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Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin.

ABSTRACT: Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in ?-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain - which contains all the genetic modifications detailed above - demonstrated a 53% and 200% increase in mcl-PHA titre (g l^-1 ) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compounds and lignin.

SUBMITTER: Salvachua D

PROVIDER: S-EPMC6922519 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin.

Salvachúa Davinia D Rydzak Thomas T Auwae Raquel R De Capite Annette A Black Brenna A BA Bouvier Jason T JT Cleveland Nicholas S NS Elmore Joshua R JR Huenemann Jay D JD Katahira Rui R Michener William E WE Peterson Darren J DJ Rohrer Holly H Vardon Derek R DR Beckham Gregg T GT Guss Adam M AM

Microbial biotechnology 20190830 1

Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of meta ...[more]

PMID: 31468725

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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.

Project description:Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Project description:Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process. Here, we developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, the genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy- and/or carbon-consuming traits were removed: mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50%. Variation of time of induction resulted in an 18% increase in titers. A scale-up from shake flasks was carried out using a 1-L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or antifoaming agents. Subsequent liquid-liquid extraction was optimized by using a suitable minimal medium during fermentation to reduce undesired interphase formation. A technical-scale production process was designed and evaluated by a life-cycle assessment (LCA). Different process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The present work underlines the need for multidisciplinary approaches to address the challenges associated with achieving sustainable production of microbial secondary metabolites. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale.

Dataset Information

Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin.

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Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin.

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