Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.

B. Overall, the study is well-planned, logically presented, and provides sufficiently novel and interesting results. The manuscript should be appreciated among the metabolic engineering community.
C. adequate D. adequate F. Line 27, 215, 217, & 241 Claims of "highest" or "first" should in general be deleted. The authors have no way of knowing what is or is not reported in conferences, in press, or in patents under examination.
Line 74 Since it is already known that this pathway plays an important role in increasing acetyl-CoA for lipid accumulation in oleaginous yeasts the pathway should not be described as a novel pathway.
Line 115 Please expand the explanation on why this is interesting.
G. adequate H. well written

Reviewer #2
In this study, the authors engineered S. cerevisiae to overproduce free fatty acids and then transformed into alkanes and fatty alcohols via different metabolic engineering stategies. Through screening of specific pathway enzymes and endogenous alcohol dehydrogenases/aldehyde reductases, the authors obtained the highest titer of fatty acids, alkanes and fatty alcohols in S. cerevisaie. This is a significant milestone and breakthrough in fatty acid overproduction works in last five years. The quality of data and presentation is very high in this manuscript. The manuscript also well summarized the previous work and the conclusion is robustness. Overall, this paper is easy to read and worth publishing in Nature Communication.

Response to reviewer comments Dear Editor and Reviewers
Thanks for all your constructive comments and suggestions. Accordingly, we have modified our manuscript, and the changes are shown in red in the revised text. Here below is our point to point response to the reviewers' comments.

Responses to Reviewer 1
Question/Comment 1: A&E The manuscript by Zhou et al. describes production of fatty acids, fatty alcohols, and alkanes in Saccharomyces cerevisiae. First they use various previously developed modifications to improve free fatty acid production in S. cerevisiae. Additionally, they install a pathway to produce acetyl-CoA from citrate, which is an important pathway to increase acetyl-CoA supply in oleaginous yeasts. The best strain produces 10.8 g/L free fatty acids in 120 hr under fed-batch fermentation conditions. This titer is very impressive. A glucose consumption profile should be added to Fig. 2 so that readers can estimate the yields during the production. Next, they extend the pathway to alkanes. The titers are very low (~1 mg/L) due to low activity of ADO, which catalyzes the conversion of fatty aldehydes to alkanes. Finally, they extend the pathway to fatty alcohols. They install two types of fatty alcohol production pathways (1. FaCoAR, 2. CAR-ADH) and remove several competing pathways. The best strain produces 1.5 g/L fatty alcohols under fed-batch fermentation conditions. For Fig. 5, a glucose consumption profile should be added. The fed-batch fermentation experiment in Fig. 2b produces 10 g/L free fatty acids. However, the fed-batch fermentation experiment in Fig. 5c produces only 1.5 g/L fatty alcohols and 0.8 g/L free fatty acids (total 2.3 g/L). Please comment on why the free fatty acid (+ fatty alcohol) production in the fatty alcohol strain is much lower than that in the free fatty acid strain.

Reply 1:
We thank the reviewer for the positive comments.
1) The glucose consumption profile for the fed batch experiment was added in Fig 2c  and Fig 5d. 2) Concerning the comment about the lower titer of free fatty acid + fatty alcohol than that in the free fatty acid strain: the Free fatty acid production strain YJZ47 carries a genome integrated chimeric ACL pathway, RtFAS and overexpressed ACC1 with the deletion of HFd1, FAA1/4 and POX1, while fatty alcohol producing strain FOH33 just carries the quadruple deletion without enhancing precursor supply and fatty acid synthesis. Because we found that the fatty acid reduction (CAR activity) is limiting in fatty alcohol production the fatty acid synthesis is sufficient even without enhancing precursor supply and fatty acid synthesis (high level FFA accumulated, Fig. 5C). In future, the FFA reduction toward fatty alcohol should be further enhanced and then the fatty acid biosynthesis might need to be improved.