Sex, light and carotenes: the development of Phycomyces.

Phycomyces blakesleeanus is known for the elaborate behaviour of its sporangiophores (fruiting bodies). Sporangiophore development is exquisitely sensitive to blue light, easy to describe quantitatively, pliable to genetic and biochemical research, and reminiscent in many details of other photoresponses in the same and in other organisms. The developmental and behavioural processes of Phycomyces share a number of genes. A combinatorial use of gene expression appears to be the basis for the complexities observed in this fungus.     

Modification of sexual development and carotene production by acetate and other small carboxylic acids in Blakeslea trispora and Phycomyces blakesleeanus

In Phycomyces blakesleeanus and Blakeslea trispora (order Mucorales, class Zygomycetes), sexual interaction on solid substrates leads to zygospore development and to increased carotene production (sexual carotenogenesis). Addition of small quantities of acetate, propionate, lactate, or leucine to mated cultures on minimal medium stimulated zygospore production and inhibited sexual carotenogenesis in both Phycomyces and Blakeslea. In Blakeslea, the threshold acetate concentration was <1 mmol/liter for both effects, and the concentrations that had one-half of the maximal effect were <2 mmol/liter for carotenogenesis and >7 mmol/liter for zygosporogenesis. The effects on Phycomyces were similar, but the concentrations of acetate had to be multiplied by ca. 3 to obtain the same results. Inhibition of sexual carotenogenesis by acetate occurred normally in Phycomyces mutants that cannot use acetate as a carbon source and in mutants whose dormant spores cannot be activated by acetate. Small carboxylic acids may be signals that, independent of their ability to trigger spore germination in Phycomyces, modify metabolism and development during the sexual cycle of Phycomyces and Blakeslea, uncoupling two processes that were thought to be linked and mediated by a common mechanism.     

Ubiquinone and carotene production in the <Emphasis Type="Italic">Mucorales Blakeslea</Emphasis> and <Emphasis Type="Italic">Phycomyces</Emphasis>

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of β-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.     

Effect of light on several metabolites of carbohydrate metabolism in Phycomyces blakesleeanus.

The concentrations of all metabolites studied, except fructose 1,6-bisphosphate from wild-type Phycomyces blakesleeanus, were light dependent. This photoregulation appears to be independent of the mad gene product(s) and also independent of carotene biosynthesis regulation. However, the photoregulation of glyceraldehyde 3-phosphate, 2-phosphoglycerate, and phosphoenolpyruvate may be assigned to these mad and car S gene products.     

Photomorphogenesis in<Emphasis Type="Italic"> Phycomyces</Emphasis>: differential display of gene expression by PCR with arbitrary primers

The zygomycete fungus Phycomyces blakesleeanus develops two types of fruiting bodies of very different size, macrophores and microphores. Blue light stimulates macrophorogenesis and inhibits microphorogenesis. I have adapted a method based on the polymerase chain reaction with arbitrary primers to investigate the role of differential gene expression during photophorogenesis in Phycomyces. Several cDNAs for genes induced in vegetative mycelium have been observed, but only one gene induced by blue light has been detected. I have demonstrated the feasibility of this approach by the isolation of a cDNA segment for the heat-shock protein HSP100 that is induced by blue light at the onset of sporangiophore development. The heat-shock protein HSP100 is an ATP-binding protein that has the ability to disassemble protein complexes. In plants, the gene for HSP100 is induced by light. The cDNA segment for HSP100 obtained from Phycomyces is 686 bp long and the predicted amino acid sequence contains one of the ATP-binding sites.     

Intersexual partial diploids of phycomyces

Sexual interaction between strains of opposite sex in many fungi of the order Mucorales modifies hyphal morphology and increases the carotene content. The progeny of crosses of Phycomyces blakesleeanus usually include a small proportion of anomalous segregants that show these signs of sexual stimulation without a partner. We have analyzed the genetic constitution of such segregants from crosses that involved a carF mutation for overaccumulation of beta-carotene and other markers. The new strains were diploids or partial diploids heterozygous for the sex markers. Diploidy was unknown in this fungus and in the Zygomycetes. Random chromosome losses during the vegetative growth of the diploid led to heterokaryosis in the coenocytic mycelia and eventually to sectors of various tints and mating behavior. The changes in the nuclear composition of the mycelia could be followed by selecting for individual nuclei. The results impose a reinterpretation of the sexual cycle of Phycomyces. Some of the intersexual strains that carried the carF mutation contained 25 mg beta-carotene per gram of dry mass and were sufficiently stable for practical use in carotene production.     

