Background Reducing fresh water consumption and nutrient addition will be an effective way to reduce the whole cost of bioethanol production. On the other hand, treatment of biogas slurry derived from anaerobic digestion (AD), in which a great amount of nutrients is still left, costs too much to remove these pollutants. It would be beneficial for both digestate valorization and ethanol production if biogas slurry is used for producing bioethanol. However, both hyperosmosis and potential biotoxic components of the biogas slurry can severely inhibit fermentation. Results In this study, two rounds of atmospheric and room temperature plasma (ARTP) mutagenesis combined with adaptive laboratory evolution (ALE) were applied to improve the adaptability and genetic stability of Zymomonas mobilis in biogas slurry. Mutants D95 and S912 were identified. Growth of the mutants was remarkably improved in biogas slurry. The highest ethanol productivity reached 0.63 g/L/h which was 61.7% higher than ZM4 (0.39 g/L/h). Genomic re-sequencing results also revealed that single nucleic variations (SNVs) and Indels occurred in the mutants, which are likely related to inhibitor in biogas slurry and low pH tolerance. Conclusions Our study demonstrated that these mutant strains have great potential to produce ethanol using biogas slurry to replace fresh water and nutrients. Electronic supplementary material The online version of this article (10.1186/s13068-019-1463-2) contains supplementary material, which is available to authorized users.

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Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

Backgroud: Acetic acid and furfural are two major inhibitors during lignocellulosic ethanol production. In our previous study, we successfully constructed an engineered Zymomonas mobilis ZM532 strain tolerant these double inhibitors by genome shuffling, but the molecular mechanisms of tolerance to these inhibitors are still unknown. This study investigated the responses of ZM532 and wild-type ZM4 to acetic acid and furfural using genomics, transcriptomics and label free quantitative proteomics. Results: By Sanger sequencing technology we re-verified of previously identified 19 mutations in ZM532, but we found a total of 23 single nucleotide polymorphisms (SNPs) in the coding sequence (CDS; 4) and intergenic region (19) in ZM532. Six SNPs were novel in this study. We also identified a total of 1865 and 14 novel differentially expressed genes (DEGs) in ZM532 and wild-type ZM4. Among these, 352 DEGs were up-regulated; while and 393 were down-regulated in AF_ZM532 vs RM_532, respectively. However, 442 DEGs were up while 463 were down-regulated in AF_ZM4 vs RM_ZM4. Moreover, 2 up and 8 down-regulated genes were identified in AF_ZM532 vs AF_ZM4; while 7 up and 1 down-regulated genes were found in RM_ZM532 vs RM_ZM. We also identified 1,532 proteins among 107 up and 204 down-regulated proteins detected in ZM4_AF vs ZM4_RM, 123 up and 205 down regulated proteins were identified in ZM532_AF vs ZM532_RM, respectively. In addition, a total of 16 up and 5 down-regulated proteins were identified out of 1462 in ZM4_AF vs ZM532_AF, while 8 up and 5 down-regulated proteins were observed out of 1491 in ZM4_RM vs ZM532_RM. These proteins and genes are involved in amino acid biosynthesis, macromolecules repair, glycolysis, flagella assembly, ABC transporter, fermentation, and ATP synthesis pathways and stress response. These mentioned genes and proteins confirmed and help to unravel the acetic acid and furfural tolerance mechanism between ZM532 and wild-type ZM4. May be these proteins and genes play key roles in ZM532 regulation with strong expressions under acids stress conditions. Furthermore, we knocked-out and overexpressed two differentially expressed genes (DEGs), ZMO_RS02740 up-regulated and ZMO_RS06525 down-regulated to investigate their roles in acetic acid and furfural tolerance. Our knockout and complementary experiments revealed that up-regulated expression gene ZMO_RS02740 and the down-regulated expression gene ZMO_RS06525 play important roles in dealing with furfural and acetic acid stress. Conclusion: ZM532 can be used to substitute ZM4 as a biocatalyst for bioethanol under acetic acid and furfural condition, with a shorter fermentation time and higher productivity. Further studies may be required to clarify the relationship between the acid resistance and the genetic disparity of mutant strains.

Zymomonas mobilis ( Z. mobilis ) is a potential candidate for consolidated bioprocessing (CBP) strain in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion affected by cell wall peptidoglycan is also not well understood. Here we constructed several Penicillin Binding Proteins (PBPs)-deficient strains derivated from Z. mobilis S192 to perturb the cell wall peptidoglycan network and investigated the effects of peptidoglycan on the endoglucanase secretion. Results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBPs mutant strains, notably, △1089/0959 (4.09-fold) and △0959 (5.76-fold) in comparison to parent strains. Besides, for PBPs-deficient strains, the growth performance was not significantly inhibited but with enhanced antibiotic sensitivity and reduced inhibitor tolerance, otherwise, cell morphology was altered obviously. The concentration of intracellular soluble peptidoglycan was increased, especially for single gene deletion. Outer membrane permeability of PBPs-deficient strains was also improved, notably, △1089/0959 (1.14-fold) and △0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our finding indicated that PBPs-deficient Z. mobilis is capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness, and also acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacterias. Herein, we perturb the peptidoglycan synthesis network via knocking out PBPs ( ZMO0197 , ZMO0959 , ZMO1089 ) in order to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study can not only lay the foundation for understanding the regulatory network of cell wall synthesis but also provide guidance for the construction of CBP strains in Z. mobilis .

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