Expression of alcohol-forming fatty acyl-CoA reductases in E. coli can result in the biosynthesis of fatty alcohols from endogenous E. coli fatty acids, but the levels were quite low . To improve the production of fatty alcohols, Steen et al. carried out a further genetic modification of E. coli, and achieved an increased titer (~60 mg/L) of the medium chain fatty alcohols (C12 or C14 alcohols) . In their strategy, they employed thioesterases with different substrate specificities to tailor the composition of the FFAs, and used the aldehyde-forming fatty acyl-CoA reductase Acr1 for the conversion of fatty acyl-CoAs to fatty aldehydes. The synthesized fatty aldehydes can be further converted to fatty alcohols by an unknown alcohol dehydrogenase/aldehyde reductase of E. coli .
However, Steen et al. just obtained a small quantity of C16/18 alcohol, though they used the thioesterase ‘TesA, which was capable of yielding a large proportion of C16/18 FFA . As aforementioned, expression of Acr1 in E. coli can only obtain a predominant C12/14 alcohol, even if the longer FFAs are supplied. Therefore, their poor C16/18 alcohol production is probably attributed to the Acr1 they employed. However, Reiser and Somerville spectroscopically assayed the substrate specificity of an unpurified Acr1 protein by measuring the acyl-CoA-dependent oxidation of NADPH, and found Acr1 had the biggest substrate preference towards C16/18 acyl-CoAs . It suggests that the endogenous alcohol dehydrogenase/aldehyde reductase of E. coli may have substrate preference for fatty aldehydes with shorter chain lengths, and thus block the conversion of C16/18 aldehydes to corresponding alcohols. This bioconversion process will be much clearer if the purified Acr1 protein can be characterized by determining its synthesized products.
We are cognizant of the fact that it is impossible to obtain all our desired fatty alcohols just by tailoring the composition of FFAs. We thus replaced Acr1 with FAR as the fatty acyl-CoA reductase for the production of long chain fatty alcohols, given FAR preferred the longer acyl groups. To data, no research was performed to optimize the long chain fatty alcohol biosynthesis pathway. Therefore, we combined the expression of thioesterase and acyl-CoA synthase with FAR to enhance the long chain fatty alcohol production from glucose. Our achieved titer of 101.5 mg/L represents the highest C16/18 alcohol production ever reported [4, 19].
In addition, Steen et al. found the importance of acyl-CoA synthase in improving the fatty alcohol production . It is of significance to explore and find a proper acyl-CoA synthase that capable of enhancing the production of C12/14 or C16/18 alcohol. We found FadD possessed broad substrate specificity and high catalytic activity, based on the investigation of three different acyl-CoA synthases from E. coli (FadD), B. subtilis (Yngl) and S. cerevisiae (FAA2), respectively. FadD can convert most of the C12-C18 FFAs into their activated forms – fatty acyl-CoAs, and it was suitable for the production of both C12/14 and C16/18 alcohols.
The level of free CoA may also play an important role in the conversion of FFAs to fatty acyl-CoAs, since free CoA directly participates the reaction as a substrate. Once the fatty acyl-CoAs accumulate, that will lead to a reduction of free CoA. The decreased level of free CoA may further block the activation of FFAs. In contrast, increased level of free CoA will benefit the conversion of FFAs to fatty acyl-CoAs.
With a series of combination across different thioesterases, acyl-CoA synthases and fatty acyl-CoA reductases, we constructed two engineered strains (Zh072 and Zh054) that were capable of high-specificity production of C12/14 and C16/18 alcohols, respectively. Lennen et al. achieved a high level of fatty acid production by using a medium-strength PBAD . Therefore, it is possible to enhance the fatty alcohol production by optimization of the expression level of fatty alcohol biosynthesis pathway. No enhanced fatty alcohol production was obtained when either BTE or acr1 was under the control of PBAD. However, an obviously improved fatty alcohol production was achieved by optimization of the plasmid copy number. This result provides a useful clue for enhancing the fatty alcohol production. Of course, a more detailed optimization of the expression level is still needed to further improve the production of fatty alcohols.
Given no process data is available on the fatty alcohol production by engineered E. coli, we evaluated the performances of three well-performed strains in the fed-batch fermentation. The maximum fatty alcohol productivity was observed in the early stage of the post-induction. Over this phase, fatty alcohol production ceased. Given FFAs can be also converted to fatty aldehydes [6, 21–23], maybe the released FFAs were predominately transformed to fatty aldehydes instead of fatty alcohols in the latter stage. To make the fermentation processes be clearer, it is needed to perform some further investigations focusing on the production of fatty aldehydes.
The lower titer in our shake-flask study was probably attributed to the low buffering capacity of our culture medium, whose pH decreased rapidly with the growth of cells. The resultant lower biomass caused the decreased fatty alcohol production. The fatty alcohol production dramatically increased in the fed-batch fermentation with pH adaption.
In addition, given that fatty acyl-CoA reductases need the participation of coenzyme NADPH , the production of fatty alcohols may be enhanced by expressing the NADPH regeneration system such as phosphite dehydrogenase and glucose-6-phosphate dehydrogenase in E. coli.