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Process for producing n-propanol and propionic acid using metabolically engineered propionibacteria

A technology of propionic acid bacteria and n-propanol, applied in chemical instruments and methods, biochemical equipment and methods, oxidoreductases, etc., can solve the problem of lack of research on bacterial genetic engineering, difficulties in transforming gram-positive bacteria, and lack of cloning tools And other issues

Inactive Publication Date: 2016-04-13
DOW GLOBAL TECH LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although Propionibacterium have received attention to date, however, little research has been done on the genetic engineering of these bacteria due to the high GC content in their genomes, the difficulty of transforming Gram-positive bacteria, and the lack of cloning tools

Method used

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  • Process for producing n-propanol and propionic acid using metabolically engineered propionibacteria
  • Process for producing n-propanol and propionic acid using metabolically engineered propionibacteria
  • Process for producing n-propanol and propionic acid using metabolically engineered propionibacteria

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example 1 and comparative example 1

[0043] Bacterial Strains, Plasmids, and Media:

[0044] All bacterial strains and plasmids used in this example are listed in Table 1. coli DH5α (a A facultative anaerobic organism with a GC content of 40% or higher) to obtain transformants thereof. For comparative purposes, Clostridium acetobutylicum (Clostridium acetobutylicum, C. acetobutylicum) American Type Culture Collection (American Type Culture Collection, ATCC) 824 (which is also a facultative anaerobic organism) was grown at 37°C in an enhanced Clostridium medium (RCM, Difco). Propionibacterium was grown anaerobically in Northern Lights BioScience (NLB) medium containing 10g / L sodium lactate, 10g / L yeast extract, and 10g / L tryptic casein soybean culture medium at 32°C to prepare Competent cells and for recovery of cells after electroporation. For fermentation kinetics studies, cells were cultured in the presence of 10 g / L yeast extract, 5 g / L tryptic casein, 0.25 g / L K 2 HPO 4 , 0.05g / LMnSO 4 , 25g / L glucose ...

example 2 and comparative example 2

[0064] Kinetics of Batch Fermentation Using Glycerol as Substrate

[0065] Batch fermentation kinetics of various strains were also investigated with glycerol as carbon source and the results are shown in Figure 6 , Figure 7 as well as Figure 8 , the key kinetic parameters are summarized in Table 2. Again, the mutant produced significant amounts of n-propanol, while the WT did not produce any detectable amount. Compared to glucose fermentation, 50% to 65% more n-propanol was produced from glycerol at a faster rate, reaching a maximum propanol titer of approximately 500 mg / L at the end of the fermentation. It is particularly noteworthy that the mutant was able to use glycerol much faster and more efficiently, consuming all 20 g / L of glycerol in 200 h (consumption rate: roughly 0.10 g / L h), while WT used only Approximately 10 g / L glycerin (consumption rate: approximately 0.032 g / L·h). Thus, more propionate was produced by the mutant with a higher productivity of 0.047 g / ...

example 3 and comparative example 3

[0069] The effect of propanol on propionic acid fermentation:

[0070] Propionibacterium fresnerei DSM4209 wild-type cells were grown in culture medium with glycerol as carbon source and various amounts of n-propanol (0 g / L, 0.5 g / L, 1 g / L, 5 g / L and 10g / L) to illustrate the effect of n-propanol on cell growth and fermentation. as in Figure 9 , Figure 10 as well as Figure 11 As can be seen in , propanol does not significantly affect cell growth or fermentation at low concentrations of 0.1 g / L to 1 g / L, but inhibits cell growth with a significantly longer lag period at higher concentrations of 10 g / L. However, in the presence of 5 g / L propanol, cells were able to consume glycerol and produce propionate at a significantly higher rate after a short-term adaptation in the stasis phase. Specific growth rate, glycerol consumption rate, and propionate production rate and yield were estimated from the time course data and summarized in Table 4. The results showed that in the p...

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Abstract

A metabolically engineered propionibacteria, genomically modified to express a bifunctional aldehyde / alcohol dehydrogenase identified as GenBank Gene ID: 6062148; GI: 170080868, having activity on propionyl-CoA, is prepared and used in a fermentative process to biosynthetically prepare n-propanol, propionic acid, or a combination thereof.

Description

[0001] This application claims priority to US Provisional Patent Application 61 / 740,490, filed December 21, 2012, which is hereby incorporated by reference in its entirety. technical field [0002] The present invention relates to metabolically engineered bacteria, their preparation and their use in a method for the bioconversion of biological substrates into products comprising n-propanol, propionic acid or combinations thereof. Background technique [0003] Concerns about the future scarcity, cost and environmental impact of fossil fuels have fueled interest in the exploitation of cheap, renewable biomass as an alternative source of fuels and chemicals. As crude oil prices have risen, bio-based chemicals and industrial products have become attractive alternatives to petroleum-derived counterparts. Fermentation methods using anaerobic microorganisms offer promising avenues for the conversion of biomass and agricultural waste into chemicals and fuels. There are large quanti...

Claims

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Application Information

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IPC IPC(8): C12P7/04C07K14/245C12P7/52A23L19/00
CPCC12P7/04C12P7/52C12N9/0008C12Y101/01001C12Y102/0101C07K14/245A23L19/09A23L19/01C12Y102/01003C12N9/0006C12Y102/01
Inventor S-T·杨E·阿马尔C·C·斯托瓦斯B·A·罗德里格兹
Owner DOW GLOBAL TECH LLC
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