Genes from a Dietzia sp. for synthesis of C40 and C50 beta-cyclic carotenoids.

Dietzia sp. CQ4 accumulated the C(40) beta-cyclic carotenoids (canthaxanthin and echinenone) and the C(50) beta-cyclic carotenoid (C.p.450 monoglucoside). A plant-type lycopene beta-cyclase gene crtL was identified for beta-cyclization of the C(40) carotenoids. A carotenoid synthesis gene cluster was identified away from the crtL gene, which contained the crtEBI genes for the synthesis of lycopene followed by the lbtABC genes for lycopene elongation and beta-cyclization of the C(50) carotenoids. This C(50) beta-cyclic carotenoid synthesis gene cluster from Dietzia sp. CQ4 showed high homology with the gene clusters for synthesizing the C(50) epsilon-cyclic carotenoids (decaprenoxanthin and glucosides) from Corynebacterium glutamicum and Agromyces mediolanus. One unique feature of the C(50) beta-cyclic carotenoid synthesis genes in Dietzia sp. CQ4 was that the gene encoding a C(50) carotenoid beta-cyclase subunit and the gene encoding the lycopene elongase appeared to be fused as a single gene (lbtBC). Expression of the gene (lbtA) encoding another subunit of the C(50) carotenoid beta-cyclase and the lbtBC gene in lycopene-accumulating Escherichia coli produced almost exclusively the C(50) beta-cyclic carotenoid C.p.450. One gene (crtX) with high homology to glycosyl transferases was transcribed in the opposite orientation downstream of the lbtBC gene. The crtX gene was likely involved in C.p.450 glucosylation in Dietzia sp. CQ4. The pathway analogous to the synthesis of the C(50) epsilon-cyclic carotenoids was proposed for the synthesis of the C(50) beta-cyclic carotenoids.

A carotenoid synthesis gene cluster from a non-marine Brevundimonas that synthesizes hydroxylated astaxanthin.

A Brevundimonas vesicularis strain DC263 isolated from surface soil was shown to produce hydroxylated astaxanthin. A carotenoid synthesis gene cluster containing ten genes was cloned from strain DC263, among which eight genes were involved in carotenoid synthesis. In addition to the crtW gene encoding the 4,4'-beta-ionone ring ketolase and the crtZ gene encoding the 3,3'-beta-ionone ring hydroxylase that were responsible for astaxanthin synthesis, the cluster also contained a novel gene crtG identified recently encoding the 2,2'-beta-ionone ring hydroxylase that further hydroxylate astaxanthin. The individual genes in the DC263 cluster showed the highest sequence similarities to the corresponding genes reported in Brevundimonas sp. strain SD212, a marine isolate that also produced hydroxylated astaxanthin. The genetic organization of the carotenoid synthesis gene clusters in the two Brevundimonas strains was identical. It is likely that the two Brevundimonas strains were evolved from the same ancestor and adapted later to growth in different environments. Expression of the crtW and crtZ from DC263 in a beta-carotene-accumulating E. coli produced astaxanthin as the predominant carotenoid. The crtG from DC263 and the crtG from another Brevundimonas aurantiaca strain were expressed in E. coli producing different carotenoid substrates. Both CrtG showed low activity on beta-carotene and high activity on zeaxanthin. The main difference was that the CrtG from B. aurantiaca worked well on canthaxanthin or astaxanthin, but the CrtG from DC263 did not work on either of the ketocarotenoids.

Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids.

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.

Engineering Escherichia coli for canthaxanthin and astaxanthin biosynthesis.

Escherichia coli is a non-carotenogenic bacterium that could synthesize farnesyl pyrophosphate precursor through the isoprenoid pathway. Carotenoid production in E. coli requires heterologous expression of carotenoid synthesis genes. The carotenoid synthesis operons are assembled from genes isolated from carotenogenic bacterial sources. Expression of the different operons yields different carotenoid titers. The operons containing the idi gene give more than fivefold higher carotenoid titers than the operons lacking the idi gene. The carotenoid modification genes encoding ketolases and hydroxylases are incorporated into the operons for canthaxanthin and astaxanthin production. The ketolases and hydroxylases from different bacterial sources produce astaxanthin of different purity relative to the total carotenoids. Expression of the ketolases and hydroxylases closer to the promoter appears to give higher astaxanthin purity than expression farther from the promoter at the end of the operons. Balanced expression of ketolases and hydroxylases is critical to achieve high astaxanthin purity. Here, we describe methods to assemble carotenoid biosynthesis operons from carotenogenic gene clusters isolated from different bacterial sources and evaluate canthaxanthin or astaxanthin production in E. coli.

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 C(40) 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.

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp.

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1-crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.

Improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in Methylomonas sp.

Two types of non-homologous beta-carotene ketolases (CrtW and CrtO) were previously described. We report improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in a methylotrophic bacterium, Methylomonas sp. 16a, which could use the C1 substrate (methane or methanol) as sole carbon and energy source. The crtO gene from Rhodococcus erythropolis was improved for canthaxanthin production in an E. coli strain engineered to produce high titer carotenoids by error-prone PCR mutagenesis followed by in vitro recombination. The best mutants from protein engineering could produce approximately 90% of total carotenoids as canthaxanthin in the high titer E. coli strain compared to approximately 20% canthaxanthin produced by the starting gene. Canthaxanthin production in Methylomonas was also significantly improved to approximately 50% of total carotenoids by the mutant genes. Further improvement of canthaxanthin production to approximately 93% in Methylomonas was achieved by increased expression of the best mutant gene. Some mutations were found in many of the improved genes, suggesting that these sites, and possibly the regions around these sites, were important for improving the crtO's activity for canthaxanthin production.

