Biology subjects

Mrksich, M.

Publications and source records attributed to Mrksich, M..

3 recordsLinked to original sources

bioRxiv2022

Cell-free prototyping enables implementation of optimized reverse beta-oxidation pathways in heterotrophic and autotrophic bacteria

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse {beta}-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimized and implemented r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screened 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generated designer strains for the selective production of butanoic acid (4.9 {+/-}0.1 gL-1), hexanoic acid (3.06 {+/-} 0.03 gL-1) and 1-hexanol (1.0 {+/-} 0.1 gL-1) at the best performance reported to date in this bacterium. We also generated Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5-L continuous fermentation. Our strategy enables optimization of rBOX derived products for biomanufacturing and industrial biotechnology.

synthetic biology↗

bioRxiv2019

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer protein glycosylation remains a bottleneck. To address this limitation, we describe a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude cell lysates are enriched with glycosyltransferases by cell-free protein synthesis and then glycosylation pathways are assembled in a mix-and-match fashion to elaborate a single glucose priming handle installed by an N-linked glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs. We then use selected pathways to design a one-pot cell-free system to synthesize a vaccine protein with an -galactose motif and engineered Escherichia coli strains to produce human antibody constant regions with minimal sialic acid motifs. We anticipate that our work will facilitate glycoscience and make possible new glycoengineering applications.

synthetic biology↗

bioRxiv2019

Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-function modification on cells where the products of individual glycosyltransferases can be selectively characterized or manipulated at will.

synthetic biology↗