Modulating ilvA encoding threonine deaminase for balanced growth and PHB synthesis by Halomonas grown in rich nitrogen source.

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.

BackgroundMicrobial polyesters, also known as polyhydroxyalkanoates (PHAs), closely resemble physical and mechanical features of petroleum derived plastics. Recombinant Escherichia coli strains are being used in industrial production of PHAs in Stirred Tank Bioreactors (STRs). However, use of Air-Lift Reactors (ALRs) has been known to offer numerous technical operating options over STRs, and as such has been successfully implemented in many bioprocesses. Halomonas boliviensis is a halophilic bacterium that is known to assimilate various carbohydrates and convert them into a particular type of PHA known as poly(3-hydroxybutyrate) (PHB). Owing to this capability, it has been used to synthesize the polyester using hydrolysates of starch or wheat bran in stirred tank bioreactors.ResultsThis research article firstly describes the production of PHB in shake flasks by H. boliviensis using different combinations of carbohydrates and partially hydrolyzed starch as carbon sources. The highest PHB yields, between 56 and 61 % (wt.), were achieved when either starch hydrolysate or a mixture of glucose and xylose were used as carbon sources. The starch hydrolysate obtained in this study was then used as carbon source in an ALR. The largest amount of PHB, 41 % (wt.), was attained after 24 hrs of cultivation during which maltose in the hydrolysate was assimilated more rapidly than glucose during active cell growth; however, the rate of assimilation of both the carbohydrates was found to be similar during synthesis of PHB. An incomplete pentose phosphate pathway, which lacks 6-phosphogluconate dehydrogenase, was deduced from the genome sequence of this bacterium and may result in the characteristic assimilation of glucose and maltose by the cells.ConclusionsThis study showed that the production of PHB by H. boliviensis using cheap substrates such as starch hydrolysate in a simple production system involving an ALR is feasible. Both maltose and glucose in the hydrolysate induce cell growth and PHB synthesis; most likely the cells balance adequately CoA and NAD(P)H during the assimilation of these carbohydrates. The combination of cheap substrates, simple production systems and the use of non-strict sterile conditions by the halophile H. boliviensis are desirable traits for large scale production of PHB, and should lead to a competitive bioprocess.Electronic supplementary materialThe online version of this article (doi:10.1186/s40709-015-0031-6) contains supplementary material, which is available to authorized users.

Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.

PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene

Oleaginous microalgae have gained increasing attention as an alternative feedstock for biodiesel production due to the increasing demand of fuel, climate change, and global warming. This study aimed to isolate and screen robust microalgal strains from hot springs for cultivation in subtropical and tropical areas. The newly isolated oleaginous microalgae were cultivated at 30, 35 and 40 °C. Among mesophilic and thermophilic strains tested, Chlamydomonas sp. HP59 is considered the most robust strain as it showed high cell growth in a broad range of temperatures (30 - 40 °C), with the maximum dried cell weight at 40 °C. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. To increase lipid production, the 2-stage cultivation, in which nitrogen-rich was applied to promote cell growth in the 1st stage and nitrogen-limitation was applied to stimulate lipid accumulation in the 2nd stage, was performed. The high temperature combined with nitrogen-limitation did improve lipid production by all microalgae. With this strategy, Chlamydomonas sp. HP59 showed the highest dried cell weight of 4.33 g/L and lipid production of 1.72 g/L. This study has shown the potential use of the newly isolated oleaginous microalgae from hot springs to be cultivated at high temperatures and under nitrogen-limited conditions for the production of biodiesel feedstocks. HIGHLIGHTS Mesophilic and thermophilic oleaginous microalgae were isolated from hot springs. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. Combined effect of high temperature and nitrogen limitation effectively increased lipid content. Two-stage cultivation gave high dried cell weight and hence overall high lipid production. GRAPHICAL ABSTRACT

Protein phase separation for enhanced production of 3-hydroxypropionate and polyhydroxybutyrate by Halomonas.

Poly-(R)-3-hydroxybutyric acid (PHB) was synthesized anaerobically in recombinant Escherichia coli. The host anaerobically accumulated PHB to more than 50% of its cell dry weight during cultivation in either growth or nongrowth medium. The maximum specific PHB production rate during growth-associated synthesis was approximately 2.3 +/- 0.2 mmol of PHB/g of residual cell dry weight/h. The by-product secretion profiles differed significantly between the PHB-synthesizing strain and the control strain. PHB production decreased acetate accumulation for both growth and nongrowth-associated PHB synthesis. For instance under nongrowth cultivation, the PHB-synthesizing culture produced approximately 66% less acetate on a glucose yield basis as compared to a control culture. A theoretical biochemical network model was used to provide a rational basis to interpret the experimental results like the fermentation product secretion profiles and to study E. coli network capabilities under anaerobic conditions. For example, the maximum theoretical carbon yield for anaerobic PHB synthesis in E. coli is 0.8. The presented study is expected to be generally useful for analyzing, interpreting, and engineering cellular metabolisms.

Oleaginous microorganisms can produce polyunsaturated fatty acids beneficial to human health through adjusting the nitrogen content in the medium. The target of rapamycin complex 1 (TORC1) is important for nitrogen sensing and then regulates lipid metabolism. However, the function of Kog1, a subunit of TORC1, in TORC1-regulated lipid metabolism in oleaginous microorganisms remains unclear. In this study, the gene kog1 was knocked out to explore the mechanism of lipid accumulation in the oleaginous fungus M. circinelloides under nitrogen-limited and nitrogen-rich conditions. The results showed that the cell dry weight (CDW) of the kog1 deletion mutant was obviously decreased from 22.2 to 15.4 g/L under nitrogen-limited conditions; however, the lipid content markedly increased by 43.2% compared to the control, from 20.8% of CDW to 29.9%. A similar trend was observed under nitrogen-rich conditions; the cell growth was significantly inhibited, the CDW was decreased from 28.6 to 23.0 g/L, and the lipid content increased by 79.6% compared to the control strain, reaching 9.7% of CDW. The addition of rapamycin further enhanced lipid accumulation in the kog1 knockout mutant but not in the tor knockout mutant, indicating that Kog1 is the upstream target of rapamycin (TOR) in regulating lipid regulation. Transcriptional analysis under both nitrogen-limited and nitrogen-rich conditions notably suggested that nitrogen stress may activate Snf1/AMPK to inhibit Kog1, facilitating SREBP-1c nuclear translocation and activating fatty acid biosynthesis genes.

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

The Vam6 GEF Controls TORC1 by Activating the EGO Complex

Statistical evaluation and discrimination of competing kinetic models and hypothesis for the mathematical description of poly-3(hydroxybutyrate) synthesis by Cupriavidus necator DSM 545

Masterbatch plant in South East Asia

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB), is one of the most valuable biopolymers because of its flexible mechanical properties. In this study, the goal is to establish a scaled-up process of low cost P(3HB-co-4HB) from a 7.5-L fermentor to 1- and 5-m3 industrial bioreactors, respectively, using Halomonas bluephagenesis TD40 grown on glucose, γ-butyrolactone, and waste corn steep liquor (CSL) as substrates, under open non-sterile and fed-batch or continuous conditions. The non-sterile process enables the energy reduction for less steam consumption. Moreover, waste gluconate is successfully utilized to replace glucose as a carbon source for cell growth and PHA accumulation in 7.5-L fermentor, which opens the possibility of 60% of raw material cost reduction for recycling the waste resources. A mathematical model and rational calculation is established to help guide the feeding strategy and scale-up, respectively, leading to 100 g L-1 cell dry weight (CDW) containing 60.4% P(3HB-co-mol 13.5% 4HB) after 36 h of growth in the 5 m3 vessel. An even higher P(3HB-co-4HB) content of 74% is achieved by decreasing the use of waste CSL. A stable and continuous open process for efficient low-cost production of P(3HB-co-4HB) is successfully developed coupling fermentation with the downstream extraction processing.

The study carried out during the present Ph.D. program aimed at investigating the PHB production process in autotrophic cultures of cyanobacteria. The work was carried out at the Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale of the University of Naples ‘Federico II’ and at Molecular Microbial Physiology Group of the Swammerdam Institute for Life Sciences in Universiteit van Amsterdam. The activities were articulated according to three paths: i) the characterization of PHB production process as regards kinetics and yields using different growth media characterized by different nitrate concentration and screening cyanobacteria strains; ii) the characterization of the PHB production process by cyanobacteria according to kinetic models; iii) the development of genetic engineering approach on cyanobacteria to improve PHB production. 1)Selection of the optimal growth media to produce PHB and screening of cyanobacteria strains: characterization in terms of kinetics and yields. The study was aimed at the assessment of both the kinetics and the yields of the cell growth and PHB produced during the growth on different growth media characterized by different nitrate concentrations: BG11 (optimal nitrate concentration), BG1/2 (half of optimal nitrate concentration), BG1/4 (one fourth of the optimal nitrate concentration) and BG0 (nitrogen-starved conditions). Batch tests were focused on the preliminary characterization of the PHB production process from cyanobacteria with the aim of highlighting the relevant features of the process. Synechocystis PCC6803 was growth as model strains on the different culture media. The investigation was carried out in photobioreactors using CO2 as carbon source. The best media for PHB production was BG1/2 (half of optimal nitrate concentration). Five cyanobacteria strains were screened using BG1/2 medium, to select a high PHB producer: Synechocystis PCC6803, Synechocystis aquatilis, Synechocystis fuscopigmentosa, Synecoccoccus nidulans and Chlorogloeopsis Fritshii. 2)Characterization of the PHB production process: kinetic models. An adequate kinetic model to describe the PHB production process is a key issue to address the conditions to maximize the PHB fraction in the cell. A kinetic model of PHB production by Synechocystis PCC6803 from CO2 was proposed using the biochemical networks simulator COPASI. Data form growth tests under dynamic light system (light/dark cycle) carried out at different initial nitrate concentration were used to assess the kinetic and stoichiometric parameters of the proposed model. Two types of cells were considered in the cultures: growing cells (X), which are able to grow, and PHB producing cells (XPHB). The specific velocity (kT) of XPHB formation was considered dependent from the initial concentration of nitrate. The proposed model includes the nitrate and phosphate utilization rate, the PHB production rate, the cell growth and lysis rate. The model adequately predicted the experimental data, indeed the square correlation coefficient of metabolite concentrations, calculated by comparing experiments and simulations, ranged between 0.81 and 0.99. 3)Genetic modified cyanobacteria strain to improve PHB production. In this study Synechocystis sp. PCC6803 was genetically modified with aim to improve PHB production from CO2. Seven mutant strains were created by single and combined mutations: deletion of phosphotransacetylase (pta); the deletion of acetyl-CoA hydrolase (ach); the overexpression of phosphoketolase (xfpk). The tests were carried out on BG11 medium. The mutant strain that gave best performance on BG11 medium was tested on optimized medium (BG1/2 medium).

BackgroundDue to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production.ResultsWe constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJΔphaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJΔphaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJΔphaZ (phaA3), HJΔphaZ (phaB2), and HJΔphaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔphaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔphaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88 ± 0.08 g l−1 and with 100 mM ammonium sulfate; 0.99 ± 0.05 g l−1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively.ConclusionIn this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.