Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering

ABSTRACTThe development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeastYarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported forY. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters inY. lipolyticaare enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

Download Full-text

Genetic Tools for Streamlined and Accelerated Pathway Engineering in Yarrowia lipolytica

Methods in Molecular Biology - Microbial Metabolic Engineering ◽

10.1007/978-1-4939-9142-6_11 ◽

2019 ◽

pp. 155-177 ◽

Cited By ~ 6

Author(s):

Lynn Wong ◽

Benjamin Holdridge ◽

Jake Engel ◽

Peng Xu

Keyword(s):

Yarrowia Lipolytica ◽

Pathway Engineering ◽

Genetic Tools

Download Full-text

Recent Advances in Producing Sugar Alcohols and Functional Sugars by Engineering Yarrowia lipolytica

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.648382 ◽

2021 ◽

Vol 9 ◽

Author(s):

Abdul Rahman Abbasi ◽

Jinle Liu ◽

Zhi Wang ◽

Anqi Zhao ◽

Hanjie Ying ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Pathway Engineering ◽

Sugar Alcohol ◽

Breeding Methods ◽

Biological Functions ◽

Sugar Alcohols ◽

Bioprocess Engineering ◽

Chemical Structures ◽

Recent Advances ◽

Catalyst Production

The sugar alcohols and functional sugars have wide applications in food, pharmaceutical, and chemical industries. However, the smaller quantities of natural occurring sugar alcohols and functional sugars restricted their applications. The enzymatic and whole-cell catalyst production is emerging as the predominant alternatives. The properties of Yarrowia lipolytica make it a promising sugar alcohol and functional sugar producer. However, there are still some issues to be resolved. As there exist reviews about the chemical structures, physicochemical properties, biological functions, applications, and biosynthesis of sugar alcohols and/or functional sugars in Y. lipolytica, this mini review will not only update the recent advances in enzymatic and microbial production of sugar alcohols (erythritol, D-threitol, and xylitol) and functional sugars (isomaltulose, trehalose, fructo-oligosaccharides, and galacto-oligosaccharides) by using recombinant Y. lipolytica but also focus on the studies of gene discovery, pathway engineering, expanding substrate scope, bioprocess engineering, and novel breeding methods to resolve the aforementioned issues.

Download Full-text

Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607295113 ◽

2016 ◽

Vol 113 (39) ◽

pp. 10848-10853 ◽

Cited By ~ 206

Author(s):

Peng Xu ◽

Kangjian Qiao ◽

Woo Suk Ahn ◽

Gregory Stephanopoulos

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Free Fatty Acids ◽

Chain Length ◽

Yarrowia Lipolytica ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Ethyl Esters ◽

Pathway Engineering ◽

Efficient Production

Harnessing lipogenic pathways and rewiring acyl-CoA and acyl-ACP (acyl carrier protein) metabolism in Yarrowia lipolytica hold great potential for cost-efficient production of diesel, gasoline-like fuels, and oleochemicals. Here we assessed various pathway engineering strategies in Y. lipolytica toward developing a yeast biorefinery platform for sustainable production of fuel-like molecules and oleochemicals. Specifically, acyl-CoA/acyl-ACP processing enzymes were targeted to the cytoplasm, peroxisome, or endoplasmic reticulum to generate fatty acid ethyl esters and fatty alkanes with tailored chain length. Activation of endogenous free fatty acids and the subsequent reduction of fatty acyl-CoAs enabled the efficient synthesis of fatty alcohols. Engineering a hybrid fatty acid synthase shifted the free fatty acids to a medium chain-length scale. Manipulation of alternative cytosolic acetyl-CoA pathways partially decoupled lipogenesis from nitrogen starvation and unleashed the lipogenic potential of Y. lipolytica. Taken together, the strategies reported here represent promising steps to develop a yeast biorefinery platform that potentially upgrades low-value carbons to high-value fuels and oleochemicals in a sustainable and environmentally friendly manner.

Download Full-text