Engineering Escherichia coli for co-production of acetaldehyde and hydrogen from glucose

Biomass is widely accepted as a good alternative to petroleum due to concerns about global warming and energy security. Microbial conversion of renewable resources into fuel molecules and important chemicals has been achieved through efforts in genetic engineering of the biocatalysts. In this work, we demonstrated efficient production of an important bulk chemical in glucose fermentation by recombinant Escherichia coli strains. Escherichia coli K12 strain MG1655 was engineered to co-produce acetaldehyde and hydrogen during glucose fermentation using an exogenous acetyl-CoA reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 y 1.63 ymol min-1 mg-1) and the desired acetyl-CoA reductase activity (49.23 y 2.88 ymol min-1 mg-1). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, ΔfrdC). The engineered strain (ZH88) produced of 4.91 y 0.29 mM acetaldehyde while consuming 11.05 mM glucose, but also produced 6.44 y 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated and all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g/L yeast extract and lowering the pH to 6.0 further increased the co-production of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 y 0.20 g·h-1·g-1 dry cell weight, and in 86% of theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient no-distill ethanol fermentation based on the co-production of acetaldehyde and hydrogen is discussed

Challenges of scale down model for disposable bioreactors: Case studies on growth & product quality impacts

Despite wide-spread use of disposable bioreactors, there is a lack of well-established scale-down model for larger scale SUBs. Here we report a case of NS0 cell culture process transfer from 2000L stainless steel bioreactor (SST) to 2000L disposable bioreactor (SUB). Initial attempts in trying to grow the NS0 cells in the small scale 2D bags yielded non-satisfactory results, as growth was impacted by bag material type as well as by suppliers of the same bag material type. However, 3D bags of 50L and above proved to be supportive of the NS0 cell line growth. Even for cell lines that do not have growth issues in SUBs, surprising product quality difference between SUBs and traditional bench top glass bioreactors are still being observed, thus making the bench top glass bioreactors non-ideal as scale down models. We report two cases where glycan profiles of the expressed antibody products show such dramatic differences. In one case, extensive testing of glass bioreactors from various suppliers led to a particular type being able to mimic the glycan profiles from the SUB, whereas in the other case, alternative scale down model had to be identified and the process had to be modified to maintain the glycan profiles when scaling up to the 200L SUB

Challenges of scale down model for disposable bioreactors: Case studies on growth & product quality impacts

Despite wide-spread use of disposable bioreactors, there is a lack of well-established scale-down model for larger scale SUBs. Here we report a case of NS0 cell culture process transfer from 2000L stainless steel bioreactor (SST) to 2000L disposable bioreactor (SUB). Initial attempts in trying to grow the NS0 cells in the small scale 2D bags yielded non-satisfactory results, as growth was impacted by bag material type as well as by suppliers of the same bag material type. However, 3D bags of 50L and above proved to be supportive of the NS0 cell line growth. Even for cell lines that do not have growth issues in SUBs, surprising product quality difference between SUBs and traditional bench top glass bioreactors are still being observed, thus making the bench top glass bioreactors non-ideal as scale down models. We report two cases where glycan profiles of the expressed antibody products show such dramatic differences. In one case, extensive testing of glass bioreactors from various suppliers led to a particular type being able to mimic the glycan profiles from the SUB, whereas in the other case, alternative scale down model had to be identified and the process had to be modified to maintain the glycan profiles when scaling up to the 200L SUB

Experimental Investigation on Failure Modes and Mechanical Properties of Rock-Like Specimens with a Grout-Infilled Flaw under Triaxial Compression

Flaws existing in rock mass are one of the main factors resulting in the instability of rock mass. Epoxy resin is often used to reinforce fractured rock mass. However, few researches focused on mechanical properties of the specimens with a resin-infilled flaw under triaxial compression. Therefore, in this research, epoxy resin was selected as the grouting material, and triaxial compression tests were conducted on the rock-like specimens with a grout-infilled flaw having different geometries. This study draws some new conclusions. The high confining pressure suppresses the generation of tensile cracks, and the failure mode changes from tensile-shear failure to shear failure as the confining pressure increases. Grouting with epoxy resin leads to the improvement of peak strengths of the specimens under triaxial compression. The reinforcement effect of epoxy resin is better for the specimens having a large flaw length and those under a relatively low confining pressure. Grouting with epoxy resin reduces the internal friction angle of the samples but improves their cohesion. This research may provide some useful insights for understanding the mechanical behaviors of grouted rock masses.National Natural Science Foundation of China [41672258, 41102162]; Postgraduate Research & Practice Innovation Program of Jiangsu Province [KYCX18_0622]; Fundamental Research Funds for the Central Universities [2018B695X14]; Chinese Scholarship CouncilOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]