Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a

Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the β-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g⁻¹ of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.     

Use of Transposon Promoter-Probe Vectors in the Metabolic Engineering of the Obligate Methanotroph Methylomonas sp. Strain 16a for Enhanced C40 Carotenoid Synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C₄₀ carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations—the fliCS, hsdM, ccp-3, cysH, and nirS regions—were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.     

A carotenoid synthesis gene cluster from Algoriphagus sp. KK10202C with a novel fusion-type lycopene β-cyclase gene

A carotenoid synthesis gene cluster was isolated from a marine bacterium Algoriphagus sp. strain KK10202C that synthesized flexixanthin. Seven genes were transcribed in the same direction, among which five of them were involved in carotenoid synthesis. This cluster had a unique gene organization, with an isoprenoid gene, ispH (previously named lytB), being present among the carotenoid synthesis genes. The lycopene β-cyclase encoded by the crtY _cd gene appeared to be a fusion of bacterial heterodimeric lycopene cyclase CrtY_c and CrtY_d. This was the first time that a fusion-type of lycopene β-cyclase was reported in eubacteria. Heterologous expression of the Algoriphagus crtY _cd gene in lycopene-accumulating Escherichia coli produced bicyclic β-carotene. A biosynthesis pathway for monocyclic flexixanthin was proposed in Algoriphagus sp. strain KK10202C, though several of the carotenoid synthesis genes not linked with the cluster have not yet been cloned.     

Characterization of β -Carotene Ketolases, CrtW, from Marine Bacteria by Complementation Analysis in Escherichia coli

A complementation analysis was performed in Escherichia coli to evaluate the efficiency of β-carotene ketolases (CrtW) from the marine bacteria Brevundimonas sp. SD212, Paracoccus sp. PC1 (Alcaligenes PC-1), and Paracoccus sp. N81106 (Agrobacterium aurantiacum), for astaxanthin production. Each crtW gene was expressed in Escherichia coli synthesizing zeaxanthin due to the presence of plasmid pACCAR25ΔcrtX. Carotenoids that accumulated in the resulting E. coli transformants were examined by chromatographic and spectroscopic analyses. The transformant carrying the Paracoccus sp. PC1 or N81106 crtW gene accumulated high levels of adonixanthin, which is the final astaxanthin precursor for CrtW, and astaxanthin, while the E. coli transformant with crtW from Brevundimonas sp. SD212 did not accumulate any adonixanthin and produced a high level of astaxanthin. These results show efficient conversion by CrtW of Brevundimonas sp. SD212 from adonixanthin to astaxanthin, which is a new-found characteristic of a bacterial CrtW enzyme. The phylogenetic positions between CrtW of the two genera, Brevundimonas and Paracoccus, are distant, although they fall into α-Proteobacteria.     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

Tailoring cyanobacteria as a new platform for highly efficient synthesis of astaxanthin

With the ability to recycle CO₂ into value-added chemicals, cyanobacteria have been considered as renewable microbial cell factories. Astaxanthin, a highly valued carotenoid with potent antioxidant activity, could be beneficial to human health. Astaxanthin biosynthesis in engineered chassis has been achieved previously, but it generated a relatively low yield. Here, we successfully constructed a highly efficient astaxanthin biosynthetic pathway in cyanobacterium Synechocystis sp. PCC 6803, and achieved more than a 500-fold increase in astaxanthin production via stepwise reconstruction of the biosynthetic pathway and rational rewiring of the endogenous metabolism. The engineered strain produced up to 29.6 mg/g of astaxanthin (dry cell weight), which is the highest yield reported in the engineered chassis to date. Moreover, multi-omics analyses revealed that establishing a high astaxanthin flux may enhance photosynthesis and central metabolism in the engineered strain to compensate for the depleted pigments, which could be valuable for astaxanthin overproduction. This study presents a novel alternative for high-efficiency biosynthesis of astaxanthin directly from CO₂.     

The present perspective of astaxanthin with reference to biosynthesis and pharmacological importance

Astaxanthin is an important natural pigment, a diketo carotenoid that besides being a food ingredient has importance as a nutraceutical. Astaxanthin is a fat-soluble nutrient with a molecular weight of 596.8 Da (Dalton) and a molecular formula of C₄₀H₅₂O_4. It is water insoluble and lipophilic. Organisms that produce astaxanthin include the basidiomycetous yeast; Phaffia rhodozyma, the green alga; Haematococcus pluvialis and the Gram-negative bacteria; Agrobacterium aurantiacum, Paracoccus marcusii, P. carotinifaciens, Paracoccus sp. strain MBIC 01143, and P. haeundaensis. Xanthophyllomyces dendrorhous and Haematococcus pluvialis, which are potential sources of astaxanthin. The antioxidant properties of astaxanthin are believed to have a key role in the medicinal, pharmaceutical, and food industries. Astaxanthin acts as a free-radical scavenger and an immunomodulator. It is a medicinal ingredient against degenerative diseases such as cancer, skin related illness, and heart disease. Presently, this carotenoid is used as a major pigmentation source and a feed supplement in aquaculture, primarily salmon, trout, crabs, shrimp, chickens, and red sea bream. The present review focuses on the pharmacological connotations of astaxanthin and specifies the natural sources and pathways of its production along with other relevant aspects.     

