Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device

Abstract

Aspartate kinase (AK) is a key enzyme involved in catalyzing the first step of the aspartate-derived amino acid biosynthesis, including L-lysine and L-threonine, which is regulated by the end-metabolites through feedback inhibition. In order to accumulate the end-metabolites in the host, the feedback inhibition of AK has to be released. In this study, a chimeric aspartate kinase, which is composed of the N-terminal catalytic region from Bacillus subtilis AKII and the C-terminal region from Thermus thermophilus, was evolved through random mutagenesis and then screened using a high-throughput synthetic RNA device which comprises of an L-lysine-sensing riboswitch and a selection module. Of three evolved aspartate kinases, the best mutant BT3 showed 160 % increased in vitro activity compared to the wild-type enzyme. Recombinant Escherichia coli harboring BT3 produced 674 mg/L L-lysine in batch cultivation, similar to that produced by the strain harboring the typical commercial widely used feedback resistant aspartate kinase AKC (fbr) from E. coli. The results suggested that this strategy can be extended for screening of other key enzymes involved in lysine biosynthesis pathways.

Deep scanning lysine metabolism in Escherichia coli

“…Transferring barcodes from plasmids to genomes (Roy et al , ) could also decrease cell‐to‐cell variation and hence decrease noise in barcode enrichments. In the specific case of lysine metabolism, comparing mutations identified in the presence of different antimetabolites or with screening‐based approaches using lysine biosensors (Yang et al , ; Wang et al , , ) would be a valuable contribution.…”

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…; Wang et al . ). Other riboswitches, such as theophylline, flavin mononucleotide (FMN) and c‐di‐GMP riboswitches—all controlling GFP expression—have successfully been used to screen enzyme libraries of caffeine demethylase, vitamin B2‐producing bacterial strains and c‐di‐GMP‐metabolizing enzymes, respectively (Michener and Smolke ; Gao et al .…”

“…Finally, riboswitches have been useful when screening for different traits and directed evolution, such as selecting for lysine production using lysine riboswitches. This was done by having a lysine riboswitch controlling an antibiotic resistance gene while applying a selective pressure (the antibiotic), allowing one to screen for and select high lysine-producing cells and enzyme mutations (Yang et al 2013;Wang et al 2015). Other riboswitches, such as theophylline, flavin mononucleotide (FMN) and c-di-GMP riboswitches-all controlling GFP expressionhave successfully been used to screen enzyme libraries of caffeine demethylase, vitamin B2-producing bacterial strains and c-di-GMP-metabolizing enzymes, respectively (Michener and Smolke 2012;Gao et al 2014;Meyer et al 2015).…”

Using metabolite-responsive gene regulators to improve microbial biosynthesis

“…Growth competition was implemented, and the top three resulting strains with optimized ppc expression levels significantly improved lysine production. The same lysine-responsive riboswitch was also used to evolve a chimeric aspartate kinase (AK) for improved lysine production in E. coli [28]. The best AK variant (BT3) showed 160% increased activity compared with the wildtype enzyme.…”