Background: Pichia pastorishas been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensivein silicomodel ofP. pastoriscan be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.Results: A fully compartmentalized metabolic model ofP. pastoris(iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype ofP. pastorisobserved during chemostat experiments. Subsequentin silicoanalysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinantP. pastorisstrains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability ofP. pastorissystem to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.Conclusion: The genome-scale metabolic model characterizes the cellular physiology ofP. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement throughin silicosimulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design ofP. pastorismetabolic network to enhance humanized glycoprotein production.

Sebacic acid (SA) is an aliphatic ten-carbon dicarboxylic acid (1,10-decanedioic acid) with a variety of industrial applications, including the production of plasticizers, lubricants, cosmetics, and plastics. Currently, SA is produced exclusively from alkaline pyrolysis of castor oil. Herein, we present an environmentally friendly green route of SA production from plant oil-derived sources by microbial ω-oxidation. We genetically engineered β-oxidation-blocked diploid yeast Candida tropicalis, and created an effective microbial cell factory with an increase of 46% in SA production by overexpression of genes involved in ω-oxidation of hydrocarbons compared to the original strain. A biotransformation process of SA production from decanoic acid methyl ester was developed to overcome the challenges of high-density cell culture, substrate feed, substrate/intermediate toxicity, and foam generation. Fed-batch production of engineered C. tropicalis resulted in a molar yield of above 98%, a productivity of 0.57 g L⁻¹ h⁻¹, and a final titre of 98.3 g L⁻¹ in a 5-litre fermenter and the results were successfully reproduced using a larger scale 50-litre fermenter. The produced SAs were successfully purified to >99.8% using acid precipitation and recrystallization. Finally, bio-nylon 610 was successfully synthesized by polymerization of the purified SA with hexamethylenediamine and showed thermal properties very similar to those of commercially available nylon 610. The processes developed and described in this study can be employed to produce and isolate SA for the synthesis of bio-nylons, using environmentally friendly procedures based on microbial biotransformation with potential industrial applications.

To develop a functional phosphate-regulated promoter in Pichia pastoris , a phosphate-responsive gene, PHO89 , which encodes a putative sodium (Na ⁺ )-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter ( P _PHO89 ) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P _PHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1α and the glyceraldehyde-3-phosphate dehydrogenase promoter, P _PHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P _PHO89 -based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P _PHO89 for controlled production of recombinant proteins in P. pastoris .

As a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast Candida tropicalis has been used as a host for DCA production, additional strains are continually investigated to meet productivity thresholds and industrial needs. In this regard, the yeast Wickerhamiella sorbophila, a potential candidate strain, has been screened. However, the lack of genetic and physiological information for this uncommon strain is an obstacle that merits further research. To overcome this limitation, we attempted to develop a method to facilitate genetic recombination in this strain and produce high amounts of DCAs from methyl laurate using engineered W. sorbophila. In the current study, we first developed efficient genetic engineering tools for the industrial application of W. sorbophila. To increase homologous recombination (HR) efficiency during transformation, the cell cycle of the yeast was synchronized to the S/G2 phase using hydroxyurea. The HR efficiency at POX1 and POX2 loci increased from 56.3% and 41.7%, respectively, to 97.9% in both cases. The original HR efficiency at URA3 and ADE2 loci was nearly 0% during the early stationary and logarithmic phases of growth, and increased to 4.8% and 25.6%, respectively. We used the developed tools to construct W. sorbophila UHP4, in which β-oxidation was completely blocked. The strain produced 92.5 g/l of dodecanedioic acid (DDDA) from methyl laurate over 126 h in 5-l fed-batch fermentation, with a productivity of 0.83 g/l/h. Wickerhamiella sorbophila UHP4 produced more DDDA methyl laurate than C. tropicalis. Hence, we demonstrated that W. sorbophila is a powerful microbial platform for vegetable oil-based DCA production. In addition, by using the developed genetic engineering tools, this emerging yeast could be used for the production of a variety of fatty acid derivatives, such as fatty alcohols, fatty aldehydes, and ω-hydroxy fatty acids.

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.