Microalgae can produce high-value-added products such as lipids and carotenoids using light or sugars, and their biosynthesis mechanism can be triggered by various stress conditions. Under nutrient deprivation or environmental stresses, microalgal cells accumulate lipids as an energy-rich carbon storage battery and generate additional amounts of carotenoids to alleviate the oxidative damage induced by stress conditions. Though stressful conditions are unfavorable for biomass accumulation and can induce oxidative damage, stress-based strategies are widely used in this field due to their effectiveness and economy. For the overproduction of different target products, it is required and meaningful to deeply understand the effects and mechanisms of various stress conditions so as to provide guidance on choosing the appropriate stress conditions. Moreover, the underlying molecular mechanisms under stress conditions can be clarified by omics technologies, which exhibit enormous potential in guiding rational genetic engineering for improving lipid and carotenoid biosynthesis.

Escherichia coli, one of the most efficient expression hosts for recombinant proteins (RPs), is widely used in chemical, medical, food and other industries. However, conventional expression strains are unable to effectively express proteins with complex structures or toxicity. The key to solving this problem is to alleviate the host burden associated with protein overproduction and to enhance the ability to accurately fold and modify RPs at high expression levels. Here, we summarize the recently developed optimization strategies for the high-level production of RPs from the two aspects of host burden and protein activity. The aim is to maximize the ability of researchers to quickly select an appropriate optimization strategy for improving the production of RPs.

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (P_lacUV5), which is leakier and more active than wild-type lac promoter (P_lacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced P_lacUV5 with other inducible promoters respectively, including arabinose promoter (P_araBAD), rhamnose promoter (P_rhaBAD), tetracycline promoter (P_tet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.