Production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains.

BACKGROUND: Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications. RESULTS: Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases. CONCLUSIONS: This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.

Monitoring the plant epiphyte Methylobacterium extorquens DSM 21961 by real-time PCR and its influence on the strawberry flavor.

Besides its influence on plant growth and health, plant-associated bacteria exert an impact on fruit quality. Methylotrophic bacteria can enhance the biosynthesis of strawberry flavor compounds, especially the two furanoid compounds 2,5-dimethyl-4-hydroxy-2H-furanone (DMHF) and 2,5-dimethyl-4-methoxy-2H-furanone in vitro. Here, we report the selection and characterization of Methylobacterium extorquens DSM 21961, a strain that was able to enhance the furanone content ad planta under greenhouse conditions. For monitoring the colonization of strawberry plants, a strain-specific quantification system for M. extorquens DSM 21961 was developed. Specificity, linear range and quantitative limit of the system were shown, and successful application was demonstrated in a monitoring experiment of M. extorquens DSM 21961 on strawberry leaves under greenhouse conditions. Furthermore, the quantification of DMHF in strawberry fruits via GC indicated an increased biosynthesis of this compound in strawberry plants. The colonization behavior analyzed by confocal laser scanning microscopy using GFP-tagged cells revealed high colonization of the upper and the lower leaf surfaces, with a specific accumulation of bacterial cells on trichomes. The results support a biotechnological application of this promising flavor-stimulating agent.

Novel, versatile, and tightly regulated expression system for Escherichia coli strains.

A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-beta-D-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTG-induced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.

Phylogenetic analysis of methanotrophic communities in cover soils of a landfill in Ontario.

We examined the methanotrophs in the Trail Road Landfill soils, Ottawa, Ontario, through cultivation-independent molecular assay and the culturing approach. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified methanotroph-specific 16S rDNA gene fragments revealed a more diverse type I (RuMP pathway) methanotrophic community than type II (serine pathway) in 17 soil samples taken along a 50 m transect. The type II methanotrophic community was less diverse, with the dominance of Methylocystis in almost all samples, and clustering with high similarity (85%-88%). Also, the results showed that the C/N ratio of soil organic matter could significantly affect the methanotrophic community structures. The DGGE results were supported by sequence analysis of cloned pmoA. Members of the genera Methylobacter (type I), Methylocaldum (type X), and Methylocystis (type II) appeared to be the dominant methanotrophs. From methanotrophic enrichments, we isolated type I Methylobacter sp., and 3 type II Methylocystis spp.,which appeared to be one of the dominant bacteria species in the soil sample from which isolates were obtained.

Production of an insecticidal crystal protein from Bacillus thuringiensis by the methylotroph Methylobacterium extorquens.

The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, P(mxaF). Transmission electron microscopy revealed characteristic bipyramidal intracellular delta-endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed.

Bestowing inducibility on the cloned methanol dehydrogenase promoter (PmxaF) of Methylobacterium extorquens by applying regulatory elements of Pseudomonas putida F1.

PmxaF is a strong methanol-inducible promoter in Methylobacterium extorquens. When this promoter is cloned in expression vectors and used to drive heterologous gene expression, methanol inducibility is either greatly reduced or entirely lost. In order to bestow inducibility upon the cloned PmxaF promoter in expression vectors, we adopted combinational methods (regulatory elements of the Pseudomonas putida F1 cym and cmt operons and Tn7 transposon system) to control reporter gene expression at the transcriptional level in M. extorquens. An operator fragment (26 nucleotides) of the cmt operon was inserted downstream of the cloned PmxaF promoter in the broad-host-range expression vector (pCHOI3). The repressor gene (cymR) located upstream of the cym operon in P. putida F1 was amplified by PCR. To avoid cellular toxicity for M. extorquens caused by the overexpression of CymR, single and/or double copies of cymR were integrated into the chromosome of M. extorquens using the mini-Tn7 transposon system. Cultures containing the chromosomally integrated cymR gene were subsequently transformed with pCHOI3 containing modified PmxaF (i.e., PmxaF plus operator). In this construct, inducibility is afforded by cumate (p-isopropylbenzoate). In this report, we describe the inducible and tightly regulated expression of heterologous genes (bgl [for beta-galactosidase], est [for esterase], and gfp [for green fluorescent protein]) in M. extorquens. This is the first documented example of an inducible/regulated heterologous gene expression system in M. extorquens.

Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach.

BACKGROUND: In the interest of generating large amounts of recombinant protein, inducible systems have been studied to maximize both the growth of the culture and the production of foreign proteins. Even though thermo-inducible systems were developed in the late 1970's, the number of studies that focus on strategies for the implementation at bioreactor scale is limited. In this work, the bacteriophage lambda PL promoter is once again investigated as an inducible element but for the production of green fluorescent protein (GFP). Culture temperature, induction point, induction duration and number of inductions were considered as factors to maximize GFP production in a 20-L bioreactor. RESULTS: It was found that cultures carried out at 37 degrees C resulted in a growth-associated production of GFP without the need of an induction at 42 degrees C. Specific production was similar to what was achieved when separating the growth and production phases. Shake flask cultures were used to screen for desirable operating conditions. It was found that multiple inductions increased the production of GFP. Induction decreased the growth rate and substrate yield coefficients; therefore, two time domains (before and after induction) having different kinetic parameters were created to fit a model to the data collected. CONCLUSION: Based on two batch runs and the simulation of culture dynamics, a pre-defined feeding and induction strategy was developed to increase the volumetric yield of a temperature regulated expression system and was successfully implemented in a 20-L bioreactor. An overall cell density of 5.95 g DW l(-1) was achieved without detriment to the cell specific production of GFP; however, the production of GFP was underestimated in the simulations due to a significant contribution of non-growth associated product formation under limiting nutrient conditions.

Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis.

Stable isotope probing (SIP) allows the isolation of nucleic acids from targeted metabolically active organisms in environmental samples. In previous studies, DNA-SIP has been performed with the one-carbon growth substrates methane and methanol to study methylotrophic organisms. The methylotrophs that incorporated the labelled substrate were identified with polymerase chain reaction and sequencing of 16S rRNA and 'functional genes' for methanotrophs (mxaF, pmoA, mmoX). In this study, a SIP experiment was performed using a forest soil sample incubated with (13)CH(4), and the (13)C-DNA was purified and cloned into a bacterial artificial chromosome (BAC) plasmid. A library of 2300 clones was generated and most of the clones contained inserts between 10 and 30 kb. The library was probed for key methylotrophy genes and a 15.2 kb clone containing a pmoCAB operon, encoding particulate methane monooxygenase, was identified and sequenced. Analysis of the pmoA sequence suggested that the clone was most similar to that of a Methylocystis sp. previously detected in this forest soil. Twelve other open reading frames were identified on the clone, including the gene encoding beta-ribofuranosylaminobenzene 5'-phosphate synthase, which is involved in the biosynthesis of the 'archaeal' C(1)-carrier, tetrahydromethanopterin, which is also found in methylotrophs. This study demonstrates that relatively large DNA fragments from uncultivated organisms can be readily isolated using DNA-SIP, and cloned into a vector for metagenomic analysis.

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments.

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.

Multicopy integration and expression of heterologous genes in Methylobacterium extorquens ATCC 55366.

High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (PmxaF) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [beta-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.

Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2.

The molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small-scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC, were adjacent to two genes, mmoR and mmoG, encoding a sigma54 transcriptional activator and a putative GroEL-like chaperone, located downstream (3') of mmoC. Transcriptional analysis revealed that the genes were all cotranscribed from a sigma54-dependent promoter located upstream (5') of mmo X. The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane-bound methane monooxygenase enzyme in M. silvestris BL2 and the copper-mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material.

Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure-function research. Following various cloning strategies, a KM71H (Mut(s)) strain was selected. A significant concentration (74 microg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7-8, 11, 20 kDa vs. 4.6 kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some "collagen-like" material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The "collagen-like" material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat "collagen-like" nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils.

Heterologous extracellular production of enterocin P from Enterococcus faecium P13 in the methylotrophic bacterium Methylobacterium extorquens.

