Genome replication engineering assisted continuous evolution (GREACE) to improve microbial tolerance for biofuels production

BACKGROUND: Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were engineered through evolutionary engineering using the principle of “Mutagenesis followed-by Selection”. The iterative rounds of mutagenesis-selection and frequent manual interventions resulted in discontinuous and inefficient strain improvement processes. This work aimed to develop a more continuous and efficient evolutionary engineering method termed as “Genome Replication Engineering Assisted Continuous Evolution” (GREACE) using “Mutagenesis coupled-with Selection” as its core principle. RESULTS: The core design of GREACE is to introduce an in vivo continuous mutagenesis mechanism into microbial cells by introducing a group of genetically modified proofreading elements of the DNA polymerase complex to accelerate the evolution process under stressful conditions. The genotype stability and phenotype heritability can be stably maintained once the genetically modified proofreading element is removed, thus scarless mutants with desired phenotypes can be obtained. Kanamycin resistance of E. coli was rapidly improved to confirm the concept and feasibility of GREACE. Intrinsic mechanism analysis revealed that during the continuous evolution process, the accumulation of genetically modified proofreading elements with mutator activities endowed the host cells with enhanced adaptation advantages. We further showed that GREACE can also be applied to engineer n-butanol and acetate tolerances. In less than a month, an E. coli strain capable of growing under an n-butanol concentration of 1.25% was isolated. As for acetate tolerance, cell growth of the evolved E. coli strain increased by 8-fold under 0.1% of acetate. In addition, we discovered that adaptation to specific stresses prefers accumulation of genetically modified elements with specific mutator strengths. CONCLUSIONS: We developed a novel GREACE method using “Mutagenesis coupled-with Selection” as core principle. Successful isolation of E. coli strains with improved n-butanol and acetate tolerances demonstrated the potential of GREACE as a promising method for strain improvement in biofuels production

Adopting a Theophylline-Responsive Riboswitch for Flexible Regulation and Understanding of Glycogen Metabolism in Synechococcus elongatus PCC7942

Cyanobacteria are supposed to be promising photosynthetic microbial platforms that recycle carbon dioxide driven into biomass and bioproducts by solar energy. Glycogen synthesis serves as an essential natural carbon sink mechanism, storing a large portion of energy and organic carbon source of photosynthesis. Engineering glycogen metabolism to harness and rewire carbon flow is an important strategy to optimize efficacy of cyanobacteria platforms. ADP-glucose pyrophosphorylase (GlgC) catalyzes the rate-limiting step for glycogen synthesis. However, knockout of glgC fails to promote cell growth or photosynthetic production in cyanobacteria, on the contrary, glgC deficiency impairs cellular fitness and robustness. In this work, we adopted a theophylline-responsive riboswitch to engineer and control glgC expression in Synechococcus elongatus PCC7942 and achieved flexible regulation of intracellular GlgC abundance and glycogen storage. With this approach, glycogen synthesis and glycogen contents in PCC7942 cells could be regulated in a range from about 40 to 300% of wild type levels. In addition, the results supported a positive role of glycogen metabolism in cyanobacteria cellular robustness. When glycogen storage was reduced, cellular physiology and growth under standard conditions was not impaired, while cellular tolerance toward environmental stresses was weakened. While when glycogen synthesis was enhanced, cells of PCC7942 displayed optimized cellular robustness. Our findings emphasize the significance of glycogen metabolism for cyanobacterial physiology and the importance of flexible approaches for engineering and understanding cellular physiology and metabolism

Rescuing ethanol photosynthetic production of cyanobacteria in non-sterilized outdoor cultivations with a bicarbonate-based pH-rising strategy

Abstract Background Ethanol photosynthetic production based on cyanobacteria cell factories utilizing CO2 and solar energy provides an attractive solution for sustainable production of green fuels. However, the scaling up processes of cyanobacteria cell factories were usually threatened or even devastated by biocontaminations, which restricted biomass or products accumulations of cyanobacteria cells. Thus it is of great significance to develop reliable biocontamination-controlling strategies for promoting ethanol photosynthetic production in large scales. Results The scaling up process of a previously developed Synechocystis strain Syn-HZ24 for ethanol synthesis was severely inhibited and devastated by a specific contaminant, Pannonibacter phragmitetus, which overcame the growths of cyanobacteria cells and completely consumed the ethanol accumulation in the cultivation systems. Physiological analysis revealed that growths and ethanol-consuming activities of the contaminant were sensitive to alkaline conditions, while ethanol-synthesizing cyanobacteria strain Syn-HZ24 could tolerate alkaline pH conditions as high as 11.0, indicating that pH-increasing strategy might be a feasible approach for rescuing ethanol photosynthetic production in outdoor cultivation systems. Thus, we designed and evaluated a Bicarbonate-based Integrated Carbon Capture System (BICCS) derived pH-rising strategy to rescue the ethanol photosynthetic production in non-sterilized conditions. In lab scale artificially simulated systems, pH values of BG11 culture medium were maintained around 11.0 by 180 mM NaHCO3 and air steam, under which the infection of Pannonibacter phragmitetus was significantly restricted, recovering ethanol production of Syn-HZ24 by about 80%. As for outdoor cultivations, ethanol photosynthetic production of Syn-HZ24 was also successfully rescued by the BICCS-derived pH-rising strategy, obtaining a final ethanol concentration of 0.9 g/L after 10 days cultivation. Conclusions In this work, a novel product-consuming biocontamination pattern in cyanobacteria cultivations, causing devastated ethanol photosynthetic production, was identified and characterized. Physiological analysis of the essential ethanol-consuming contaminant directed the design and application of a pH-rising strategy, which effectively and selectively controlled the contamination and rescued ethanol photosynthetic production. Our work demonstrated the importance of reliable contamination control systems and strategies for large scale outdoor cultivations of cyanobacteria, and provided an inspiring paradigm for targeting effective solutions