Mutants of the carotene cyclase domain of al-2 from Neurospora crassa

Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.     

Carotenes and Retinal in Phycomyces Mutants

Three different types of beta-carotene mutants of Phycomyces have been studied. In 2 mutants (Type I) beta-carotene is still the principal carotene but scaled down or up relative to wild type. The carotene mixture of 2 mutants (Type II) consists mainly of phytoene and phytofluene. In Type III (2 mutants) beta-carotene is replaced by lycopene.The examination of the mutants reveals that the receptor pigment is very likely neither beta-carotene nor retinal. Transmission spectra through the growing zone of live sporangiophores of 1 of these mutants which contains less than one-thousandth of the beta-carotene content of wild type show that the receptor pigment extinction is less than 0.003 at its maximum.     

New Mutants of Phycomyces blakesleeanus for (beta)-Carotene Production

The accumulation of (beta)-carotene by the zygomycete Phycomyces blakesleeanus is increased by mutations in the carS gene. The treatment of spores of carS mutants with N-methyl-N(prm1)-nitro-N-nitrosoguanidine led to the isolation, at very low frequencies, of mutants that produced higher levels of (beta)-carotene. Strain S556 produced about 9 mg of (beta)-carotene per g of dry mass when it was grown on minimal agar. Crosses involving strain S556 separated the original carS mutation from a new, unlinked mutation, carF. The carF segregants produced approximately as much carotene as did carS mutants, but they were unique in their ability to produce zygospores on mating and in their response to agents that increase carotenogenesis in the wild type. The carotene contents of carF segregants and carF carS double mutants were increased by sexual interaction and by dimethyl phthalate but were not increased by light or retinol. Mixed opposite-sex cultures of carF carS mutants contained up to 33 mg of (beta)-carotene per g of dry mass. Another strain, S444, produced more (beta)-carotene than did S556 but was marred by slow growth, defective morphology, and bizarre genetic behavior. In all the strains tested, the carotene concentration was minimal during the early growth phase and became higher and constant for several days in older mycelia.     

Carotenoid and fatty acid metabolism in light‐stressed Dunaliella salina

β‐Carotene is overproduced in the alga Dunaliella salina in response to high light intensities. We have studied the effects of a sudden light increase on carotenoid and fatty acid metabolism using a flat panel photobioreactor that was run in turbidostat mode to ensure a constant light regime throughout the experiments. Upon the shift to an increased light intensity, β‐carotene production commenced immediately. The first 4 h after induction were marked by constant intracellular levels of β‐carotene (2.2 g LCV^?1), which resulted from identical increases in the production rates of cell volume and β‐carotene. Following this initial phase, β‐carotene productivity continued to increase while the cell volume productivity dropped. As a result, the intracellular β‐carotene concentration increased reaching a maximum of 17 g LCV^?1 after 2 days of light stress. Approximately 1 day before that, the maximum β‐carotene productivity of 30 pg cell^?1 day^?1 (equivalent to 37 mg LRV^?1 day^?1) was obtained, which was about one order of magnitude larger than the average productivity reported for a commercial β‐carotene production facility, indicating a vast potential for improvement. Furthermore, by studying the light‐induced changes in both β‐carotene and fatty acid metabolism, it appeared that carotenoid overproduction was associated with oil globule formation and a decrease in the degree of fatty acid unsaturation. Our results indicate that cellular β‐carotene accumulation in D. salina correlates with accumulation of specific fatty acid species (C16:0 and C18:1) rather than with total fatty acid content. Biotechnol. Bioeng. 2010;106: 638–648. © 2010 Wiley Periodicals, Inc.     

Regulation of carotene synthesis in Phycomyces

Summary Threeindependent mutations of Phycomyces blakesleeanus resulting in overaccumulation of -carotene are recessive and belong to the same complementation group. The corresponding gene has been named carS. Evidence is presented that gene carS is not the same as gene carA, previously defined by mutations blocking carotene production. Vitamin A increases carotenogenesis in wild types and in carS mutants to about the same extent. Intersexual heterokaryosis increases carotenogenesis most prominently in carS genetic backgrounds (up to 300 times the production of the wild type in the same conditions). Vitamin A, intersexual heterokaryosis and carS mutations are thought to stimulate carotenogenesis through different mechanisms. It is suggested that the carS gene product participates in end-product regulation of the pathway.     

