Methanol bioconversion in Methylotuvimicrobium buryatense 5GB1C through self-cycling fermentation.

Methanol is an abundant and low-cost next-generation carbon source. While many species of methanotrophic bacteria can convert methanol into valuable bioproducts in bioreactors, Methylotuvimicrobium buryatense 5GB1C stands out as one of the most promising strains for industrialization. It has a short doubling time compared to most methanotrophs, remarkable resilience against contamination, and a suite of tools enabling genetic engineering. When approaching industrial applications, growing M. buryatense 5GB1C on methanol using common batch reactor operation has important limitations; for example methanol toxicity leads to mediocre biomass productivity. Advanced bioreactor operation strategies, such as fed-batch and self-cycling fermentation, have the potential to greatly improve the industrial prospects of methanotrophs growing on methanol. Herein, implementation of fed-batch operation led to a 26-fold increase in biomass density, while two different self-cycling fermentation (SCF) strategies led to 3-fold and 10-fold increases in volumetric biomass productivity. Interestingly, while synchronization is a typical trait of microbial populations undergoing SCF, M. buryatense 5GB1C cultures growing under this mode of operation led to stable, reproducible cycles but no significant synchronization.

The influence of self-cycling fermentation long- and short-cycle schemes on Saccharomyces cerevisiae and Escherichia coli.

Self-cycling fermentation (SCF), a cyclic process in which cells, on average, divide once per cycle, has been shown to lead to whole-culture synchronization and improvements in productivity during bioconversion. Previous studies have shown that the completion of synchronized cell replication sometimes occurs simultaneously with depletion of the limiting nutrient. However, cases in which the end of cell doubling occurred before limiting nutrient exhaustion were also observed. In order to better understand the impact of these patterns on bioprocessing, we investigated the growth of Saccharomyces cerevisiae and Escherichia coli in long- and short-cycle SCF strategies. Three characteristic events were identified during SCF cycles: (1) an optimum in control parameters, (2) the time of completion of synchronized cell division, and (3) the depletion or plateau of the limiting nutrient. Results from this study and literature led to the identification of three potential trends in SCF cycles: (A) co-occurrence of the three key events, (B) cell replication ending prior to the co-occurrence of the other two events, and (C) depletion or plateau of the limiting nutrient occurring later than the co-occurrence of the other two events. Based on these observations, microbial physiological differences were analyzed and a novel definition for SCF is proposed.

Transcriptomic analysis of synchrony and productivity in self-cycling fermentation of engineered yeast producing shikimic acid.

Industrial fermentation provides a wide variety of bioproducts, such as food, biofuels and pharmaceuticals. Self-cycling fermentation (SCF), an advanced automated semi-continuous fermentation approach, has shown significant advantages over batch reactors (BR); including cell synchrony and improved production. Here, Saccharomyces cerevisiae engineered to overproduce shikimic acid was grown under SCF operation. This led to four-fold increases in product yield and volumetric productivity compared to BR. Transcriptomic analyses were performed to understand the cellular mechanisms leading to these increases. Results indicate an up-regulation of a large number of genes related to the cell cycle and DNA replication in the early stages of SCF cycles, inferring substantial synchronization. Moreover, numerous genes related to gluconeogenesis, the citrate cycle and oxidative phosphorylation were significantly up-regulated in the late stages of SCF cycles, consistent with significant increases in shikimic acid yield and productivity.

The VapBC1 toxin-antitoxin complex from Mycobacterium tuberculosis: purification, crystallization and X-ray diffraction analysis.

Mycobacterium tuberculosis, a major human pathogen, encodes at least 88 toxin-antitoxin (TA) systems. Remarkably, more than half of these modules belong to the VapBC family. Under normal growth conditions, the toxicity of the toxin VapC is neutralized by the protein antitoxin VapB. When bacteria face an unfavourable environment, the antitoxin is degraded and the free toxin VapC targets important cellular processes in order to inhibit cell growth. TA systems function in many biological processes, such as in the stringent response, in biofilm formation and in drug tolerance. To explore the structure of the VapBC1 complex, the toxin VapC1 and the antitoxin VapB1 were separately cloned, co-expressed and crystallized. The best crystal was obtained using a crystallization solution consisting of optimized solution with commercial sparse-matrix screen solutions as additives. The crystal diffracted to a resolution of 2.7 Šand belonged to space group P21, with unit-cell parameters a = 59.3, b = 106.7, c = 250.0 Å, ß = 93.75°.

Crystallization and preliminary crystallographic study of Porcine epidemic diarrhea virus main protease in complex with an inhibitor.

Porcine epidemic diarrhea virus (PEDV) mainly infects neonatal pigs, resulting in significant morbidity and mortality. Owing to problems such as long periods of virus shedding, existing vaccines cannot provide complete protection from PEDV infection. The PEDV genome encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of Porcine epidemic diarrhea virus in complex with a Michael acceptor was crystallized. The complex crystals diffracted to 2.5 Šresolution and belonged to space group R3, with unit-cell parameters a = 175.3, b = 175.3, c = 58.7 Å. Two molecules were identified per asymmetric unit.

Crystallization and preliminary crystallographic study of Feline infectious peritonitis virus main protease in complex with an inhibitor.

Feline infectious peritonitis virus (FIPV) causes a lethal systemic granulomatous disease in wild and domestic cats around the world. Currently, no effective vaccines or drugs have been developed against it. As a member of the genus Alphacoronavirus, FIPV encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of FIPV in complex with a Michael acceptor-type inhibitor was crystallized. The complex crystals diffracted to 2.5 Šresolution and belonged to space group I422, with unit-cell parameters a = 112.3, b = 112.3, c = 102.1 Å. There is one molecule per asymmetric unit.

Expression, crystallization and preliminary crystallographic study of the functional mutant (N60K) of nonstructural protein 9 from Human coronavirus HKU1.

Human coronavirus HKU1 (HCoV-HKU1), which mainly causes acute self-limited respiratory-tract infections, belongs to group A of the Betacoronavirus genus. Coronavirus genomes encode 16 nonstructural proteins (nsp1-16), which assemble into a large replication-transcription complex mediating virus propagation. Nonstructural protein 9, which binds to the single-stranded DNA/RNA, has been shown to be indispensible for viral replication. Interestingly, a functional mutant (N60K) of nsp9 was identified to compensate for a 6 nt insertion mutation of the 3'-untranslated region (UTR), which is critical for viral RNA synthesis. It has been proposed that the N60K mutation may cause certain conformational changes of nsp9 to rescue the defective insertion mutant. To further investigate the underlying structural mechanism, the N60K mutant of nsp9 from HCoV-HKU1 was successfully crystallized in this study. The crystals diffracted to 2.6 Šresolution and belonged to space group P212121, with unit-cell parameters a = 31.9, b = 85.0, c = 95.0 Å. Two molecules were identified per asymmetric unit.

Crystallization and preliminary crystallographic study of human coronavirus NL63 main protease in complex with an inhibitor.

Human coronavirus NL63 mainly infects younger children and causes cough, fever, rhinorrhoea, bronchiolitis and croup. It encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of human coronavirus NL63 was crystallized in complex with a Michael acceptor. The complex crystals diffracted to 2.85 Šresolution and belonged to space group P41212, with unit-cell parameters a = b = 87.2, c = 212.1 Å. Two molecules were identified per asymmetric unit.