Isobutanol production by combined in vivo and in vitro metabolic engineering

Mamta Gupta; Matthew Wong; Kamran Jawed; Kamil Gedeon; Hannah Barrett; Marcelo Bassalo; Clifford Morrison; Danish Eqbal; Syed Shams Yazdani; Ryan T Gill; Jiaqi Huang; Marc Douaisi; Jonathan Dordick; Georges Belfort; Mattheos A G Koffas

doi:10.1016/j.mec.2022.e00210

Isobutanol production by combined in vivo and in vitro metabolic engineering

Metab Eng Commun. 2022 Oct 23:15:e00210. doi: 10.1016/j.mec.2022.e00210. eCollection 2022 Dec.

Authors

Mamta Gupta^{1

2}, Matthew Wong^{1

3}, Kamran Jawed^{1

4}, Kamil Gedeon¹, Hannah Barrett¹, Marcelo Bassalo⁵, Clifford Morrison¹, Danish Eqbal⁴, Syed Shams Yazdani⁴, Ryan T Gill⁵, Jiaqi Huang¹, Marc Douaisi¹, Jonathan Dordick^{1

3}, Georges Belfort^{1

3}, Mattheos A G Koffas^{1

6

3}

Affiliations

¹ Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
² Department of Botany and Environmental Studies, DAV University, Jalandhar, 144 001, Punjab, India.
³ Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
⁴ DBT-ICGEB Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India.
⁵ Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, 80309, USA.
⁶ Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.

Abstract

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L^-1 h^-1, and titers of 5.6 and 1.78 g L^-1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

Keywords: Bicistronic; CRISPR; CRISPRi; Fed-batch; Isobutanol; α-Ketoisovalerate.