Expressing 2-keto acid pathway enzymes significantly increases photosynthetic isobutanol production

Hao Xie,Peter Lindblad

doi:10.1186/s12934-022-01738-z

Abstract

BackgroundCyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO2 and solar energy in a direct process.ResultsHeterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L−1 in short-term screening experiments. Through modulating kivdS286T copy numbers as well as the composition of the 5′-region, a final Synechocystis strain HX47 with three copies of kivdS286T showed a significantly improved isobutanol production of 144 mg L−1, an 177% increase compared to the previously reported best producing strain under identical conditions.ConclusionsThis work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.Graphic abstract

Highlights

As an effective approach to alleviate the increased demand of energy and the concerns of global climate change caused by CO2 emissions, there is a great urgency to develop biofuels as new energy carriers to replace presently used fossil resources [1]
Expressing acetolactate synthase (AlsS) has an effect on isobutanol biosynthesis Acetolactate synthase (AlsS), catalyzing condensation of pyruvate into 2-acetolactate, is the first enzyme of the 2-keto acid pathway, which plays a vital role in converting more carbon from the central metabolite pyruvate towards isobutanol synthesis
AlsS functions as acetolactate synthase to condense two pyruvate molecules, and it was experimentally verified that it functions as α-ketoisovalerate decarboxylase [20]

Summary

Results

Heterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxyacid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L−1 in short-term screening experiments. Through modulating kivdS286T copy numbers as well as the composition of the 5′-region, a final Synechocystis strain HX47 with three copies of kivdS286T showed a significantly improved isobutanol production of 144 mg L−1, an 177% increase compared to the previously reported best producing strain under identical conditions

Conclusions

Introduction

Materials and methods

Results and discussion

Conclusion