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Collagen, the highest content protein in the body, has irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding the recombinant type III human-like collagen α1 chain fragment was integrated into P. pastoris genome after partial amino acids were substituted. Combined with promoter engineering and high-density fermentation technology, soluble secretory expression with the highest yield of 1.05 g L−1 was achieved using two-stage feeding method, and the purity could reach 96% after affinity purification. The determination of N/C-terminal protein sequence were consistent with the theoretical expectation and showed the characteristics of Gly-X-Y repeated short peptide sequence. In amino acid analysis, glycine shared 27.02% and proline 23.92%, which were in accordance with the characteristics of collagen. Ultraviolet spectrum combined with Fourier transform infrared spectroscopy as well as mass spectrometry demonstrated that the target product conformed to the characteristics of collagen spectrums and existed as homologous dimer and trimer in the broth. This work provided a sustainable and economically viable source of the recombinant type III human-like collagen. [ABSTRACT FROM AUTHOR]

Dextranase (DEX) is an important hydrolase in the food industry, which can degrade the dextran into low-molecular-weight polysaccharides. However, its commercial applications are limited by ineffective heterologous recombination system. Here, we build an efficient Pichia pastoris system for DEX following two different engineering strategies. First, through increasing the gene dosage, the DEX activity increased from 40.13 U/mL to 121.02 U/mL. The up-regulation of the endoplasmic reticulum stress gene ERO1 was detected by the qPCR. Since chaperones are involved in the protein folding and modification, co-expression of chaperones was applied to relieve the endoplasmic reticulum stress and facilitated further increase in DEX activity to 164.78 U/mL. Then, high cell density cultivation of P. pastoris was performed in a 5 L fermenter (cell dry weight reached 105.37 g/L), resulting in the DEX activity to 619.78 U/mL. Finally, under the conditions of 25 °C, pH 5.0, 5% sorbitol as co-substrates, and a feed rate of 7.2 mL/h, a DEX yield of 3257.23 U/mL was obtained through 5-L fed-batch fermentation. DCFH-DA staining showed that the reactive oxygen species level decreased by 68.83% due to sorbitol co-feeding. Our study thus represents an important step for building a robust P. pastoris expression system for recombinant DEX production. • The recombinant P. pastoris harboring 7 copies of dextranase gene was constructed. • Effect of constitutive and inducible co-expression of chaperones was investigated. • High-density fermentation was achieved through sorbitol co-feeding. [ABSTRACT FROM AUTHOR]