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. The engineered strain overexpressing the keto-acid degradation pathway was able to produce 284 mg/L of isobutanol when supplemented with 2-ketoisovalerate. To improve the production of isobutanol and eliminate the need to supplement the production media with the expensive 2-ketoisovalerate intermediate, we overexpressed a portion of the amino acid l-valine biosynthetic pathway in the engineered strain. While heterologous expression of the pathway genes from the yeast Saccharomyces cerevisiae did not lead to improvement in isobutanol production in the engineered P. pastoris, overexpression of the endogenous l-valine biosynthetic pathway genes led to a strain that is able to produce 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes by employing an episomal plasmid-based expression system further improved the production titer of isobutanol to 2.22 g/L, a 43-fold improvement from the levels observed in the original strain. Finally, heterologous expression of a broad-substrate-range alcohol-O-acyltransferase led to the production of isobutyl acetate ester and isopentyl acetate ester at 51 and 24 mg/L, respectively. In this study, we engineered high-level production of the biofuel isobutanol and the corresponding acetate ester by P. pastoris from readily available carbon sources. We envision that our work will provide an economic route to this important class of compounds and establish P. pastoris as a versatile production platform for fuels and chemicals.

Removal of non-cellulosic impurities from cotton fabric, known as scouring, by conventional alkaline treatment causes environmental problems and reduces physical strength of fabrics. In this study, an endo-polygalacturonase (EndoPG) from Aspergillus aculeatus produced in Pichia pastoris was evaluated for its efficiency as a bioscouring agent while most current bioscouring process has been performed using crude pectinase preparation. The recombinant EndoPG exhibited a specific activity of 1892.08 U/mg on citrus pectin under the optimal condition at 50 °C, pH 5.0 with a V max and K m of 65,451.35 μmol/min/mL and 15.14 mg/mL, respectively. A maximal activity of 2408.70 ± 26.50 U/mL in the culture supernatant was obtained by high cell density batch fermentation, equivalent to a 4.8 times greater yield than that from shake-flask culture. The recombinant enzyme was shown to be suitable for application as a bioscouring agent, in which the wettability of cotton fabric was increased by treatment with enzyme at 300 U/mL scouring solution at 40 °C, pH 5.0 for 1 h. The bio-scoured fabric has comparable wettability to that obtained by conventional chemical scouring, but has higher tensile strength. The work has demonstrated for the first time functions of A. aculeatus EndoPG on bioscouring in eco-textile processing. EndoPG alone was shown to possess effective scouring activity. High expression level and homogeneity could be achieved in bench-scale bioreactor.

The sustainable production of natural compounds is increasingly important in today’s industrial landscape. This study investigates the metabolic engineering of Saccharomyces cerevisiae for the efficient biosynthesis of valuable carotenoids: canthaxanthin, zeaxanthin, and astaxanthin. Utilizing a tailored parental yeast strain, Sp_Bc, we optimized the carotenoid pathway by screening and identifying CrtW and CrtZ enzymatic variants. The CrtW variant from Bradyrhizobium sp. achieved a canthaxanthin titer of 425.1 ± 69.1 µg/L, while the CrtZ variant from Pantoea ananatis achieved a zeaxanthin titer of 70.5 ± 10.8 µg/L. Additionally, we optimized carotenoid production by exploring enzyme fusion strategies for all three studied carotenoids and organelle compartmentalization specifically for enhancing astaxanthin synthesis. We further improved carotenoid production by integrating the optimal gene constructs into the yeast genome and deleting the GAL80 gene, enabling the use of sucrose as a carbon source. The engineered strain Sp_Bc-Can001 ∆gal80 was evaluated in a 5 L bioreactor fermentation, achieving a notable canthaxanthin titer of 60.36 ± 1.51 mg/L using sucrose. This research conclusively establishes S. cerevisiae as a viable platform for efficient carotenoid biosynthesis and, for the first time in this yeast system, illustrates sucrose’s viability as a carbon source for canthaxanthin production. These findings pave the way for sustainable, cost-effective carotenoid production at an industrial scale.

Biopolymers have various applications in medicine, food and petroleum industries. The ascomycetous fungus Ophiocordyceps dipterigena BCC 2073 produces an exobiopolymer, a (1→3)-β-D-glucan, in low quantity under screening conditions. Optimization of O. dipterigena BCC 2073 exobiopolymer production using experimental designs, a scale-up in 5 liter bioreactor, analysis of molecular weight at different cultivation times, and levels of induction of interleukin-8 synthesis are described in this study.

Phomalactone, an antibacterial, insecticidal, and herbicidal compound, was produced by insect pathogenic fungi, Ophiocordyceps communis BCC 1842 and BCC 2763, in bioreactors using different carbon and nitrogen sources. Glucose and fructose were preferable for growth and phomalactone production. The highest specific growth rate (μ) of 0.012 hr(-1), the highest biomass yield (Ysx) of 0.38 g DW g(-1) sugar, the highest volumetric sugar consumption rate (qs) of 0.036 g (L hr)(-1), the maximum phomalactone concentration ([Formula: see text]) of 93.30 mg L(-1) at 127 hr, and the highest volumetric production rate of phomalactone (qp) of 0.46 ± 0.12 mg (L d)(-1) were obtained on glucose and sodium nitrate as the sole carbon and nitrogen sources, respectively, by O. communis BCC 1842. In contrast, O. communis BCC 2763 gave lower phomalactone production. This mass phomalactone production is useful for the biological synthesis of a precursor for more broad-range potent analogs such as antitumor, antifungal, and others and for its further biological studies.