, combinatorial expression, and fermentation processes play important roles. The advent of synthetic biology has served to accelerate the construction and improvement of microbial “isoprenoid factories” by removing the barriers to strain construction including gene synthesis, combinatorial library generation, and rapid molecular cloning. Ample precedence exists for cases where these principles have been applied. This review will deconstruct the processes by which microbial production of certain isoprenoids was achieved. The molecules chosen in this review, artemisinin, farnesnene, farnesol, taxol, and isoprene, represent a wide range of functionalities and applications, and also allow us to highlight the different routes taken for their successful biosyntheses.

Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia , respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae , plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4- O -desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1′,2′ double bond. The glutathione S -transferase catalyzed cis-trans isomerization of the 7′,8′ double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans -7′,8′-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1′,2′-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.

Yeast central carbon metabolism has been engineered to achieve a more efficient isoprenoid biosynthesis pathway, an advance that brings commodity-scale production of such compounds a step closer. These authors have re-engineered the central carbon metabolism of Saccharomyces cerevisiae to improve redox balance and eliminate carbon and energy waste associated with acetyl-CoA biosynthesis. The resulting strains can produce the acetyl-CoA-based hydrocarbon β-farnesene—an important precursor to many fragrances, fuels and therapeutics—in greater quantities than the starting yeast strain while consuming less oxygen. Cultures can be grown effectively in 200,000-litre industrial bioreactors. This system points the way towards a platform for high-productivity, feedstock-efficient production for all isoprenoids and other acetyl-CoA-derived compounds. A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments1. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (C15H24), a plant sesquiterpene with versatile industrial applications2,3,4,5, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO2-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained6. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.

SummaryAmbruticins and jerangolids are structurally related antifungal polyketides produced by Sorangium cellulosum strains. Comparative analysis of the gene clusters and characterization of compounds produced by gene knockout strains suggested hypothetical schemes for biosynthesis of these compounds. Polyketide synthase (PKS) architecture suggests that the pyran ring structure common to ambruticins and jerangolids forms by an intramolecular reaction on a PKS-bound intermediate. Disrupting ambM, encoding a discrete enzyme homologous to PKS C-methyltransferase domains, gave 15-desmethylambruticins. Thus, AmbM is required for C-methylation, but not pyran ring formation. Several steps in the post-PKS modification of ambruticin involve new enzymology. Remarkably, the methylcyclopropane ring and putative carbon atom excision during ambruticin biosynthesis apparently occur on the PKS assembly line. The mechanism probably involves a Favorskii rearrangement, but further work is required to elucidate these complex events.

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.

Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.

Recently, the epothilone polyketide synthase (PKS) was successfully introduced into a heterologous production host for the large-scale production of epothilone D. We have found that at least three other epothilones can also be produced as the major fermentation product of this recombinant strain by supplementation of specific substrates to the production media. Addition of acetate or propionate to the media results in modulation of the epothilone D:C ratio, whereas addition of l-serine with either acetate or propionate yields epothilone H1 or H2 as the major product. This strategy permits production of at least four novel epothilones by culturing a single host with a genetically modified epothilone PKS in various media. Journal of Industrial Microbiology & Biotechnology (2002) 28, 17–20 DOI: 10.1038/sj/jim/7000209

FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using thephi;C31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., König, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.

Thienamycin non-producing mutants of Streptomydes cattleya were identified that displayed a cross-feeding relationship. A diffusible product from one of these mutants (RK-11) resulted in restoration of thienamycin production when fed to cultures of another mutant (RK-4). In vivo radiolabeling experiments were conducted to test whether the RK-11 mutant produced a late biosynthetic intermediate which contained a carbapenem ring and a cysteaminyl and/or a hydroxyethyl side chain. Both [35S]cystine and [methyl-3H]methionine were used to label the RK-11 product which was then fed to RK-4 cultures. None of the thienamycin subsequently produced by RK-4 converter cells was labeled, implying the lack of either side chain of the thienamycin molecule in the RK-11 product. Further stability studies suggested that the RK-11 product does not contain a carbapenem ring. Additional feeding experiments with RK-4 cells also ruled out the possibility that the RK-11 product is a co-factor necessary for thienamycin production. It is concluded that the RK-11 product may regulate expression of the thienamycin gene cluster.

Polyketides are pharmaceutically important and structurally diverse natural products. Creating analogues for drug development can be done with chemistry, but this is generally restricted to a few accessible functional groups. Analogues can also be made by genetic engineering, which is particularly effective for polyketides synthesized by a modular polyketide synthase (PKS). Such a PKS displays colinearity, which means that the structural features along the polyketide chain are determined by the catalytic specificities in corresponding modules along a molecular assembly line. The assembly line can be genetically engineered through addition, deletion, or mutation of catalytic domains or the reorganization of whole modules. Chemically synthesized precursors also can be fed to engineered assembly lines to further expand the repertoire of analogues. These various methods are discussed with an aim of providing a guide to the strategies most likely to succeed in a given circumstance. Recent information that could be relevant to future polyketide engineering projects is also discussed.

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.

Polyketides are pharmaceutically important and structurally diverse natural products. Creating analogues for drug development can be done with chemistry, but this is generally restricted to a few accessible functional groups. Analogues can also be made by genetic engineering, which is particularly effective for polyketides synthesized by a modular polyketide synthase (PKS). Such a PKS displays colinearity, which means that the structural features along the polyketide chain are determined by the catalytic specificities in corresponding modules along a molecular assembly line. The assembly line can be genetically engineered through addition, deletion, or mutation of catalytic domains or the reorganization of whole modules. Chemically synthesized precursors also can be fed to engineered assembly lines to further expand the repertoire of analogues. These various methods are discussed with an aim of providing a guide to the strategies most likely to succeed in a given circumstance. Recent information that could be relevant to future polyketide engineering projects is also discussed.

Combinatorial biosynthesis can refer to any strategy for the genetic engineering of natural product biosynthesis to obtain new molecules, including the use of genetics for medicinal chemistry. However, it also implies the possibility that large libraries of complex compounds might be produced to feed a modern high-throughput screening operation. This review focuses on the multi-modular enzymes that produce polyketides, nonribosomal peptides, and hybrid polyketide-peptide compounds, which are the enzymes that appear to be most amenable to truly combinatorial approaches. The recent establishment of a high-throughput strategy for testing the activity of many non-natural combinations of modules from these enzymes should help speed the advance of this technology.

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15- R -6-dEB analogues were produced. The simpler “monoketide” acyl-thioester substrates required for this route of 15- R -6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1 o for the production of 15- R -6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N -acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor .

Combinatorial biosynthesis involves the genetic manipulation of natural product biosynthetic enzymes to produce potential new drug candidates that would otherwise be difficult to obtain. In either a theoretical or practical sense, the number of combinations possible from different types of natural product pathways ranges widely. Enzymes that have been the most amenable to this technology synthesize the polyketides, nonribosomal peptides, and hybrids of the two. The number of polyketide or peptide natural products theoretically possible is huge, but considerable work remains before these large numbers can be realized. Nevertheless, many analogs have been created by this technology, providing useful structure-activity relationship data and leading to a few compounds that may reach the clinic in the next few years. In this review the focus is on recent advances in our understanding of how different enzymes for natural product biosynthesis can be used successfully in this technology.

The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/520-calcineurin ternary complex. They also have calcineurin-independent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13- and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13- and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. A representative compound was also shown to accelerate nerve regeneration and functional recovery in the rat sciatic nerve crush model.

Five contiguous genes in the rapamycin gene cluster, rapQONML, of Streptomyces hygroscopicus ATCC29253 were replaced with a neomycin resistance marker by double homologous recombination. The resulting strain, if fed pipecolate, produced the analog 16-O-desmethyl-27-desmethoxyrapamycin instead of rapamycin. This indicates that the P450 hydroxylase encoded by rapN is specific for C-27, and that the O-methyltransferases encoded by rapQ and rapM methylate the hydroxyl groups on C-16 and C-27. By inference, the remaining P450 hydroxylase and methyltransferase genes (rapI and rapJ) are responsible for hydroxylation of C-9 and methylation of the C-39 hydroxyl, consistent with their homology to fkbD and fkbM, respectively, in the FK506 cluster. The relatively high level of 16-O-desmethyl-27-desmethoxyrapamycin produced indicates that the reactions at C-9 and C-39 do not require previous modification of the macrolactone precursor at either C-16 or C-27.

Background Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus , is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. Results An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. Conclusions The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.

Western blots of soluble protein from wheat, rice, and corn showed that ADPglucose pyrophosphorylase subunits have a size of 50 kilodaltons from endosperm tissue and 43 and 46 kilodaltons from leaf. Antisera to ADPglucose pyrophosphorylase precipitated in vitro translation products of 73 and 76 kilodaltons when leaf poly(A)+ RNA was used, whereas endosperm mRNA directed the synthesis of 50 and 56 kilodalton polypeptides. To further study the nature of these mRNA species, an ADPglucose pyrophosphorylase cDNA clone from rice endosperm polyadenylated RNA was obtained and used as a hybridization probe. Northern blots showed that ADPglucose pyrophosphorylase mRNA was slightly larger in leaf (2100 bases) than in endosperm tissue (1900 bases). These studies indicated that in cereals there are at least two tissue specific forms of ADPglucose pyrophosphorylase that are encoded by distinct mRNA transcripts. Analysis of genomic DNA by Southern blotting suggested that ADPglucose pyrophosphorylase is encoded by a small gene family.

