scholarly journals Enhancement of polyhydroxyalkanoate production by co-feeding lignin derivatives with glycerol in Pseudomonas putida KT2440

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Zhangyang Xu ◽  
Chunmei Pan ◽  
Xiaolu Li ◽  
Naijia Hao ◽  
Tong Zhang ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Stress Responses ◽  
Vanillic Acid ◽  
Feeding Strategy ◽  
Carbon Sources ◽  
Reducing Power ◽  
Value Added ◽  
Carbon Substrate ◽  
Pha Production ◽  
First Time

Abstract Background Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy—co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440—for the first time, which simultaneously improved both cell biomass and PHA production. Results Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4–16.1% and PHA content by 29.0–63.2%, respectively, compared with feeding glycerol alone. GC–MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4–4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8–3.5%. The 1H–13C HMBC, 1H–13C HSQC, and 1H–1H COSY NMR analysis confirmed that the PHA monomers (C6–C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD+/NADH and NADPH/NADP+ ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner–Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis. Conclusions This work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

scholarly journals Enhancement of Polyhydroxyalkanoate Production by Co-feeding Lignin Derivatives With Glycerol in Pseudomonas Putida KT2440

2020 ◽  
Author(s):  
Zhangyang Xu ◽  
Chunmei Pan ◽  
Xiaolu Li ◽  
Naijia Hao ◽  
Tong Zhang ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Stress Responses ◽  
Vanillic Acid ◽  
Feeding Strategy ◽  
Carbon Sources ◽  
Reducing Power ◽  
Value Added ◽  
Carbon Substrate ◽  
Pha Production ◽  
First Time

Abstract Background: Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and Polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy - co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440 - for the first time, which simultaneously improved both cell biomass and PHA production. Results: Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4 %-16.1 % and PHA content by 29.0 %-63.2 %, respectively, compared with feeding glycerol alone. GC-MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long chain monomers (C10 and C12) by 0.4 %-4.4 % and increased the distribution of short chain monomers (C6 and C8) by 0.8 %-3.5 %. The 1H -13C HMBC, 1H-13C HSQC, and 1H-1H COSY NMR analysis confirmed that the PHA monomers (C6-C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses enhanced glycerol consumption and altered the intracellular NAD(P)+/NAD(P)H ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner-Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty-acid β-oxidation, and PHA biosynthesis.Conclusions: This work demonstrated an effective co-carbon feeding strategy to improve PHA yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin derivativeswith other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favors further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

scholarly journals Mevalonate production from ethanol by direct conversion through acetyl-CoA using recombinant Pseudomonas putida, a novel biocatalyst for terpenoid production

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Jeongmo Yang ◽  
Ji Hee Son ◽  
Hyeonsoo Kim ◽  
Sukhyeong Cho ◽  
Jeong-geol Na ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Batch Fermentation ◽  
Fossil Fuels ◽  
Fatty Acid Biosynthesis ◽  
Carbon Sources ◽  
Value Added ◽  
Product Yield ◽  
Cellulosic Biomass ◽  
Ethanol Metabolism ◽  
Acetyl Coa

