Biology subjects

Cardiff, R.

Publications and source records attributed to Cardiff, R..

3 recordsLinked to original sources

bioRxiv2024

Carbon-conserving Bioproduction of Malate in an E. coli-based Cell-Free System

Formate, a biologically accessible form of CO2, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an E. coli-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that in situ regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 M malate after 8 hours. This system conserves 43% of carbon otherwise lost as CO2 and incorporates 0.13 mol CO2 equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals. HighlightsO_LISuccessfully engineered the carbon-conserving reductive TCA and formate assimilation pathways in a lysate-based cell-free system for production of the C4 industrial di-acid malate from C1 and C2 feedstocks. C_LIO_LIAchieved a 6-fold reduction in competition from the endogenous cell-free metabolism by blocking TCA activity using small-molecule inhibitors and lysate dilution. C_LIO_LIIncreased accumulation of malate by 15-fold in a single-step reaction using cell-free expression of an enzymatic cofactor regeneration system. C_LIO_LITechno-economic analysis identified routes for economically feasible production of malate from renewable feedstocks in a cell-free system by improving conversion efficiency and reducing lysate cost. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/623433v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@3c89adorg.highwire.dtl.DTLVardef@72d9b3org.highwire.dtl.DTLVardef@327e2eorg.highwire.dtl.DTLVardef@fa0964_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

Engineering bacterial metabolism to efficiently produce chemicals and materials from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important metabolic engineering tools for programming multi-gene expression regulation. However, poor predictability of guide RNA folding can disrupt enzyme balance through unreliable expression control. We devised a set of computational parameters that can describe guide RNA folding, and we expect them to be broadly applicable across CRISPR-Cas9 systems. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a kinetic parameter describing folding rate into the active structure. This parameter also enables forward design of new scRNAs, with no observed failures in our screen. We use CRISPRa target sequences from this set to design a system of three synthetic promoters that can orthogonally activate and tune expression of chosen outputs over a >35-fold dynamic range. Independent activation tuning allows experimental exploration of a three-dimensional expression design space via a 64-member combinatorial triple-scRNA library. We apply these CRISPRa programs to two biosynthetic pathways, demonstrating production of valuable pteridine and human milk oligosaccharide products in E. coli. Profiling these design spaces indicated expression combinations producing up to 2.3-fold higher titer than that produced by maximal expression. Mapping production can also identify bottlenecks as targets for pathway redesign, improving titer of the oligosaccharide lacto-N-tetraose by 6-fold. Aided by computational scRNA efficacy prediction, the combinatorial CRISPRa strategy enables effective optimization of multi-step metabolic pathways. More broadly, the guide RNA design rules uncovered here may enable the routine design of effective multi-guide programs for a wide range of model- and data-driven applications of CRISPR gene regulation in bacterial hosts.

synthetic biology↗

bioRxiv2023

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In E. coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of both genes. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find that dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/561958v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1263909org.highwire.dtl.DTLVardef@6aa156org.highwire.dtl.DTLVardef@863e59org.highwire.dtl.DTLVardef@167496e_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