Summary: Patterns of in vitro translation products from isolated mRNA and in vivo polypeptide accumulation in synchronized cultures of Cylindrotheca fusiformis were analysed by two-dimensional gel electrophoresis. The way in which the availability of silicon, the specific cell cycle stage, or the illumination conditions affected the pattern of gene expression was distinguished by comparing the timing of polypeptide and mRNA accumulation in cultures synchronized by two different methods. A rapid and dramatic shift in the relative abundance of in vitro translation products from mRNA followed either the removal or the readdition of silicate to the media as well as the transition from dark to light. Eleven mRNAs appeared to be expressed specifically between mid-S phase and cell separation, as their increase was observed at this stage in both synchronies. In addition, three mildly acidic polypeptides from the soluble protein fraction of C. fusiformis, each representing about 0·05% of the total protein, increased several-fold between mid-S and cell separation. Thus, silicon appears to affect gene expression both directly and, due to its effect on cell cycle progression, indirectly. Both effects are primarily at a level before translation.

The developmental accumulation pattern of messenger RNA transcripts and polypeptides for wheat gliadins and ADPglucose pyrophosphorylase was determined using cDNA and antibody probes. Gliadin mRNA was detected on Northern and RNA dot blots at 3 days after flowering, it increased 100-fold by 10 days and decreased subsequent to 14 days. The abundant mRNAs encoding alpha/beta- and gamma-type gliadins and mRNA for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, accumulated coordinately. Despite the coordinate accumulation of their mRNA transcripts, the accumulation of gliadin and ADPglucose pyrophosphorylase polypeptides, as determined by Western blot, differed significantly. The time at which gliadin and ADPglucose pyrophosphorylase mRNAs began accumulating was also the time when the overall pattern of gene expression, as seen by two-dimensional gel electrophoresis of in vitro translation products, changed most significantly. However, the accumulation of a number of other mRNAs or polypeptides having unknown function occurred at other times during endosperm development. The pattern of expression in the earliest stages of development was strikingly similar to that of coleoptile, another rapidly growing, nonphotosynthetic tissue. Thus, the pattern of gene expression reflects the program of development observed cytologically.

Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn 2+ for activity, though Mg 2+ gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes. Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5′-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium. Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear.

The alpha/beta-gliadin genes isolated from both hexaploid wheat (cv. Yamhill) and the diploid A genome progenitor Triticum urartu had remarkably similar sequences and differ by only a few point mutations. Primer extension analysis indicated that the transcriptional start points for individual genes in the family cluster within a few nucleotides. Comparison of the promoter region of several alpha/beta-gliadin and B-hordein genes reveals two conserved regions at about -130 and -250 bp. DNA from the hexaploid cultivars, Cheyenne and Chinese Spring, and the diploid progenitors T. urartu and Aegilops squarrosa was analysed by Southern blotting. Restriction fragment lengths of the alpha/beta-gliadin genes varied only slightly between the various wheats, although the overall copy number varied significantly. A region between approx. -1700 and -700 bp upstream from the TATA box was highly repeated in all three wheat genomes. For the hexaploid-derived gene, over 1700 bp of sequence upstream from the TATA box was determined, revealing an additional open reading frame between approx. -1550 and -1250 bp relative to the gliadin TATA box. Northern blot analysis indicated that RNA homologous to this repeated sequence family was present only in developing seed and accumulated to a maximum at late stages of maturation.

Seed formation in cereals is accompanied by the mobilization and transport of nitrogen from leaves and other source tissues to the developing cereal grain. Nitrogen enters the seed primarily as amino acids, of which glutamine appears to be the predominant constituent.1 It has been estimated that about 50% of the total nitrogen available in leaf and stem tissue of wheat is catabolized and transported to the developing grains2 where it is converted mainly into storage proteins. Storage proteins (defined here as any protein which serves as a nitrogen store and is packaged into protein bodies) are synthesized during the mid-stages of seed development and normally constitute about 80–90% of the total protein present in seeds. These proteins are subsequently utilized by the young developing seedling as a source of nitrogen and carbon and sometimes sulfur.