Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803.

Background: Cyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of α-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (KivdS286T ). However, isobutanol production still remained low.

Results: In the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 μmol photons m-2  s-1 ) showed the highest isobutanol production with an in-flask titer of 194 mg l-1 after 10 days and 435 mg l-1 at day 40. This corresponds to a cumulative isobutanol production of 911 mg l-1 , with a maximal production rate of 43.6 mg l-1  day-1 observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of KivdS286T was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803.

Conclusion: The present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.