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Production and Characterization of Polyhydroxyalkanoate from Lignin Derivatives by Pandoraea sp. ISTKB.

ABSTRACT: The present study investigates polyhydroxyalkanoate (PHA) production from lignin and its derivatives by a previously reported lignin-degrading bacterial strain Pandoraea sp. ISTKB. PHA production was screened by fluorescence microscopy and flow cytometry using a Nile red stain. PHA and biomass accumulation, while screening, was found to be maximum on 4-hydroxybenzoic acid followed by p-coumaric acid, vanillic acid, 2,6-dimethoxyphenol, and kraft lignin after 96 h. Monomer composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and was followed by Fourier transform infrared and 1H NMR analysis, indicating PHA to be a copolymer of P(hydroxybutyrate-co-hydroxyvalerate). Genomic analysis of Pandoraea sp. ISTKB also complemented the results of GC-MS and NMR, and the relevant genes responsible for the synthesis of small chain length PHA were discovered in the genome. Process parameters were optimized by response surface methodology for enhanced production of PHA and biomass on 4-hydroxybenzoate. Optimization results showed 30 and 66% increase in the biomass and PHA production, respectively. The results obtained were promising and indicated that if lignin is depolymerized into low-molecular-weight intermediates, then it can easily be utilized and converted into value-added products like PHA by microbes.

SUBMITTER: Kumar M

PROVIDER: S-EPMC6045365 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Production and Characterization of Polyhydroxyalkanoate from Lignin Derivatives by Pandoraea sp. ISTKB.

Kumar Madan M Singhal Anjali A Verma Praveen Kumar PK Thakur Indu Shekhar IS

ACS omega 20171221 12

The present study investigates polyhydroxyalkanoate (PHA) production from lignin and its derivatives by a previously reported lignin-degrading bacterial strain Pandoraea sp. ISTKB. PHA production was screened by fluorescence microscopy and flow cytometry using a Nile red stain. PHA and biomass accumulation, while screening, was found to be maximum on 4-hydroxybenzoic acid followed by p-coumaric acid, vanillic acid, 2,6-dimethoxyphenol, and kraft lignin after 96 h. Monomer compositi ...[more]

PMID: 30023602

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Project description:BackgroundLignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysaccharide-based biorefinery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to find the important enzymes for polymer utilization.ResultsGenomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profile on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase-peroxidase for lignin degradation. A strong stress response and detoxification mechanism was discovered. The two important gene clusters for lignin degradation and three PHA polymerase spanning gene clusters were identified and all the clusters were functionally active on KL-VA.ConclusionsThe unusual aerobic '-CoA'-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4-5% of total sequenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chemistry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profile. Genomic and proteomic approaches provided validation to this strain's robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future.

Project description:BackgroundEfficient 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.ResultsCo-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 ?-oxidation, and PHA biosynthesis.ConclusionsThis 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.

Project description:Cell growth and polyhydroxyalkanoate (PHA) biosynthesis are two key traits in PHA production from lignin or its derivatives. However, the links between them remain poorly understood. Here, the transcription levels of key genes involved in PHA biosynthesis were tracked in Pseudomonas putida strain A514 grown on vanillic acid as the sole carbon source under different levels of nutrient availability. First, enoyl-coenzyme A (CoA) hydratase (encoded by phaJ4) is stress induced and likely to contribute to PHA synthesis under nitrogen starvation conditions. Second, much higher expression levels of 3-hydroxyacyl-acyl carrier protein (ACP) thioesterase (encoded by phaG) and long-chain fatty acid-CoA ligase (encoded by alkK) under both high and low nitrogen (N) led to the hypothesis that they likely not only have a role in PHA biosynthesis but are also essential to cell growth. Third, 40 mg/liter PHA was synthesized by strain AphaJ4C1 (overexpression of phaJ4 and phaC1 in strain A514) under low-N conditions, in contrast to 23 mg/liter PHA synthesized under high-N conditions. Under high-N conditions, strain AalkKphaGC1 (overexpression of phaG, alkK, and phaC1 in A514) produced 90 mg/liter PHA with a cell dry weight of 667 mg/liter, experimentally validating our hypothesis. Finally, further enhancement in cell growth (714 mg/liter) and PHA titer (246 mg/liter) was achieved in strain Axyl_alkKphaGC1 via transcription level optimization, which was regulated by an inducible strong promoter with its regulator, XylR-PxylA, from the xylose catabolic gene cluster of the A514 genome. This study reveals genetic features of genes involved in PHA synthesis from a lignin derivative and provides a novel strategy for rational engineering of these two traits, laying the foundation for lignin-consolidated bioprocessing.IMPORTANCE With the recent advances in processing carbohydrates in lignocellulosics for bioproducts, almost all biological conversion platforms result in the formation of a significant amount of lignin by-products, representing the second most abundant feedstock on earth. However, this resource is greatly underutilized due to its heterogeneity and recalcitrant chemical structure. Thus, exploiting lignin valorization routes would achieve the complete utilization of lignocellulosic biomass and improve cost-effectiveness. The culture conditions that encourage cell growth and polyhydroxyalkanoate (PHA) accumulation are different. Such an inconsistency represents a major hurdle in lignin-to-PHA bioconversion. In this study, we traced and compared transcription levels of key genes involved in PHA biosynthesis pathways in Pseudomonas putida A514 under different nitrogen concentrations to unveil the unusual features of PHA synthesis. Furthermore, an inducible strong promoter was identified. Thus, the molecular features and new genetic tools reveal a strategy to coenhance PHA production and cell growth from a lignin derivative.

Dataset Information

Production and Characterization of Polyhydroxyalkanoate from Lignin Derivatives by Pandoraea sp. ISTKB.

Publications

Production and Characterization of Polyhydroxyalkanoate from Lignin Derivatives by <i>Pandoraea</i> sp. ISTKB.

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