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

Abstract

Synthesis of poly-[3-hydroxybutyrate] (PHB) by Cupriavidus necator H16 in batch cultures was evaluated using three biodiesel-derived by-products as the sole carbon sources: waste glycerol (REG-80, refined to 80 % purity with negligible free fatty acids); glycerol bottom (REG-GB, with up to 65 % glycerol and 35 % free fatty acids), and free fatty acids (REG-FFA, with up to 75 % FFA and no glycerol). All the three substrates supported growth and PHB production by C. necator, with polymer accumulation ranging from 9 to 84 % cell dry weight (cdw), depending on the carbon source. To help understand these differences, proteomic analysis indicated that although C. necator H16 was able to accumulate PHB during growth on all three biodiesel by-products, no changes in the levels of PHB synthesis enzymes were observed. However, significant changes in the levels of expression were observed for two Phasin proteins involved with PHB accumulation, and for a number of gene products in the fatty acid β-oxidation pathway, the Glyoxylate Shunt, and the hydrogen (H2) synthesis pathways in C. necator cells cultured with different substrates. The glycerol transport protein (GlpF) was induced in REG-GB and REG-80 glycerol cultures only. Cupriavidus necator cells cultured with REG-GB and REG-FFA showed up-regulation of β-oxidation and Glyoxylate Shunt pathways proteins at 24 h pi, but H2 synthesis pathways enzymes were significantly down-regulated, compared with cells cultured with waste glycerol. Our data confirmed earlier observations of constitutive expression of PHB synthesis proteins, but further suggested that C. necator H16 cells growing on biodiesel-derived glycerol were under oxidative stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0206-z) contains supplementary material, which is available to authorized users.

Data Driven Modelling of a Chemically Defined Growth Medium for Cupriavidus necator H16

“…Such low growth was attributed to oxidative stress resulting from high levels of reactive oxygen species (ROS) formed as a result of elevated level of hydrogenases, leading to cell destruction by damaging DNA and other cell components (32). Formulations of chemically defined media previously described for the cultivation of C. necator H16 are typically prepared without amino acids (3,7,11,12,22,33). In this study, bacterial growth is improved when a small number of amino acids (arginine, histidine, leucine and methionine) are included in the medium.…”

“…Chemically defined media are important to enable experimental reproducibility, to reliably characterise the genetics of the organism, to determine genotype by environment interactions, and to facilitate fundamental research of bacterial physiology that underpins bioengineering efforts. While different chemically defined media have been deployed for the cultivation of C. necator H16 (3,(10)(11)(12)(20)(21)(22) there is no consensus regarding the components that are required, the concentration of each component, or how each component interacts to affect growth of the bacterium (SI Table 1).…”

Microbial Polyhydroxyalkanoates and Nonnatural Polyesters

“…[129] The omics analyses were conducted under different nutrient conditions (PHA accumulating and PHA nonaccumulating), [128] growth phases (exponential and stationary), [130] and carbon sources (styrene, biodiesel-derived fatty acids, and glycerol). [126,131,132] Also, the PHA producers were compared with their mutant strains such as PHA nonaccumulating mutant or superior producing mutant. [125,129] These studies have elucidated several important features of PHA producing microorganisms and offered a comprehensive insight into PHA metabolism.…”

Colonization and degradation of polyhydroxyalkanoates by lipase-producing bacteria

“…PHAs from canola oil was produced using Pseudomonas chlororaphis PA23 as described earlier (Sharma et al 2017). Copolymer of 3hydroxybutyrate and 3-hydroxyvalrate was produced from biodiesel fatty acid supplemented with 0.5% valeric acid using Cuprividus necator H16 (Sharma et al 2016).…”