Search

Mesoplasma florum, a fast-growing near-minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing ˜30% of its protein-coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species-specific constraints to produce the functional iJL208 genome-scale model (GEM) of metabolism. Genome-wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine iJL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. iJL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0 revealed key features of a minimal gene set. iJL208 is a stepping-stone toward model-driven whole-genome engineering.

Although the use of ionic liquids (ILs) for the pretreatment of lignocellulosic biomass has been limited due to high costs, recent efforts to develop low-cost protic ILs show promise for achieving cost-effectiveness for biorefineries. However, an additional challenge remains in that ILs present in biomass hydrolysates are toxic to most microbial hosts, resulting in poor growth phenotypes. To address this issue, we applied an adaptive laboratory evolution (ALE) approach for tolerizingPseudomonas putidaKT2440, an industrially relevant bacterial host, to two low-cost ILs (triethanolammonium acetate [TEOH][OAc] and triethylammonium hydrogen sulfate [TEA][HS]). After continuous cultivations with gradually increased IL levels, we obtained evolved strains showing significant improvements in their growth performance under high concentrations of the ILs (maximum 4% [TEOH][OAc] and 8% [TEA][HS], in w/v) at which the wild-type strain cannot grow. Sequencing of evolved strains revealed multiple regions where mutations were associated with improved performance in minimal media conditions (relA,gacS,oprB/PP_1446,fleQ,tktA, anduvrY/PP_4100) and in IL-specific conditions (PP_5350, PP_4929/emrE,oprD, and PP_5324). We further validated the causality of the PP_5350 andemrEgenes for improved IL toleranceviareverse engineering and transcriptomic analysis. A common mutation in the PP_5350 gene, encoding a RpiR family transcriptional regulator, was shown to significantly upregulate the glyoxylate cycle for efficient acetate catabolism. In addition, it was suggested that theemrEgene encodes an efflux pump which can export [TEA][HS]. Finally, the cultivation of two of the best performing evolved strains with IL-treated biomass hydrolysates demonstrated their considerable potential to be used as platform strains. Taken as a whole, this work provides strains for utilization of IL-treated biomass and a mechanistic understanding that could be further leveraged to develop efficient microbial bioprocesses.

Additional file 1: Supplementary figures and tables.

Secondary structure of tRNA His and observed hisR mutations. This file (.pdf) contains a figure showing the secondary structure of tRNA His and observed hisR mutations aligned with tRNA numbering convention. (PDF 272 kb)

Genome duplication amplification events observed in cysK, carA, and ptsI experiments. This file (.pdf) contains a figure showing the read depth coverage (y-axis) plotted against the genome position (x-axis) for flask 1 population samples for A. cysK, B. carA, C. and ptsI experiments. (PDF 316 kb)

FBA simulations for genes listed in Table 1. This file (.pdf) contains a table of simulations demonstrating the same predicted behavior in both the iJO1366 model corresponding to the MG1655 genotype and a modified iJO1366 model corresponding to the BW25113 genotype.(PDF 79 kb)

Growth curves of five false positive replicate strains (ptsI, serB, proA, proB, and carA) in separate subplots are displayed. This file (.pdf) contains a figure that is an extension of Figure 1B, as the same curves for these cases are displayed; however, specific labeling of each curve with their associated experiment (exp.) number is provided in the legend of each subplot. The experiment number corresponds to those numbers listed in Table 2. (PDF 183 kb)

Contingency table examples showing the likelihood of mutational convergence. This file (.pdf) contains a figure of contingency table examples showing the likelihood of mutational convergence between two samples. p-values calculated from Fisherâ s exact test are reported for four examples. (PDF 97 kb)

All FP KO strains considered in this study. This file (.pdf) contains a table listing all false positive KO strains considered for extended growth tests. Additional information regarding proposed conditional essentiality is also provided as well as a comparison to a previous study [22]. (PDF 67 kb)

Adaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated using Escherichia coli K-12 MG1655 on glucose minimal media at 37°C. By keeping E. coli in constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription gene rpoB, an 82-bp deletion between the metabolic pyrE gene and rph, and an IS element between the DNA structural gene hns and tdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.

Mutations observed in starting strains grown in LB rich medium. This file (.pdf) contains a table listing mutations identified in the PCR-confirmed Keio strains that were used in the extended growth tests on minimal medium. (PDF 49 kb)