Biology subjects

Dudley, Q. M.

Publications and source records attributed to Dudley, Q. M..

6 recordsLinked to original sources

bioRxiv2023

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report the assembly of an improved N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated 3-4 times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and opens the possibility that BBLs are the sought-after coupling enzymes in nicotine biosynthesis.

plant biology↗

bioRxiv2022

A mini-TGA protein, lacking a functional DNA-binding domain, modulates gene expression through heterogeneous association with transcription factors

TGA transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigate the role of StTGA2.1, a potato TGA lacking the full bZIP, which we name a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirm that StTGA2.1 overexpression compensates for SA-deficiency. To understand the underlying mechanisms, we show that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigate the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirm that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases, which are known to play role in immune response. Finally, via structural modelling and molecular dynamics simulations, we hypothesise that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to novel functions and that such events should be studied carefully in other protein families.

plant biology↗

bioRxiv2021

Reconstitution of monoterpene indole alkaloid biosynthesis in Nicotiana benthamiana

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally significant compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. We used transcriptomic analysis to identify glycosyltransferases that were upregulated in response to biosynthetic intermediates and produced plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produced a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases did not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

synthetic biology↗

bioRxiv2021

Biofoundry-assisted expression and characterisation of plant proteins

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterisation has remained a technical bottleneck, often requiring significant effort to optimise expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimisation and enables rapid progression to characterisation. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using E. coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification, and improved expression/folding. We next optimised automated assembly of miniaturised cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11 aa tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional characterisation experiments can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

synthetic biology↗

bioRxiv2020

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/057737v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19aa729org.highwire.dtl.DTLVardef@547698org.highwire.dtl.DTLVardef@13604b6org.highwire.dtl.DTLVardef@16f6718_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIApplied the iPROBE framework to build the nine-enzyme pathway to produce limonene C_LIO_LIAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance C_LIO_LIIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions C_LIO_LIExtended pathway to biofuel precursors pinene and bisabolene C_LI

synthetic biology↗

bioRxiv2019

In vitro prototyping and rapid optimization of biosynthetic enzymes for cellular design

Microbial cell factories offer an attractive approach for production of biobased products. Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform for in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improving in vivo production in Clostridium by 20-fold to 14.63 {+/-} 0.48 g/L and identifying a new biosynthetic route to (S)-(+)-1,3-butanediol. Second, we used iPROBE and data-driven design to optimize a 6-step n-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

synthetic biology↗