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 beta-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.

A carotenoid synthesis gene cluster from Algoriphagus sp. KK10202C with a novel fusion-type lycopene beta-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 beta-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 beta-cyclase was reported in eubacteria. Heterologous expression of the Algoriphagus crtY ( cd ) gene in lycopene-accumulating Escherichia coli produced bicyclic beta-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.

Engineering a beta-carotene ketolase for astaxanthin production.

A new beta-carotene ketolase gene (crtW) was cloned from an environmental isolate Sphingomonas sp. DC18. A robust and reliable color screen was developed for protein engineering to improve its activity on hydroxylated carotenoids for astaxanthin production. Localized random mutagenesis was performed on the crtW gene including the upstream ribosomal binding site (RBS). Six mutations (H96L, R203W, A205V, A208V, F213L and A215T) in the crtW gene were isolated multiple times that showed improved astaxanthin production. These mutations were localized near the conserved histidine motifs, which were proposed for binding iron required for enzymatic activity. Combination of two of the mutations (R203W/F213L) further improved astaxanthin production. One mutation at the RBS (a438t) was shown to have additional effect on improving astaxanthin production. Most of the mutants still retained high activity on beta-carotene, however, the F213L single mutant and the R203W/F213L double mutant that yielded the highest improvement for astaxanthin production showed decreased activity for canthaxanthin production.

Metabolic engineering for synthesis of aryl carotenoids in Rhodococcus.

Rhodococcus erythropolis naturally synthesizes monocyclic carotenoids: 4-keto-gamma-carotene and gamma-carotene. The genes and the pathway for carotenoid synthesis in R. erythropolis were previously described. We heterologously expressed a beta-carotene desaturase gene (crtU) from Brevibacterium in Rhodococcus to produce aryl carotenoids such as chlorobactene. Expression of the crtU downstream of a chloramphenicol resistance gene on pRhBR171 vector showed higher activity than expression downstream of a native 1-deoxyxylulose-5-phosphate synthase gene (dxs) on pDA71 vector. Expression of the crtU in the beta-carotene ketolase (crtO) knockout Rhodococcus host produced higher purity chlorobactene than expression in the wild-type Rhodococcus host. Growth of the engineered Rhodococcus strain in eight different media showed that nutrient broth yeast extract medium supplemented with fructose gave the highest total yield of chlorobactene. This medium was used for growing the engineered Rhodococcus strain in a 10-l fermentor, and approximately 18 mg of chlorobactene was produced as the almost exclusive carotenoid by fermentation.

Novel carotenoid oxidase involved in biosynthesis of 4,4'-diapolycopene dialdehyde.

Biosynthesis of C(30) carotenoids is relatively restricted in nature but has been described in Staphylococcus and in methylotrophic bacteria. We report here identification of a novel gene (crtNb) involved in conversion of 4,4'-diapolycopene to 4,4'-diapolycopene aldehyde. An aldehyde dehydrogenase gene (ald) responsible for the subsequent oxidation of 4,4'-diapolycopene aldehyde to 4,4'-diapolycopene acid was also identified in Methylomonas. CrtNb has significant sequence homology with diapophytoene desaturases (CrtN). However, data from knockout of crtNb and expression of crtNb in Escherichia coli indicated that CrtNb is not a desaturase but rather a novel carotenoid oxidase catalyzing oxidation of the terminal methyl group(s) of 4,4'-diaponeurosporene and 4,4'-diapolycopene to the corresponding terminal aldehyde. It has moderate to low activity on neurosporene and lycopene and no activity on beta-carotene or zeta-carotene. Using a combination of C(30) carotenoid synthesis genes from Staphylococcus and Methylomonas, 4,4'-diapolycopene dialdehyde was produced in E. coli as the predominant carotenoid. This C30 dialdehyde is a dark-reddish purple pigment that may have potential uses in foods and cosmetics.

Diversity of carotenoid synthesis gene clusters from environmental Enterobacteriaceae strains.

Eight Enterobacteriaceae strains that produce zeaxanthin and derivatives of this compound were isolated from a variety of environmental samples. Phylogenetic analysis showed that these strains grouped with different clusters of Erwinia type strains. Four strains representing the phylogenetic diversity were chosen for further characterization, which revealed their genetic diversity as well as their biochemical diversity. The carotenoid synthesis gene clusters cloned from the four strains had three different gene organizations. Two of the gene clusters, those from strains DC416 and DC260, had the classical organization crtEXYIBZ; the gene cluster from DC413 had the rare organization crtE-idi-XYIBZ; and the gene cluster from DC404 had the unique organization crtE-idi-YIBZ. Besides the diversity in genetic organization, these genes also exhibited considerable sequence diversity. On average, they exhibited 60 to 70% identity with each other, as well as with the corresponding genes of the Pantoea type strains. The four different clusters were individually expressed in Escherichia coli, and the two idi-containing clusters gave more than fivefold-higher carotenoid titers than the two clusters lacking idi. Expression of the crtEYIB genes with and without idi confirmed the effect of increasing carotenoid titer by the type II idi gene linked with the carotenoid synthesis gene clusters.

Directed evolution of copy number of a broad host range plasmid for metabolic engineering.

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.