In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl₂ catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl₂ catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.

Chitosanase hydrolyzes β-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.