Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides

The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.     

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.     

Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides.

This work describes the isolation and characterization of crgA, a Mucor circinelloides gene, which has a dominant-positive effect on light-regulated carotenogenesis. The crgA gene was originally identified in a transformation experiment as a 3'-truncated open reading frame which caused carotenoid overaccumulation in the dark. The complete cloning and sequencing of crgA revealed that its putative product presented several recognizable structural domains: a RING-finger zinc binding domain near the N-terminus, a putative nuclear localization signal, two stretches of acidic amino acids, glutamine-rich regions and a putative isoprenylation motif. The expression of exogenous copies of the complete crgA gene or two different 3'-truncated versions, produced a similar dominant-positive effect on the light-inducible carotenogenesis of M. circinelloides. The presence of these exogenous sequences also caused a missregulation of the endogenous crgA gene, resulting in its overexpression. Collectively, these observations suggest that crgA is involved in the regulation of carotenoid biosynthesis by light.     

Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.     

Light induction of the carotenoid biosynthesis pathway in Blakeslea trispora

A gene of Blakeslea trispora has been cloned by heterologous hybridization with the Mucor circinelloides crgA gene, a repressor of light-inducible carotenogenesis. This gene is the ortholog of the M. circinelloides crgA, since it was able to restore the wild-type phenotype of a null crgA mutant of M. circinelloides. The expression of B. trispora crgA gene is light-induced and photoadapted, as occurs for M. circinelloides crgA. Light induction and photoadaptation of B. trispora crgA was also observed in M. circinelloides, which suggests that the mechanisms involved in light regulation are basically conserved between these filamentous fungi. Conservation of the regulatory pathway that controls carotene biosynthesis was supported by the light-induced and photoadapted expression of all structural carotenogenic genes of B. trispora. Consequently, the beta-carotene content of dark grown mycelia of B. trispora increased upon illumination with white light.     

卷枝毛霉孢子原生质体的制备与再生

为了构建高产γ-亚麻酸的卷枝毛霉稳定遗传转化体系,利用酶解法对卷枝毛霉(Mucor circinelloides sp.)EIM-10的孢子进行原生质体制备。研究酶液组成、渗透压稳定剂、酶解温度、酶解时间等对卷枝毛霉孢子原生质体形成和再生的影响,建立了制备卷枝毛霉孢子原生质体的最适条件:1%纤维素酶和2%溶壁酶为酶解体系,0.5mol/L NaCl作为渗透压稳定剂,酶解温度32℃,酶解时间2.5 h,再生培养基为0.5 mol/L NaCl高渗培养基。用双层平板培养法进行原生质体再生,在此条件下原生质体的形成量为1.2×106个/mL,再生率为70.5%。     

Genetic basis of carotenoid overproduction in Fusarium oxysporum

The phytopathogenic fungus Fusarium oxysporum is a model organism in the study of plant-fungus interactions. As other Fusarium species, illuminated cultures of F. oxysporum exhibit an orange pigmentation because of the synthesis of carotenoids, and its genome contains orthologous light-regulated car genes for this biosynthetic pathway. By chemical mutagenesis, we obtained carotenoid overproducing mutants of F. oxysporum, called carS, with upregulated mRNA levels of the car genes. To identify the regulatory gene responsible for this phenotype, a collection of T-DNA insertional mutants obtained by Agrobacterium mediated transformation was screened for carotenoid overproduction. Three candidate transformants exhibited a carS-like phenotype, and two of them contained T-DNA insertions in the same genomic region. The insertions did not affect the integrity of any annotated ORFs, but were linked to a gene coding for a putative RING-finger (RF) protein. Based on its similarity to the RF protein CrgA from the zygomycete Mucor circinelloides, whose mutation results in a similar carotenoid deregulation, this gene (FOXG_09307) was investigated in detail. Its expression was not affected in the transformants, but mutant alleles were found in several carS mutants. A strain carrying a partial FOXG_09307 deletion, fortuitously generated in a targeted transformation experiment, exhibited the carS phenotype. This mutant and a T-DNA insertional mutant holding a 5-bp insertion in FOXG_09307 were complemented with the wild type FOXG_09307 allele. We conclude that this gene is carS, encoding a RF protein involved in down-regulation of F. oxysporum carotenogenesis.     

