Functional roles of multiple Ton complex genes in a Sphingobium degrader of lignin-derived aromatic compounds

TonB-dependent transporters (TBDTs) mediate outer membrane transport of nutrients using the energy derived from proton motive force transmitted from the TonB–ExbB–ExbD complex localized in the inner membrane. Recently, we discovered ddvT encoding a TBDT responsible for the uptake of a 5,5-type lignin-derived dimer in Sphingobium sp. strain SYK-6. Furthermore, overexpression of ddvT in an SYK-6-derivative strain enhanced its uptake capacity, improving the rate of platform chemical production. Thus, understanding the uptake system of lignin-derived aromatics is fundamental for microbial conversion-based lignin valorization. Here we examined whether multiple tonB-, exbB-, and exbD-like genes in SYK-6 contribute to the outer membrane transport of lignin-derived aromatics. The disruption of tonB2–6 and exbB3 did not reduce the capacity of SYK-6 to convert or grow on lignin-derived aromatics. In contrast, the introduction of the tonB1–exbB1–exbD1–exbD2 operon genes into SYK-6, which could not be disrupted, promoted the conversion of β-O-4-, β-5-, β-1-, β-β-, and 5,5-type dimers and monomers, such as ferulate, vanillate, syringate, and protocatechuate. These results suggest that TonB-dependent uptake involving the tonB1 operon genes is responsible for the outer membrane transport of the above aromatics. Additionally, exbB2/tolQ and exbD3/tolR were suggested to constitute the Tol-Pal system that maintains the outer membrane integrity.

Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains evolved from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. Mutated DNA, added directly to growing cells, replaces homologous chromosomal regions to generate transformants that will become enriched if there is selective benefit. Thus, advantageous mutations can be rapidly identified. Here, the growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE , hcaK , and vanK , and a gene, ACIAD0482 , which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies. Importance Microbial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Although adaptive laboratory evolution (ALE) is a powerful approach to develop more tolerant strains, it is typically laborious to identify the mechanisms underlying phenotypic improvement. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of this strain can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach for rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1-derived strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

Technoeconomic Evaluation of Recent Process Improvements in Production of Sugar and High-Value Lignin Co-Products via Two-Stage Cu-Catalyzed Alkaline-Oxidative Pretreatment

Background A lignocellulose-to-biofuel biorefinery process that enables multiple product streams is recognized as a promising strategy to improve the economics of this biorefinery and to accelerate technology commercialization. We recently identified an innovative pretreatment technology that enables of the production of sugars at high yields while simultaneously generating a high-quality lignin stream that has been demonstrated as both a promising renewable polyol replacement for polyurethane applications and is highly susceptible to depolymerization into monomers. This technology comprises a two-stage pretreatment approach that includes an alkaline pre-extraction followed by a metal-catalyzed alkaline-oxidative pretreatment. Our recent work demonstrated that H2O2 and O2 act synergistically as co-oxidants during the alkaline-oxidative pretreatment and could significantly reduce the pretreatment chemical input while maintaining high sugar yields, high lignin yields, and improvements in lignin usage. Results This study considers the economic impact of these advances and provides strategies that could lead to additional economic improvements for future commercialization. The results of the technoeconomic analysis (TEA) demonstrated that adding O2 as a co-oxidant at 50 psig for the alkaline-oxidative pretreatment and reducing the raw material input reduced the minimum fuel selling price from $1.08/L to $0.85/L, assuming recoverable lignin is used as a polyol replacement. If additional lignin can be recovered and sold as more valuable monomers, the minimum fuel selling price (MFSP) can be further reduced to $0.73/L. Conclusions The present work demonstrated that high sugar and lignin yields combined with low raw material inputs and increasing the value of lignin could greatly increase the economic viability of a poplar-based biorefinery. Continued research on integrating sugar production with lignin valorization is thus warranted to confirm this economic potential as the technology matures.