Biomanufacturing of value-added chemicals from lignin.

Lignin valorization faces persistent biomanufacturing challenges due to the heterogeneous and toxic carbon substrates derived from lignin depolymerization. To address the heterogeneous nature of aromatic feedstocks, plant cell wall engineering and 'lignin first' pretreatment methods have recently emerged. Next, to convert the resulting aromatic substrates into value-added chemicals, diverse microbial host systems also continue to be developed. This includes microbes that (1) lack aromatic metabolism, (2) metabolize aromatics but not sugars, and (3) co-metabolize both aromatics and sugars, each system presenting unique pros and cons. Considering the intrinsic complexity of lignin-derived substrate mixtures, emerging and non-model microbes with native metabolism for aromatics appear poised to provide the greatest impacts on lignin valorization via biomanufacturing.

Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

NgAgo possesses guided DNA nicking activity.

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.

High-throughput screening for efficient microbial biotechnology.

Microbial systems have been widely studied and exploited through genetic engineering to address industrial needs and societal challenges. However, owing to their complexity, singular approaches often do not yield desired or optimal results, pushing researchers to explore combinatorial strategies. With advances in synthetic biology, various methods can readily be employed to generate large and comprehensive libraries. To serve as tractable tools, however, this capability necessitates the development of high-throughput screening (HTS) techniques to identify the best performing strain and/or those carrying the desired trait. Owing to their miniaturization, time efficiency, potential for automation, and so on, HTS enables comprehensive exploration of diverse experimental landscapes. Herein, we review the recent and novel HTS approaches and applications in the realm of microbial biotechnology.