A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Effects of Adding Vindoline and MeJA on Production of Vincristine and Vinblastine, and Transcription of their Biosynthetic Genes in the Cultured CMCs of Catharanthus roseus.

Vincristine and vinblastine were found by Liquid Chromatography-Mass Spectrometry (LC-MS) in Catharanthus roseuscambial meristem cells (CMCs) jointly treated with 0.25 mM vindoline and methyl jasmonate (MeJA), suggesting that C. roseus CMCs contain a complete set of the enzymes which are in response to convert vindoline into vincristine and vinblastine. Based on the facts that the transcript levels of vindoline-biosynthetic genes (STR, SGD and D4H) were up-regulated instead of being down-regulated by adding itself to the culture, and that the transcriptional factor ORCA3 was up-regulated simultaneously, we further confirmed that the transcription of STR, SGD, D4H was manipulated by ORCA3.

The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots.

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don.

Dimeric indole alkaloids are used extensively for cancer therapy. Agrobacterium tumefaciens C58 strain was used for induction of shooty teratoma in Catharanthus roseus using epicotyl and stem node explants. The transformed nature of shooty teratomas was confirmed by nopaline assay. Growth kinetics of shooty teratomas depicted maximum growth during 21-24 days of culture. Dimeric alkaloid vincristine in the transformed cultures was present at a concentration of 0.011 that was tenfold higher compared to untransformed control cultures.

[Effects of NaCl on the growth and alkaloid content of Catharanthus roseus seedlings].

Catharanthus roseus seedlings were grown in 1/2 Hoagland solution containing 0-250 mmol x L(-1) of NaCl, and their fresh and dry mass, malondialdehyde (MDA) and chlorophyll contents, tryptophan decarboxylase (TDC) and peroxidase (POD) activities, and vindoline, catharanthine, vincristine and vinblastine contents were measured after 7 days. The results showed that NaCl markedly decreased the fresh and dry mass but increased the MDA content. The chlorophyll content had no difference with the control when the concentration of NaCl was 50 mmol x L(-1), but decreased with increasing NaCl concentration when the NaCl concentration was above 50 mmol x L(-1). There was a significant enhancement of POD activity under NaCl stress. The TDC activity was the highest when the concentration of NaCl was 50 mmol x L(-1), but decreased with increasing NaCl concentration. The vindoline, catharanthine, vincristine, and vinblastine contents were the highest under 50 mmol x L(-1) NaCl stress, with the values being 4.61, 3.56, 1.19, and 2.95 mg x g(-1), respectively, and significant higher than the control and other treatments. Salt stress could restrain the growth of C. roseus seedlings, but promote the metabolism of alkaloid and increase the alkaloid content. 50 mmol x L(-1) of NaCl had the greatest promotion effect on the alkaloid content of C. roseus seedlings.

Monitoring the production yields of vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis. Semiquantitative determination by flow-injection electrospray ionization mass spectrometry.

A semiquantitative determination of two bis-indole antitumor alkaloids, vincristine and vinblastine, has been performed by flow-injection electrospray ionization mass spectrometric analysis of the extracts of Catharanthus roseus. Leaves and flowers of two different phenotypes (pink flower and white flower) obtained from somatic embryogenesis were thus examined and compared with the field-grown mother plant. Different amounts of vincristine and vinblastine were detected depending on the examined samples.

Enzymology of indole alkaloid biosynthesis in Catharanthus roseus.

Indole alkaloids in Catharanthus roseus have been in focus because of their medicinal value. These alkaloids consist of an indole moiety provided by tryptamine and a terpenoid portion provided by the secologanin. The most important catharanthus alkaloids are vinblastine (VLB), vincristine (VCR) and ajmalicine. VLB and VCR are clinically useful anticancer agents whereas ajmalicine is used for the treatment of circulatory diseases. VCR and VLB are the most expensive because of their low abundance in the plant, and are formed by the coupling of monomeric indole alkaloids vindoline and catharanthine, catalysed by peroxidases. The pathway that lead to monomeric indole alkaloids involves more than 20 enzymes of which 16 enzymes have been isolated and characterized biochemically, and only three at the molecular level. The present state of knowledge on enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their regulation has been discussed.

Divergent regulation of the class II P-glycoprotein gene in primary cultures of hepatocytes versus H35 hepatoma by glucocorticoids.

We investigated whether the glucocorticoid-mediated mechanisms controlling P-glycoprotein (pgp2 or mdr1b) are similar in normal hepatocytes compared with the H35 hepatoma cell line. In primary rat hepatocytes, dexamethasone (DEX) caused a dose- and time-dependent decrease in the amount of the pgp2 mRNA, which correlated with functional pgp2 expression (intracellular accumulation of [3H]vincristine). The suppression of pgp2 mRNA was specific for glucocorticoids because a representative estrogen and progestin were without effect, and DEX suppression of pgp2 mRNA could be reversed by cotreatment with an anti-glucocorticoid. DEX suppression of pgp2 mRNA appears to be posttranscriptional because following actinomycin D inhibition of new RNA synthesis, the pgp2 transcript disappeared at a faster rate in DEX treated versus untreated hepatocytes. Moreover, transcriptional activity of chloramphenicol acetyltransferase plasmids containing the pgp2 promoter in primary rat hepatocytes was unaffected by DEX treatment. Thus, suppression of pgp2 mRNA by glucocorticoids in primary hepatocytes is due to a decrease in pgp2 mRNA stability. In contrast, in the H35 hepatoma cell line, DEX dose dependently increased pgp protein and pgp2 mRNA, effects which parallel transcriptional activation of the pgp2 promoter. Activation of the pgp2 promoter was specific for glucocorticoids since a representative estrogen had no significant effect on transcription of the pgp2 promoter and RU486 blocked DEX activation of pgp2 transcription. Transcriptional activation of the pgp2 promoter was not due to a global up-regulation of basal transcription factors because DEX treatment did not activate either a herpes simplex virus thymidine kinase promoter or the SV40 early gene promoter. Further studies with a panel of pgp2 5' sequence deletion plasmids revealed that the minimal promoter (-66 bp) was not activated by DEX. In contrast, inclusion of sequences up to -177 bp restored DEX-dependent transcriptional activation. These are the first studies to demonstrate that glucocorticoids regulate pgp2 by different mechanisms in normal rat hepatocytes compared to the H35 hepatoma cell line.