A RING-finger protein regulates carotenogenesis via proteolysis-independent ubiquitylation of a white collar-1-like activator

Protein ubiquitylation plays a major role in the regulation of cellular processes mainly through proteasome-dependent degradation, although it has become increasingly clear that it is also involved in other processes. In the fungus Mucor circinelloides, blue light regulates carotene biosynthesis, with this response controlled by crgA and mcwc-1c genes. CrgA shows characteristics of ubiquitin ligases and represses carotenogenesis in the dark, whereas mcwc-1c is a white collar 1-like gene required for its light induction. Another two white collar 1-like genes have been identified in M. circinelloides: mcwc-1a, which is involved in phototropism, and mcwc-1b, of unknown function. Analysis of double knockout mutants generated for crgA and every mcwc-1 gene demonstrated that crgA and mcwc-1c regulate carotenogenesis by independent pathways. It was also shown that the effect of crgA on carotenogenesis is mediated by mcwc-1b, which acts as a carotenogenesis activator. CrgA is involved in proteolysis-independent mono- and di-ubiquitylation of MCWC-1b, which results in its inactivation. Regulation of carotenogenesis in M. circinelloides by proteolysis-independent ubiquitylation suggests that this mechanism of control could be more widespread than previously thought.     

卷枝毛霉pyrG基因缺陷突变株的诱变筛选与鉴定

为制备新的遗传筛选标记用于构建高产番茄红素的工程菌株,实验运用化学诱变的手段,以N-甲基-N'-硝基-N-亚硝基胍为诱变剂,以番茄红素生产菌株卷枝毛霉MU616为出发菌株,诱变获得5株尿嘧啶缺陷型突变株,突变株在基本培养基中培养5天后仍不能生长。通过同源转化带有pyrG基因(编码乳清酸核苷-磷酸盐脱羧酶)的质粒pEPM1确定突变株Mt1、Mt4和Mt5为pyrG基因缺陷突变株。随之对pyrG突变株进行生长特性的研究和产番茄红素性能的检测,结果表明,其中突变株Mt4的生物量为(9.0±0.6)g/L,番茄红素产量在黑暗和光照条件下分别为(1 648±185)μg/g和(3 234±281)μg/g,均与出发菌株相似,适宜作为进行卷枝毛霉转化的带有遗传标记的受体菌。pyrG基因缺陷突变菌株的获得对构建高产番茄红素的卷枝毛霉工程菌具有重要的意义。     

Cloning,characterization and heterologous expression of the Blakeslea trispora gene encoding orotidine-5'-monophosphate decarboxylase

The pyrG gene of the fungus Blakeslea trispora, encoding orotidine-5'-monophosphate decarboxylase (OMPD) enzyme, was cloned by heterologous hybridization of a genomic library with the Mucor circinelloides pyrG gene. The deduced amino acid sequence of the B. trispora pyrG gene is highly similar to the OMPD from other organisms. Hybridization analyses revealed that the only copy of this gene present in the genome of B. trispora is constitutively expressed. Heterologous complementation of a mutant of M. circinelloides deficient in OMPD activity with the B. trispora pyrG gene and promoter sequence confirmed the function of this gene. This functional complementation demonstrates that heterologous expression in M. circinelloides might be used to investigate the function of genes of B. trispora.     

牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。     

Distinct white collar-1 genes control specific light responses in Mucor circinelloides

Light regulates many developmental and physiological processes in a large number of organisms. The best-known light response in the fungus Mucor circinelloides is the biosynthesis of beta-carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue-light photoreceptors in M. circinelloides. Three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. When knockout mutants for each mcwc-1 gene were generated to characterize gene functions, only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways, although cross-talk between both pathways probably exists because mcwc-1a is involved in the light regulation of mcwc-1c expression.