Messina, E. | Search bioRxiv

bioRxiv2026

Synthetic biology-guided modular engineering of the secreted cutinase FoCut5a with a carbohydrate-binding module improves poly(ester-urethane) depolymerization in Yarrowia lipolytica

Poly(ester-urethane) bioconversion requires efficient polymer-degrading enzymes and microbial hosts capable of processing the resulting monomers. However, the optimal modular design of secreted cutinases for depolymerizing insoluble polyester-polyurethanes remains largely unexplored. Here, we established a combinatorial synthetic biology strategy in Yarrowia lipolytica based on the fungal cutinase FoCut5a from Fusarium oxysporum. A library of 96 Golden Gate-assembled constructs, combining four secretion signals, four interdomain linkers, and six substrate-binding domains, was integrated site-specifically into the genome and screened for depolymerization of the polyester-polyurethane dispersion Impranil DLN-SD. The best-performing variant fused FoCut5a to the carbohydrate-binding module TrCBM1. LC-MS analysis demonstrated release of adipic acid, 1,6-hexanediol, and neopentyl glycol, confirming ester-bond hydrolysis within the polyester domains of the polymer and additionally detected 6-hydroxycaproate, indicating oxidation of polyurethane-derived 1,6-hexanediol by Y. lipolytica. Purified FoCut5a-TrCBM1 displayed unchanged activity on p-nitrophenyl butyrate but reduced Impranil turbidity 5.6-fold faster and increased release of 1,6-hexanediol and adipic acid by 76-fold and 220-fold, respectively, compared to the unfused enzyme. These results highlight modular enzyme engineering as an effective strategy to enhance polyester-polyurethane deconstruction and support Y. lipolytica as a platform for polymer bioconversion and upcycling.

synthetic biology↗

bioRxiv2019

Differential polysaccharide utilization is the basis for a nanohaloarchaeon : haloarchaeon symbiosis.

Nanohaloarchaeota, a clade of diminutive archaea, with small genomes and limited metabolic capabilities, are ubiquitous in hypersaline habitats, which they share with the extremely halophilic and phylogenetically distant euryarchaea. Some of these nanohaloarchaeota and euryarchaea appear to interact with each other. In this study, we investigate the genetic and physiological nature of their relationship. We isolated the nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh and the haloarchaeon Halomicrobium sp. LC1Hm from a solar saltern, reproducibly co-cultivated these species, sequenced their genomes, and characterized their metabolic/trophic interactions. The nanohaloarchaeon is a magnesium-dependent aerotolerant heterotrophic anaerobe of the DPANN superphylum; it lacks respiratory complexes and its energy production relies on fermentative metabolism of sugar derivatives, obtained by depolymerizing alpha-glucans or by acquiring the chitin monomer N-acetylglucosamine from the chitinolytic haloarchaeal host. Halomicrobium is a member of the class Halobacteria and a chitinotrophic aerobe. The nanohaloarchaeon lacks key biosynthetic pathways and is likely to be provided with amino acids, lipids, nucleotides and cofactors via physical contact with its host Halomicrobium. In turn, the ability of Ca. Nanohalobium to hydrolyse alpha-glucans boosts the hosts growth in the absence of a chitin substrate. These findings suggest that at least some members of the nanohaloarchaea, previously considered ecologically unimportant given their limited metabolic potential, in fact may play significant roles in the microbial carbon turnover, food chains, and ecosystem function. The behaviour of Halomicrobium, which accommodates the colonization by Ca. Nanohalobium, can be interpreted as a bet-hedging strategy, maximizing its long-term fitness in a habitat where the availability of carbon substrates can vary both spatially and temporarily.

microbiology↗