Cell-Free Mixing of <i>Escherichia coli</i> Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis

Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L^–1 (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L^–1·hr^–1. We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD⁺ and demonstrates the ability to rapidly prototype and debug enzymatic pathways <i>in vitro</i> for compelling metabolic engineering and synthetic biology applications

Supporting Information from Leveraging genome-wide datasets to quantify the functional role of the anti-Shine–Dalgarno sequence in regulating translation efficiency

10 total supplementary figures that support and extend the results from the Main Tex

Supplementary Figure 1; Supplementary Figure 2; Supplementary Figure 3; Supplementary Table 1; SI text from A novel framework for evaluating the performance of codon usage bias metrics

Spearman's ρ between the calculated CUB of 3740 genes in the E. coli genome using various metrics. All correlations are statistically significant with p < 10⁻¹⁰; Performance of individual pair-wise differentiation task for six different CUB metrics for one bootstrapped sample using E. coli -like genome. Each block in the heatmap represents the AUC when using that metric to differentiate sequences created using different number of codons.; Performance of individual pair-wise differentiation task for six different CUB metrics for one bootstrapped sample under the simplest case. Each block in the heatmap represents the AUC when using that metric to differentiate sequences created using different number of codons.; P-value of the Wilcoxon signed-rank test for gene expression correlations between different metrics. Bold face values indicate when the test statistic is positive (i.e. when the metric listed at the left is higher than the metric listed at the top).; Implementation of previous CUB metric

The GC ratio of random sequences generated using the maximum entropy approach coincides exactly with desired GC content over a wide range of GC ratios.

<p>When generating nucleotide sequences from an amino acid sequence with uniform amino acid usage, we can accurately achieve a GC content between the range of 30% and 64% (top). By altering the amino acid composition of the translated sequence, a lower and higher range of GC contents can be obtained (middle and bottom). At each GC content, the average GC content of 500 randomly generated sequences with amino acid length of 2500 was taken. The <i>y</i> = <i>x</i> line (shown in gray dotted line) indicates the ideal case. The simulated results for the multinomial and maximum entropy method are shown in black jagged and solid lines respectively.</p

Random sequences generated using the maximum entropy approach are unbiased with a mean equal to the target GC content.

<p>We generated 500 random sequences, with equiprobable amino acid usage and 2500 amino acids in length. We used matching colors for target GC content (dashed line) and observed GC content distribution.</p

Characterizing and Alleviating Substrate Limitations for Improved <i>in vitro</i> Ribosome Construction

Complete cell-free synthesis of ribosomes could make possible minimal cell projects and the construction of variant ribosomes with new functions. Recently, we reported the development of an integrated synthesis, assembly, and translation (iSAT) method for <i>in vitro</i> construction of Escherichia coli ribosomes. iSAT allows simultaneous rRNA synthesis, ribosome assembly, and reporter protein expression as a measure of ribosome activity. Here, we explore causes of iSAT reaction termination to improve efficiency and yields. We discovered that phosphoenolpyruvate (PEP), the secondary energy substrate, and nucleoside triphosphates (NTPs) were rapidly degraded during iSAT reactions. In turn, we observed a significant drop in the adenylate energy charge and termination of protein synthesis. Furthermore, we identified that the accumulation of inorganic phosphate is inhibitory to iSAT. Fed-batch replenishment of PEP and magnesium glutamate (to offset the inhibitory effects of accumulating phosphate by repeated additions of PEP) prior to energy depletion prolonged the reaction duration 2-fold and increased superfolder green fluorescent protein (sfGFP) yield by ∼75%. By adopting a semi-continuous method, where passive diffusion enables substrate replenishment and byproduct removal, we prolonged iSAT reaction duration 5-fold and increased sfGFP yield 7-fold to 7.5 ± 0.7 μmol L^–1. This protein yield is the highest ever reported for iSAT reactions. Our results underscore the critical role energy substrates play in iSAT and highlight the importance of understanding metabolic processes that influence substrate depletion for cell-free synthetic biology

Target nucleotide composition of test sequences.

<p>Target nucleotide composition of test sequences.</p

Point-of-Care Peptide Hormone Production Enabled by Cell-Free Protein Synthesis

In resource-limited settings, it can be difficult to safely deliver sensitive biologic medicines to patients due to cold chain and infrastructure constraints. Point-of-care drug manufacturing could circumvent these challenges since medicines could be produced locally and used on-demand. Toward this vision, we combine cell-free protein synthesis (CFPS) and a 2-in-1 affinity purification and enzymatic cleavage scheme to develop a platform for point-of-care drug manufacturing. As a model, we use this platform to synthesize a panel of peptide hormones, an important class of medications that can be used to treat a wide variety of diseases including diabetes, osteoporosis, and growth disorders. With this approach, temperature-stable lyophilized CFPS reaction components can be rehydrated with DNA encoding a SUMOylated peptide hormone of interest when needed. Strep-Tactin affinity purification and on-bead SUMO protease cleavage yield peptide hormones in their native form that are recognized by ELISA antibodies and that can bind their respective receptors. With further development to ensure proper biologic activity and patient safety, we envision that this platform could be used to manufacture valuable peptide hormone drugs in a decentralized way

Incorporation of Nonproteinogenic Amino Acids in Class I and II Lantibiotics

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. These methods complement recent alternative approaches to incorporate nonproteinogenic amino acids into lantibiotics

<i>In Vitro</i> Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-free protein synthesis has not previously been used to study nonribosomal peptide biosynthesis, and provides a tunable platform with advantages over conventional methods for protein expression. Here, we demonstrate the use of cell-free protein synthesis to biosynthesize a cyclic dipeptide with correct absolute stereochemistry. From a single-pot reaction, we measured the expression of two nonribosomal peptide synthetases larger than 100 kDa, and detected high-level production of a diketopiperazine. Using quantitative LC–MS and synthetically prepared standard, we observed production of this metabolite at levels higher than previously reported from cell-based recombinant expression, approximately 12 mg/L. Overall, this work represents a first step to apply cell-free protein synthesis to discover and characterize new natural products