用于厌氧生产异丁醇的代谢工程。

Metabolic engineering of for anaerobic isobutanol production.

作者信息

Qiu Mengyue, Shen Wei, Yan Xiongyin, He Qiaoning, Cai Dongbo, Chen Shouwen, Wei Hui, Knoshaug Eric P, Zhang Min, Himmel Michael E, Yang Shihui

机构信息

1State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, and School of Life Sciences, Hubei University, Wuhan, 430062 China.

2Biosciences Centers, National Renewable Energy Laboratory, Golden, CO 80401 USA.

出版信息

Biotechnol Biofuels. 2020 Jan 25;13:15. doi: 10.1186/s13068-020-1654-x. eCollection 2020.

BACKGROUND

Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery.

RESULTS

In this study, the effect of isobutanol on was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as from is required for isobutanol production in . Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the gene driven by the tetracycline-inducible promoter . In addition, we determined that overexpression of a heterologous gene and two native genes ( and ) involved in valine metabolism in a recombinant strain expressing can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, --, driven by and the gene driven by the constitutive strong promoter, , were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks.

CONCLUSIONS

This study demonstrated that overexpression of in combination with a synthetic heterologous operon, --, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in .

背景

通过生物化学转化从可再生生物质中获得的生物燃料和增值生物化学品，已引起人们极大关注，以实现全球可持续能源和环境目标。异丁醇是一种四碳醇，具有诸多优势，使其成为极具吸引力的化石燃料替代品。[具体细菌名称]是一种高效的厌氧产乙醇细菌，使其成为用于生物精炼厂的有前景的工业平台。

结果

在本研究中，研究了异丁醇对[具体细菌名称]的影响，并构建了各种产异丁醇的重组菌株。结果表明，[具体细菌名称]亲本菌株能够在低于12 g/L的异丁醇存在下生长，而浓度大于16 g/L会抑制细胞生长。在[具体细菌名称]中生产异丁醇需要整合编码2-酮异戊酸脱羧酶的异源基因，如来自[具体来源]的[具体基因名称]。此外，在含有由四环素诱导型启动子[具体启动子名称]驱动的[具体基因名称]的重组菌株中，异丁醇产量从几乎为零增加到100 - 150 mg/L。此外，我们确定在表达[具体基因名称]的重组[具体细菌名称]菌株中，参与缬氨酸代谢的异源[具体基因名称]和两个天然基因（[具体基因1名称]和[具体基因2名称]）的过表达可将丙酮酸从乙醇生产转向异丁醇生物合成。这种工程改造将异丁醇产量提高到1 g/L以上。最后，含有由[具体启动子名称]驱动的合成操纵子[具体操纵子名称] - [具体基因名称] - [具体基因名称]和由组成型强启动子[具体启动子名称]驱动的[具体基因名称]的重组菌株，被确定能极大提高异丁醇产量，最高滴度约为4.0 g/L。最后，通气对异丁醇生产有负面影响，通气较差的烧瓶中产生的异丁醇更多。