Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

James Kirby,Anna Fuhrmann,John M Gladden,Joonhoon Kim,Veronica T Benites,Deepti Tanjore,Carrie Nicora ,Jon Magnuson ,Nathalie Munoz Munoz ,Jan‐Philip Prahl ,Bobbie‐Jo Webb‐Robertson ,Eric Sundström ,Kristin Burnum-Johnson ,Gina M Geiselman,Katharina Seibel,Junko Yaegashi,Yuqian Gao,Jeremy Zucker,Jeffrey M Skerker,Mary Bao Tran-Gyamfi,Xun Zhuang,Edward E K Baidoo

doi:10.1186/s13068-021-01950-w

Abstract

BackgroundMitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates.ResultsThis study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement.ConclusionsThis work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

Highlights

Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible
Enhancing α‐bisabolene titer in R. toruloides Previous work on engineering R. toruloides for production of α-bisabolene indicated that flux through the native mevalonate pathway is relatively high in this species
Since heterologous DNA was introduced into R. toruloides by Agrobacterium tumefaciens-mediated transformation (ATMT), which results in DNA integration at random loci with a variable copy number, it was of interest to investigate the correlation between copy number and α-bisabolene titer for a range of PGAPDH-bisabolene synthase (BIS) transformants

Summary

Introduction

Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. A number of C15 sesquiterpenes such as α-bisabolene [3] and farnesene [4] can function as diesel fuels, following reduction to their respective alkanes by hydrogenation Other terpenes such as the C 20 diterpene, ent-kaurene may provide bio-based replacements for petrochemicals with a variety of industrial applications [5]. This work builds upon previous efforts to produce 1,8-cineole and α-bisabolene in the single-celled basidiomycete, Rhodosporidium toruloides This fungus has previously been identified as an attractive host for production of biofuels and bioproducts from lignocellulosic biomass, in part due to its ability to co-metabolize multiple carbon sources, including C 5 and C 6 sugars and aromatic compounds found in lignocellulosic hydrolysates [6]

Methods

Results

Conclusion