Background: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose. Results: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines. Conclusion: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.
Background: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. Results: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, P emrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters P vtac, P vtrc, and P vtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, P emrR. In the presence of vanillic acid, P vtac, P vtrc, and P vtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with P emrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. Conclusions: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.
Efficient lignin valorization could add more than 10-fold the value gained from burning it for energy and is critical for economic viability of future biorefineries. However, lignin-derived aromatics from biomass pretreatment are known to be potent fermentation inhibitors in microbial production of fuels and other value-added chemicals. In addition, isopropyl-β-D-1-thiogalactopyranoside and other inducers are routinely added into fermentation broth to induce the expression of pathway enzymes, which further adds to the overall process cost. An autoregulatory system that can diminish the aromatics' toxicity as well as be substrate-inducible can be the key for successful integration of lignin valorization into future lignocellulosic biorefineries. Toward that goal, in this study an autoregulatory system is demonstrated that alleviates the toxicity issue and eliminates the cost of an external inducer. Specifically, this system is composed of a catechol biosynthesis pathway coexpressed with an active aromatic transporter CouP under induction by a vanillin self-inducible promoter, ADH7, to effectively convert the ligninderived aromatics into value-added chemicals using Escherichia coli as a host. The constructed autoregulatory system can efficiently transport vanillin across the cell membrane and convert it to catechol. Compared with the system without CouP expression, the expression of catechol biosynthesis pathway with transporter CouP significantly improved the catechol yields about 30% and 40% under promoter pTrc and ADH7, respectively. This study demonstrated an aromaticinduced autoregulatory system that enabled conversion of ligninderived aromatics into catechol without the addition of any costly, external inducers, providing a promising and economically viable route for lignin valorization.
Background: First generation bioethanol production utilizes the starch fraction of maize, which accounts for approximately 60% of the ash-free dry weight of the grain. Scale-up of this technology for fuels applications has resulted in a massive supply of distillers' grains with solubles (DGS) coproduct, which is rich in cellulosic polysaccharides and protein. It was surmised that DGS would be rapidly adopted for animal feed applications, however, this has not been observed based on inconsistency of the product stream and other logistics-related risks, especially toxigenic contaminants. Therefore, efficient valorization of DGS for production of petroleum displacing products will significantly improve the techno-economic feasibility and net energy return of the established starch bioethanol process. In this study, we demonstrate 'one-pot' bioconversion of the protein and carbohydrate fractions of a DGS hydrolysate into C4 and C5 fusel alcohols through development of a microbial consortium incorporating two engineered Escherichia coli biocatalyst strains. Results: The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type E. coli strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars. Up to 12 g/L fusel alcohols was produced from glucose or xylose synthetic medium by E. coli BLF2. The second strain, E. coli AY3, was dedicated for utilization of proteins in the hydrolysates to produce mixed C4 and C5 alcohols. To maximize conversion yield by the co-culture, the inoculation ratio between the two strains was optimized. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fusel alcohol titer of up to 10.3 g/L from DGS hydrolysates. The engineered E. coli co-culture system was shown to be similarly applicable for biofuel production from other biomass sources, including algae hydrolysates. Furthermore, the co-culture population dynamics revealed by quantitative PCR analysis indicated that despite the growth rate difference between the two strains, co-culturing didn't compromise the growth of each strain. The q-PCR analysis also demonstrated that fermentation with an appropriate initial inoculation ratio of the two strains was important to achieve a balanced co-culture population which resulted in higher total fuel titer. Conclusions: The efficient conversion of DGS hydrolysates into fusel alcohols will significantly improve the feasibility of the first generation bioethanol process. The integrated carbohydrate and protein conversion platform developed here is applicable for the bioconversion of a variety of biomass feedstocks rich in sugars and proteins.