Engineered Zymomonas mobilis tolerant to acetic acid and low pH via multiplex atmospheric and room temperature plasma mutagenesis

Abstract

BackgroundCellulosic biofuels are sustainable compared to fossil fuels. However, inhibitors, such as acetic acid generated during lignocellulose pretreatment and hydrolysis, would significantly inhibit microbial fermentation efficiency. Microbial mutants able to tolerate high concentration of acetic acid are needed urgently to alleviate this inhibition.ResultsZymomonas mobilis mutants AQ8-1 and AC8-9 with enhanced tolerance against acetic acid were generated via a multiplex atmospheric and room temperature plasma (mARTP) mutagenesis. The growth and ethanol productivity of AQ8-1 and AC8-9 were both improved in the presence of 5.0–8.0 g/L acetic acid. Ethanol yield reached 84% of theoretical value in the presence of 8.0 g/L acetic acid (~ pH 4.0). Furthermore, a mutant tolerant to pH 3.5, named PH1-29, was generated via the third round of ARTP mutagenesis. PH1-29 showed enhanced growth and ethanol production under both sterilized/unsterilized conditions at pH 4.0 or 3.5. Intracellular NAD levels revealed that mARTP mutants could modulate NADH/NAD+ ratio to respond to acetic acid and low pH stresses. Moreover, genomic re-sequencing revealed that eleven single nucleic variations (SNVs) were likely related to acetic acid and low pH tolerance. Most SNVs were targeted in regions between genes ZMO0952 and ZMO0956, ZMO0152 and ZMO0153, and ZMO0373 and ZMO0374.ConclusionsThe multiplex mutagenesis strategy mARTP was efficient for enhancing the tolerance in Z. mobilis. The ARTP mutants generated in this study could serve as potential cellulosic ethanol producers.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1348-9) contains supplementary material, which is available to authorized users.

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

“…Basically, mutants with designed mutations can be readily obtained in a single round of transformation, spending about only three days, whereas it normally takes at least half a month to achieve so in the routine genetic manipulations. Certainly, the CRISPR-based toolkit established here will largely facilitate further elucidation of the underlying mechanism of inhibitor-tolerant mutants with single-nucleotide polymorphisms (SNPs) (47,48,61), which cannot be easily studied by classical genetics approaches to correlate the robustness phenotypes with their corresponding genotypes. In addition, this toolkit will expedite the development of Z. mobilis as a synthetic chassis for sustainable economic biofuel and biochemical productions.…”

“…This strategy usually changed the copy number and hence the expression level of the target gene. In addition, another approach is to replace the single-nucleotide polymorphisms (SNPs) using homologous recombination containing a selective marker (47)(48)(49), which is time consuming or adding heterologous antibiotics gene into the chromosome. In many cases, point mutagenesis was required to be completed in an in situ manner.…”

Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress

“…Through forward and reverse genetics, the reported highest concentration of acetic acid that Z. mobilis could tolerate was 8.0 g/L [10]. Therefore, in this study, four different gradient concentrations (3.0, 6.0, 9.0, and 12.0 g/L) of acetic acid were employed.…”

“…Furthermore, furfural can form synergistic inhibition with acetic acid [6]. Many methods that have been studied in the past focused on removing inhibitors before fermentation (e.g., physical, physicochemical and biological) [7,8] or developing inhibitor-tolerant ethanologenic strains [9,10].…”

“…Apart from removing inhibitors before fermentation, creating mutants capable of tolerating furfural and acetic acid is efficient for the economic production of cellulosic biofuels [14,15]. However, although several efforts have been applied to improve these inhibitors' tolerance in Z. mobilis, including forward and reverse genetics, so far, the reported highest concentrations of furfural and acetic acid that Z. mobilis could tolerate were 3.0 g/L and 8.0 g/L [10,16], respectively. But further efforts are still needed to meet the requirement of practical lignocellulose biorefinery with high inhibitor contents in the pretreated feedstock, and due to the complex mechanism of furfural and acetic acid stress in Z. mobilis, developing a robust strain will also be a difficult challenge [13][14][15].…”