Very high gravity ethanol and fatty acid production of Zymomonas mobilis without amino acid and vitamin

Abstract

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.

Zymomonas mobilis as a model system for production of biofuels and biochemicals

“…For example, solid submerged fermentation, advanced solid‐state fermentation technology, simultaneous saccharification and fermentation, and simultaneous saccharification and co‐fermentation have been used for ethanol production (Lawford et al ., ; Zhang and Lynd, ; Das et al ., ; Saharkhiz et al ., ). VHG fermentation is the mainstream technology in the ethanol industry with fermenting medium containing sugar more than 250 g l −1 , and recombinant Z. mobilis strain TMY‐HFPX can produce ethanol up to 136 g l −1 from 295 g l −1 glucose with a theoretical yield of 90% in VHG fermentation (Wang et al ., ). However, if biomass feedstocks are used, high solid loading will increase the concentration of lignocellulose‐derived inhibitors and also the osmolarity.…”

“…Since then, recombinant and evolved Z. mobilis strains for xylose and arabinose utilization have been developed through metabolic engineering and adaptation or similar methods. Importantly, some recombinant strains of Z. mobilis have been improved to utilize glucose, xylose and arabinose derived from lignocellulosic feedstock simultaneously for the fermentation of bioethanol (Chou et al ., ; Deanda et al ., ; Mohagheghi et al ., , ; Jeon et al ., ; Agrawal et al ., ; Ma et al ., ; Yanase et al ., ; Zhang et al ., ; Dunn and Rao, ; Wang et al ., ).…”

“…Recently, a recombinant Z. mobilis strain was developed to incorporate multiple gene modules, including the xylA/xlyB/tktA/talB operon for xylose utilization, the metB/yfdZ operon for lysine and methionine biosynthesis, the thioesterase gene tesA to enhance free fatty acid biosynthesis for increasing ethanol tolerance, a proton‐buffering peptide operon for acid stress tolerance, and a small heat shock protein operon for heat stress tolerance (Wang et al ., ). The final recombinant strain TMY‐HFPX can produce ethanol up to 136 g l −1 from 295 g l −1 glucose in very high gravity (VHG) fermentation conditions without the supplementation of exogenous amino acids and vitamins with a theoretical yield of 90% (Wang et al ., ).…”

“…A significant collection of classical genetics tools have been explored and are now routine metabolic engineering practices in Z. mobilis , including stable and transferable plasmids, shuttle vectors, promoters, transformation methods such as conjugation and electroporation, reporter genes such as green fluorescent protein (GFP) and ice nucleation activity, and transposon mutagenesis strategies (Skotnicki et al ., ; Carey et al ., ; Browne et al ., ; Conway et al ., ,b; Arfman et al ., ; Delgado et al ., ; Drainas et al ., ; Zhang et al ., , ; Pappas et al ., ; Douka et al ., ; Yang et al ., ,b, ; Dong et al ., , ; Pappas, ; Jia et al ., ; Dunn and Rao, ; Yi et al ., ; Wang et al ., ). These methods have been widely reviewed (Panesar et al ., ; He et al ., ) and are not further described here.…”

Dynamic cell responses in Thermoanaerobacterium sp. under hyperosmotic stress

“…High substrate concentration leads to elevated osmotic pressure and can thus cause cell dehydration 5 , plasmolysis 6 , and even cell inactivation 7 . These effects results in extended lag phase, metabolic disturbance, low productivity, and stagnation of fermentation 8 , 9 .…”