Controlled biotechnological production of polyhydroxyalkanoates

Předložená diplomová práce se zabývá produkcí polyhydroxyalkanoátů (PHA) bakterií Cupriavidus necator H16. Cílem práce byla příprava, selekce a charakterizace mutantních kmenů schopných vyšší produkce PHA. V teoretické části byla zpracována literární rešerše zabývající se nejdůležitějšími typy PHA, bakterií Cupriavidus necator a způsoby indukce mutageneze. V experimentální části byly připraveny mutantní kmeny pomocí fyzikální a chemické mutageneze. Mutantní kmeny schopné nadprodukce PHA byly selektovány pomocí kultivace na minerálním médium s olejem. Pro další studium byly vybrány 4 mutantní kmeny schopné nadprodukce PHA. Tyto mutantní kmeny byly dále podrobeny biochemické charakterizaci. Byly naměřeny specifické aktivity vybraných intracelulárních enzymů včetně enzymů podílejících se na biosyntéze PHA. Také byla naměřena resistence mutantů vůči oxidačnímu stresu. Bylo zjištěno, že mutantní kmeny schopné nadprodukce PHA mají vyšší aktivity enzymů produkujících NADPH. NADPH je jeden z klíčových substrátů ovlivňujících směr toku acetyl-CoA metabolizmem. Vyšší intracelulární koncentrace NADPH parciálně inhibuje Krebsův cyklus a aktivuje akumulaci PHA. Aktivity acetoacetyl-CoA reduktázy a PHA syntázy, enzymů zapojených do syntézy PHA, těchto mutantů proto byly také vyšší stejně jako molekulová hmotnost připravených polymerů. Aplikace fyzikálních a chemických mutagenů je způsob, kterým lze připravit biotechnologicky perspektivní mutantní kmeny schopné nadprodukce PHA.This diploma thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Cupriavidus necator H16. Goal of this work was preparation, selection and characterization of mutant strains overproducing PHA. Theoretical focuses on the most important PHA, bacteria Cupriavidus necator and mutagenesis techniques. In practical part mutant strains were prepared through physical and chemical mutagenesis. Mutant strains overproducing PHA were selected by cultivation in mineral medium with oil. For further study, 4 mutant strains overproducing PHA were selected. These mutants were biochemically characterized. Specific activities of several intracellular enzymes including enzymes involved in PHA biosynthesis were measured. Resistance of mutants against oxidative stress was measured as well. Mutant strains overproducing PHA revealed higher enzymatic activities of NADPH producing enzymes. Generally, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH partially inhibits TCA cycle and activates accumulation of PHA. Therefore, activities of acetoacetyl-CoA reductase and PHB synthase, enzymes directly involved in PHA synthesis were higher as compared to wild strain as well as molecular weight of produced materials. It can be concluded that biotechnologically perspective mutagens capable of PHA overproduction can be prepared by application of chemical and physical mutagens.

Poly(hydroxyalkanoate) production by Cupriavidus necator from fatty waste can be enhanced by phaZ1 inactivation

PHA production from waste oils or fats requires microorganisms that should be both excellent PHA producers and equipped with enzymatic activities allowing hydrolysation of triglycerides. Unfortunately, microbes with the combination of substrate-utilization and PHA production are not currently available, and the strategies to be adopted are the use of costly commercial enzymes, or genetic modification of microorganisms exhibiting high PHA product yields. In the present work, after a general investigation on the ability of Cupriavidus necator to grow on a number of fatty substrates, the possibility to enhance PHA production by limiting intracellular depolymerisation, was investigated. By knocking out the related phaZ1 gene, the construction of C. necator recombinant strains impaired in depolymerase (PhaZ1) activity was achieved. The polymer yield of the recombinant strain was finally compared to that of the parental C. necator DSM 545

Global changes in the proteome of Cupriavidus necator H16 during poly-(3-hydroxybutyrate) synthesis from various biodiesel by-product substrates

Additional file 1: Table S1. P-scores of proteomic runs of C. necator H16 grown with different substrates

Metabolism of Poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and Sequences of Genes and Function of the Encoded Proteins in the Synthesis and Degradation of PHA

Pseudomonas oleovorans accumulates poly(3-hydroxyalkanoates) (PHAs) after growth on medium chain length hydrocarbons. Large amounts of this polyester are synthesized when cells are grown under nitrogen-limiting conditions. When nitrogen is resupplied in the medium, the accumulated PHA is degraded. In this paper, we describe mutants which are defective in the synthesis or in the degradation of PHA. These mutants were used to select DNA fragments which encode PHA polymerases and a PHA depolymerase. A 25-kilobase (kb) DNA fragment was isolated from P. oleovorans that complements a Pseudomonas putida mutant unable to accumulate PHA. Subcloning resulted in the assignment of a 6.4-kb EcoRI fragment as the pha locus, containing genetic information of PHA synthesis. Mutants in the PHA degradation pathway were also complemented by this fragment, indicating that genes encoding PHA biosynthetic and degradative enzymes are clustered. Analysis of the DNA sequence of the 6.4-kb fragment revealed the presence of two open reading frames encoding PHA polymerases based on homology to the poly(3-hydroxybutyrate) polymerase from Alcaligenes eutrophus. A third open reading frame complemented the PHA degradation mutation and is likely to encode a PHA depolymerase. The presence of two PHA polymerases is due to a 2098-base pair DNA duplication. The PHA polymerases are 53% identical and show 35-40% identity to the poly(3-hydroxybutyrate) polymerase. No clear difference in specificity was found for the PHA polymerases. However, with the pha locus cloned on a multicopy vector, a polymer was accumulated that contains a significantly higher amount of substrate-derived monomers. An increase in the rate of polyester synthesis versus oxidation of the monomers in the beta-oxidation explains these findings

Elucidation of Beta-Oxidation Pathways in Ralstonia Eutropha H16 by Examination of Global Gene Expression

Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid β-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single β-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.Malaysia-MIT Biotechnology Partnership Programm

Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silico analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases. NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Structural Biology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Structural Biology, 2015. http://dx.doi.org/10.1016/j.jsb.2015.04.00

Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery:The case of the polyhydroxyalkanoates

This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains. B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA productio

Second-generation functionalized mediumchain- length polyhydroxyalkanoates: the gateway to high-value bioplastic applications

Polyhydroxyalkanoates (PHAs) are biodegradable biocompatible polyesters, which accumulate as granulesin the cytoplasm of many bacteria under unbalanced growth conditions. Medium-chain-length PHAs (mcl-PHAs), characterizedby C6-C14 branched monomer chains and typically produced by Pseudomonas species, are promising thermoelastomers,as they can be further modified by introducing functional groups in the side chains. Functionalized PHAs areobtained either by feeding structurally related substrates processed through the β-oxidation pathway, or using specificstrains able to transform sugars or glycerol into unsaturated PHA by de novo fatty-acid biosynthesis. Functionalized mcl-PHAs provide modified mechanical and thermal properties, and consequently have new processing requirements andhighly diverse potential applications in emergent fields such as biomedicine. However, process development and sampleavailability are limited due to the toxicity of some precursors and still low productivity, which hinder investigation. Conversely,improved mutant strains designed through systems biology approaches and cofeeding with low-cost substratesmay contribute to the widespread application of these biopolymers. This review focuses on recent developments in theproduction of functionalized mcl-PHAs, placing particular emphasis on strain and bioprocess design for cost-effectiveproduction. [Int Microbiol 2013; 16(1):1-15