Independence of the carotene and sterol pathways of Phycomyces.

Light, chemicals, and mutations that affect the carotene content of the fungus Phycomyces blakesleeanus had practically no effect on the ergosterol content. Lovastatin, a specific inhibitor of hydroxymethylglutaryl coenzyme A reductase, blocked growth at 1 microM; sodium DL-mevalonate (10 mM) fully reversed this inhibition. In the presence of [14C]mevalonate, a carS mutant accumulated 16 times more beta-carotene than the wild-type with a specific radioactivity five times lower. The specific radioactivity of ergosterol was different from that of beta-carotene, even when calculated in terms of the constituent isoprene units, and unaffected by the carS mutation. The carotene and sterol pathways of Phycomyces are independently regulated and physically separated in different subcellular compartments.     

Photocarotenogenesis in Phycomyces: Expression of the carB Gene Encoding Phytoene Dehydrogenase

Phycomyces blakesleeanus the biosynthesis of β-carotene is under the control of blue light. The light-controlled expression of the carB gene encoding phytoene dehydrogenase was investigated with slot blot and Northern analyses. After irradiation of mycelia with short pulses of blue light the amount of carB mRNA was stimulated transiently in the subsequent dark period. Depending on the energy fluence of the light pulse the mRNA increased about 3 to 5-fold after a latency of 2-5 min. Twenty minutes after the irradiation the amount of carB mRNA decreased again and reached, after 60 min, the dark level it had prior to the irradiation. A Northern analysis showed that the carB mRNA is unstable. After irradiation the full-length mRNA (2.2 kb) as well as degraded carB mRNA increased. The degradation was specific for the carB gene, because no degradation was observed for 25S rRNA or the mRNA of the pyrG gene. The rapid turnover as well as the degradation of the carB mRNA provide for an adaptive light control and appear to represent an essential feature of the regulation of the carotene pathway. Received 11 April 2000/ Accepted in revised form 30 November 2000     

Non‐endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4′ β‐carotene oxygenase (CrtW) and 3,3′ β‐carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD‐212 under the control of a temperature‐inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non‐endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of β‐carotene (β‐car). When both Brevundimonas sp. SD‐212 genes were coexpressed, significant amounts of non‐endogenous Asx were obtained accompanied by a strong decrease in β‐car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added‐value compounds.     

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Relationships among the biosyntheses of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of β-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [¹⁴C]acetyl-CoA, L-[¹⁴C]leucine, or R-[¹⁴C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), β-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway.     

Light Induced Concentration Changes of Adenosine-Triphosphate in Phycomyces Sporangiophores

The concentrations of extractable adenosine triphosphate (ATP) following the induction of positive light-growth responses in Phycomyces sporangiophores by blue light stimuli have been measured by means of the luciferin-luciferase assay. The ATP concentration in the light-sensitive growing zone increases 30 to 50% within 30 seconds after the start of a light stimulus and returns to the normal adapted level within 1 minute after stimulation. The ATP concentration is constant for any level of light adaptation and is uniform along the length of sporangiophores even though the light sensitivity is confined to a growing zone less than 5 mm long. These results suggest that one of the initial biochemical steps after a light stimulus is the production of extractable ATP.     

A RING-finger photocarotenogenic repressor involved in asexual sporulation in Mucor circinelloides.

Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids and bending its sporangiophores towards the light source. The CrgA protein product acts as a repressor of carotene biosynthesis, as its inactivation leads to the overaccumulation of carotenoids in both the dark and the light. We show here that asexual sporulation in Mucor is also stimulated by light and that the crgA gene is involved in sporulation, given that lack of crgA function affects both carotenogenesis and the normal production of spores. A small interference RNA (siRNA) gene silencing approach was used to block the biosynthesis of carotenoids and to demonstrate that abnormal sporulation in crgA mutants is not a consequence of a defective production of carotenes. These results reveal an active role for the predicted CrgA product, a RING-finger protein, in the control of cellular light-regulated processes in Mucor.