Abstract Background Bioethanol is one of the most representative eco-friendly fuels developed to replace the non-renewable fossil fuels and is the most successful commercially available bio-conversion technology till date. With the availability of inexpensive carbon sources, such as cellulosic biomass, bioethanol production has become cheaper and easier to perform, which can facilitate the development of methods for converting ethanol into higher value-added biochemicals. In this study, a bioconversion process using Pseudomonas putida as a biocatalyst was established, wherein ethanol was converted to mevalonate. Since ethanol can be converted directly to acetyl-CoA, bypassing its conversion to pyruvate, there is a possibility that ethanol can be converted to mevalonate without producing pyruvate-derived by-products. Furthermore, P. putida seems to be highly resistant to the toxicity caused by terpenoids, and thus can be useful in conducting terpenoid production research. Results In this study, we first expressed the core genes responsible for mevalonate production (atoB, mvaS, and mvaE) in P. putida and mevalonate production was confirmed. Thereafter, through an improvement in genetic stability and ethanol metabolism manipulation, mevalonate production was enhanced up to 2.39-fold (1.70 g/L vs. 4.07 g/L) from 200 mM ethanol with an enhancement in reproducibility of mevalonate production. Following this, the metabolic characteristics related to ethanol catabolism and mevalonate production were revealed by manipulations to reduce fatty acid biosynthesis and optimize pH by batch fermentation. Finally, we reached a product yield of 0.41 g mevalonate/g ethanol in flask scale culture and 0.32 g mevalonate/g ethanol in batch fermentation. This is the highest experimental yield obtained from using carbon sources other than carbohydrates till date and it is expected that further improvements will be made through the development of fermentation methods. Conclusion Pseudomonas putida was investigated as a biocatalyst that can efficiently convert ethanol to mevalonate, the major precursor for terpenoid production, and this research is expected to open new avenues for the production of terpenoids using microorganisms that have not yet reached the stage of mass production.

scholarly journals What Is New in the Field of Industrial Wastes Conversion into Polyhydroxyalkanoates by Bacteria?

Polymers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1731
Author(s):  
Paulina Marciniak ◽  
Justyna Możejko-Ciesielska
Keyword(s):  
Food Industry ◽  
Scale Up ◽  
Carbon Sources ◽  
Food Service ◽  
Value Added ◽  
Industrial Wastes ◽  
Recent Developments ◽  
Tremendous Amount ◽  
Pha Production ◽  
The Cost

The rising global consumption and industrialization has resulted in increased food processing demand. Food industry generates a tremendous amount of waste which causes serious environmental issues. These problems have forced us to create strategies that will help to reduce the volume of waste and the contamination to the environment. Waste from food industries has great potential as substrates for value-added bioproducts. Among them, polyhydroxyalkanaotes (PHAs) have received considerable attention in recent years due to their comparable characteristics to common plastics. These biodegradable polyesters are produced by microorganisms during fermentation processes utilizing various carbon sources. Scale-up of PHA production is limited due to the cost of the carbon source metabolized by the microorganisms. Therefore, there is a growing need for the development of novel microbial processes using inexpensive carbon sources. Such substrates could be waste generated by the food industry and food service. The use of industrial waste streams for PHAs biosynthesis could transform PHA production into cheaper and more environmentally friendly bioprocess. This review collates in detail recent developments in the biosynthesis of various types of PHAs produced using waste derived from agrofood industries. Challenges associated with this production bioprocess were described, and new ways to overcome them were proposed.

scholarly journals Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Duoduo Hu ◽  
Hao Wu ◽  
Yingying Zhu ◽  
Wenli Zhang ◽  
Wanmeng Mu
Keyword(s):  
Feeding Strategy ◽  
Carbon Sources ◽  
Value Added ◽  
Lactose Hydrolysis ◽  
Cell Factory ◽  
Efficient Production ◽  
E Coli ◽  
Maximum Titer ◽  
Related Pathway ◽  
Value Added Products

Abstract Background Lacto-N-triose II (LNT II), an important backbone for the synthesis of different human milk oligosaccharides, such as lacto-N-neotetraose and lacto-N-tetraose, has recently received significant attention. The production of LNT II from renewable carbon sources has attracted worldwide attention from the perspective of sustainable development and green environmental protection. Results In this study, we first constructed an engineered E. coli cell factory for producing LNT II from N-acetylglucosamine (GlcNAc) feedstock, a monomer of chitin, by introducing heterologous β-1,3-acetylglucosaminyltransferase, resulting in a LNT II titer of 0.12 g L−1. Then, lacZ (lactose hydrolysis) and nanE (GlcNAc-6-P epimerization to ManNAc-6-P) were inactivated to further strengthen the synthesis of LNT II, and the titer of LNT II was increased to 0.41 g L−1. To increase the supply of UDP-GlcNAc, a precursor of LNT II, related pathway enzymes including GlcNAc-6-P deacetylase, glucosamine synthase, and UDP-N-acetylglucosamine pyrophosphorylase, were overexpressed in combination, optimized, and modulated. Finally, a maximum titer of 15.8 g L−1 of LNT II was obtained in a 3-L bioreactor with optimal enzyme expression levels and β-lactose and GlcNAc feeding strategy. Conclusions Metabolic engineering of E. coli is an effective strategy for LNT II production from GlcNAc feedstock. The titer of LNT II could be significantly increased by modulating the gene expression strength and blocking the bypass pathway, providing a new utilization for GlcNAc to produce high value-added products.

