Use of Transporter Plug-Ins for Enhanced Productivity and Reduced Byproduct Formation of Bioalkanes and Related Compounds

Synthetic biology hopes to predictably design from scratch or reprogram cells into cellular factories for the production of important chemicals, pharmaceuticals and fuels from cheap renewable feedstocks. A critical, yet understudied, element of this is the control of compound transport across the cell membrane both into and out of the cell as a means of avoiding issues such as substrate access limitations, substrate and product inhibition and aiding product recovery ¹.

We report here a successful strategy for the contextual evaluation of this topic using a library of transport modifying plug-ins combined with multifactorial characterisation. Auxiliary plasmids designed for analogue expression of membrane proteins (pUMP) were used to express a library of transport proteins as plug-ins alongside two biosynthesis plasmids pGEC41, which oxidises hydrocarbons into primary alkanols and pADAR7942 which synthesizes bioalkanes from metabolic free fatty acid precursors. We demonstrate here benefits of the transporter plug-in approach for:

(i)                    Facilitated delivery of bulky and hydrophobic substrates to improve whole-cell biocatalysis rates by up to 70 fold for hydrocarbon substrates

(ii)                   Industrially relevant product yields of over 40g/L_{organic phase} (8g/L_total) achieved using this strategy

(iii)                  Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols

(iv)                 Improving bioalkane synthesis yields from glycerol by >5 fold

(v)                  Reducing alkanol and aldehyde intermediate formation in biosynthesis of bioalkanes by >10 fold

(vi)                 The integration with in-situ product removal strategies to improve bioalkane yields by 10 fold compared to the starting process.

This library and plug-in approach is of broad appeal for biological production of hydrophobic compounds and could be a key enabling technology for biological routes for producing a wider range of hydrophobic compounds such as biofuels, fine and specialty chemicals and pharmaceutical intermediates.

1.         Chen, R. R. Permeability issues in whole-cell bioprocesses and cellular membrane engineering. Appl. Microbiol. Biotechnol. 74, 730–738 (2007).