Engineering Terpene Biosynthesis in Streptomyces for Production of the Advanced Biofuel Precursor Bisabolene

Abstract

The past decade has witnessed a large influx of research toward the creation of sustainable, biologically derived fuels. While significant effort has been exerted to improve production capacity in common hosts, such as Escherichia coli or Saccharomyces cerevisiae, studies concerning alternate microbes comparatively lag. In an effort to expand the breadth of characterized hosts for fuel production, we map the terpene biosynthetic pathway in a model actinobacterium, Streptomyces venezuelae, and further alter secondary metabolism to afford the advanced biofuel precursor bisabolene. Leveraging information gained from study of the native isoprenoid pathway, we were able to increase bisabolene titer nearly 5-fold over the base production strain, more than 2 orders of magnitude greater than the combined terpene yield in the wild-type host. We also explored production on carbon sources of varying complexity to, notably, define this host as one able to perform consolidated bioprocessing.

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

“…The process of cell-extract preparation can be divided into five stages in the order of (1) cell-growth, (2) washing, (3) sonication, (4) run-off and (5) dialysis. We merged the Streptomyces method for stages (1-2) with the E. coli TX-TL methodology for stages (3)(4)(5). This new protocol provided a significant baseline level of sfGFP fluorescence (154 nM), whereas by following the separate protocols on their own, only trace levels of sfGFP fluorescence (≈5 nM) were observed (data not shown).…”

“…Recently, Streptomyces venezuelae, the chloramphenicol producer, has been adopted by synthetic biology for its use in metabolic engineering [2], since it is relatively well characterized, has strong promoter tools and genome engineering plasmids for integration [3][4][5]. Whilst it is not as characterized as Streptomyces coelicolor A3(2), in contrast, S. venezuelae provides significant advantages such as a fast growth (≈T D = 40 min) and no aggregation during liquid culture [3].…”

Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes

“…Strain engineering efforts have provided an array of new opportunities to engineer alternate microbial strains as hosts for the nonnatural production of isoprenoid-based biofuels (13,15,58,59). Engineering alternate microbial hosts also enables researchers to explore their ability to utilize a vast array of carbon sources for biofuel production.…”

“…Both enzymes have been introduced in microbes either with a heterologous MVA pathway (73,74) or with an optimized endogenous DXP pathway (13). Modulation of the DXP pathway in alternate microbial hosts, such as cyanobacteria, Corynebacterium glutamicum, and Streptomyces sp., has improved biofuel yields in such hosts (13,15,58,59). Combinatorial gene expression of Table 1).…”

“…Various endogenous (the host's genes and pathways), homologous (genes and pathways of other organisms distantly related to the host's), and heterologous (genes and pathways of other organisms that do not correspond in evolutionary origin to the host's) genes and pathways need to be optimized inside a host to enhance biofuel production. The plenteous genetic modifications not only improve substrate utilization, product titers, and yields but also extend the variety of biofuels from microbial hosts (10)(11)(12)(13)(14)(15)(16)(17).…”