Utilization of mustard waste isolates for improved production of astaxanthin by Xanthophyllomyces dendrorhous

Astaxanthin production in the wild strain Xanthophyllomyces dendrorhous TISTR 5730 was investigated using different mustard waste media, including mustard waste residue extract (MRE), mustard waste residue hydrolysate (MRH), mustard waste precipitated extract (MPE), and mustard waste precipitated hydrolysate (MPH). The growth of X. dendrorhous and the production of astaxanthin were dependent on the type and initial concentrations of mustard waste media. The MPH medium was the best substrate resulting in yields of biomass and astaxanthin of 19.6 g/L and 25.8 mg/L, respectively, under optimal conditions. MPH medium improved astaxanthin production 11-fold compared to the commonly used commercial yeast malt medium, and 1.3–2.1-fold compared to other mustard waste media.     

Utilization of the recombinant human β-carotene-15,15′-monooxygenase gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> and mammalian cells

In animals, β-carotene 15,15′-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant β-carotene to retinal. In the present study, we utilized β-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from β-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for β-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of β-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from β-carotene.     

Molecular sequencing and analysis of soluble methane monooxygenase gene clusters from methanotroph <Emphasis Type="Italic">Methylomonas</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> GYJ3

Summary A methanotroph Methylomonas sp. GYJ3 was isolated, whose sMMO genes and 16S rDNA were sequenced and analysed, demonstrating that the bacterium might be a type I methanotroph (γ-Proteobacteria) and was closer to Methylomonas sp. KSWIII/KSPIII. This result was consistent with the result previously determined by biochemistry and morphological taxonomy. Sequence comparison among six open reading frames and the deduced amino acid sequences of the sMMO genes from six strains revealed that the strain GYJ3 had highly conserved regions in MMOX with other strains, amounting to 78–99% homology at protein level and 71–97% homology at DNA level. Highly conserved sequences lay in two iron-binding regions. Furthermore, scanning electron microscopy of the strain GYJ3 showed rod shapes with a slightly bent configuration on the even surfaces and with plump bodies.     

Cloning and Expression of the Vitreoscilla Hemoglobin Gene in Enterobacter Aerogenes: Effect on Cell Growth and Oxygen Uptake

The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxi-dative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5–2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2–5-fold higher activity for these enzymes than cells at the exponential phase.     

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.     

Organisation of the bph gene cluster of transposon Tn 4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds

  Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzo-ate derivatives. We constructed a cosmid library of Tn4371 DNA. The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon. Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp. KKS102, i.e. the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD. Consensus sequences for σ54-associated RNA polymerase were found upstream of bphA1 and bphEGF. Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking σ54. In contrast to wild-type H16 exconjugants, the σ54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371bph gene expression on σ54. The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium. Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC. Received: 13 May 1996 / Accepted: 16 September 1996     

The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor

The mycoparasite Trichoderma harzianum has been extensively used in the biocontrol of a wide range of phytopathogenic fungi. Hydrolytic enzymes secreted by the parasite have been directly implicated in the lysis of the host. Dual cultures of Trichoderma and a host, with and without contact, were used as means to study the mycoparasitic response in Trichoderma. Northern analysis showed high-level expression of genes encoding a proteinase (prb1) and an endochitinase (ech42) in dual cultures even if contact with the host was prevented by using cellophane membranes. Neither gene was induced during the interaction of Trichoderma with lectin-coated nylon fibres, which are known to induce hyphal coiling and appressorium formation. Thus, the signal involved in triggering the production of these hydrolytic enzymes by T. harzianum during the parasitic response is independent of the recognition mediated by this lectin-carbohydrate interaction. The results showed that induction of prb1 and ech42 is contact-independent, and a diffusible molecule produced by the host is the signal that triggers expression of both genes in vivo. Furthermore, a molecule that is resistant to heat and protease treatment, obtained from Rhizoctonia solani cell walls induces expression of both genes. Thus, this molecule is involved in the regulation of the expression of hydrolytic enzymes during mycoparasitism by T. harzianum. Received: 8 June 1998 / Accepted: 28 July 1998     

Production of β-glucosidase using immobilised<Emphasis Type="Italic"> Piromyces</Emphasis> sp. KSX1 and<Emphasis Type="Italic"> Orpinomyces</Emphasis> sp. 478P1 in repeat-batch culture

Two anaerobic fungi, one a monocentric strain (Piromyces sp. KSX1) and the other a polycentric strain (Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. β-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, β-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more β-glucosidase than strain 478P1, the maximum specific β-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor. Electronic Publication     

Screening and identification of yeast sequences that cause growth inhibition when overexpressed

To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter. In all, 240 000 yeast transformants were screened for lethality on galactose medium. From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained. Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII. Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition. The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule. Overexpression of the BNI1ΔN and BOI1ΔN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation. Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively. The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA. Received: 3 June 1996 / Accepted: 1 October 1996     

Effective production of retinal from β-carotene using recombinant mouse β-carotene 15,15′-monooxygenase

The gene encoding β-carotene 15,15′-monooxygenase from Mus musculus (house mouse), which cleaves β-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg⁻¹. The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37°C, 200 mg l⁻¹ β-carotene, 5% (w/v) Tween 40, and 0.2 U ml⁻¹ enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l⁻¹ retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported.