Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

Abstract

L-serine is a promising building block biochemical with a high theoretical production yield from glucose.Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100 g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Other mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium.Production using the tolerant strains resulted in 37 g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology. AbbreviationsAdaptive Laboratory Evolution (ALE); Mutiplex Genome Engineering (MAGE).

Control of metabolite efflux in microbial cell factories: current advances and future prospects

“…), but that it was due to a change in membrane tension that activated a mechanosensitive glutamate efflux pump encoded by a gene called NCgl1221 (a homologue of the E. coli yggB gene, now known as mscS, the mechanosensitive channel of small conductance) [84][85][86][87][88][89]. Similar efflux pumps are now known to be involved in the export of product during a variety of other amino acid fermentations [79,90], such as those for lysine [91][92][93][94][95], isoleucine [96,97], serine [98], threonine [99][100][101][102], cysteine [103], methionine [104], alanine [105] and others [103,106,107]. Thus, the general role of product exporters in enhancing fermentation yields (as per Figure 1) is clear [2,103,108].…”

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

“…Nowadays, methods for generation of sequence-variant libraries often include custom-designed DNA oligo library pools, multiplexed oligo assembly, or more or less randomized mutagenesis of template DNA fragments (Mutalik et al, 2013;Plesa et al, 2018;Yang et al, 2017). Screening and selection assays based on conditional growth, genetically-encoded biosensors, and reporter gene activities, has recently been applied to complement the efficiency of library generation Mundhada et al, 2016;Raman et al, 2014;Skjoedt et al, 2016). Irrespective of the origin of input library and the screening assays applied, when aiming to infer functional consequences of regulatory or genic DNA variants, it is important to acknowledge the impact of gene dosage and the genomic context of the gene encoding the protein of interest, as both overexpression and epigenetic regulation can impact downstream regulation arising from genetic variants when expressed from non-native plasmid-based cassettes or when using heterologous promoters (Gibson et al, 2013).…”

Fast growth phenotype of E. coli K-12 from adaptive laboratory evolution does not require intracellular flux rewiring

“…ALE has been applied to increasing chemical tolerance (Atsumi et al, 2010; Horinouchi et al, 2010; Mundhada et al, 2017; Reyes et al, 2012), rates of growth on diverse substrates (Cordova et al, 2016; Herring et al, 2006; Lee and Palsson, 2010; Sandberg et al, 2017), and gaining general insight into microbial responses to environmental or genetic perturbations (Charusanti et al, 2010; Fong and Palsson, 2004; Tenaillon et al, 2012). Following an ALE experiment, the resulting strains are sequenced to identify genetic mutations (Herring et al, 2006).…”