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

Abstract

Background Cyanobacteria, 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. 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), 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. Conclusions This 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

Sustainable production of photosynthetic isobutanol and 3-methyl-1-butanol in the cyanobacterium Synechocystis sp. PCC 6803

“…Natural transformation of integrative plasmids and conjugation of self-replicating plasmids were performed as described previously [ 15 ]. All generated engineered Synechocystis strains in this study are listed in Table 1 .…”

“…PCC 6803 strains used in this study Strain Relevant genotypes a References WT Wild-type Synechocystis sp. PCC 6803 [ 12 ] HX29 Δddh ::(P trc BCD- kivd S286T -T)-Cm R , Δsll1564 ::(P trc BCD- kivd S286T -T)-Sp R , pEEK2-(P trc BCD- kivd S286T -T)-Km R [ 15 ] HX42 Δddh ::(P trc BCD- slr1192 OP - alsS -T)-Cm R , Δslr0168 ::(P trc BCD- ilvC - ilvD -T)-Sp R , pEEK2-(P trc BCD- kivd S286T -T)-Km R [ 15 ] HX56 Δddh ::(P trc BCD- kivd S286T -T)-Cm R , Δsll1564 ::(P trc BCD- kivd S286T -T)-Sp R , Δslr0168 ::(P trc BCD- kivd S286T -T)-Em R , pEEK2-(P trc BCD- kivd S286T -T)-Km R This study HX61 Δddh ::(P trc BCD- kivd S286T -T)-Cm R , Δsll1564 ::(P trc BCD- kivd S286T -T)-Sp R , Δslr0168 ::Em R , pEEK2-(P trc BCD- kivd S286T -T)-Km R This study HX62 Δddh ::(P trc BCD- kivd S286T -T)-Cm R , ΔPEPc ::(P trc BCD- kivd ...…”

“…Crude protein extraction and SDS-PAGE/Western-immunoblot were performed as previously detailed [ 15 ]. Ten micrograms (Strep-tagged proteins) and 20 μg (His-tagged and Flag-tagged proteins) of soluble crude proteins were loaded for protein expression analysis.…”

“…This engineered Kivd S286T has been used throughout following studies. In a more recent study, photosynthetic IB production was further enhanced by either increased Kivd S286T expression level or integration of a complete 2-keto acid pathway [ 15 ]. Even with substantial progress reported on 2-keto acid pathway for photosynthetic IB and 3M1B production, the achieved production is still far behind to that of heterotrophic microorganisms [ 10 ] or cell-free system using a synthetic biochemistry approach [ 16 ].…”

Whole-genome sequence of the filamentous diazotrophic cyanobacterium Tolypothrix sp. PCC 7712 and its comparison with non-diazotrophic Tolypothrix sp. PCC 7601

“…These features form the basis for a sustainable biotech-workhorse being independent of organic carbon and reduced nitrogen compounds, which usually add significantly to the ecological footprint of biotech processes. Although numerous proof-of-concept studies show the feasibility of using cyanobacteria as solar cell factories for producing commodity products ( Angermayr et al, 2015 ; Betterle and Melis, 2019 ; Hoschek et al, 2019 ; Xie and Lindblad, 2022 ), only a few examples exist where cyanobacterial biocatalysts are applied at an economic scale. All these processes are based on biomass, which needs to be harvested to extract, e.g., pigments or lipids ( Jones and Mayfield, 2012 ; Garlapati et al, 2019 ; Nagappan et al, 2020 ).…”