7-Dehydrocholesterol (7-DHC) has attracted increasing attentions due to its great medical value and the enlarging market demand of its ultraviolet-catalyzed product vitamin D3. Microbial production of 7-DHC from simple carbon has been recognized as an attractive complement to the traditional sources. Even though our previous work realized 7-DHC biosynthesis in Saccharomyces cerevisiae, the current productivity of 7-DHC is still too low to satisfy the demand of following industrialization. As increasing the compatibility between heterologous pathway and host cell is crucial to realize microbial overproduction of natural products with complex structure and relative long pathway, in this study, combined efforts in tuning the heterologous Δ24-dehydrocholesterol reductase (DHCR24) and manipulating host cell were applied to promote 7-DHC accumulation.

Butanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol.

Limonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene.

Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources.

Pseudomonas putida is a promising candidate for the industrial production of biofuels and biochemicals because of its high tolerance to toxic compounds and its ability to grow on a wide variety of substrates. Engineering this organism for improved performances and predicting metabolic responses upon genetic perturbations requires reliable descriptions of its metabolism in the form of stoichiometric and kinetic models.

Bioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. 13C metabolic flux analysis (13C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied 13C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by Escherichia coli. Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.

Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose.

Functional sugar alcohols have been widely used in the food, medicine, and pharmaceutical industries for their unique properties. Among these, erythritol is a zero calories sweetener produced by the yeast Yarrowia lipolytica. However, in wild-type strains, erythritol is produced with low productivity and yield and only under high osmotic pressure together with other undesired polyols, such as mannitol or d-arabitol. The yeast is also able to catabolize erythritol in non-stressing conditions.

Interests in renewable fuels have exploded in recent years as the serious effects of global climate change become apparent. Microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered Pichia pastoris, an industrial workhorse in heterologous enzyme production, to produce the biofuel isobutanol from two renewable carbon sources, glucose and glycerol. Our strategy exploited the yeast's amino acid biosynthetic pathway and diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production. To further demonstrate the versatility of our yeast platform, we incorporated a broad-substrate-range alcohol-O-acyltransferase to generate a variety of volatile esters, including isobutyl acetate ester and isopentyl acetate ester.

Inefficient utilization of glycerol by Clostridium beijerinckii (Cb) is a major impediment to adopting glycerol metabolism as a strategy for increasing NAD(P)H regeneration, which would in turn, alleviate the toxicity of lignocellulose-derived microbial inhibitory compounds (LDMICs, e.g., furfural), and improve the fermentation of lignocellulosic biomass hydrolysates (LBH) to butanol. To address this problem, we employed a metabolic engineering strategy to enhance glycerol utilization by Cb.

Yarrowia lipolytica, an oleaginous yeast, is a promising platform strain for production of biofuels and oleochemicals as it can accumulate a high level of lipids in response to nitrogen limitation. Accordingly, many metabolic engineering efforts have been made to develop engineered strains of Y. lipolytica with higher lipid yields. Genome-scale model of metabolism (GEM) is a powerful tool for identifying novel genetic designs for metabolic engineering. Several GEMs for Y. lipolytica have recently been developed; however, not many applications of the GEMs have been reported for actual metabolic engineering of Y. lipolytica. The major obstacle impeding the application of Y. lipolytica GEMs is the lack of proper methods for predicting phenotypes of the cells in the nitrogen-limited condition, or more specifically in the stationary phase of a batch culture.

The steadily increasing demand for diesel fuels calls for renewable energy sources. This has attracted a growing amount of research to develop advanced, alternative biodiesel worldwide. Several major disadvantages of current biodiesels are the undesirable physical properties such as high viscosity and poor low-temperature operability. Therefore, there is an urgent need to develop novel and advanced biodiesels.

Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi.

Cellulosic biomass is a promising resource for bioethanol production. However, various sugars in plant biomass hydrolysates including cellodextrins, cellobiose, glucose, xylose, and arabinose, are poorly fermented by microbes. The commonly used ethanol-producing microbe Saccharomyces cerevisiae can usually only utilize glucose, although metabolically engineered strains that utilize xylose have been developed. Direct fermentation of cellobiose could avoid glucose repression during biomass fermentation, but applications of an engineered cellobiose-utilizing S. cerevisiae are still limited because of its long lag phase. Bioethanol production from biomass-derived sugars by a cellulolytic filamentous fungus would have many advantages for the biorefinery industry.

The filamentous fungus Trichoderma reesei, the most widely used cellulase producer, also has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high value-added products. However, such applications are thwarted by the time-consuming metabolic engineering processes (design-build-test-learn cycle) for T. reesei, resulted from (i) the spore separation-mediated purification as the multinucleate hyphae, (ii) transformant screening for high expression levels since unavailable of episomal expression system, and (iii) cases of inexpressible heterologous proteins.

Production of the versatile bulk chemical 1,2-propanediol and the potential biofuel 1-propanol is still dependent on petroleum, but some approaches to establish bio-based production from renewable feed stocks and to avoid toxic intermediates have been described. The biotechnological workhorse Corynebacterium glutamicum has also been shown to be able to overproduce 1,2-propanediol by metabolic engineering. Additionally, C. glutamicum has previously been engineered for production of the biofuels ethanol and isobutanol but not for 1-propanol.

Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance.

Triacylglycerols (TAGs) rich in medium-chain fatty acids (MCFAs, C10-14 fatty acids) are valuable feedstocks for biofuels and chemicals. Natural sources of TAGs rich in MCFAs are restricted to a limited number of plant species, which are unsuitable for mass agronomic production. Instead, the modification of seed or non-seed tissue oils to increase MCFA content has been investigated. In addition, microbial oils are considered as promising sustainable feedstocks for providing TAGs, although little has been done to tailor the fatty acids in microbial TAGs.

Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica.

l-Histidine biosynthesis is embedded in an intertwined metabolic network which renders microbial overproduction of this amino acid challenging. This is reflected in the few available examples of histidine producers in literature. Since knowledge about the metabolic interplay is limited, we systematically perturbed the metabolism of Corynebacterium glutamicum to gain a holistic understanding in the metabolic limitations for l-histidine production. We, therefore, constructed C. glutamicum strains in a modularized metabolic engineering approach and analyzed them with LC/MS-QToF-based systems metabolic profiling (SMP) supported by flux balance analysis (FBA).