Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1

Abstract

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 μg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 μg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

Keywords: cell-free protein synthesis; genome engineering; non-standard amino acids; release factor 1; synthetic biology.

Figure 1

Strain construction, verification, and cell-free protein synthesis performance. A) Genomic locations of five nuclease-encoding genes (black circles), prfA (gray circle), and 13 genes with stop codons re-coded from TAG to TAA (white circles). Numbers in inner circle indicate millions of bases. B) Verification of nuclease gene mutations by using multiplex allele-specific PCR. Mutant alleles were amplified by using the mutant forward and reverse primer sets (-mut-F and -R; Table S1). Mutant strain numbers are indicated at the top of the gels. A table in (D) details mutations per each strain. M: DNA ladder. C) Scheme of combined transcription–translation (TX–TL) reaction for sfGFP CFPS. D) Comparison of CFPS efficiency of different cell extracts. Active wild-type sfGFP was synthesized by using cell extracts derived from genomically recoded E. coli with single and multiple inactivation of nucleases. At least three independent reactions for each sample were performed for 20 h at 30°C, and one standard deviation is shown.

Figure 2

The impact of functionally inactivating nucleases on cell-free transcription and translation. A) Cell-free translation (TL)-only reactions of wild-type sfGFP from purified mRNA in different single RNase-deficient cell extracts. At least three independent reactions for each sample were performed for 120 min at 30°C. sfGFP synthesis was monitored by sfGFP fluorescence (left), and mRNA levels were assessed by an RNA gel (right). B) Cell-free Spinach aptamer synthesis by using endonuclease I-deficient (MCJ.495) and -present (rEc.E13.ΔprfA) extracts. After preincubation (0 ( ), 60 ( ), and 180 min (■)) of Spinach aptamer plasmid DNA with cell extract, CFPS reagents were added and incubated at 30°C. Maximum mRNA synthesis levels from the mRNA synthesis time course (Figure S2) were compared. At least three independent reactions for each sample were performed, and one standard deviation is shown.

Figure 3

Assessing stability of the MCJ.559 strain. A) Growth of MCJ.559 before (●) and after (○) 250 generations was assayed in LB medium at 32°C in 96-well plates. Each data point is the average of ten replicate wells from two independent cultures. B) CFPS of sfGFP by using an MCJ.559 crude extract before and after 250 generations shows that extract performance was the same. At least three independent CFPS reactions for each sample were performed for 20 h at 30°C. C) PCR verification of prfA, csdA, and endA mutation. “specR” indicates spectinomycin resistance gene in replacement of prfA. On the right is a trace of DNA sequencing results for the csdA and endA mutation showing the introduction of the TAA stop codon at the desired location.

Figure 4

pAcF incorporation at single and multiple amber sites by using the improved cell extract from the MCJ.559 strain. A) Yields of active wild-type sfGFP (WT-sfGFP) and modified sfGFP proteins containing one, two, and five pAcFs. B) Spectrum of the 32+ charge state of sfGFP, obtained by top-down mass spectrometry and illustrating site-specific incorporation of pAcF at single and multiple sites. Major peaks (gray) in each spectrum coincide with the theoretical peaks for each species (Figure S6). “Exper” indicates experimentally obtained protein mass, and “Theor” indicates theoretically calculated protein mass (Table S4). Smaller peaks to the right of the major peaks are due to oxidation of the protein—a common electrochemical reaction occurring during electrospray ionization. Water loss events from the intact sfGFP were detected at minor levels to the left of the major peaks. C) Comparison of total ( ), soluble ( ), and active (■) protein yields of sfGFP and CAT with and without single pAcF. At least three independent CFPS reactions for each sample were performed for 20 h at 30°C for (A) and (C), and one standard deviation is shown.

Figure 5

Scaled-up and semicontinuous CFPS by using an MCJ.559 extract. A) Production of wild-type sfGFP (WT-sfGFP) and modified sfGFP with pAcF (sfGFP-1pAcF) in different reaction volumes by using a microcentrifuge tube (MT) and a flat-bottom 24-well plate (FB). Time course semicontinuous and batch CFPS for B) WT-sfGFP and C) sfGFP-1pAcF. In the semicontinuous reaction ( ), CFPS reagents from the substrate reservoir passively diffuse into the CFPS reaction (inward arrows) through the microdialysis membrane, while by-products are removed from the CFPS reaction (outward arrows). Batches were of 15 (●), 30 ( ), 60 ( ), 120 (⋄), and 240 μL (▲). At least three independent reactions for each sample were performed at 30°C, and one standard deviation is shown.