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

Produção biotecnológica de poli-hidroxialcanoatos para a geração de polímeros biodegradáveis no Brasil

In recent years, several studies have been developed in Brazil to produce biodegradable materials. A particular family of bacterial polymers, the polyhydroxyalkanoates (PHA), has received special attention. PHAs are thermoplastic, biodegradable, biocompatible, are synthesised from renewable resources and can substitute petrochemical plastics in some applications. Different aspects have been focused to increase productivity and to reduce the cost of PHA production: bacterial improvement, use of industrial by-products as raw material, bioreactor design, process operation strategies, downstream process, mathematical modelling, polymer characterisation, application and biodegradability of blends. A production process was transferred to industry and studies to produce new PHA by controlling monomer composition are in progress. All these aspects are presented in this review

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g(-1) and PHB productivity (P(PHB)) 0.10 and 0.06 g l(-1) h(-1). This 40% P(PHB) difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol(-1) for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol(-1)). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Enzimas hidrolíticas (DNAses, lipases e proteases) secretadas por Cladosporium cladosporioides isolado de solo e seu potencial de aplicação em biotecnologia

Os microrganismos habitam todos os tipos de solos e apresentam uma enorme diversidade metabólica o que os faz uma importante fonte de bioprodutos, dentre eles, as enzimas. O objetivo deste trabalho foi isolar pelo menos um microrganismo capaz de secretar as enzimas hidrolíticas lipases, proteases e com ênfase DNAses, a partir de uma amostra de terra de jardim e usá-las como fonte produtora destas enzimas, para a formulação de um detergente. Foi encontrado o fungo filamentoso, denominado inicialmente F6, produtor de tais enzimas. Estudos com os extratos de cada enzima sobre seus substratos (azeite de oliva, caseína e DNA extraído de diferentes organismos) mostraram forte atividade a 30 °C e pH 7,5, e estabilidade quando estocados sob refrigeração a 8 °C por dois meses. O fungo F6 foi identificado como Cladosporium cladosporioides por morfologia observada à microscopia óptica e por sequenciamento da região ITS pelo método de Sanger. A importância deste achado inédito é o fato de obter as enzimas de interesse a partir de um mesmo microrganismo, o que facilita e economizam condições de cultivo, temperatura de incubação e teor de aeração, dentre outros, para desenvolver no futuro um detergente enzimático

PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)International Mobility of Santander BankInternational Mobility of Santander Ban

The diversity of polyketide synthase genes from sugarcane-derived fungi

The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with beta-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential633565577CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPThe Academy of Science for the Developing World (TWAS

Increasing PHB production with an industrially scalable hardwood hydrolysate as a carbon source

In this study, we report an industrially feasible conversion of wood hydrolysate from a pilot-scale forest biorefinery. The wood hydrolysate was compared to a synthetic hydrolysate with a similar content of carbon sources (glucose, xylose, and acetate). To maximize the PHB concentration, high-cell density cultivations were conducted in bioreactors for 52 h using the bacterium Paraburkholderia sacchari IPT 101 LMG 19450. The conversion of wood hydrolysate yielded a maximum PHB concentration of 34.5 g/L, which is among the highest reported for lignocellulose hydrolysates. In comparison, the use of a synthetic hydrolysate resulted in substantially lower PHB concentration (22.0 g/L). This could be attributed to a higher maximum specific growth rate of 0.36 vs. 0.33 per h for cells grown in wood hydrolysate vs. synthetic hydrolysate. For the wood hydrolysate, the final PHB content per cell mass reached 58 % g/g and a maximum PHB productivity of 0.72 g/(Lh), while the synthetic hydrolysate reached 55 % g/g and 0.46 g/(Lh) respectively. Given the increase in bacterial growth and PHB productivity when using the wood hydrolysate, the chosen lignocellulose conversion process is beneficial for subsequent biotechnological conversion to key bioproducts

The Diversity of Polyketide Synthase Genes from Sugarcane-Derived Fungi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase (PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26 shared sequence homology with beta-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase domains. A neighbour-joining phylogenetic analysis of the translated sequences could group the domains into previously established chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that, in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical ecology and likely biotechnological potential.633565577The Academy of Science for the Developing World (TWAS)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other\ud venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including conesnails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.King’s College LondonSISBIO license 15031-2FAPESP (grant 2010/50174-7)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq