Affiliation:
1. School of Environmental Science and Engineering, Shandong University, Qingdao, China
2. Sino-French Research Institute for Ecology and Environment, Shandong University, Qingdao, China
3. Weihai Research Institute of Industrial Technology, Shandong University, Weihai, China
Abstract
ABSTRACT
Biological valorization of lignin, the second most abundant biopolymer on Earth, is an indispensable sector to build a circular economy and net-zero future. However, lignin is recalcitrant to bioupcycling, demanding innovative solutions. We report here the biological valorization of lignin-derived aromatic carbon to value-added chemicals without requesting extra organic carbon and freshwater via reprogramming the marine
Roseobacter
clade bacterium
Roseovarius nubinhibens
. We discovered the unusual advantages of this strain for the oxidation of lignin monomers and implemented a CRISPR interference (CRISPRi) system with the
lacI
-P
trc
inducible module, nuclease-deactivated Cas9, and programmable gRNAs. This is the first CRISPR-based regulatory system in
R. nubinhibens
, enabling precise and efficient repression of genes of interest. By deploying the customized CRISPRi, we reprogrammed the carbon flux from a lignin monomer, 4-hydroxybenzoate, to achieve the maximum production of protocatechuate, a pharmaceutical compound with antibacterial, antioxidant, and anticancer properties, with minimal carbon to maintain cell growth and drive biocatalysis. As a result, we achieved a 4.89-fold increase in protocatechuate yield with a dual-targeting CRISPRi system, and the system was demonstrated with real seawater. Our work underscores the power of CRISPRi in exploiting novel microbial chassis and will accelerate the development of marine synthetic biology. Meanwhile, the introduction of a new-to-the-field lineage of marine bacteria unveils the potential of blue biotechnology leveraging resources from the ocean.
IMPORTANCE
One often overlooked sector in carbon-conservative biotechnology is the water resource that sustains these enabling technologies. Similar to the “food-versus-fuel” debate, the competition of freshwater between human demands and bioproduction is another controversial issue, especially under global water scarcity. Here, we bring a new-to-the-field lineage of marine bacteria with unusual advantages to the stage of engineering biology for simultaneous carbon and water conservation. We report the valorization of lignin monomers to pharmaceutical compounds without requesting extra organic substrate (e.g., glucose) or freshwater by reprogramming the marine bacterium
Roseovarius nubinhibens
with a multiplex CRISPR interference system. Beyond the blue lignin valorization, we present a proof-of-principle of leveraging marine bacteria and engineering biology for a sustainable future.
Funder
MOST | National Natural Science Foundation of China
Department of Science and Technology of Shandong Province
National Science Foundation of Shandong Province
Shandong University
Taishan Scholars Project of Shandong Province
Publisher
American Society for Microbiology
Cited by
1 articles.
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