Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

Noemi Ruiz‐Lopez,Richard P Haslam,Frédéric Beaudoin,Johnathan A Napier,Sarah Usher,Richard Broughton,Joaquin J Salas

doi:10.1111/pbi.12679

Noemi Ruiz‐Lopez, Richard P Haslam + Show 5 more

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https://doi.org/10.1111/pbi.12679

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Abstract

SummaryThe functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl‐CoA substrate pool, through the co‐expression of acyl‐ACP thioesterases, to direct the accumulation of medium‐chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl‐CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non‐native WEs in the seeds of transgenic Camelina plants. A total of fourteen constructs were prepared containing selected FAR and WS genes in combination with an acyl‐ACP thioesterase. All enzyme combinations led to the successful production of wax esters, of differing compositions. The impact of acyl‐CoA thioesterase expression on wax ester accumulation varied depending on the substrate specificity of the WS. Hence, co‐expression of acyl‐ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co‐expression of the same acyl‐ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition. This was despite substantial remodelling of the acyl‐CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds.

Highlights

There is a growing recognition of the potential of so-called green factories to help address some of the major societal challenges that face the human race (Yuan and Grotewold, 2015)
Acyl-ACP products, primarily 16:0-and 18:1-ACP, synthesized by the plastidial fatty acid synthase (FAS) are hydrolysed by the FAtB and FATA thioesterases, respectively, and activated to CoA esters by long-chain acyl-CoA synthetases (LACS) during their export from the plastid. They enter the endoplasmic reticulum (ER)-associated acyl-CoA pool where they become available as the primary substrates for wax ester synthesis. This can be achieved by introduction and seed-specific expression of a fatty acyl reductase (FAR) and a wax synthase (WS) leading the production of novel wax esters
Seed-specific expression in Camelina of Wax synthase (WS), Fatty acyl-CoA reductase (FAR) and thioesterase combinations was examined as a strategy to effectively tailor wax ester composition and thereby produce industrial oils with properties mimicking those of spermaceti oil

Summary

The functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. Co-expression of acyl-ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co-expression of the same acyl-ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition This was despite substantial remodelling of the acyl-CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. This was despite substantial remodelling of the acyl-CoA pool, suggesting that these substrates were not efficiently incorporated into WEs These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds

Introduction

Results and discussion

Conclusion

Experimental procedures