Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid

Shinjinee Sengupta; Damini Jaiswal; Annesha Sengupta; Shikha Shah; Shruti Gadagkar; Pramod P Wangikar

doi:10.1186/s13068-020-01727-7

Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid

Biotechnol Biofuels. 2020 May 18:13:89. doi: 10.1186/s13068-020-01727-7. eCollection 2020.

Authors

Shinjinee Sengupta^{1

2}, Damini Jaiswal¹, Annesha Sengupta¹, Shikha Shah^{1

2}, Shruti Gadagkar¹, Pramod P Wangikar^{1

2

3}

Affiliations

¹ 1Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076 India.
² 2DBT-Pan IIT Center for Bioenergy, Indian Institute of Technology Bombay, Powai, Mumbai, 400076 India.
³ 3Wadhwani Research Center for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076 India.

Abstract

Background: Cyanobacteria, a group of photosynthetic prokaryotes, are being increasingly explored for direct conversion of carbon dioxide to useful chemicals. However, efforts to engineer these photoautotrophs have resulted in low product titers. This may be ascribed to the bottlenecks in metabolic pathways, which need to be identified for rational engineering. We engineered the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801 to produce succinate, an important platform chemical. Previously, engineering of the model cyanobacterium S. elongatus PCC 7942 has resulted in succinate titer of 0.43 g l^-1 in 8 days.

Results: Building on the previous report, expression of α-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and phosphoenolpyruvate carboxylase yielded a succinate titer of 0.6 g l^-1 in 5 days suggesting that PCC 11801 is better suited as host for production. Profiling of the engineered strains for 57 intermediate metabolites, a number of enzymes and qualitative analysis of key transcripts revealed potential flux control points. Based on this, we evaluated the effects of overexpression of sedoheptulose-1,7-bisphosphatase, citrate synthase and succinate transporters and knockout of succinate dehydrogenase and glycogen synthase A. The final construct with seven genes overexpressed and two genes knocked out resulted in photoautotrophic production of 0.93 g l^-1 succinate in 5 days.

Conclusion: While the fast-growing strain PCC 11801 yielded a much higher titer than the model strain, the efficient photoautotrophy of this novel isolate needs to be harnessed further for the production of desired chemicals. Engineered strains of S. elongatus PCC 11801 showed dramatic alterations in the levels of several metabolites suggesting far reaching effects of pathway engineering. Attempts to overexpress enzymes deemed to be flux controlling led to the emergence of other potential rate-limiting steps. Thus, this process of debottlenecking of the pathway needs to be repeated several times to obtain a significantly superior succinate titer.

Keywords: Cyanobacteria; Flux control; Metabolites; Succinic acid.