Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties

Polyethylene terephthalate (PET), the most common synthetic polyester today, is largely produced from fossil resources, contributing to global warming. Consequently, sustainable sources must be developed to meet the increasing demand for this useful polymer. Here, we demonstrate a cascaded value chain that provides green PET from lignin, the world's most underutilized renewable, via fermentative production of cis, cis-muconate (MA) from lignin-based aromatics as a central step. Catechol, industrially the most relevant but apparently also a highly toxic lignin-related aromatic, strongly inhibited MA-producing Pseudomonas putida MA-1. Assessed by 13C metabolic flux analysis, the microbe substantially redirected its carbon core fluxes, resulting in enhanced NADPH supply for stress defense but causing additional ATP costs. The reconstruction of MA production in a genome-reduced P. putida chassis yielded novel producers with superior pathway fluxes and enhanced robustness to catechol and a wide range of other aromatics. Using the advanced producer P. putida MA-10 catechol, MA could be produced in a fed-batch process from catechol (plus glucose as additional growth substrate) up to an attractive titer of 74 g L-1 and a space-time-yield of 1.4 g L-1 h-1. In terms of co-consumed sugar, the further streamlined strain MA-11 achieved the highest yield of 1.4 mol MA (mol glucose)-1, providing a striking economic advantage. Following fermentative production, bio-based MA was purified and used to chemically synthetize the PET monomer terephthalic acid and the comonomer diethylene glycol terephthalic acid through five steps, which finally enabled the first green PET from lignin

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2 -eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 10 ⁶-10 ⁸gsoilDW ⁻¹. Methanol dissimilation was measured based on conversion of supplemented 14C-methanol, and occurred at concentrations down to 0.002 μmol methanol gsoilDW ⁻¹. Tested soils exhibited specific affinities to methanol (a ⁰ s=0 ⁻¹) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (≤0.6 μmol methanol gsoilDW ⁻¹) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant-methylotroph interactions were determinative for soil methylotrophs