Cell growth and protein production of recombinant strain with modified α-factor signal peptide (mα-1pRML-X33), strain with original α-factor signal peptide (zα-1pRML-X33), and two control strains (mα-X33 and zα-X33). A: OD600 of the recombinants in flask fermentation. Cell growth did not differ notably among the four strains. B: Enzyme activity of the recombinants during fermentation. Activity of mα-1pRML-X33 (600 U/ml) was higher than that of zα-1pRML-X33 (430 U/ml). Activity increased 1.4-fold after optimization of signal peptide codons. C: Extracellular protein expression in fermentation supernatant detected by Western blotting. Lane 1: protein markers (same as in Figure 1). Lane 2: recombinant strain containing original α-factor signal peptide. Lane 3: strain containing modified α-factor signal peptide. The concentration of extracellular target protein for mα-1pRML-X33 (0.22 mg/mL) was higher than that for zα-1pRML-X33 (0.16 mg/mL). D: The transcription level of prml (detected by qPCR) in mα-1pRML-X33 vs. zα-1pRML-X33. Optimization of signal peptide (mα-1pRML-X33) increased target gene transcription at both 48 and 96 hours.