Chitin is the most abundant renewable nitrogenous material on earth and is accessible to humans in the form of crustacean shell waste. Such waste has been severely underutilized, resulting in both resource wastage and disposal issues. Upcycling chitin-containing waste into value-added products is an attractive solution. However, the direct conversion of crustacean shell waste-derived chitin into a wide spectrum of nitrogen-containing chemicals (NCCs) is challenging via conventional catalytic processes. To address this challenge, in this study, we developed an integrated biorefinery process to upgrade shell waste-derived chitin into two aromatic NCCs that currently cannot be synthesized from chitin via any chemical process (tyrosine andl-DOPA). The process involves a pretreatment of chitin-containing shell waste and an enzymatic/fermentative bioprocess using metabolically engineeredEscherichia coli. The pretreatment step achieved an almost 100% recovery and partial depolymerization of chitin from shrimp shell waste (SSW), thereby offering water-soluble chitin hydrolysates for the downstream microbial process under mild conditions. The engineeredE. colistrains produced 0.91 g/L tyrosine or 0.41 g/Ll-DOPA from 22.5 g/L unpurified SSW-derived chitin hydrolysates, demonstrating the feasibility of upcycling renewable chitin-containing waste into value-added NCCs via this integrated biorefinery, which bypassed the Haber–Bosch process in providing a nitrogen source.

Here we report GT (Guanin/Thymine) standard (GTS) for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We develop a technology that can efficiently add any two barcodes to two ends of any fragment without leaving scars in most cases. We can assemble up to seven such barcoded fragments into one plasmid by using one of the existing DNA assembly methods, including CLIVA, Gibson assembly, In-fusion cloning, and restriction enzyme-based methods. Plasmids constructed under GTS can be easily edited, and/or be further assembled into more complex plasmids by using standard DNA oligonucleotides (oligos). Based on 436 plasmids we constructed under GTS, the averaged accuracy of the workflow was 85.9%. GTS can also construct a library of plasmids from a set of fragments and barcodes combinatorically, which has been demonstrated to be useful for optimizing metabolic pathways.

No abstract

No abstract

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a twostep heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA.