Secretion of Acetylxylan Esterase From Chlamydomonas reinhardtii Enables Utilization of Lignocellulosic Biomass as a Carbon Source.

Microalgae offer a promising biological platform for sustainable biomanufacturing of a wide range of chemicals, pharmaceuticals, and fuels. The model microalga Chlamydomonas reinhardtii is thus far the most versatile algal chassis for bioengineering and can grow using atmospheric CO2 and organic carbons (e.g., acetate and pure cellulose). Ability to utilize renewable feedstock like lignocellulosic biomass as a carbon source could significantly accelerate microalgae-based productions, but this is yet to be demonstrated. We observed that C. reinhardtii was not able to heterotrophically grow using wheat straw, a common type of lignocellulosic biomass, likely due to the recalcitrant nature of the biomass. When the biomass was pretreated with alkaline, C. reinhardtii was able to grow using acetate that was released from the biomass. To establish an eco-friendly and self-sustained growth system, we engineered C. reinhardtii to secrete a fungal acetylxylan esterase (AXE) for hydrolysis of acetylesters in the lignocellulosic biomass. Two transgenic strains (CrAXE03 and CrAXE23) secreting an active AXE into culture media were isolated. Incubation of CrAXE03 with wheat straw resulted in an eight-fold increase in the algal cell counts with a concomitant decrease of biomass acetylester contents by 96%. The transgenic lines showed minor growth defects compared to the parental strain, indicating that secretion of the AXE protein imposes limited metabolic burden. The results presented here would open new opportunities for applying low-cost renewable feedstock, available in large amounts as agricultural and manufacturing by-products, for microalgal cultivation. Furthermore, acetylesters and acetate released from them, are well-known inhibitors in lignocellulosic biofuel productions; thus, direct application of the bioengineered microalga could be exploited for improving renewable biofuel productions.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins.

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria.

BACKGROUND: Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons. RESULTS: Here we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system. CONCLUSIONS: An easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.

Tratamiento endovascular de las fístulas arteriovenosas piales no galénicas / No disponible

Introducción y objetivos Las fístulas arteriovenosas piales son malformaciones vasculares infrecuentes. Generalmente son congénitas y su historia natural es ominosa. El objetivo es describir nuestra experiencia en su manejo endovascular y analizar la literatura. Pacientes y métodos Es un estudio retrospectivo descriptivo de pacientes tratados por vía endovascular durante 3 años en 3 instituciones latinoamericanas. Resultados Fueron 6 pacientes con edad media de 22 años. Un caso fue resultado de un traumatismo. El 50% presentó hemorragia intracraneal, el 66% desarrollaron clínica secundaria a efecto de masa y al flujo retrógrado. En los estudios de imagen se observaron varices intracraneales en el 83% de los casos. La angiografía cerebral mostró arterias fistulosas provenientes de la circulación anterior en el (..) (AU)

[Endovascular treatment of non-galenic pial arteriovenous fistulas]. / Tratamiento endovascular de las fístulas arteriovenosas piales no galénicas.

INTRODUCTION AND OBJECTIVES: Pial arteriovenous fistulas are infrequent vascular malformations. They are generally congenital and their natural history is ominous. The objective of this work is to describe our experience in their endovascular management and to review the existing literature. PATIENTS AND METHODS: This is a retrospective and descriptive study of patients treated by endovascular approach during 3 years at 3 Latin-American hospitals. RESULTS: The study included 6 patients with a mean age of 22 years. One case was caused by cranial trauma. In total, 50% suffered intracranial haemorrhage and 66% developed symptoms attributable to volume effect or retrograde blood flow. Intracranial varices were identified by CT and MRI scans in 83% of cases. Digital subtraction angiography showed arteriovenous fistulas from anterior circulation in 67% of cases and deep venous drainage in 50%. One endovascular procedure was performed in 5 cases (83%), while 2 procedures were required in one case. A single embolic agent was used to occlude fistulas in 67% of cases; whilst 33% required a combination. Coils were used in 4 cases (67%) and onyx was injected in another 4 (67%). One case required stent and balloon deployment. The fistulas were uneventfully occluded in all cases. The follow-up period was one year in 5 cases and 6 months in one case. All patients remained symptom-free. CONCLUSIONS: Endovascular management can be considered as the treatment of choice. It consists in the embolisation of arterial pedicles with one or more embolic agents and should be performed as close as possible to the drainage vein, avoiding migration of the embolic agent towards the venous side.

Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene.

Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme.