13C-assisted metabolic flux analysis to investigate heterotrophic and mixotrophic metabolism in Cupriavidus necator H16

Introduction. Cupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO2) through Calvin-Benson-Bassham (CBB) cycle. The potential to utilize synthesis gas (Syngas) and the prospects of rerouting carbon from polyhydroxybutyrate synthesis to value-added compounds makes C. necator an excellent chassis for industrial application. Objectives. In the context of lack of sufficient quantitative information of the metabolic pathways and to advance in rational metabolic engineering for optimized product synthesis in C. necator H16, we carried out a metabolic flux analysis based on steady-state 13C-labelling. Methods. In this study, steady-state carbon labelling experiments, using either D-[1-13C]fructose or [1,2-13C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in C. necator H16 under heterotrophic and mixotrophic growth conditions, respectively. Results. We found that the CBB cycle is active even under heterotrophic condition, and growth is indeed mixotrophic. While Entner-Doudoroff (ED) pathway is shown to be the major route for sugar degradation, tricarboxylic acid (TCA) cycle is highly active in mixotrophic condition. Enhanced flux is observed in reductive pentose phosphate pathway (redPPP) under the mixotrophic condition to supplement the precursor requirement for CBB cycle. The flux distribution was compared to the mRNA abundance of genes encoding enzymes involved in key enzymatic reactions of the central carbon metabolism. Conclusion. This study leads the way to establishing 13C-based quantitative fluxomics for rational pathway engineering in C. necator H16

Proteomic Examination for Gluconeogenesis Pathway-Shift during Polyhydroxyalkanoate Formation in Cupriavidus necator Grown on Glycerol

Because of availability and inexpensiveness, glycerol can be considered as a suitable raw material for polyhydroxyalkanoate (PHA) production with bacterial fermentation. Nevertheless, compared to the production of glucose as a raw precursor, PHA produced from glycerol by Cupriavidus necator was found to produce lower PHA with low bacterial growth rates. According to our study, C. necator was able to synthesize glucose-like intermediates from glycerol via gluconeogenesis. This resulted in a decrease of the cell dry weight and the yield of PHA polymers, especially in the active cell growth phase. It was indicated that glycerol used as a carbon source of the PHA synthesis pathway has glucogenesis-shift, which causes a decrease of the PHA content and productivity. In this research, we investigated the proteins that were closely expressed with the increase of the intracellular PHA and glucose content. For solving the above problem, the proteins inside the bacterial cells were analyzed and compared to the database proteins via mass spectrometry. The proteins were isolated by 1-D SDS-polyacrylamide gel electrophoresis (PAGE) technique and identified by the liquid chromatography mass spectrometry (LC-MS) technique. By using bioinformatics validation, a total number of 1361 proteins were examined and found in the culture bacterial cells. Selective protein expression was correlated with the amount of PHA at each cultivation time and generating glucose by studying the 1361 proteins was elucidated in proteomic information. The results of the cluster of proteins were found to contain 93 proteins using the multiple array viewer (MEV) program with the KMS data analysis model. Protein species with the same expression pattern for PHA and six proteins with similar expression patterns were found to be correlated with generating glucose content. The associations of the two protein groups were then determined through a Stitch program. The protein and chemical associations were analyzed both directly and indirectly through different databases. The proteins of interest were found with research data linked between glycerol and glucose. Five protein types are connecting to glucose and glycerol shift pathway, two of which are glycosyl hydrolase (H16_B1563) and short-chain dehydrogenase (H16_B0687), both of which are enzymes used to break the bonds of complex sugars, possibly related to the partial conversion of glycerol to glucose. The two proteins found in the strains used in the Cupriavidus necator H16 experiment give rise to the break down the bonds of α,α-1,1-glucoside of malto-oligosyltrehalose and short-chain sugar molecules such as mannitol (C6H14O6), respectively. In this research, finding the associated expression proteins which is involved in changing the pathway of gluconeogenesis shift to PHA synthesis will be useful information on genetically modifying microorganisms to produce PHA more efficiently, leading to reduction of the production costs

Evaluation of Biodegradabilities of Biosynthetic Polyhydroxyalkanoates in Thailand Seawater and Toxicity Assessment of Environmental Safety Levels

Every year, thousands of tons of non-biodegradable plastic products are dumped into marine environments in Thailand’s territorial seawater, impacting various marine animals. Recently, there has been a surge in interest in biodegradable plastics as a solution for aquatic environments. However, in Thailand’s coastal waters, no suitable biodegradable plastic has been used as ocean-biodegradable packaging. Among them, polyhydroxyalkanoates (PHAs) have excellent biodegradability even in seawater, which is the desired property for packaging applications in tourist places such as plastic bags and bottles. In this report, we assess the environment’s safety and study the biodegradation in Thailand seawater of polyhydroxybutyrate (PHB) and PHA copolymer (PHBVV) that were successfully synthesized by bacteria with similar molecular weight. The two types of extracted PHA samples were preliminary biodegradability tested in the marine environment compared with cellulose and polyethylene. Within 28 days, PHB and PHBVV could be biodegraded in both natural and synthetic seawater with 61.2 and 96.5%, respectively. Furthermore, we assessed residual toxicity after biodegradation for environmental safety using seawater samples containing residual digested compounds and the standard guide for acute toxicity tests. It was discovered that marine water mites (Artemia franciscana) have 100 percent viability, indicating that they are non-toxic to the marine environment

Evaluation of 3D-Printing Scaffold Fabrication on Biosynthetic Medium-Chain-Length Polyhydroxyalkanoate Terpolyester as Biomaterial-Ink

Currently, the selection of materials for tissue engineering scaffolds is still limited because some tissues require flexible and compatible materials with human cells. Medium-chain-length polyhydroxyalkanoate (MCL-PHA) synthesized in microorganisms is an interesting polymer for use in this area and has elastomeric properties compatible with the human body. MCL-PHAs are elastomers with biodegradability and cellular compatibility, making them an attractive material for fabricating soft tissue that requires high elasticity. In this research, MCL-PHA was produced by fed-batch fermentation that Pseudomonas Putida ATCC 47054 was cultured to accumulate MCL-PHA by using glycerol and sodium octanoate as carbon sources. The amounts of dry cell density, MCL-PHA product per dry cells, and MCL-PHA productivity were at 15 g/L, 27%, and 0.067 g/L/h, respectively, and the components of MCL-PHA consisting of 3-hydroxydecanoate (3HD) 64.5%, 3-hydroxyoctanoate (3HO) 32.2%, and 3-hydroxyhexanoate (3HHx) 3.3%. The biosynthesized MCL-PHA terpolyester has a relatively low melting temperature, low crystallinity, and high ductility at 52 °C, 15.7%, and 218%, respectively, and considering as elastomeric polyester. The high-resolution scaffold of MCL-PHA terpolyester biomaterial-ink (approximately 0.36 mm porous size) could be printed in a selected condition with a 3D printer, similar to the optimum pore size for cell attachment and proliferation. The rheological characteristic of this MCL-PHA biomaterial-ink exhibits shear-thinning behavior, leading to good shape fidelity. The study results yielded a condition capable of fabricating an elastomer scaffold of the MCL-PHA terpolyester, giving rise to the ideal soft tissue engineering application

Electrospun Fibers of Polybutylene Succinate/Graphene Oxide Composite for Syringe-Push Protein Absorption Membrane

The adsorption of proteins on membranes has been used for simple, low-cost, and minimal sample handling of large volume, low protein abundance liquid samples. Syringe-push membrane absorption (SPMA) is an innovative way to process bio-fluid samples by combining a medical syringe and protein-absorbable membrane, which makes SPMA a simple, rapid protein and proteomic analysis method. However, the membrane used for SPMA is only limited to commercially available protein-absorbable membrane options. To raise the method’s efficiency, higher protein binding capacity with a lower back pressure membrane is needed. In this research, we fabricated electrospun polybutylene succinate (PBS) membrane and compared it to electrospun polyvinylidene fluoride (PVDF). Rolling electrospinning (RE) and non-rolling electrospinning (NRE) were employed to synthesize polymer fibers, resulting in the different characteristics of mechanical and morphological properties. Adding graphene oxide (GO) composite does not affect their mechanical properties; however, electrospun PBS membrane can be applied as a filter membrane and has a higher pore area than electrospun PVDF membrane. Albumin solution filtration was performed using all the electrospun filter membranes by the SPMA technique to measure the protein capture efficiency and staining of the protein on the membranes, and these membranes were compared to the commercial filter membranes—PVDF, nitrocellulose, and Whatman no. 1. A combination of rolling electrospinning with graphene oxide composite and PBS resulted in two times more captured protein when compared to commercial membrane filtration and more than sixfold protein binding than non-composite polymer. The protein staining results further confirmed the enhancement of the protein binding property, showing more intense stained color in compositing polymer with GO