scholarly journals Biosynthesis of Polyhydroxyalkanoates (PHAs) by the Valorization of Biomass and Synthetic Waste

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5539
Author(s):  
Hadiqa Javaid ◽  
Ali Nawaz ◽  
Naveeda Riaz ◽  
Hamid Mukhtar ◽  
Ikram -Ul-Haq ◽  
...  
Keyword(s):  
Uv Light ◽  
Nile Blue ◽  
Carbon Sources ◽  
Culture Broth ◽  
Plastic Bags ◽  
Pha Production ◽  
Fermentation Parameters ◽  
Effective Use ◽  
First Time ◽  
Microbial Strain

Synthetic pollutants are a looming threat to the entire ecosystem, including wildlife, the environment, and human health. Polyhydroxyalkanoates (PHAs) are natural biodegradable microbial polymers with a promising potential to replace synthetic plastics. This research is focused on devising a sustainable approach to produce PHAs by a new microbial strain using untreated synthetic plastics and lignocellulosic biomass. For experiments, 47 soil samples and 18 effluent samples were collected from various areas of Punjab, Pakistan. The samples were primarily screened for PHA detection on agar medium containing Nile blue A stain. The PHA positive bacterial isolates showed prominent orange–yellow fluorescence on irradiation with UV light. They were further screened for PHA estimation by submerged fermentation in the culture broth. Bacterial isolate 16a produced maximum PHA and was identified by 16S rRNA sequencing. It was identified as Stenotrophomonas maltophilia HA-16 (MN240936), reported first time for PHA production. Basic fermentation parameters, such as incubation time, temperature, and pH were optimized for PHA production. Wood chips, cardboard cutouts, plastic bottle cutouts, shredded polystyrene cups, and plastic bags were optimized as alternative sustainable carbon sources for the production of PHAs. A vital finding of this study was the yield obtained by using plastic bags, i.e., 68.24 ± 0.27%. The effective use of plastic and lignocellulosic waste in the cultivation medium for the microbial production of PHA by a novel bacterial strain is discussed in the current study.

scholarly journals Functional analysis of CfSnf1 in the development and pathogenicity of anthracnose fungus Colletotrichum fructicola on tea-oil tree

BMC Genetics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Shengpei Zhang ◽  
Yuan Guo ◽  
Sizheng Li ◽  
Guoying Zhou ◽  
Junang Liu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Protein A ◽  
Carbon Sources ◽  
Critical Factor ◽  
Economic Losses ◽  
Colletotrichum Fructicola ◽  
Amp Activated Protein Kinase ◽  
First Time ◽  
Fungicide Target ◽  
Kinase Pathway