Enterocin P (EntP), a strong antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, was produced by Methylobacterium extorquens. For heterologous expression of EntP in the methylotrophic bacterium M. extorquens, a recombinant plasmid was constructed. The gene encoding the EntP structural gene (entP) was cloned into the plasmid vector pCM80, under control of the methanol dehydrogenase promoter (P(mxaF)), to generate plasmid pS25. When M. extorquens ATCC 55366 was transformed with pS25, EntP was detected and quantified in supernatants of the recombinant M. extorquens S25 strain by using specific anti-EntP antibodies and a non-competitive indirect enzyme-linked immunosorbent assay (NCI-ELISA). Purification of EntP by hydrophobic adsorption and reverse-phase (RP-FPLC) chromatographies, permitted recovery of active EntP from the supernatants of M. extorquens S25 grown in a synthetic defined medium.

Production of ribonucleotides by autolysis of Pichia anomala mutant and some physiological activities.

Various mutants of Pichia anomala were isolated by ethyl methanesulfonate (EMS) treatment and UV irradiation through cycloheximide resistance and KCl sensitivity. The selected mutant HA-2 accumulated a higher content of RNA and grew faster than the wild-type strain in yeast extract-malt (YM) broth. Autolysis of the HA-2 mutant at 60 degrees C and pH 7.0 for 6 h was the best condition to obtain maximum yields of 5'-ribonucleotides, inosinic monophosphate (IMP) (6.2 mg/g biomass) and guanylic monophosphate (GMP) (35.5 mg/g biomass). The yield of adenylic monophosphate (AMP) (7.8 mg/g biomass) was optimal at 60 degrees C at pH 6.5 for 6 h. The inhibitory activity of the angiotensin-converting enzyme and the nitrite-scavenging activity for autolysates of the HA-2 mutant were about 13.0% and 47.0% higher than those of native strain, respectively.

Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96.

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S. tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.

Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

Screening for soluble methane monooxygenase in methanotrophic bacteria using combined molecular and biochemical methods for hydroxylase detection.

Three well known methanotrophic bacteria (Methylosinus trichosporium OB3b, Methylocystis sp. WI 14, and Methylocystis sp. GB 25) and three newly isolated methanotrophic bacteria (Methylocystis sp. WI 11, Methylocystis sp. X, and FI-9) were screened for sMMO considering the existence of hydroxylase (component A) genes as well as its gene expression. For these purposes monoclonal antibodies that specifically recognize each subunit of the hydroxylase of Methylocystis sp. WI 14 (alpha-subunit [9E5/F2], beta-subunit [4E2/G11], gamma-subunit [10G3/D7]) were produced. PCR amplification using well known primers showed that the hydroxylase encoding genes appear to be only present in M. trichosporium OB3b, Methylocystis sp. WI 11 and WI 14, and in the isolate FI-9. Western and ELISA analysis using the monoclonal antibodies revealed that all subunits of hydroxylase were present. However, in FI-9, only the alpha-subunit of the hydroxylase might be expressed. Surprisingly, in Methylocystis sp. GB 25, where no sMMO activity and no amplification with sMMO specific primers was obtained, the antibody 4E2/G11 recognized a protein band with exactly the same molecular mass as the beta-subunit of the hydroxylase. Methylocystis sp. X showed no positive reaction in any of the tests. In combination with the detection methods currently used, the described antibodies provide a powerful tool for detecting even partially expressed hydroxylase genes.

Detection of methanotrophs with highly divergent pmoA genes from Arctic soils.

Tundra soil samples from the Canadian Arctic community, Kuujjuaq, were analyzed for the presence of the soluble (sMMO) and particulate (pMMO) methane monooxygenase genes. Total genomic DNA extracted from these soils was used as template for PCR using sMMO- and pMMO-specific primers, mmoX1-mmoX2 and A189-A682, respectively. pMMO and sMMO genes were detected in the Kuujjuaq soil samples. Isolation of sMMO-possessing methanotrophic microorganisms from the three soils, as determined by the colony naphthalene oxidation assay, was carried out using direct plating (5 degrees C) and methane enrichment studies (5 degrees C and 25 degrees C). Direct plating did not yield sMMO-possessing methanotrophic bacteria, whereas methane enrichments yielded isolates possessing and expressing sMMO activity. Analysis of derived amino acid sequences of pmoA genes and partial 16S rRNA genes obtained by PCR, using DNA isolated directly from this environment and from isolates, revealed the presence of highly divergent PmoA/AmoA sequences and 16S rRNA sequences that cluster closely with but are distinct from the genes from the genera Methylosinus and Methylocystis.