A Marine Gas Turbine Fault Diagnosis Method Based on Endogenous Irreversible Loss

When a malfunction occurs in a marine gas turbine, its thermal efficiency will decrease slightly, and the gas path fault is often difficult to distinguish. In order to solve this problem, based on the second law of thermodynamics, the endogenous irreversible loss (EIL) model of the marine gas turbine is established, and the exergy loss analysis under normal conditions is carried out to verify the accuracy of the model. The fault diagnosis of gas turbine gas path based on EIL is proposed, and a simulation experiment conducted on a three-shaft marine gas turbine demonstrated that the proposed approach can detect and isolate gas path fault accurately under different operating conditons and enviroments

Synthesis of Novel Chitin Derivatives Bearing Amino Groups and Evaluation of Their Antifungal Activity

Chemical modification is one of the most effective methods to improve the biological activity of chitin. In the current study, we modified C3-OH and C6-OH of chitin (CT) and successfully synthesized 6-amino-chitin (NCT) and 3,6-diamino-chitin (DNCT) through a series of chemical reactions. The structure of NCT and DNCT were characterized by elemental analyses, FT-IR, 13C NMR, XRD, and SEM. The inhibitory effects of CT, NCT, and DNCT against six kinds of phytopathogen (F. oxysporum f. sp. cucumerium, B. cinerea, C. lagenarium, P. asparagi, F. oxysporum f. niveum, and G. zeae) were evaluated using disk diffusion method in vitro. Meanwhile, carbendazim and amphotericin B were used as positive controls. Results revealed that 6-amino-chitin (NCT) and 3,6-diamino-chitin (DNCT) showed improved antifungal properties compared with pristine chitin. Moreover, DNCT exhibited the better antifungal property than NCT. Especially, while the inhibition zone diameters of NCT are ranged from 11.2 to 16.3 mm, DNCT are about 11.4–20.4 mm. These data demonstrated that the introduction of amino group into chitin derivatives could be key to increasing the antifungal activity of such compounds, and the greater the number of amino groups in the chitin derivatives, the better their antifungal activity was

The evaluation of antioxidant and antifungal properties of 6-amino-6-deoxychitosan in vitro

We synthesized 6-amino-6-deoxychitosan (NCS) through a series of reactions from chitosan (CS). The antioxidant ability of CS and NCS were investigated in vitro, including that of DPPH-radical and hydrogen peroxide, reducing power, chelating abilities and inhibition of lipid peroxidation. As expected, after the introduction of amino groups, antioxidant ability had improved significantly. Especially, scavenging effect against DPPH-radical and hydrogen peroxide of NCS were 97% and 92% at 1.6 mg/mL, respectively. Moreover, inhibition of lipid peroxidation was 57% at 0.1 mg/mL. And the reducing power of NCS was 0.68 at 0.8 mg/mL. Meanwhile, inhibitory effects against four fungi were also tested. Antifungal activity of NCS were better than those of CS and antifungal activity had improved more than 20% at 0.5 mg/mL against these four kinds of plant pathogens. Based on the above results, it was reasonable to speculate that the obtained antioxidant ability and antifungal activity of NCS might benefit from amino groups on chitosan backbone. These in vitro results suggest the possibility that NCS as antioxidant compound could be effectively alleviate oxidative stress and thus inhibit the oxidative mechanisms that lead to degenerative diseases. (C) 2017 Elsevier B.V. All rights reserved

MOESM1 of Rescuing ethanol photosynthetic production of cyanobacteria in non-sterilized outdoor cultivations with a bicarbonate-based pH-rising strategy

Additional file 1: Figure S1. Microscopic analysis of the outdoor non-sterilized cultivation system for photosynthetic production of ethanol. Figure S2. Structure schematic of the ethanol recovery system for the MPBR system. Figure S3. Total ethanol production and distribution of Syn-HZ24 cultivated in MPBR under non-sterilized outdoor conditions

Formic Acid Triggers the “Acid Crash” of Acetone-Butanol-Ethanol Fermentation by Clostridium acetobutylicum▿

Solvent production by Clostridium acetobutylicum collapses when cells are grown in pH-uncontrolled glucose medium, the so-called “acid crash” phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium, C. acetobutylicum DSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanol-ethanol fermentation