Abstract Background Tea-oil tree (Camellia oleifera) is a unique edible-oil tree in China, and anthracnose occurs in wherever it is cultivated, causing great economic losses each year. We have previously identified the Ascomycete fungus Colletotrichum fructicola as the major pathogen of anthracnose in Ca.oleifera. The purpose of this study was to characterize the biological function of Snf1 protein, a key component of the AMPK (AMP-activated protein kinase) pathway, for the molecular pathogenic-mechanisms of C. fructicola. Results We characterized CfSnf1 as the homolog of Saccharomyces cerevisiae Snf1. Targeted CfSNF1 gene deletion revealed that CfSnf1 is involved in the utilization of specific carbon sources, conidiation, and stress responses. We further found that the ΔCfSnf1 mutant was not pathogenic to Ca.oleifera, resulting from its defect in appressorium formation. In addition, we provided evidence showing crosstalk between the AMPK and the cAMP/PKA pathways for the first time in filamentous fungi. Conclusion This study indicate that CfSnf1 is a critical factor in the development and pathogenicity of C. fructicola and, therefore, a potential fungicide target for anthracnose control.

Oxidation-reduction processes in cultures of bacteria - I. The reducing power of Bact. lactis aerogenes

1950 ◽  
Vol 137 (889) ◽  
pp. 524-534 ◽  
Keyword(s):  
Growth Rate ◽  
Carbon Sources ◽  
Reducing Power ◽  
Optimal Growth ◽  
Carbon Substrate ◽  
Rate Limiting Step ◽  
Oxidation Reduction ◽  
Methylene Blue Test ◽  
High Concentrations ◽  
Stable Characteristic

Cultures of Bact. lactis aerogenes show a reducing power towards oxygen (as measured by a modification of the Thunberg methylene-blue test) which is a rather stable characteristic of the growth under specified conditions. During adaptation of cells to utilize a given carbon substrate the reducing power and the growth rate increase in parallel (over considerable ranges) to their respective optima. In general the reducing power of fully adapted cultures is nearly constant for a considerable variety of carbon sources (12 to 15 units) in spite of a nearly fourfold variation of optimal growth rates. The regularity is masked with certain substrates by abnormal enhancements or inhibitions occurring at high concentrations but reappears when the measurements are made in the low concentration range. The result suggests that there is normally a rate-limiting step in the part of the cell mechanism which consumes molecular oxygen. This is not altered by adaptation to a new carbon source, though when the cells are first transferred to a new medium the proportions of various enzymes are unsuitable for the optimum utilization of the substrate, and the amount of oxygen which can be consumed is far below the maximum. Until these proportions are finally readjusted growth rate and the degree to which the oxidizing mechanism can be used increase in parallel, a limit to the adaptive process being set by the relatively unchanging maximum oxygen uptake.

Oxidation-reduction processes in cultures of bacteria I. The reducing power of Bact. lactis aerogenes

1950 ◽  
Vol 204 (1077) ◽  
pp. 295-295
Keyword(s):  
Carbon Sources ◽  
Reducing Power ◽  
Optimal Growth ◽  
Carbon Substrate ◽  
Rate Limiting Step ◽  
Oxidation Reduction ◽  
Methylene Blue Test ◽  
High Concentrations ◽  
Rate Limiting ◽  
Made In

Cultures of Bact. lactis is aerogenes show a reducing power towards oxygen (as measured by a modification of the Thunberg methylene-blue test) which is a rather stable characteristicof the growth under specified conditions. During adaptation of cells to utilize a given carbon substrate the reducing power and the grow the rate increase in parallel (over considerable ranges) to their respective optima. In general the reducing pow er of fully adapted cultures is nearly constant for a considerable variety of carbon sources (12 to 15 units) in spite of a nearly fourfold variation of optimal growth rates. This regularity is masked with certain substrates by abnormal enhancements or inhibitions occurring at high concentrations but reappears when the measurements are made in the low concentration range. The result suggests that there is normally a rate-limiting step in the part of the cell mechanism which consumes molecular oxygen. This is not altered by adaptation to a new carbon source, though when the cells are first transferred to a new medium the proportions of various enzymes are unsuitable for the optimum utilization of the substrate, and the amount of oxygen which can be consumed is far below the maximum. Until these proportions are finally readjusted growth rate and the degree to which the oxidizing mechanism can be used increase in parallel, a limit to the adaptive process being set by the relatively unchanging maximum oxygen uptake.

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