Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.

BACKGROUND: Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. RESULTS: We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. CONCLUSION: A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

Bionanotechnology application of polypeptides in a hair color product: self-assembly enables expression, processing, and functionality.

Bionanotechnology aims to impart new properties to materials from unique functionalities present in biomolecules. However, the promise of bionanotechnology has not materialized beyond the biomedical field due in large part to issues of scalability, purity, and cost of manufacturing. In this work we demonstrate an approach to co-engineer production and system functionality into a single polypeptide. We designed a system to anchor particles onto hair via a multifunctional polypeptide composed of two domains, one with affinity to hair and the other capable of strong interactions with the particle surface. These strong interactions, exemplified by resistance to anionic surfactants, stem from the ability to self-assemble into higher order structures, which were observed by atomic force microscopy. At the same time, the controlled solubility properties of the particle binding domain permit the scalable production in Escherichia coli via inclusion bodies and cost effective purification. We believe this is a significant advance toward the development of bionanotechnology for industrial applications.

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.

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.

High-density sampling differential display of prokaryotic mRNAs with RAP-PCR.

A high-throughput approach to prokaryotic differential display has been developed. A large number of reverse transcription polymerase chain reactions (RT-PCR) are performed on total RNA isolated from induced and control bacterial cultures. Each RT-PCR reaction uses a single oligonucleotide primer and constitutes an independent sampling of the mRNA population. The large number of reactions performed allows the repeated sampling of the targeted polycistronic mRNA, which is clearly identified among possible false positives.

Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli.

For metabolic engineering it is advantageous in terms of stability, genetic regulation, and metabolic burden to modulate expression of relevant genes on the chromosome rather than relying on over-expression of the genes on multi-copy vectors. Here we have increased the production of beta-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (P(T5)). We recombined PCR fragments with the lambda-Red recombinase to effect chromosomal promoter replacement, which allows direct integration of a promoter along with a selectable marker that can subsequently be excised by the Flp/FRT site-specific recombination system. The resulting promoter-engineered isoprenoid genes were combined by serial P1 transductions into a host strain harboring a reporter plasmid containing beta-carotene biosynthesis genes allowing a visual screen for yellow color indicative of beta-carotene accumulation. Construction of an E. coli P(T5)-dxs P(T5)-ispDispF P(T5)-idi P(T5)-ispB strain resulted in producing high titers (6mg/g dry cell weight) of beta-carotene. Surprisingly, over-expression of the ispB gene, which was expected to divert carbon flow from the isoprenoid pathway to quinone biosynthesis, resulted in increased beta-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of beta-carotene in a non-carotenogenic E. coli. The high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes.

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.

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.

Proposed involvement of a soluble methane monooxygenase homologue in the cyclohexane-dependent growth of a new Brachymonas species.

High-throughput mRNA differential display (DD) was used to identify genes induced by cyclohexane in Brachymonas petroleovorans CHX, a recently isolated beta-proteobacterium that grows on cyclohexane. Two metabolic gene clusters were identified multiple times in independent reverse transcription polymerase chain reactions (RT-PCR) in the course of this DD experiment. These clusters encode genes believed to be required for cyclohexane metabolism. One gene cluster (8 kb) encodes the subunits of a multicomponent hydroxylase related to the soluble butane of Pseudomonas butanovora and methane monooxygenases (sMMO) of methanotrophs. We propose that this butane monooxygenase homologue carries out the oxidation of cyclohexane into cyclohexanol during growth. A second gene cluster (11 kb) contains almost all the genes required for the oxidation of cyclohexanol to adipic acid. Real-time PCR experiments confirmed that genes from both clusters are induced by cyclohexane. The role of the Baeyer-Villiger cyclohexanone monooxygenase of the second cluster was confirmed by heterologous expression in Escherichia coli.

A gene for a dioxygenase-like protein determines the production of the DF signal in Xanthomonas campestris pv. campestris.

SUMMARY The DF signal molecule regulates the production of both yellow pigments (xanthomonadins) and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris. These two bacterial products are crucial to the epiphytic survival and pathogenicity of this pathogen on its plant hosts. Previous work suggested that DF is a butyrolactone, which the Streptomyces bacteria are known to utilize as signals. pigB is one of seven transcriptional units in the X. c. pv. campestris xanthomonadin gene cluster, and its inactivation results in the loss of DF signal, xanthomonadin and EPS production. Here, determination and analysis of the pigB DNA sequence reveals the presence of two open reading frames, the first (xanB1) encoding a putative reductase/halogenase, and the second (xanB2) showing the highest level of identity to Streptomyces genes encoding putative pteridine-dependent dioxygenase-like proteins. We show that xanB2 (but not xanB1) is needed for production of the DF signal, and that some Streptomyces strains produce functional analogues of DF. A role for xanB2 in the biosynthesis of DF is proposed.

Isolation of Brachymonas petroleovorans CHX, a novel cyclohexane-degrading beta-proteobacterium.

A new bacterium that grows aerobically on cyclohexane was isolated from the wastewater plant of a petroleum refinery. This strain grows on a range of light hydrocarbons (C5-C10) as well as on some aromatic compounds such as toluene and m-cresol. Growth on hydrocarbons requires the presence of yeast extract and other complex media components that are not substrates for growth themselves. Strain CHX is resistant to cyclohexane and grows at concentrations up to 2 g l(-1). Strain CHX branches deeply within the Comamonadeae family of beta-proteobacteria and is tentatively assigned to the Brachymonas genus as Brachymonas petroleovorans CHX.

mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.