Creative Enzymes conducts DNA expression and enzyme production in a wide range of hosts, serving the whole enzyme industry with high-quality custom expression. Our advanced technology allows us to develop various expression systems in addition to the most commonly used ones. Despite the technical difficulties with some expression hosts such as methylotrophic yeasts, these systems can benefit enzyme production in special needs. With years of experiences in testing and optimizing enzyme production in methylotrophic yeasts, Creative Enzymes has demonstrated high yields and strong genetic skills in the host.

Methylotrophic yeasts, such as Pichia, Hansenula, Candida, and Torulopsis, have become attractive hosts for the industrial production of recombinant enzymes since the expression of these genes are under control of the strongest and most strictly regulated promoters. These yeasts share a common metabolic pathway that enables them to use methanol as a sole carbon source.

Promoted by the success of ecallantide, Pichia pastoris expression system has become the preference choice for some enzyme expression. Both the quantity and quality of enzymes expressed in P. pastoris are beyond those in the prokaryotic expression system. Under the strongest induce AOX1 promoter, P. pastoris can achieve precise and tight regulation of recombinant proteins using methanol. During the fermentation, the growth is very prosperous and high cell densities can be reached. A wide variety of enzymes are expressed yield exceed g L^-1, with the highest up to dozens of g L^-1. Other methylotrophic yeasts such as Hansenula polymorpha also attract growing interests, due to the commercial examples including phytase, hexose oxidase, and lipase, showing advantages of strong and tunable promoters. Compared to S. cerevisiae, it is a notable that methylotrophic hosts tend to secret the enzymes to extracellular, which simplify the purification process and the save the cost. Moreover, the existence of post-translational modification mechanism allows the correct formation of disulfide bonds and glycosylation in enzymes, which is important for enzymatic structures and functions. The absence of α-1,3-liked mannosyl transferase avoids extensive hyper-mannosylation in P. pastoris compared to S. cerevisiae, which is another advantage of this expression system.

There are, however, some disadvantages of using Pichia as a host for heterologous expression. Compared with the bacteria, yeasts have a longer lifespan, meaning longer fermentation period. Also, a common concern about methylotrophs is connected with the inducer methanol, which could be toxic and flammable. In addition, the genetic engineering techniques have not been developed in methylotrophs as deeply as in bacteria, and regulation of expression could be difficult in some cases. Regardless these potential challenges, Creative Enzymes employs rich resources and strong scientist teams to develop and perform reliable expression services through multiple targeted strategies:

• Vectors: Most vectors in P. pastoris are inserted into the genome, which guarantees the stability of the system. We have a variety of vectors to choose from, which can satisfy different research demands such as multi-copies selection and secret expression.

• Promoters: In order to avoid the potential dangers of using AOX1 promoter, we also offer constitutive promoters such as GAP promoter, which has a strength equal to AOX1 promoter but uses glucose as the inducer. Other inducible or constitutive promoters are also available.

• Strains: Usually, Mut⁺ strains such as GS115 and X-33 are used in enzyme production. However, some studies have shown that using Mut^s strains such as KM71 may result in higher yields. Besides, protease-deficient strains like SMD1168 could significantly improve the production yield. These stains are all available from Creative Enzymes.

• Fermentation process optimization: Bioprocess operation parameters can highly influence the product yield and quality. Optimization of these parameters, including culture medium composition, temperature, pH, induction, and feeding strategies, are covered by the service packaged provided by Creative Enzymes.

At Creative Enzymes, our experienced scientists keep detailed studies on the rational design of system construction and optimal fermentation. We can produce small, trial batches or large quantities of enzymes according to specific requests. The expression, purification, and enzyme activity are guaranteed. Our reliable expression service is also performed in a fast turnaround. We sincerely look forward to receiving your inquiry of the enzyme expression service.