Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis.

Furong Tan,Mingxiong He,Qili Zhu,Jingli Wang,Han Qin,Guoquan Hu,Lichun Dai,Bo Wu,Zongxia Shui

doi:10.1186/s12934-015-0398-y

Abstract

BackgroundWith the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. One of the major challenges faced during the process of biofuel production is the low tolerance of the microbial host towards increasing biofuel concentrations.ResultsHere, we demonstrate that the ethanol tolerance of Zymomonas mobilis can be greatly enhanced through the random mutagenesis of global transcription factor RpoD protein, (σ70). Using an enrichment screening, four mutants with elevated ethanol tolerance were isolated from error-prone PCR libraries. All mutants showed significant growth improvement in the presence of ethanol stress when compared to the control strain. After an ethanol (9 %) stress exposure lasting 22 h, the rate of glucose consumption was approximately 1.77, 1.78 and 1.39 g L−1 h−1 in the best ethanol-tolerant strain ZM4-mrpoD4, its rebuilt mutant strain ZM4-imrpoD and the control strain, respectively. Our results indicated that both ZM4-mrpoD4 and ZM4-imrpoD consumed glucose at a faster rate after the initial 9 % (v/v) ethanol stress, as nearly 0.64 % of the initial glucose remained after 54 h incubation versus approximately 5.43 % for the control strain. At 9 % ethanol stress, the net ethanol productions by ZM4-mrpoD4 and ZM4-imrpoD during the 30–54 h were 13.0–14.1 g/l versus only 6.6–7.7 g/l for the control strain. The pyruvate decarboxylase activity of ZM4-mrpoD4 was 62.23 and 68.42 U/g at 24 and 48 h, respectively, which were 2.6 and 1.6 times higher than the control strain. After 24 and 48 h of 9 % ethanol stress, the alcohol dehydrogenase activities of ZM4-mrpoD4 were also augmented, showing an approximate 1.4 and 1.3 times increase, respectively, when compared to the control strain. Subsequent quantitative real-time PCR analysis under these stress conditions revealed that the relative expression of pdc in cultured (6 and 24 h) ZM4-mrpoD4 increased by 9.0- and 12.7-fold when compared to control strain.ConclusionsCollectively, these results demonstrate that the RpoD mutation can enhance ethanol tolerance in Z. mobilis. Our results also suggested that RpoD may play an important role in resisting high ethanol concentration in Z. mobilis and manipulating RpoD via global transcription machinery engineering (gTME) can provide an alternative and useful approach for strain improvement for complex phenotypes.

Highlights

With the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important
Our results indicated that the net ethanol production by ZM4-mrpoD4 and ZM4-imrpoD during the 30–54 h were 13.0–14.1 g/l versus only 6.6–7.7 g/l for the control strain, indicating that ZM4-mrpoD4 and ZM4-imrpoD can produce more ethanol than the control strain under the condition of 9 % ethanol stress, which was consistent with its higher cell growth and faster glucose consumption under the ethanol stress condition
Our results suggest that sigma 70 may play an important role in resisting high ethanol concentration in Z. mobilis, with manipulation of σ70 allowing for another avenue for strain improvement

Summary

Introduction

With the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. With the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from. Z. mobilis, a Gramnegative facultative anaerobic bacterium, has attracted considerable interest It has a unique metabolism and ability to produce ethanol and/or other valuable chemicals from simple sugars via its unique Entner–Doudoroff (ED) pathway [3,4,5,6]. High ethanol concentrations, osmotic pressure, and oxidative stresses are all major stress that can impede the specific growth rate and viability of Z. mobilis cells as well as its ethanol production [7,8,9].

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Conclusion